EMBO/soda-nel huggingface dataset:
The nucleoporin ELYS regulates nuclear size by controlling NPC number and nuclear import capacity (2019)
Figure 3

Figure 3: ELYS overexpression increases nuclear size, NPC density, and nuclear levels of lamin B2, Ran, and NTF2. (A) MCF-10AT1k.cl2 cells were co-transfected with control or ELYS siRNA and plasmids expressing eGFP alone or siRNA-resistant eGFP-ELYS. Cell lysates were analyzed by western blot and probed for ELYS and tubulin. ELYS band intensity was normalized to tubulin and then normalized to the eGFP/negative siRNA control. ELYS concentrations are indicated in the row labeled [ELYS] (average ± SD). Quantification was performed on two biological replicates. Cells were stained with an antibody against lamin B1. Representative images are shown. (B) For each experiment, nuclear cross-sectional areas were quantified for 68-298 nuclei per condition (162 nuclei on average) and averaged. Three biological replicates, data from one representative experiment shown. (C) For each experiment, nuclear lamin B2 staining intensities were quantified for 29-81 nuclei per condition (60 nuclei on average) and normalized to the eGFP/negative siRNA control. Two biological replicates, data from one representative experiment shown. (D) Cells with and without cytoplasmic lamin B2 aggregates were counted and the percentage of cells with aggregates was calculated. For each experiment, 58-299 cells were examined per condition. Three biological replicates, data from one representative experiment shown. (E-F) Cells were stained with antibodies against Ran and NTF2. For each experiment, 62-130 nuclei were quantified per condition (92 nuclei on average). Two biological replicates, data from one representative experiment shown. (E) Nuclear Ran and NTF2 staining intensities were quantified and normalized to the eGFP control. (F) To measure the N/C staining intensity ratio of a given cell, the average fluorescence intensity of a nuclear region was divided by the average fluorescence intensity of a cytoplasmic region. (G) Transfections were performed as in (A) and cells were stained with an antibody against FG-Nups (mAb414) and Nup133. Representative images for mAb414 staining are shown. Confocal imaging was performed through nuclear cross-sections as well as on the surface of nuclei. (H) To measure NPC densities, confocal NE surface images were acquired for mAb414 and Nup133 stained nuclei. NPC numbers were counted per unit area for 25-116 nuclei per condition (53 nuclei on average) and normalized to the negative control. Two biological replicates, data from one representative experiment shown.

Two-tailed Student's t-tests assuming equal variances: ***, p<0.005; **, p<0.01; *, p<0.05. Scale bars are 10 µm. Error bars represent SD.
The nucleoporin ELYS regulates nuclear size by controlling NPC number and nuclear import capacity (2019)
Figure 4

Figure 4: Increasing nuclear import by overexpressing importin α rescues nuclear size and lamin aggregation phenotypes in ELYS knockdown cells. (A) MCF-10AT1k.cl2 cells were co-transfected with control or ELYS siRNA and plasmids expressing mCherry alone or mCherry-importin α. Cells were stained with an antibody against lamin B2. Representative images are shown. Scale bar, 10 µm. (B) For each experiment, nuclear cross-sectional areas were quantified for 30-172 nuclei per condition (84 nuclei on average) and averaged. Two biological replicates, data from one representative experiment shown. (C) Cells with and without cytoplasmic lamin B2 aggregates were counted and the percentage of cells with aggregates was calculated. For each experiment, 74-218 cells were examined per condition. Two biological replicates, data from one representative experiment shown. (D) For each experiment, nuclear lamin B2 staining intensities were quantified for 34-187 nuclei per condition (85 nuclei on average) and normalized to the mCherry/negative siRNA control. To measure bulk import capacity for importin α/β cargos, cells were co-transfected as in (A) along with a plasmid expressing GFP-3x SV40 NLS. For each experiment, nuclear GFP-NLS intensities were quantified for 22-188 nuclei per condition (73 nuclei on average) and normalized to the mCherry/negative siRNA control. Two biological replicates, data from one representative experiment shown.

