EMBO/SourceData huggingface dataset:
Figure 4B GCN2 coimmunoprecipitates with PP1α and γ. HeLa cells stably transfected with GFP-PP1α or GFP-PP1γ were transduced to express either GCN2-siR (wild-type) or GCN2-siR-AAXA. Cells were arrested in prometaphase with Eg5i, GFP-PP1α and GFP-PP1γ were immunoprecipitated using GFP-trap and the immunoprecipitates were immunoblotted for GCN2. The vertical line in the PP1α and PP1γ blots indicates that different exposures of the same blot are shown for the inputs and the IP-s.D GCN2 can phosphorylate PP1α and γ in vitro. HeLa cells carrying GFP-PP1α or GFP-PP1γ were arrested in prometaphase with an Eg5 inhibitor (Lane 3), then GFP-tagged PP1α or PP1γ were immunoprecipitated using GFP-trap (input, Lane 2). The immunoprecipitates were incubated in kinase buffer and ATP with constitutively active, recombinant GCN2. The samples were run on phos-tag gels and immunoblotted with PP1γ or PP1α antibody.F PP1α and PP1γ bandshifts are diminished in cells depleted for GCN2 and arrested in mitosis. HeLa cells were transfected with control or GCN2-targeting siRNA and arrested in prometaphase using a PLK1 inhibitor. Representative quantifications of the ratios of the phospho-PP1/PP1 signal (average and SEM) from four independent experiments are shown. *p=0.0217 (PP1α) and **p=0.0069 (PP1γ) (paired two-tailed t-tests).
Figure 5HeLa cells were transfected with control or GCN2-targeting siRNA and fixed for immunofluorescence. Representative image of mitotic cells stained for A ChTOG (magenta), tubulin (green) and DNA (blue), Scale bars represent 5 µm. Percentage of cells displaying the aberrant localizations Averages and SEM from three independent experiments are shown. For chTOG n= 29, 37, 44 for siCtr; n= 14, 41, 28 for siGCN2; **p=0.0063 (paired two-tailed t-tests).HeLa cells were transfected with control or GCN2-targeting siRNA and fixed for immunofluorescence. Representative image of mitotic cells stained for B CENPE (magenta), P-Aurora B (yellow), tubulin (green) and DNA (blue) are shown. Scale bars represent 5 µm. D Percentage of cells displaying the aberrant localizations Averages and SEM from three independent experiments are shown. For CENPE n= 15, 35, 43 for siCtr; n= 11, 21, 20 for siGCN2; **p=0.001, for P-Aurora B n=29, 37, 44 for siCtr; n= 14, 41, 28 for siGCN2; **p=0.0053 (paired two-tailed t-tests).
Figure 6HeLa cells were transfected with control or GCN2-targeting siRNA, synchronized in the cell cycle by thymidine block-and-release and samples were collected for flow cytometry at the indicated time points. A Cell-cycle progression in the cells synchronized by thymidine block-and-release, three independent experiments, averages and SEM are shown.HeLa cells were transfected with control or GCN2-targeting siRNA, synchronized in the cell cycle by thymidine block-and-release and samples were collected for immunoblotting at the indicated time points. B Representative immunoblots of TACC3-S558 phosphorylation and Aurora A-T288 phosphorylationF Dephosphorylation of PP1 substrates after Aurora A inhibition in the presence and absence of GCN2 activity. HeLa cells were treated with Aurora Ai (MLN8237, MedChemTronica HY-10971), and with both Aurora Ai and GCN2i. Cell suspensions were incubated at 37oC and at the indicated times samples were removed, mixed with Laemmli sample buffer and boiled. F The immunoblots were probed with the indicated antibodies.
