EMBO/soda-nel huggingface dataset:
miR-10b expression in breast cancer stem cells supports self-renewal through negative PTEN regulation and sustained AKT activation (2016)
Figure 4

Figure 4. The effects of miR-10b on stemness are mediated by the PI3K pathway.

(A) AKT phosphorylation in CSCs from MCF-7 cells sorted by CD44. GAPDH protein levels were used as a loading control.

(B) Upper panel. Cultures were serum-starved for 18 h and then stimulated with insulin or exposed to the PI3K pathway inhibitors Wortmannin or LY294002. PTEN, PI3KCA, GSK3 and phospho-AKT Ser-473 protein levels were analyzed by Western blot. GAPDH was used as a loading control. AKT kinase activity was analyzed in these cells by immunoprecipitating phosphorylated AKT proteins with a p-AKT-Ser-473 antibody and using chimeric GSK-3 as a substrate. A two-tailed Student’s t test was used to compare the two groups (p<0.05). SEM, standard error of the mean. Lower panel. SKBR3 cells were transiently transfected with a plasmid containing an open reading frame of PTEN, miR-10b or both and phospho-AKT, AKT or tubulin levels analyzed by Western Blot assays. Control is empty vector-transfected cells.

(C) Clonogenic ability of miR-10b-depleted CD44+ MCF-7 cells after PI3K pathway inhibition. Cultures were serum-starved for 18 h and then stimulated with insulin or exposed to the PI3K pathway inhibitors wortmannin or LY294002. Cells transfected with anti-miR-10b or a scrambled control (anti-miR-ctl) were seeded in soft agar at clonal density and exposed to insulin or PIK3CA inhibitors. After 3 weeks, colonies were quantified. Bars show the mean ± SEM for 3 independent experiments.

(D) miR-10b overexpression does not induce additional changes in cell colony numbers in response to insulin or PI3K inactivation. MCF-7 stable miR-Scr-OE or miR-10b-OE cells were seeded in soft agar at clonal density and exposed to insulin or PIK3CA inhibitors. After 3 weeks, colonies were quantified. Bars show the mean ± SEM for 3 independent experiments.
Mechanical strain stimulates COPII-dependent secretory trafficking via Rac1 (2022)
Figure 1

Figure 1. Mechanical strain stimulates ERES and ER-to-Golgi transport

(A) Representative immunofluorescence images of Sec31A marked ERES in HeLa cells cultured on crossbow shaped micropatterned surface of small (upper panel) or large (lower panel) size for 4 hours. Cells were treated with DMSO (Ctrl) or 50 µM NSC23766 (Rac1 inhibitor) for 4 hours prior to fixation and immunostaining. Scale bars: 5 µm

(B-C) The number of ERES per cell for each pattern size in HeLa (B) and RPE-1 (C) cells. The number of ERES was quantified from at least 74 cells per condition for HeLa (B) and at least 42 cells per condition for RPE-1 (C) from 3 independent experiments. Each dot represents an individual cell.

(D) HeLa cells transiently expressing GFP-Sec16A were grown on PDMS membranes, treated as indicated and subjected to 7.5% equibiaxial stretch. The effect of stretch on ERES was then monitored by live cell imaging. Representative still images are shown. Scale bar: 5 µm. See also movieEV1.

(E) Quantification of the experiments in (C). ERES increase in HeLa cells before and during equibiaxial stretch were quantified from a total of 19 cells in 4 independent live cell imaging experiments. Baseline GFP-Sec16A count from control (Ctrl) before strain was used to normalize GFP-Sec16A counts for each condition. Shading represents standard deviation.

(F) HeLa cells transfected with Rac1 FRET biosensor were cultured on crossbow micropatterns and allowed to grow for 4 hours before Rac1 FRET ratio was measured in living cells. The dot plot shows FRET ratio for individual cells (small = 28, large = 33 cells) quantified from 3 independent experiments.

