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Journal Articles

γ-tubulin mediates DNA double-strand break repair

In collection:
Nuclear structure and function
Abhishikt David Solomon, Odjo G. Gouttia, Ling Wang, Songli Zhu, Feifei Wang, Yanqui Li, Mohammadjavad Paydar, Tadayoshi Bessho, Benjamin H. Kwok, Aimin Peng
Journal: Journal of Cell Science
J Cell Sci (2025) 138 (6): jcs262255.
https://doi.org/10.1242/jcs.262255
Published: 26 March 2025
View article titled, γ-tubulin mediates DNA double-strand break repair
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Includes: Supplementary data
Journal Articles

Direct neutrophil and T cell contact with macrophages induces release of phagosomally processed PAMPs via eructophagy

In collection:
Cell biology of the immune system
Jenny A. Nguyen, Tanis L. Orsetti, Philip Vernon, Catherine J. Greene, Neil McKenna, Robin M. Yates
Journal: Journal of Cell Science
J Cell Sci (2025) 138 (6): jcs263731.
https://doi.org/10.1242/jcs.263731
Published: 26 March 2025
View article titled, Direct neutrophil and T cell contact with macrophages induces release of phagosomally processed PAMPs via eructophagy
Open the PDF for Direct neutrophil and T cell contact with macrophages induces release of phagosomally processed PAMPs via eructophagy in another window
Includes: Supplementary data
Journal Articles

First person – Abhishikt David Solomon

Journal: Journal of Cell Science
J Cell Sci (2025) 138 (6): jcs263871.
https://doi.org/10.1242/jcs.263871
Published: 26 March 2025
View article titled, First person – Abhishikt David Solomon
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Journal Articles

Eructophagy boosts immune communication via ICAM1

Journal: Journal of Cell Science
J Cell Sci (2025) 138 (6): e138_e0603.
Published: 26 March 2025
View article titled, Eructophagy boosts immune communication via ICAM1
Images
The recruitment of γ-tubulin to laser-induced DNA damage sites.

The recruitment of γ-tubulin to laser-induced DNA damage sites.

in First person – Abhishikt David Solomon > Journal of Cell Science
Published: 26 March 2025
The recruitment of γ-tubulin to laser-induced DNA damage sites. More about this image found in The recruitment of γ-tubulin to laser-induced DNA damage sites.
Images
γ-tubulin associates with DSB-mimicking DNA in vitro, and is recruited to DNA damage sites in cells. (A) Pulldown with biotin-labeled dsDNA (500 bp with free ends, as described in Zhu et al., 2020) mimicking double-strand breaks (DSBs) was performed using HeLa cell lysates. A control (ctr) pulldown was performed using blank beads. The input, control pulldown and dsDNA pulldown samples were analyzed by immunoblotting (IB). n=3 independent experiments. (B) SCC38 cells were laser-microirradiated and analyzed by immunofluorescence for γ-tubulin and phospho-ATM S1981, as described in the Materials and Methods. The images were taken 3 min post laser treatment. The region of laser microirradiation is denoted by the white arrow. n=3 independent experiments. (C,D) HeLa cells with transient expression of mCherry–γ-tubulin and GFP–polymerase λ were laser microirradiated after pre-sensitization and imaged, as described in the Materials and Methods. The images were taken 5 min post laser treatment. Colocalization of mCherry–γ-tubulin and GFP–polymerase λ after laser microirradiation was quantified in panel C and shown in panel D. Arrows in the inset images in C indicate the points chosen for colocalization analysis. The regions of laser microirradiation are denoted by white arrows in D. n=3 independent experiments. (E) HeLa cells were treated with etoposide (1 µM, 10 min) and analyzed by immunofluorescence for γ-tubulin and 53BP1. The colocalization is shown. n=3 independent experiments. (F) Whole-cell extracts (WCE) and nuclear fractions were isolated from HeLa cells with or without etoposide treatment (1 µM, 3 h), as described in the Materials and Methods, and analyzed by IB for γ-tubulin and histone H3. (G) The IB results in panel F were quantified. Band intensities of γ-tubulin were normalized to those of histone H3. n=3 independent experiments. Bars show mean±s.d. Two-tailed unpaired Student's t-test was performed; ns, not significant; *P<0.05.