Figure 7B HeLa cells were transfected with control or GCN2-targeting siRNA or treated with the indicated concentrations of GCN2 inhibitor, cells were fixed after 72 h incubation, and the number of cells containing micronuclei was determined by DAPI staining. Four independent experiments, averages with SD are shown. n= 187, 72, 162, 531 for siCtr; n= 159, 112, 200, 656 for siGCN2; p= 0.009; n=721, 821, 682 for 0 µM; n= 607, 585, 635 for 1 µM, n= 414, 510, 723 for 2 µM; n= 123, 147, 280 for 3 µM; p=0.004 for 1 µM, **p=0.005 for 2 µM, **p=0.005 for 3 µM (paired two-tailed t-tests).C Progression from prophase to anaphase in the presence of the GCN2i at the indicated concentrations was observed by live-cell imaging in C hTert-RPE1 cells. Averages (lines) and SEM (bands) from three (hTert-RPE1) independent experiments are shown t=0 denotes entry into prophase. for hTert-RPE1 cells n= 276, 284, 231 and 228 for 0, 1, 2, and 3 µM GCN2i, respectively. *p=0.0168; ****<0.0001 (Kolmogorov-Smirnov test).E PP1α and PP1γ bandshifts are diminished in cells arrested in mitosis upon pharmacological inhibition of GCN2. HeLa or hTert-RPE1 cells were arrested in mitosis using a PLK1 inhibitor and GCN2-IN1 was added to 2 µM for 3 h before harvesting the cells. Representative E quantifications of the ratios of the phospho-PP1/PP1 signal (average and SEM) from four independent experiments are shown. HeLa: ***p=0.0007 (PP1α); **p=0.0023 (PP1γ); htert-RPE1: *p=0.0111 (PP1α) and ***p=0.0007(PP1γ), paired two-tailed t-tests.
Figure 8Progression from prophase to anaphase in the presence of the Aurora A and GCN2 inhibitors was observed by live-cell imaging in HeLa and hTert-RPE1 cells. GCN2 inhibitor ("G") and Aurora A inhibitor ("A") was added C, D Representative images from experiments shown in A and B, respectively.E, F Percentages of cells with micronuclei and mitotic slippage in the experiments shown in A and B. Averages and SEM from three independent experiments are shown. HeLa mitotic slippage *p=0.0137, HeLa micronculei p=0.0774, hTert-RPE1 mitotic slippage **p=0.0011, hTert-RPE1 micronuclei p=0.0640; (two-tailed t-tests).
Figure 9B HeLa and Tert-RPE1 cells stably expressing the cell-cycle markers CDT1-Ruby and GMNN-Neongreen were treated with the Aurora A and GCN2 inhibitors and cell-cycle progression was followed by live-cell imaging. Representative examples of HeLa and hTert-RPE1 cells are shown from the supplementary videos 4 and 5. Scale bars represent 5 µM.C HeLa and hTert-RPE1 cells were seeded in triplicate and incubated in the presence of the GCN2 and Aurora A inhibitors for 7 (HeLa) or 10 (RPE1) days, then fixed and colonies were counted. Shown are averages and SEM from three biological replicates. For HeLa cells *p=0.0267 for A versus G2A; *p= 0.0432 A versus G3A (paired two-tailed t-tests). For hTert-RPE1 cells *p=0.0391 A versus G2A; **p=0.021 A versus G3A (paired two-tailed t-tests). D HeLa and Tert-RPE1 cells were treated with the indicated inhibitors for 72 h, stained with Hoechst and analyzed by flow cytometry. The graph shows the percentage of Hoechst-positive (dead) cells in four independent experiments. Averages and SEM are shown. For HeLa cells **p=0.0087 A versus AG; **p=0.0027 Ctr versus AG; *p= 0.0237 Ctr versus G; p= 0.0809 Ctr versus A (paired two-tailed t-test). For hTert-RPE1 cells ***p=0.0005 A versus AG; **p=0.0036 Ctr versus AG; *p=0.0210 Ctr versus G; p= 0.29184 Ctr versus A (paired two-tailed t-tests).E, F HeLa and Tert-RPE1 cells were treated with the indicated inhibitors for 72 h, stained with the CellEvent™ Caspase-3/7 green detection reagent and Hoechst and analyzed by flow cytometry. Representative 2D plots of Alexa-Fluor 488 (a measure of Caspase-3/7 activity) and Hoechst intensity are shown for E HeLa and F RPE1 cells. All single cells are plotted in magenta, Hoechst-negative cells are overlaid in grey. Note that all Hoechst-positive (dead) cells are also stained with the Caspase-3/7 green reagent, indicating apoptosis.