(G) HeLa cells transfected with Rac1 FRET biosensor were cultured on PDMS membranes, subjected to 7.5% equibiaxial stretch and Rac1 FRET ratio was measured in living cells for indicated time points. Graph shows average FRET ratio calculated from a total from 16 cells from 3 independent experiments.

(H-I) Quantification of ERES in HeLa cells seeded on large (H) and small (I) micropatterned surfaces (described in A). A total of 90-100 cells from 3 independent experiments were used for ERES quantification.

(I) Golgi arrival kinetics of GFP-ManII-RUSH in HeLa cells stably expressing Str-KDEL-SBP-GFP-ManII (RUSH system). Cell were grown on PDMS membranes, treated as indicated and subjected to 7.5% equibiaxial stretch. See also movieEV2. ManII arrival at the Golgi was monitored in nineteen cells in two independent experiments. See also movieEV2.

Data information: In all graphs, asterisks (*) mark statistical significance (p-value < 0.05; Student's unpaired t test) and error bars show standard deviation.
Mechanical strain stimulates COPII-dependent secretory trafficking via Rac1 (2022)
Figure 2

Figure 2. Dysfunctional ERES affects ability of cells to adapt to mechanical tension

(A) Representative immunofluorescence images of HeLa cells cultured on micropatterned surface of multiple geometries. Cells were transfected with 10 nM non-targeting control (siCtrl) or co-transfected with siRNAs targeting Sar1A and Sar1B. After 48 h, cells were trypsinized and 80,000 cells were seeded on micropatterned chips. Cells were allowed to attach for 3 h, stained with CellMask for 5 min and processed for microscopy. Scale bars: 5 µm

(B) Quantification graph showing the percentage of cells failing to entirely cover the different geometries (described in A). Data derived from at least 100 cells from a set of three independent experiments. Error bars represent standard deviation. Asterisks (*) denote statistical significance (p-value < 0.05; chi-squared test).

(C) Quantification graph shows the diameter of cells in non-targeting control (siCtrl) or siRNA targeting Sar1A and Sar1B (siSar1A/B). After trypsinization and resuspension of cells in culture medium, the diameter of cells was determined using the CASY cell counter. A total of 27,630 and 19,677 cells were counted for siCtrl and siSar1A/B, respectively.

(D) Representative immunofluorescence images of HeLa cells cultured on micropatterned surface of multiple geometries. Cells were treated with 50 µM of the Rac1 inhibitor NSC23766 for 4 hours prior to staining with CellMask and processing for immunofluorescence microscopy. Scale bars: 5 µm

(E) Quantification of (D). Percentage of cells failing to cover entire micropatterned surface is shown for control (ctrl) and the Rac1 inhibitor NSC23766. Between 113 - 117 cells were counted in total from a set of three independent experiments. Asterisks (*) mark statistical significance (p-value <0.05; chi-squared test). Error bars show standard deviation.
Mechanical strain stimulates COPII-dependent secretory trafficking via Rac1 (2022)
Figure 3

Figure 3. Perturbation of Rac1 activity affects ERES and ER-export

(A) HeLa cells were transfected with 10 nM non-targeting control siRNA (siCtrl) or siRNAs targeting Rac1 (siRac1). After 72 h, cells were fixed and processed for immunostaining against Sec31A to label ERES. Representative confocal microscopy images are shown.

(B) Quantification graph shows the number of ERES/cell in cells transfected with control siRNA (siCtrl) or siRNA against Rac1 (two siRNAs #1 and #2). Values are expressed as % of siCtrl. From three independent experiments, with >30 cells per condition were counted.

(C) Representative immunofluorescence images showing ERES in HeLa cells treated with DMSO (Ctrl), Rac1 inhibitor NSC23766 (50 µM, 4 h), or NSC23766 washout (50 µM for 4h, then washout for 2h).

(D) Graph shows the number of ERES per cell (displayed as % of Ctrl) derived from at least 30 cells per condition from three experiments.

(E) Fluorescence recovery after photobleaching (FRAP) of GFP-Sec16A marked ERES in HeLa cells. Images show an individual ERES before (-1), immediately after (0) and 28 sec after photobleaching. The graph illustrates FRAP analysis of individual ERES from 23 (for control) and 24 (for NSC23766) regions in 3 experiments. Line connects individual time points. Data are ± SD.