γ-tubulin associates with DSB-mimicking DNA in vitro , and is recruited to...

in γ-tubulin mediates DNA double-strand break repair > Journal of Cell Science
Published: 26 March 2025
Fig. 1. γ-tubulin associates with DSB-mimicking DNA in vitro , and is recruited to DNA damage sites in cells. (A) Pulldown with biotin-labeled dsDNA (500 bp with free ends, as described in Zhu et al., 2020 ) mimicking double-strand breaks (DSBs) was performed using HeLa cell lysates. A control... More about this image found in γ-tubulin associates with DSB-mimicking DNA in vitro , and is recruited to...
Images
γ-tubulin associates with DNA repair proteins. (A) As described in the Materials and Methods, γ-tubulin immunoprecipitation (IP) was performed in lysates of HeLa cells that were untreated or treated with bleomycin (20 µM, 3 h) to induce DNA damage. The γ-tubulin IP products and control IP products with blank beads were subjected to proteomic analysis for identification of associated proteins. Poly-ADP-ribose polymerase 1 (PARP1) and the catalytic subunit (DNA-PKcs) of the DNA-dependent protein kinase complex were identified as γ-tubulin-associated proteins, as shown by the numbers of identified peptides, percentage of coverage and Mascot scores (via RAW MS data to MGF conversion and Mascot Server analysis). (B) γ-tubulin IP was performed in HeLa cells with or without etoposide. The input (10%), control IP using blank beads and γ-tubulin IP products were analyzed by IB for the indicated proteins. (C–E) Quantification of control or γ-tubulin IP products, as shown in panel B, was shown as the percentage of the input. Bars show mean±s.d. Two-tailed unpaired Student's t-test was performed from >3 independent experiments; *P<0.05; **P<0.01; ***P<0.001. (F) Ku80 IP was performed in HeLa cells with bleomycin treatment. The input, control IP using blank beads, and Ku80 IP products were analyzed by IB. n=3 independent experiments.

γ-tubulin associates with DNA repair proteins. (A) As described in the Mat...

in γ-tubulin mediates DNA double-strand break repair > Journal of Cell Science
Published: 26 March 2025
Fig. 2. γ-tubulin associates with DNA repair proteins. (A) As described in the Materials and Methods, γ-tubulin immunoprecipitation (IP) was performed in lysates of HeLa cells that were untreated or treated with bleomycin (20 µM, 3 h) to induce DNA damage. The γ-tubulin IP products and control I... More about this image found in γ-tubulin associates with DNA repair proteins. (A) As described in the Mat...
Images
Inhibition of γ-tubulin leads to accumulation of endogenous DNA damage and delayed DNA repair. (A) HeLa cells were treated with control or γ-tubulin siRNA for 1 day. The cells were harvested and analyzed by IB. n=3 independent experiments. (B) HeLa cells were treated with control or γ-tubulin siRNA, and with or without transfection of a γ-tubulin-expressing vector, for 1 day. The cells were harvested and analyzed by IB. n=3 independent experiments. (C) For the repair kinetics analysis, HeLa cells with control or γ-tubulin siRNA were treated with 1 μM etoposide for 1 h, followed by repair/recovery for 0, 3, 6 and 9 h. The cells were harvested and analyzed by IB. n=3 independent experiments. (D) HeLa cells were treated with or without 6 μM gatastatin G2 and irradiated (IR), followed by repair/recovery for 0, 3, 6 and 9 h. The cells were harvested and analyzed by IB. n=3 independent experiments.

Inhibition of γ-tubulin leads to accumulation of endogenous DNA damage and ...