Figure 1E and F, Naïve WT CD4+CD62Lhi T cells were cultured under Th17 polarizing conditions. On day 3, cells were transfected with control or siRNA targeting Batf, rested overnight, used for total RNA isolation to assess gene expression (E), or restimulated with anti-CD3 for 24 h to measure cytokine production by ELISA (F). Data information: , mean ± s.e.m. of 3-5 independent experiments (*p<0.05; two-sided t-test or one-way ANOVA followed by Tukey's test). RQ, relative quantification.Naïve CD4+CD62Lhi T cells from IL-17a reporter mice (Il17aeGFP) were cultured under Th17 polarizing conditions. On day 5, eGFP+/IL-17a+ cells were sorted and cultured for two additional rounds in the presence of anti-CD3 alone (TCR(R3)) or Th17 polarizing cytokines (Th17(R3)) (G) Data information: Data are representative of 3-5 independent experimentsWT and Batf KO mice were orally infected by gavage with 2x109 cfu/ml of Citrobacter rodentium (Cr) expressing GFP quantification of the signal (O) at the indicated time points. Data information: mean ± s.e.m. of n=5-6 mice (O) (*p<0.05; two-sided t-test or one-way ANOVA followed by Tukey's test).WT and Batf KO mice were orally infected by gavage with 2x109 cfu/ml of Citrobacter rodentium (Cr) expressing GFP Cr infected WT and Batf KO were sacrificed on day 4 and day 10. Cells were isolated from the colon, re-stimulated with PMA and ionomycin, and stained intracellularly for flow cytometric analysis of cytokine production and Foxp3 expression presented as density plots (P). Data are gated on viable CD4+TCRβ+. Data information: Data are representative of two independent experiments
Figure 2Naïve WT and Batf KO CD4+CD62Lhi T cells were cultured under Th17 polarizing conditions for 5 days. ChIP-seq for transcription factor (Batf) and histone modifications (H3K4me3, H3K27me3, and H3K27ac) was performed using day 5 WT and Batf KO Th17 cells (A Data information: Data are representative of two independent experiments with similar results (A,J, Naïve WT-2BiT and Batf KO-2BiT CD4+CD62Lhi T cells were cultured under Th17 polarizing conditions. IL-2-Thy1.1 production comparing WT-2BiT and Batf KO-2BiT cells was measured by flow cytometry at the indicated time points and frequencies of positive cells determined. Data information: Data are representative of or mean ± s.e.m. of 3-5 independent experiments (*p<0.05; two-sided t-test).L and M, ELISA was used to quantify IL-2 production in supernatants derived from cells in Figures 1G (L) and 1J (M) after 24 h of anti-CD3 stimulation. Data information: Data are representative of or mean ± s.e.m. of 3-5 independent experiments L-M) (*p<0.05; two-sided t-test).
Figure 3ChIP-seq for Stat3 and Stat5 was performed after 1 h of IL-6 and IL-2 stimulation, respectively, using WT and Batf KO cells that were cultured under Th17 polarizing conditions for 5 days. Histograms show Batf, Stat3, and Stat5 binding occupied around ±1 kb of peak centers of co-bound Stat3-Stat5 sites that were annotated near Th1, Th17, or Treg-specific genes (C). Data information: Data are mean ± s.e.m. representative of two independent experiments with similar results (c (*p<0.05; two-sided t-test).D, ChIP-seq for Stat3 and Stat5 was performed after 1 h of IL-6 and IL-2 stimulation, respectively, using WT and Batf KO cells that were cultured under Th17 polarizing conditions for 5 days. Tracks show Batf, Stat5, and Stat3 ChIP-seq data compared between WT and Batf KO Th17 in the indicated gene loci (WT, grey; Batf KO, red). Red arrows indicate differential Stat5 binding comparing WT and Batf KO cells (D). Data information: Data are mean ± s.e.m. representative of two independent experiments with similar results d), or (*p<0.05; two-sided t-test).