Two-tailed Student's t-tests assuming equal variances: ***, p<0.005; **, p<0.01; NS, not significant. Error bars represent SD.
The nucleoporin ELYS regulates nuclear size by controlling NPC number and nuclear import capacity (2019)
Figure 5

Figure 5: Inhibiting nuclear import leads to smaller nuclei and cytoplasmic lamin aggregates. (A) MCF-10AT1k.cl2 cells were transfected with plasmids expressing mCherry alone or mCherry-RanQ69L and stained with an antibody against lamin B2. Representative images are shown. (B) MCF-10AT1k.cl2 cells were treated with 20 µM importazole or an equivalent volume of DMSO as a control for 24 hours and stained with an antibody against lamin B1. Representative images are shown. (C) For each experiment, nuclear cross-sectional areas were quantified for 32-271 nuclei per condition (123 nuclei on average) and averaged. Three biological replicates, data from one representative experiment shown. (D) To measure bulk import capacity for importin α/β cargos, experiments were performed as in (A-B) except that cells were also transfected with a plasmid expressing GFP-3x SV40 NLS. For each experiment, nuclear GFP-NLS intensities were quantified for 11-75 nuclei per condition (35 nuclei on average) and normalized to the appropriate control. Two biological replicates, data from one representative experiment shown. (E) Cells with and without cytoplasmic lamin B aggregates were counted and the percentage of cells with aggregates was calculated. For each experiment, 31-130 cells were examined per condition. Three biological replicates, data from one representative experiment shown.

Two-tailed Student's t-tests assuming equal variances: ***, p<0.005; **, p<0.01. Scale bars are 10 µm. Error bars represent SD.
The nucleoporin ELYS regulates nuclear size by controlling NPC number and nuclear import capacity (2019)
Figure 6

Figure 6: Reducing nuclear export increases nuclear size. Representative images for XPO1 siRNA are shown in Figure EV4D. (A) Cell lysates from control and XPO1 siRNA transfected MCF-10AT1k.cl2 cells were analyzed by western blot and probed for XPO1 and tubulin. One representative western blot is shown. XPO1 band intensity was normalized to tubulin. Quantification from two biological replicates is shown. (B) MCF-10AT1k.cl2 cells were transfected with control or XPO1 siRNA. Lamin B1 and lamin A/C nuclear staining intensities were quantified based on experiments using two different XPO1 siRNA sequences and two biological replicates. For each experiment, 108-865 nuclei were quantified per condition (416 nuclei on average). Average and SD values based on averages from these four experiments are plotted, normalized to the negative siRNA control. (C) MCF-10AT1k.cl2 cells were transfected with the indicated siRNAs and nuclear cross-sectional areas were quantified. For each experiment, 203-604 nuclei were quantified per condition (403 nuclei on average). Average and SD values based on averages from four biological replicates are plotted, normalized to the negative siRNA control. (D) MCF-10AT1k.cl2 cells were transfected with control siRNA or with siRNA against XPO1. Cells were stained with DAPI and quantification of DNA staining intensity was used to estimate the fraction of cells in various stages of the cell cycle by high-throughput imaging as previously described [123] (see Methods). The stacked bars represent the means of the fractions for each cell cycle phase calculated over 3 biological replicates. The control data are the same shown in Fig. EV1F. (E) MCF-10AT1k.cl2 cells were transfected with control or XPO1 siRNA. For the bottom row of images, cells transfected with control siRNA were treated with 20 ng/ml leptomycin B for 24 hours. Cells were fixed and stained with 2 µg/ml FITC. Representative images are shown. Scale bar, 20 µm. (F) For each experiment, nuclear cross-sectional areas and nuclear FITC staining intensities were quantified for 105-620 nuclei per condition (267 nuclei on average) and normalized to the negative control. Two biological replicates, data from one representative experiment shown.

Two-tailed Student's t-tests assuming equal variances: ***, p<0.005; **, p<0.01; *, p<0.05. Error bars represent SD.

EXPANDED VIEW FIGURE LEGENDS
YAP1-LATS2 feedback loop dictates senescent or malignant cell fate to maintain tissue homeostasis (2019)
Figure 1

Figure 1. Ectopic expression of YAP or constitutively active YAP induce arrest of cell growth in cultured primary human ovarian surface epithelial cells (hOSE).

(A) Left panel: Representative blots showing protein levels of YAP and phosphorylated YAP (Ser127) in control (si-Ctrl) and YAP knockdown (si-YAP) hOSEs. Protein levels were detected by Western blotting. β-Actin was used as a protein loading control. Right panel: Cell proliferation (cell number) in control (si-Ctrl) and YAP knockdown (si-YAP) hOSE cells. Each bar represents the mean ± SEM (n=8). Bars with different letters are significantly different from each other (P < 0.01).