in γ-tubulin mediates DNA double-strand break repair > Journal of Cell Science
Published: 26 March 2025
Fig. 3. Inhibition of γ-tubulin leads to accumulation of endogenous DNA damage and delayed DNA repair. (A) HeLa cells were treated with control or γ-tubulin siRNA for 1 day. The cells were harvested and analyzed by IB. n =3 independent experiments. (B) HeLa cells were treated with control or γ-... More about this image found in Inhibition of γ-tubulin leads to accumulation of endogenous DNA damage and ...
Images
γ-tubulin suppression reduces DSB repair efficacy. (A,B) Chromosome-integrated, I-SceI-induced homologous recombination (HR) (A) or non-homologous end joining (NHEJ) (B) assays were performed as described in the Materials and Methods. The reporter cells, HeLa-Dr or U2OS-EJ5, were transfected with control (ctr) or γ-tubulin siRNA. I-SceI endonuclease was expressed using a lentiviral vector. Following 48 h incubation, DNA repair was measured by IB to assess GFP expression relative to SMC1 expression. The GFP/SMC1 ratio in γ-tubulin-depleted cells was normalized to that in control cells for relative repair efficiency. The mean±s.d. values, calculated from three independent experiments, are shown. Statistical significance was analyzed using an unpaired two-tailed Student's t-test; *P<0.05; **P<0.01. (C) HeLa cells treated with control or γ-tubulin siRNA were irradiated with 20 Gy of X-ray irradiation (IR), followed by repair/recovery for 30 min. Chromatin fractionation was performed as described in the Materials and Methods. Whole-cell lysates and chromatin-enriched fractions were analyzed by IB. n=5 independent experiments. (D) HeLa cells were treated with control or γ-tubulin siRNA. Cells were then control treated (‘C’) or treated with hydroxyurea (HU, 5 mM) for 2 or 4 h. Chromatin fractionation was performed. Whole-cell lysates and chromatin-enriched fractions were analyzed by IB. n=5 independent experiments. (E) HeLa cells were treated with control or γ-tubulin siRNA. Cells were then control treated (‘C’) or treated with HU at the indicated concentrations for 3 h. Chromatin fractionation was performed. Whole-cell lysates and chromatin-enriched fractions were analyzed by IB. n=5 independent experiments. (F) HeLa cells were treated with or without γ-tubulin siRNA. Cells were then control treated (‘C’) or treated with 20 Gy IR. Cells were incubated post IR for the indicated durations (in minutes) and then subjected to chromatin fractionation. Whole-cell lysates and chromatin-enriched fractions were analyzed by IB. n=5 independent experiments. (G) The chromatin recruitment of NHEJ and HR repair proteins, normalized to that of H3, was analyzed for control or γ-tubulin siRNA-treated groups. n=5 independent experiments. Bars show mean±s.d. Statistical analysis was performed using two-tailed unpaired Student's t-test; ***P<0.001.

γ-tubulin suppression reduces DSB repair efficacy. (A,B) Chromosome-integr...

in γ-tubulin mediates DNA double-strand break repair > Journal of Cell Science
Published: 26 March 2025
Fig. 4. γ-tubulin suppression reduces DSB repair efficacy. (A,B) Chromosome-integrated, I-SceI-induced homologous recombination (HR) (A) or non-homologous end joining (NHEJ) (B) assays were performed as described in the Materials and Methods. The reporter cells, HeLa-Dr or U2OS-EJ5, were transfe... More about this image found in γ-tubulin suppression reduces DSB repair efficacy. (A,B) Chromosome-integr...
Images
γ-tubulin depletion reduces the mobilization and formation of DNA damage foci. (A–D) Wild-type (WT) or Kif2C knockout (KO) U2OS cells were treated with control or γ-tubulin siRNA (knockdown, KD). (A) Mean square displacement measurements of EGFP–53BP1 foci were carried out as described in the Materials and Methods, and are shown. (B) Examples of 10 min mobility traces of EGFP–53BP1 are shown. (C) The measured distances traveled by individual 53BP1 foci are shown. The central bar shows the mean. Statistical analysis was performed using two-tailed unpaired Student's t-test; ns, not significant; **P<0.01; ***P<0.001. (D) The depletion of γ-tubulin was confirmed by IB. EGFP–53BP1 was transiently transfected, and expressed at a moderate level, as observed by immunofluorescence (Fig. S7C), relative to endogenous 53BP1. (E,F) HeLa cells expressing mApple–53BP1 were treated with control or γ-tubulin siRNA. Cells were irradiated with 20 Gy of X-ray irradiation (IR), followed by 30 min incubation. 53BP1 foci are shown in panel F and quantified in panel E (n>100). All data were collected from at least three independent experimental sets as mean±s.d. **P<0.01 by unpaired two-tailed Student's t-test.