(F) Quantification of the mobile fraction of GFP-Sec16A in control and Rac1 inhibited cells. Mobile fraction is derived from a total of 23 (Ctrl) and 24 (NSC23766) individual ERES from three independent experiments.

(G) The rate of ER-export was monitored using GFP-ManII-RUSH in HeLa cells after perturbation of Rac1 with siRNA (siRac1), or Rac1 inhibitor NSC23766, or a combination of both. Representative images show GFP-ManII-RUSH distribution in HeLa cells at indicated time points.

(H) Quantification shows ratio of ManII fluorescence intensity within Golgi to outside Golgi region after addition of biotin at indicated time points. Between 76 - 104 cells were used for measurement of ManII intensity in 3 experiments.

Data information: Scale bars in all immunofluorescence images are 5 µm. Asterisks (*) indicate statistical significance (p-value < 0.05), ns indicates non-significant. For data presented in B, D and H, one-way Anova was used, whereas Students unpaired t-test was used for F. Error bars represent standard deviation.
Mechanical strain stimulates COPII-dependent secretory trafficking via Rac1 (2022)
Figure 4

Figure 4. Manipulation of ER localized Rac1 affects ERES

(A) Co-occurrence of mCherry-Rac1 and GFP-Sec61β at the endoplasmic reticulum (ER) in HeLa cells stably expressing mCherry-Rac1 and transiently expressing GFP-Sec61β as monitored by confocal microscopy. SRRF imaging mode was used in Andor DragonFly spinning disk confocal microscope for obtaining the image. Scale bar: 5 µm and 1 µm (inset). See also movieEV3.

(B) Line profile of two marked spots from (A) showing the overlap between mCherry-Rac1 and GFP-Sec61β signal at the ER.

(C) Side-by-side comparison of cells with endogenously tagged GFP-Rac1. The image on the left shows a confocal image and the image on the right was performed using SRRF mode. Scale bar: 5 µm

(D) Cells with endogenous GFP-Rac1 were fixed and stained against Sec31A to label ERES. Arrows indicate Rac1-puncta colocalizing with ERES. Magnified view of two spots (numbered 1 and 2 in merged channel) is shown. Scale bar: 5 µm and 1 µm (inset).

(E) Representative images showing Sec31A marked ERES in HeLa cells transfected with indicated plasmids. HeLa cells were transfected with the plasmids 16 h before immunolabelling with Sec31A. Scale bar: 5 µm.

(F) Quantification of ERES number in HeLa cells from the experiment described in C. Between 90-100 cells were quantified from 4 independent experiments. Average ERES per cell ± standard deviation is shown. Each dot represents average ERES/cell from an experiment. A one-way ANOVA was used test for statistical significance. * = P-value < 0.05, ns = non-significant.

(G) Images represent ERES in HeLa cells that were transfected with the indicated plasmids and cultured on the crossbow micropattern for 4 hours. Cells were transfected with the plasmids 16 h before they were trypsinized and seeded on the micropatterns for ERES staining. Scale bar: 5 µm

(H) The dot plot shows ERES count in individual cells from (G). Mean ± standard deviation is shown for each condition. At least 58 cells were used for counting ERES number. Independent experiments were performed 3 times. Statistical significance was tested using one-way ANOVA, * = p-value <0.05, ns = non-significant.

(I) Merged confocal images show distribution of Rac1 specific GEF TIAM (magenta) in HeLa cells before (0), and after optogenetic recruitment at indicated time points. CIBN-Sec61β (green) was used to recruit Cry2-TIAM to the ER with 488 nm laser. Scale bar: 5 µm.