(B) Left panel: Representative blots showing protein levels of YAP and phosphorylated YAP (Ser127) in control (si-Ctrl), YAP knockdown (si-YAP), TAZ knockdown (si-TAZ) and YAP/TAZ double knockdown (si-YAP/TAZ) hOSE Cells. β-Actin was used as a protein loading control. Right panel: Cell proliferation (cell number) of control (si-Ctrl), YAP knockdown (si-YAP), TAZ knockdown (si-TAZ) and YAP/TAZ double knockdown (si-YAP&si-TAZ) hOSE cells. Each bar represents the mean ± SEM (n=6). Bars with different letters are significantly different from each other (P < 0.05).

(C) Representative blots showing protein levels of YAP and phosphorylated YAP (Ser127) in control hOSEs (MXIV) and hOSEs expressing wild type YAP (YAP) or constitutively active YAP (YAPS127A). Cells were collected at passage 4. β-Actin was used as a protein loading control.

(D) Growth of hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells at passage 4. Each bar represents mean ± SEM of four independent samples. Bars with different letters are significantly different from each other (P < 0.001).

(E) Growth curves of hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells at passage 7. Each point represents mean ± SEM of four independent samples.

(F) Representative images showing the morphologic changes of hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells at passage 7. Scale bar: 50 µm.

(G) Representative images showing expression and location of YAP in hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells at passage 7. YAP was visualized using an Alexa-488 (Green) conjugated secondary antibody. Nuclei were stained with DAPI. Scale bar: 50 µm.

Data information: Data were analyzed for significance using one-way ANOVA with Tukey's post-hoc tests.
YAP1-LATS2 feedback loop dictates senescent or malignant cell fate to maintain tissue homeostasis (2019)
Figure 2

Figure 2. YAP induces cellular senescence in cultured primary human OSE cells.

(A) Representative images showing SA-β-gal staining in hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells at passage 7. Scale bar: 50 µm.

(B) Quantitative data showing the ratio of SA-β-gal positive cells in hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells. Each bar represents the mean ± SEM (n=6). Bars with different letters are significantly different from each other (P < 0.05). Data were analyzed for significance using one-way ANOVA with Tukey's post-hoc tests.

(C) Representative blots showing expression of key proteins associated with cell cycle arrest in hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells analyzed using Western blotting at the 7th passage. β-Actin was used as a protein loading control.

(D) Representative images showing expression and location of phosphorylated pRB (807/811) in hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells at the 7th passage. Phosphorylated pRB (807/811) was visualized using an Alexa-488 (Green) conjugated secondary antibody. Nuclei were stained with DAPI (blue). Scale bar: 50 µm. Each experiment was repeated at least three times.
YAP1-LATS2 feedback loop dictates senescent or malignant cell fate to maintain tissue homeostasis (2019)
Figure 3

Figure 3. HPV E6/E7 oncoproteins prevent hOSEs from YAP-induced senescence.

(A) Representative blots showing expression of TP53 and RB1 expression in hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells in the presence or absence of HPV E6/E7 oncoproteins at the 7th passage. β-Actin was used as a protein loading control.

(B) Growth curves of hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells in the presence of HPV E6/E7 oncoproteins at the 9th passage. Each point represents mean ± SEM of four independent samples.

(C) Representative images showing SA-β-gal staining in hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells in the presence or absence of HPV E6/E7 oncoproteins at the 7th passage. Scale bar: 50 µm.

(D) Quantitative data showing the ratio of SA-β-gal positive cells in hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells in the presence or absence of HPV E6/E7 oncoproteins. Each bar represents the mean ± SEM (n=6). Bars with different letters are significantly different from each other (P < 0.01).

(E) Representative images showed SA-β-gal staining in cultured primary hOSE cells in the presence or absence of HPV E6/E7 oncoproteins. Cells were collected and stained at the 13th passage. Scale bar: 25 µm. Right graph showing the ratio of SA-β-gal positive cells in P13 hOSE cells transfected with empty vectors (CTRL-P13) or vectors expressing E6/E7 oncoproteins (E6/E7-P13). Each bar represents the mean ± SEM (n = 4). Bars with different letters are significantly different from each other (P < 0.05).

Data information: Data were analyzed for significance using one-way ANOVA with Tukey's post-hoc tests.
YAP1-LATS2 feedback loop dictates senescent or malignant cell fate to maintain tissue homeostasis (2019)
Figure 4

Figure 4. Involvement of RB1 signaling pathways in YAP-induced senescence.

(A) Representative blots showing the expression of YAP and RB1 in hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells with or without pRB knockdown. Cells were collected at the 7th passage and protein levels were analyzed using Western blotting. β-Actin was used as a protein loading control.

(B) Growth curves of hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells after RB1 knockdown. Each point represents mean ± SEM of four independent samples.