γ-tubulin depletion reduces the mobilization and formation of DNA damage fo...

in γ-tubulin mediates DNA double-strand break repair > Journal of Cell Science
Published: 26 March 2025
Fig. 5. γ-tubulin depletion reduces the mobilization and formation of DNA damage foci. (A–D) Wild-type (WT) or Kif2C knockout (KO) U2OS cells were treated with control or γ-tubulin siRNA (knockdown, KD). (A) Mean square displacement measurements of EGFP–53BP1 foci were carried out as described i... More about this image found in γ-tubulin depletion reduces the mobilization and formation of DNA damage fo...
Images
Inhibition of γ-tubulin enhances tumor cell responses to DNA damage. (A–D) HeLa cells were treated with control, etoposide (2 µM), gatastatin G2 (2 µM), doxorubicin (3 µM), MR-3 (15 µM) and combinations of drugs, as indicated. Cell numbers were quantified as described in the Materials and Methods. Relative cell viability was calculated by normalizing the final cell count relative to the count recorded on the initial day. The displayed results depict the mean±s.d. derived from at least three distinct experiments. *P<0.05; **P<0.01; two-way ANOVA with Tukey's multiple post hoc test. (E,F) SCC38 cells were treated with control, etoposide (3 µM), doxorubicin (4 µM), MR-3 (15 µM) and combinations of drugs, as indicated. Cell numbers were quantified, and relative cell viability was calculated by normalizing the final cell count relative to the count recorded on the initial day. The displayed results depict the mean±s.d. derived from at least three distinct experiments. *P<0.05; **P<0.01; one-way ANOVA with Dunnett's multiple post hoc test. (G–I) SCC38 cells were maintained under anchorage-independent culture conditions. Cells were then incubated with gatastatin G2 (3 µM), MR-3 (15 µM), doxorubicin (4 µM) and combinations of drugs, as indicated. (G) Representative images of spheroid formation. (H,I) The relative diameters of spheroids in treated groups were normalized to that of the control. The data were analyzed from at least three independent experimental sets using one-way ANOVA with Dunnett's multiple post hoc test and are depicted as mean±s.e.m. ***P<0.001.

Inhibition of γ-tubulin enhances tumor cell responses to DNA damage. (A–D)...

in γ-tubulin mediates DNA double-strand break repair > Journal of Cell Science
Published: 26 March 2025
Fig. 6. Inhibition of γ-tubulin enhances tumor cell responses to DNA damage. (A–D) HeLa cells were treated with control, etoposide (2 µM), gatastatin G2 (2 µM), doxorubicin (3 µM), MR-3 (15 µM) and combinations of drugs, as indicated. Cell numbers were quantified as described in the Materials an... More about this image found in Inhibition of γ-tubulin enhances tumor cell responses to DNA damage. (A–D)...
Images
Microtubule components mobilize DSBs to form repair centers and promote DNA repair. (A) γ-tubulin, Kif2C and potentially other microtubule (MT) components facilitate DSB mobilization, resulting in the clustering of DSBs and formation of repair centers. An anticipated consequence of the formation of repair centers is increased local concentration of DNA repair proteins and, subsequently, enhanced recruitment of these proteins to DSBs. Ka, association constant; Kd, dissociation constant. (B) Mathematical modeling of increased DNA damage recruitment of repair proteins due to γ-tubulin-mediated foci formation. The assumed parameters include cell nucleus size (10 µm), DNA damage focus size (0.5 µm), 5% of repair protein recruitment, 600 total DSBs induced and 15 DSBs clustering within a focus. The overall recruitment of the repair protein to each DSB is projected to be twofold higher with DSB clustering, consistent with our chromatin fractionation analyses.

Microtubule components mobilize DSBs to form repair centers and promote DNA...

in γ-tubulin mediates DNA double-strand break repair > Journal of Cell Science
Published: 26 March 2025
Fig. 7. Microtubule components mobilize DSBs to form repair centers and promote DNA repair. (A) γ-tubulin, Kif2C and potentially other microtubule (MT) components facilitate DSB mobilization, resulting in the clustering of DSBs and formation of repair centers. An anticipated consequence of the f... More about this image found in Microtubule components mobilize DSBs to form repair centers and promote DNA...
Images
Contact-induced macrophage eructophagy is ICAM1-dependent. (A) Schematic of the neutrophil–macrophage co-culture assay to assess macrophage eructophagy. Created in BioRender by Yates, R., 2025. https://BioRender.com/w70t854. Republished with permission. (B–E) Rates of eructophagy were detected by the resorufin-cellobioside reporter assay for (B–E) WT, (C,D) Lfa1−/− or Icam1−/− BMMɸs co-cultured with or without (B) live or fixed neutrophils, (C) WT neutrophils with or without the presence of a CD18 blocking antibody, (D) WT, Lfa1−/− or Icam1−/− neutrophils, or (E) naïve or activated CD4+ T cells. (F) BMMɸs were treated with or without a ICAM1 primary antibody (1° Ab; 10 μg/ml), followed with or without treatment of a goat anti-rat-IgG secondary antibody (2° Ab; 10 μg/ml), immediately prior to imaging. Rates were made relative to a WT and/or no treatment control. Error bars represent means±s.e.m. (n≥3). Each data point represents an experimental replicate. ns, not statistically different; *P≤0.05; **P≤0.005; ***P≤0.0005 [one-way ANOVA followed by a Bonferroni multiple comparisons test (B–E); two-way ANOVA with Bonferroni comparison of means (F)].