(J) Graph shows the number of SNAP-Sec16A marked ERES in HeLa cells before (0) and after (30, 60 and 90 seconds) recruitment of TIAM to the ER from 2 independent experiments. Whiskers indicate min-max range and central band indicates the median of all data. Number of cells = 9. A paired t test was run to compare t0 and t90. * = p-value <0.05.
Mechanical strain stimulates COPII-dependent secretory trafficking via Rac1 (2022)
Figure 5

Figure 5. Effect of actin disruption on ERES and ER-to-Golgi transport

(A and B) Representative immunofluorescence images showing Sec31A labelled ERES in HeLa cells treated with DMSO (Ctrl), 0.5 µM cytochalasin D (CytoD) and 0.5 µM latrunculin A (LatA) for 15 and 30 min (A). Scale bar: 5 µm. The number of ERES per cell in HeLa cells treated with different doses of CytoD and LatA for 30 min are displayed as % of control ± standard deviation (90 -100 cells from 3 experiments) (B).

(C) Distribution of ManII-RUSH in HeLa cells. Cells were pre-treated with DMSO (Ctrl) or 1 µM LatA for 15 min prior to addition of biotin to initiate ManII-trafficking. Golgi arrival for ManII-RUSH was monitored for 30 min. Scale bar: 5 µm.

(D-E) Graph showing ratio of ManII within Golgi to outside Golgi. Values were derived from HeLa cells treated with LatA or CytoD as described in C. At least 30 cells per condition were used for quantification in each experiment (n = 3).

(F) HeLa cells cultured on PDMS membranes were treated with DMSO or 1 µM cytochalasin D (CytoD) and ERES were labelled with anti-Sec31A antibody before (no strain) or after 15% equibiaxial strain (15% strain). Anti-phalloidin staining was performed to visualize actin in cells. Representative images are shown. Scale bars, 5 µm.

(G) Quantification of experiment described in (F). The graph shows average ERES/Cell. Data were derived from a total of 90 - 115 cells from 3 experiments. Each dot represents several cells and error bars show standard deviation.

(H and I) siRNA mediated depletion of INF2 does not phenocopy Rac1 effect. HeLa cells were transfected with non-targeting control siRNA (siCtrl) or siRNA against INF2 (siINF2) for 72 h before processing cells immunostaining with Sec31A to quantify ERES. Representative images showing Sec31A-labelled ERES in HeLa cells (H), and quantification of ERES per cell presented as % of control (I). In total 137 cells (siCtrl) and 113 cells (siINF2) were used for ERES quantification from three independent experiments. Scale bar in H is 5 µm .

(J and K) Knockdown of INF2 does not alter the kinetics of ER-to-Golgi transport. HeLa cells stably expressing ManII-RUSH were transfected with siRNAs as described in E, and the ER-to-Golgi transport of ManII-RUSH was followed by confocal microscopy (J). Representative images for indicated time points are shown. Scale bars, 5 µm. The ratio of ManII-RUSH within the Golgi to outside the Golgi was calculated for the indicated time points (K). Three independent experiments were performed and a total of 82 (siCtrl, 0 min), 72 (siINF2, 0 min) and 93 (siCtrl and siINF2, 20 min) were used for quantification.

Data information: In all graphs, error bars represent standard deviation, * = p-value < 0.05 and ns = not significant. One-way ANOVA was used to test for statistical significance for B and G. Students t-test was used for the rest.
Mechanical strain stimulates COPII-dependent secretory trafficking via Rac1 (2022)
Figure 6

Figure 6. Rac1 regulates ERES and ER-export by dimerizing with Sar1

(A) Co-localization of mCherry-Rac1 and GFP-Sec16A in HeLa cells as monitored by SRRF live cell imaging. See also movieS4. Scale bars: 5 µm and 0.5 µm (inset)

(B) Representative immunofluorescence images of HeLa cells co-transfected with YFP(N)-Rac1 and Sar1-YFP(C) and immunostained with Sec31A. Rac1 and Sar1 form complexes at ERES in HeLa cells, which partially overlap with Sec31A. Scale bars: 5 µm and 1 µm (insets).

(C) Box plot showing the fraction of ERES that overlap with the Rac1-Sar1 complex (number of cells = 24, number of experiments = 4). Individual data points shown in the graph represent number of images. Whiskers indicate min-max range and central band indicates the median of all data.