(C) Representative images showing SA-β-gal staining in the hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells with or without RB1 or TP53 knockdown. Cells were collected at the 7th passage. Scale bar: 50 µm.
YAP1-LATS2 feedback loop dictates senescent or malignant cell fate to maintain tissue homeostasis (2019)
Figure 5

Figure 5. Involvement of LATS2 in natural replicative senescence of hOSEs.

(A) Representative blots showing LATS and YAP protein levels in hOSEs at the fourth, ninth and thirteenth passage. β-Actin was used as a protein loading control.

(B) Representative gel photographs showing mRNA levels of LATS1 and LATS2 in hOSEs with or without LATS2 knockout. CRISPR/Cas-9 system was used to knock out LATS2 in hOSEs. LATS1/2 mRNA levels were analyzed using qRT-PCR at the fourth, ninth and thirteenth passage.

(C) Representative images showing the expression and location of YAP in control and LATS2 knockout hOSE cells. Cells were collected at the 4th passage and YAP expression was examined by fluorescent immunohistochemistry. YAP protein was visualized using an Alexa-488 (Green) conjugated secondary antibody. Nuclei were stained with DAPI. Scale bar: 50 µm.

(D) Representative images showing YAP protein expression in the 13th passage hOSE Cells with or without LATS2 knockout. YAP protein was examined by fluorescent immunohistochemistry and visualized using an Alexa-488 (Green) conjugated secondary antibody. Nuclei were stained with DAPI (blue). Scale bar: 50 µm.

(E & F) Representative images showing morphological change (E) and SA-β-gal staining (F) in the 13th passage (P13) hOSE cells with (LATS2 KO) or without (CTRL) LATS2 knockout. Scale bar: 50 µm.

(G) Quantitative data showing the ratio of SA-β-gal positive cells in the control (CTRL) and LATS2-knockout (LATS2KO) P13 hOSE cells. Each bar represents mean ± SEM (n = 4). ***: P < 0.001, compared to control group (CTRL). Data were analyzed for significance using one-way ANOVA with Tukey's post-hoc tests.
YAP1-LATS2 feedback loop dictates senescent or malignant cell fate to maintain tissue homeostasis (2019)
Figure 6

Figure 6. Involvement of LATS2 in YAP-induced senescence of hOSE cells.

(A) Representative blots showing expression levels of LATS1/2, CCNE1, CCND1 and YAP in hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells. Cells were collected at passage four (P4) and Passage seven (P7). Actin was used as internal control.

(B) mRNA levels of LATS1, LATS2 and YAP in hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells. Cells were collected at passage seven and protein levels were analyzed using quantitative real-time PCR. Each bar represents mean ± SEM (n=5). Bars with different letters are significantly different from each other (P < 0.05).

(C) Representative gel photographs showing mRNA levels of LATS1, LATS2, and YAP in the LATS2-knockout hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells examined using semi-quantitative RT-PCR at passage seven.

(D) Representative images showing the morphologic changes and SA-β-gal staining in control and LATS2-knockout hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells. Scale Bar: 50 µm.

(E) Quantitative data showing the ratio of SA-β-gal positive cells in the control and LATS2-knockout hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells at the seventh passage. Each bar represents mean ± SEM of four independent samples. Bars with different letters are significantly different from each other (P < 0.05).

(F) Growth curves of the control and LATS2-knockout hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells. Each point represents mean ± SEM of four independent samples.

Data information: Data were analyzed for significance using one-way ANOVA with Tukey's post-hoc tests.
YAP1-LATS2 feedback loop dictates senescent or malignant cell fate to maintain tissue homeostasis (2019)
Figure 7

Figure 7. Ectopic expression of LATS2 suppressed proliferation and induced senescence in cultured primary hOSE cells.

(A) Representative images showing cells with ectopic expression of LATS2, which is labelled with GFP (the control is empty vectors). Scale bar: 50 µm.

(B) Relative mRNA levels of LATS1, LATS2, and YAP in the control and LATS2-expressing hOSEs at the 4th passage. Relative mRNA levels were determined with RT-PCR. Each bar represents mean ± SEM (n=4). ***: P < 0.001, compared to control.

(C) Growth curves of hOSE cells at the 4th passage with or without ectopic expression of LATS2. Each point represents mean ± SEM of at least four independent samples.

(D) Representative images showing the effects of LATS2 overexpression on the morphology and growth of hOSE cells at the 9th passage. Scale bar: 50 µm.

(E) Growth curves of control and LATS2-overexpressing hOSE cells. Each point represents mean ± SEM of at least four independent samples.