Contact-induced macrophage eructophagy is ICAM1-dependent. (A) Schematic o...

in Direct neutrophil and T cell contact with macrophages induces release of phagosomally processed PAMPs via eructophagy > Journal of Cell Science
Published: 26 March 2025
Fig. 1. Contact-induced macrophage eructophagy is ICAM1-dependent. (A) Schematic of the neutrophil–macrophage co-culture assay to assess macrophage eructophagy. Created in BioRender by Yates, R., 2025. https://BioRender.com/w70t854 . Republished with permission. (B–E) Rates of eructophagy were ... More about this image found in Contact-induced macrophage eructophagy is ICAM1-dependent. (A) Schematic o...
Images
Lyn-deficient macrophages have ablated contact-induced eructophagy. (A) Eructophagy and knockdown efficiencies of screened selected genes in GB8 macrophages in the absence of neutrophils and T cells. Gene products with >30% reduction in eructophagy were determined as positive ‘hits’ (green). n=1 with three internal replicates. Rates were made relative to a WT and/or no treatment control. (B,C) Verification of positive ‘hits’ from the knockdown screen. (D) Quantification of eructophagy in WT or Lyn−/− GB8-Mɸs with or without added neutrophils or naïve CD4+ T cells. (E) Phagocytic index assessed by inside or outside labelling of phagocytosed ovalbumin (OVA)-conjugated beads. Images were acquired on a Leica SP5 confocal microscope. Representative images are shown. Scale bars: 10 μm. (F) Phagocytic index was determined based on the ratio of internalised beads per cell and made relative to WT control macrophages. Error bars represent means±s.e.m. (n≥3). Each data point represents an experimental replicate. ns, not statistically different; *P≤0.05; **P≤0.005; ***P≤0.0005 [two-tailed unpaired Student's t-test (B,C,F); one-way ANOVA followed by a Bonferroni multiple comparisons test (D)]. ns, not statistically different.

Lyn-deficient macrophages have ablated contact-induced eructophagy. (A) Er...

in Direct neutrophil and T cell contact with macrophages induces release of phagosomally processed PAMPs via eructophagy > Journal of Cell Science
Published: 26 March 2025
Fig. 2. Lyn-deficient macrophages have ablated contact-induced eructophagy. (A) Eructophagy and knockdown efficiencies of screened selected genes in GB8 macrophages in the absence of neutrophils and T cells. Gene products with >30% reduction in eructophagy were determined as positive ‘hits’ (... More about this image found in Lyn-deficient macrophages have ablated contact-induced eructophagy. (A) Er...
Images
Lyn-deficient macrophages fail to activate neighbouring cells through the release of phagosomally processed PAMPs. (A) Representative confocal images demonstrating p65 (a NF-κB subunit, also known as RELA)-positive nuclei in CD4+ T cells. Scale bars: 5 μm. Percentage of p65-positive nuclei in WT or Lfa1−/− CD4+ T cells after co-culture with (B) WT or Icam1−/− BMMɸs and (C) WT or Lyn−/− GB8-MØs post-phagocytosis of phagocytic targets. Five fields of views were imaged over three biologically independent experiments. Error bars represent means±s.e.m. (n=3 experimental replicates). Each data point represents a technical replicate within an experimental replicate. ns, not statistically different; ***P≤0.0005 (one-way ANOVA followed by a Bonferroni multiple comparisons test).