(D) GFP-tagged Rac1 was immunoprecipitated by GFP-trap and the fraction was immunoblotted for endogenous Sar1. Representative immunoblot showing co-immunoprecipitation of Sar1 by Rac1.

(E) Representative immunofluorescence images of HeLa cells co-transfected with YFP(N)-Rac1 and Sar1-YFP(C) and cultured on crossbow micropatterns of indicated size. Cells were co-transfected with the indicated DNA constructs and incubated overnight prior to trypsinization and seeding on the micropatterned coverslip. Cells were allowed to grow for 3h, fixed and processed for microscopy. Scale bar: 5 µm

(F) Quantification of Rac1-Sar1 puncta described in (E) (number of experiments = 3, number of cells = 28 (small) and 27 (large)). In the quantification graph, error bars represent standard deviation and asterisk (*) denotes statistical significance at p-value < 0.05. A two-tailed unpaired Students t test was used for statistical testing.
Mechanical strain stimulates COPII-dependent secretory trafficking via Rac1 (2022)
Figure 7

Figure 7. In silico modeling predicts a Rac1-Sar1-Sec12 ternary complex and its experimental validation.

(A-B) In silico model showing the complex between Rac1 (grey) and Sar1 (gold) and how Rac1 covers the GTP binding site of Sar1. GTP of Rac1 in stick model, and of Sar1 in ball-and-stick.

(C) In silico model showing the steric clash between Rac1 and Sec23 (encircled), obtained by superposing the Sar1 units of the Sar1-Sec23 (green-purple) heterodimer onto the Sar1-Rac1 heterodimer (gold-grey).

(D) In silico model showing that the Sar1-Rac1 heterodimer can interact with Sec12 by superposing the Sar1 units of Sar1-Rac1 (gold-grey) and Sar1-Sec12 heterodimers (green-blue).

(E) Cells expressing GFP-Rac1 were lysed followed by immunoprecipitation against GFP and immunoblotting against endogenous Sar1 and Sec12. Blotting against GFP was performed to determine efficiency of the IP.

(F) HeLa cells were co-transfected with the indicated plasmids and cells were incubated overnight prior to cell lysis and immunoprecipitation against GFP. Immunoblotting against anti-mCherry was performed to detect Sar1A-mCherry. The IP efficiency was determined using anti-GFP. Different exposure times are shown for anti-mCherry blot.
Mechanical strain stimulates COPII-dependent secretory trafficking via Rac1 (2022)
Figure 8

Figure 8. Rac1 stimulates the formation of active Sar1 and COPII

(A) Microsomes isolated from HeLa cells were incubated as indicated. Active Sar1 pulldown was then carried out, followed by immunoblotting against Sar1.

(B) Microsomes (M) from HeLa cells stably expressing mCherry-ManII-RUSH were incubated with ATP and an ATP-regenerating system, GTP, biotin and cytosol in the presence or absence of recombinant Rac1 for 30 min at 25°C. COPII vesicles (V) were pelleted by ultracentrifugation (100,000 xg) and immunoblotted for anti-mCherry to detect ManII-RUSH and anti-calnexin (ER marker).

(C) Quantification data of two independent vesicle budding assays. All values were normalized to 4 ℃ Ctrl condition.
Loss of the VHR dual-specific phosphatase causescell-cycle arrest and senescence (2006)
figf1

(a) Confocal microscopy of synchronized HeLa cells stained for VHR (green) and BrdU (red) to indicate S-phase entry, and DNA (DAPI; blue). The cells were synchronized using a double thymidine block and then washed and placed in culture for the indicated times. The cells shown are representative of the majority of cells in each sample. (b) Anti-VHR, anti-actin and anti-Prc1immunoblots of lysates of the same cells. (c) DNA histograms of the same cells at the indicated time points. (d) Confocal microscopy of HeLa cells at various stages of mitosis stained for tubulin (green), VHR (red) and DNA (DAPI; blue). (e) Anti-VHR immunoblot of synchronized cells from G1, S, G2 and M phase treated for 30 min with 50 μg ml−1 of the protein synthesis inhibitor cycloheximide. Note that only cells in G1 showed a rapid loss of VHR protein. The scale bars represent 5 μm in a and 2 μm in b.
Loss of the VHR dual-specific phosphatase causescell-cycle arrest and senescence (2006)
figf2