(F) Quantitative data showing the ratio of SA-β-gal positive hOSEs at their 4th passage with or without ectopic expression of LATS2. Each point represents mean ± SEM of at least four independent samples. *: P < 0.05, compared to the control.

(G) Representative images showing the effect of LATS2 on the morphology and β-galactosidase activities in cultured primary hOSE cells at the 9th passage. Scale bar: 50 µm

(H) Quantitative data showing the ratio of SA-β-gal positive hOSEs at their 9th passage with or without ectopic expression of LATS2. Each bar represents mean ± SEM of at least four independent samples. ***: P < 0.001, compared to the control.

(I) mRNA levels of major components of the DREAM complex (E2F4, E2F5, RBBP4, TFDP2) in the control and LATS2-overexpressing hOSEs at the 9th passage. Relative mRNA levels were examined by real-time PCR. Each bar represents mean ± SEM (n = 4). **: P < 0.01; ***: P < 0.001, compared to control.

(J) Growth curves of the hOSE-shRB1 cells and hOSE-shRB1-LATS2 cells at their 16th passages. Each point represents mean ± SEM of at least four independent samples.

(K - L) Representative images showing cellular morphology and β-galactosidase activities in control and LATS2-overexpressing hOSEs (K). Scale Bar: 50 µm. Right bar graph (L) showing quantitative results of SA-β-gal positive cells in the hOSE-shRB1 cells and hOSE-shRB1-LATS2 cells. Each bar represents mean ± SEM of at least four independent samples. ***: P < 0.001, compared to control.

Data information: Data were analyzed for significance using one-way ANOVA with Tukey's post-hoc tests.
YAP1-LATS2 feedback loop dictates senescent or malignant cell fate to maintain tissue homeostasis (2019)
Figure 8

Figure 8. YAP induces malignant transformation of hOSE cells with LATS2 deletion or pRB knockdown.

(A) Representative images showing colony formation in hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells with or without pRB knockdown or LATS2 knockout. hOSEs at the 7th passage were incubated in the growth medium in a soft agar culture system for 9 days before imaging. Scale bar: 500µm.

(B) Quantitative data showing colony numbers in hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells with or without pRB knockdown or LATS2 deletion. hOSE cells at the 7th passage were incubated in the growth medium in a soft agar culture system for 9 days before counting colonies under a microscope. Each bar represents means ± SEM of four independent repeats. Bars with different letters are significantly different from each other (P < 0.001). Data were analyzed for significance using one-way ANOVA with Tukey's post-hoc tests.

(C) Representative images showing tumorigenesis (red circle area) of hOSE-YAPS127A cells with or without pRB knockdown or LATS2 deletion. Tumors formed by hOSE-YAPS127A cells with RB1 knockdown or LATS2 deletion are shown in red circle area. Right panel showing representative tumors formed by hOSE-YAPS127A-LATS2KO and hOSE-YAPS127A-shRB1 cells. Please note that no tumor xenografts formed in hOSE-YAPS127A alone group.

(D) Representative images showing expression of YAP, Ki67 and LATS2 in hOSE-LATS2KO-YAPS127A cell-derived tumors examined by immunohistochemistry. Scale bar: 50 µm.
Stimulation of the insulin/mTOR pathway delays cone death in a mouse model of retinitis pigmentosa (2008)
figf1

All panels show Rho−/− mice with the exception of e. (a–d) Onset of rod death visualized by staining for cleaved nuclear envelope protein LaminA (a), cleaved caspase3 (arrowheads, magenta and red signal, b) and TUNEL (arrows, brown signal, c, d). Blue in a and b shows nuclear DAPI staining. A retinal flat mount with a view onto the photoreceptor layer is shown in d. (e–h) Progression of rod death determined by the reduction of the ONL was visualized by hematoxylin and eosin staining. (i–q) End phase of rod death was assessed by section analysis (i–l) or by retinal flat mounts (m–q). In the Rho−/− mouse, the onset of rod death was around PW5 (a) and progressed up to PW25 (l). By PW17, the ONL was reduced to one row of cells (h, j) and the remaining rods died in the following 8 weeks (j–q), as seen by immunofluorescence with an antibody to Gnat1 on sections of progressively older animals (j–l). Retinal flat mounts showing rods visualized by immunofluorescence with an antibody Gnat1 are shown in m–q. The entire retina is shown in m, higher magnifications around the optic nerve head are shown in n and o, and the peripheral region is shown in p. No signal was seen at PW25 on flat mounts (q) or sections (l). Age (in PW) is indicated in the panels. Vertical bar in a–c and e–l indicates thickness of the ONL.