Lyn-deficient macrophages fail to activate neighbouring cells through the r...

in Direct neutrophil and T cell contact with macrophages induces release of phagosomally processed PAMPs via eructophagy > Journal of Cell Science
Published: 26 March 2025
Fig. 3. Lyn-deficient macrophages fail to activate neighbouring cells through the release of phagosomally processed PAMPs. (A) Representative confocal images demonstrating p65 (a NF-κB subunit, also known as RELA)-positive nuclei in CD4 + T cells. Scale bars: 5 μm. Percentage of p65-positive nu... More about this image found in Lyn-deficient macrophages fail to activate neighbouring cells through the r...
Journal Articles

An ER-associated structure sequesters misassembled FG-rich nucleoporins to help maintain nuclear pore complex function

In collection:
Cell biology and disease , Nuclear structure and function
Madison Pletan, Emily Wang, Luke Gohmann, Billy Tsai
Journal: Journal of Cell Science
J Cell Sci (2025) 138 (6): jcs263659.
https://doi.org/10.1242/jcs.263659
Published: 25 March 2025
View article titled, An ER-associated structure sequesters misassembled FG-rich nucleoporins to help maintain nuclear pore complex function
Open the PDF for An ER-associated structure sequesters misassembled FG-rich nucleoporins to help maintain nuclear pore complex function in another window
Includes: Supplementary data
Journal Articles

Fibronectin matrix assembly at a glance

In collection:
Adhesion
Yu Sun, Aaron J. Hamlin, Jean E. Schwarzbauer
Journal: Journal of Cell Science
J Cell Sci (2025) 138 (6): jcs263834.
https://doi.org/10.1242/jcs.263834
Published: 25 March 2025
View article titled, Fibronectin matrix assembly at a glance
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Includes: Supplementary data
Images
Depletion of Nup98 causes FG-Nups to form foci in the ER. (A) COS-7 cells transfected with scrambled control siRNA (Scr) or siRNA against Nup98 (Nup98 #1 siRNA) were lysed, and the resulting whole-cell lysates analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. Blots shown are representative of three experiments. (B) COS-7 cells with siRNA treatments as in A were stained with the Nup98 antibody. Images shown are representative of three experiments. (C,D) COS-7 cells transfected with the indicated siRNAs were fixed and stained for FG-Nups (MAb414 antibody) and assessed by immunofluorescence microscopy. Representative images are shown in C. Presence of ER foci was quantified (D) by condition-masked counting of MAb414- and Bap31-colocalized puncta. Graph represents mean±s.d. from three independent experiments. The significance was determined via unpaired two-tailed Student's t-test. ***P≤0.001. (E) The relative MAb414 signal intensities in the Nuc versus Non-Nuc regions of cells as in C were determined using an automated protocol (see Materials and Methods). Graph represents mean±s.d. from three independent experiments. The significance was determined via unpaired two-tailed Student's t-test. ***P≤0.001. (F) COS-7 cells were transfected with the indicated siRNAs, harvested and subjected to nuclear fractionation (see Materials and Methods) to yield a whole-cell lysate sample, a Non-Nuc fraction and a Nuc fraction. These fractions were subjected to SDS-PAGE and immunoblotting using the indicated antibodies. Blots shown are representative of three experiments. (G) Schematic of experiments in H and I: FLAG–GFP or Nup98–GFP was transfected before cells were transfected with either Scr or Nup98 #1 siRNA (Pre-KD) or after cells were transfected with either Scr or Nup98 #1 siRNA (Post-KD). (H) Quantification of the percentage of cells with MAb414+ ER foci in the experiment depicted schematically in G. Graphs represent mean±s.d. from three independent experiments. The significance was determined via unpaired two-tailed Student's t-test. ***P≤0.001; **P≤0.01; *P≤0.05. (I) Representative images of Nup98 KD cells transfected with Nup98–GFP either Pre-KD (top row) or Post-KD (bottom row). Scale bars: 10 µm.

Depletion of Nup98 causes FG-Nups to form foci in the ER. (A) COS-7 cells ...