(a) Anti-VHR and anti-actin immunoblots of lysates of HeLa cells transfected with a control luciferase siRNA or VHR siRNA and cultured for the indicated number of days. (b) Number of cells at the start of the experiment (200,000) and on day 3 for HeLa cells transfected with a control luciferase siRNA (black bars) or with VHR siRNA (white bars). (c) Light microscopy of HeLa cells transfected with a control luciferase siRNA or VHR siRNA and cultured for three days. (d) Thymidine incorporation by HeLa cells transfected with a control luciferase siRNA or with VHR siRNA. The data represent the mean ± sd. (n = 3). (e) β-galactosidase staining of HeLa cells transfected with a control luciferase siRNA or VHR siRNA and cultured for three days. Note the increased number of blue-staining granules in many cells. (f) Telomerase activity of cells treated with the indicated concentrations of VHR siRNA. Each sample was analysed in triplicate, with RNAse added to a fourth sample. (g) Anti-VHR immunoblot of the same cells as in f. (h) Anti-actin control immunoblot of the same cells as in f. The scale bars represent 10 μm in c and 25 μm in e.
Loss of the VHR dual-specific phosphatase causescell-cycle arrest and senescence (2006)
figf3

(a) Cell-cycle distribution and cell number count of cells transfected with control or VHR siRNA and observed for 4–5 days. The cells were stained with propidium iodide for DNA content and analysed by FACS after the indicated number of days in culture. (b) Confocal microscopy of the same HeLa cells stained for VHR (green) and BrdU (red) to indicate S-phase entry, and DNA (DAPI, blue). Control siRNA-treated cells and cells treated with VHR are shown. The bottom panels represent merges of the 3 images above. (c) Quantification of the BrdU positive cells in b. The indicated numbers of cells were counted and the data are given as percentage of BrdU positive cells of this total number. (d) Quantification of cells staining for phosphoH3. The indicated number of cells were counted and the data are given as percentage of phosphoH3-positive cells of this total number. (e) Confocal microscopy of control or VHR siRNA-transfected HeLa cells stained for VHR (green) and phospho H3 (red) to indicate M-phase entry, and DNA (DAPI, blue). The bottom panels represent merges of the images above. (f) Immunoblots of total lysates of control siRNA transfected cells and VHR siRNA cells with the indicated antibodies. Note that VHR was downregulated by approximately 80%, wheras actin levels were unchanged. (g) Immunoblot with anti-p21Cip1–WAF1 of total lysates of cells transfected with control siRNA (lanes 1–2), two different VHR siRNAs cells (lanes 3–6) or another control siRNA (lanes 7–8). The scale bars represent 10 μm in b and e.
Loss of the VHR dual-specific phosphatase causescell-cycle arrest and senescence (2006)
figf4

(a) Anti-VHR immunoblots of the same cell lysates used for assessment of MAP kinase activation in b–e. The cells were stimulated with 10% serum for 0, 1, 5, 15, 30 and 60 min, as indicated at the bottom of the figure. Equal amounts of total protein were loaded into each lane. (b–e) Immunoblots of the same lysates with the indicated antibodies for assement of MAP kinase activation.
Loss of the VHR dual-specific phosphatase causescell-cycle arrest and senescence (2006)
figf5

(a) Proliferation of cells transfected 3 days earlier with a combination of control siRNA or VHR siRNA together with Mek, Jnk or Mek plus Jnk siRNAs. The data are calculated as percentage of control (control siRNA alone) and represent the mean ± s.d. from three independent experiments. The starting cell numbers were 150,000 or 200,000 and the control cells had increased by 5.3, 5.0 and 13-fold in the three experiments. (b) Immunoblots of the same cells with the indicated antibodies.