in An ER-associated structure sequesters misassembled FG-rich nucleoporins to help maintain nuclear pore complex function > Journal of Cell Science
Published: 25 March 2025
Fig. 1. Depletion of Nup98 causes FG-Nups to form foci in the ER. (A) COS-7 cells transfected with scrambled control siRNA (Scr) or siRNA against Nup98 (Nup98 #1 siRNA) were lysed, and the resulting whole-cell lysates analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. Blots s... More about this image found in Depletion of Nup98 causes FG-Nups to form foci in the ER. (A) COS-7 cells ...
Images
Nup-containing ER foci formation requires the ER morphogenic membrane proteins RTN3, ATL3 and LNP. (A) COS-7 cells transfected with the indicated siRNAs were lysed, and the resulting whole-cell lysates analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. The anti-RTN3 antibody used detects RTN3A–RTN3C; in COS7 cells the predominant form is the shorter RTN3B/C form shown in the figure. The anti-RTN4 antibody used detects RTN4A/B. Blots shown are representative of three experiments. (B,C) COS-7 cells transfected with the indicated siRNAs were fixed and stained for FG-Nups (MAb414 antibody) and assessed by immunofluorescence microscopy. Representative images are shown in B. Scale bar: 10 µm. Presence of ER foci was quantified (C) by condition-masked counting of MAb414- and Bap31-colocalized puncta. Graph represents mean±s.d. from three independent experiments. The significance was determined via unpaired two-tailed Student's t-test. ***P≤0.001; ns, not significant. (D) Cells as in B were quantified as in Fig. 1E. Graph represents mean±s.d. from three independent experiments. The significance was determined via unpaired two-tailed Student's t-test. ***P≤0.001; *P≤0.05; ns, not significant. (E) Cells with siRNA treatments as in B were analyzed as in Fig. 1F. Blots shown are representative of three experiments.

Nup-containing ER foci formation requires the ER morphogenic membrane prote...

in An ER-associated structure sequesters misassembled FG-rich nucleoporins to help maintain nuclear pore complex function > Journal of Cell Science
Published: 25 March 2025
Fig. 2. Nup-containing ER foci formation requires the ER morphogenic membrane proteins RTN3, ATL3 and LNP. (A) COS-7 cells transfected with the indicated siRNAs were lysed, and the resulting whole-cell lysates analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. The anti-RTN3 a... More about this image found in Nup-containing ER foci formation requires the ER morphogenic membrane prote...
Images
Preventing accumulation of misassembled FG-Nups at the ER foci impairs nuclear transport function. (A–C) COS-7 cells were transfected with either the scrambled control siRNA (Scr) or siRNAs targeting Nup98 (Nup98 #1 siRNA) and ER morphogenic proteins (for double KD), as indicated, for 24 h, followed by 24 h of transfection with an mCherry–NLS construct. Cells were fixed and stained, and the location of the mCherry signal was assessed. (A) Representative images. Scale bar: 10 μm. (B) Graph represents the mean±s.d. percentage of cells with the indicated mCherry localization from three independent experiments as in A. The significance was determined using unpaired two-tailed Student's t-test. ***P≤0.001; **P≤0.01; ns, not significant. (C) Graph represents per-cell ratio of nuclear to whole-cell mCherry fluorescence of n>100 cells from three independent experiments as in A. The significance was determined via unpaired two-tailed Student's t-test. ****P≤0.0001; ns, not significant. (D) As in B, except cells were transfected with indicated siRNA (single KD). (E) Cell viability under the knockdown conditions from B was assessed using an MTS absorbance assay (see Materials and Methods). Graph represents the mean±s.d. from three independent experiments. ns, not significant. The significance was determined via unpaired two-tailed Student's t-test. (F–H) COS-7 cells were transfected with either scrambled control siRNA or siRNAs targeting Nup98 and ER morphogenic proteins (double KD), as indicated, for 48 h. Cells were fixed and stained, and the location of TAg signal was assessed. (F) Representative images. Scale bar: 10 μm. (G) Graph represents mean±s.d. percentage of cells with the indicated TAg localization from three independent experiments as in F. The significance was determined using unpaired two-tailed Student's t-test. ***P≤0.001. (H) Graph represents per-cell ratio of nuclear to whole-cell TAg fluorescence of n>300 cells from three independent experiments as in F. The significance was determined via unpaired two-tailed Student's t-test. ****P≤0.0001. Violin plots in C and H show the distribution of data, with the median (dashed line) and upper and lower quartiles (dotted lines) marked.

Preventing accumulation of misassembled FG-Nups at the ER foci impairs nucl...

in An ER-associated structure sequesters misassembled FG-rich nucleoporins to help maintain nuclear pore complex function > Journal of Cell Science
Published: 25 March 2025
Fig. 3. Preventing accumulation of misassembled FG-Nups at the ER foci impairs nuclear transport function. (A–C) COS-7 cells were transfected with either the scrambled control siRNA (Scr) or siRNAs targeting Nup98 (Nup98 #1 siRNA) and ER morphogenic proteins (for double KD), as indicated, for 24... More about this image found in Preventing accumulation of misassembled FG-Nups at the ER foci impairs nucl...