Figure 4B, Naïve WT and Batf KO CD4+CD62Lhi T cells were cultured under Th17 polarizing conditions for 5 days. The kinetics of Ets1 and Runx1 gene expression was assessed by RT-PCR in the indicated time points (data normalized to naïve WT cells). Data information: Data are mean ± s.e.m. of 3 independent experiments (B) (*p<0.05; two-sided t-test). RQ, relative quantification.
Figure 5Tracks show Ets1 and Runx1 ChIP-seq data from WT and Batf KO Th17-polarized cells at the indicated gene loci (D; WT, grey; Batf KO, red). Arrows indicate differential Ets1 and Runx1 binding with (red) or without (blue) the presence of Batf binding comparing WT and Batf KO cells. Ets1 (E) or Runx1 (F) peaks from WT Th1, Treg, or both (Th1-Treg) were integrated with enriched Ets1 or Runx1 peaks in Batf KO compared to WT Th17 cells (Fig. 4C) to separate peaks into unique and overlapping clusters.
Figure 6Naïve WT and Batf KO CD4+CD62Lhi T cells cultured under Th17-polarizing conditions were treated with or without 1 uM Stat5 inhibitor. On day 5, cells were harvested and used for RNA extraction to assess gene expression (A; data were normalized to WT DMSO; Foxp3, unstimulated; Il17a and Ifng, 6 h 2 ug/ml anti-CD3 restimulation) Data information: Data are mean ± s.e.m. of 4 independent experiments. (*p<0.05; two-sided t-test or one-way ANOVA followed by Tukey's test). a.u., arbitrary unit.Naïve WT and Batf KO CD4+CD62Lhi T cells cultured under Th17-polarizing conditions were treated with or without 1 uM Stat5 inhibitor. restimulated with 2 ug/ml anti-CD3 for 24 h to assess cytokine production by ELISA (B). Data information: Data are mean ± s.e.m. of 4 independent experiments. (*p<0.05; two-sided t-test or one-way ANOVA followed by Tukey's test). a.u., arbitrary unit.Naïve WT and Batf KO CD4+CD62Lhi T cells cultured under Th17-polarizing conditions were treated with or without 1 uM Stat5 inhibitor. WT and Batf KO Th17-polarized cells were collected on day 5 of culture and used to performed ChIP using antibodies to Stat5, Batf, Ets1, Runx1, Stat3 and p300 (C). Data information: Data are mean ± s.e.m. of 4 independent experiments. (*p<0.05; two-sided t-test or one-way ANOVA followed by Tukey's test). a.u., arbitrary unit.Nuclear extracts from WT Th17 cells were incubated with biotinylated oligonucleotides containing Ets1-Runx1-Stat5-specific binding sites at the indicated genomic regions (wild type or mutant (∆) as indicated). Immunoblots of precipitated proteins (D)E, Nuclear extracts from WT Th17 cells were incubated with biotinylated oligonucleotides containing Ets1-Runx1-Stat5-specific binding sites at the indicated genomic regions (wild type or mutant (∆) as indicated). densitometry measurements (E). Data information: Data are mean ± s.e.m. of 4 independent experiments. (*p<0.05; two-sided t-test or one-way ANOVA followed by Tukey's test). a.u., arbitrary unit.
Figure 7Naïve CD4+CD62Lhi T cells from IL-17a reporter mice (Il17aeGFP) were cultured under Th17 polarizing conditions. On day 5, eGFP+/IL-17a+ cells were sorted (Th17(R1)) and cultured for two additional rounds in the presence of anti-CD3 alone (TCR(R3)) or Th17 polarizing cytokines (Th17(R3)). Ets1,Runx1, and Stat5 binding at the indicated genomic regions by ChIP-qPCR (C). Data information: Data are mean ± s.e.m. of 3 independent experiments. (*p<0.05; **p<0.01; one-way ANOVA followed by Tukey's test).
Figure 1The percentage of myofibroblast-like cells in total endometrial cells between controls and patients with IUA (n=3 samples for each group). Data are presented as mean ± SEM. Two-tailed Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001.Masson trichrome staining and immunostaining of α-SMA and Collagen 1 in endometrium of controls and patients with IUA (n=5 samples for each group). Scale bars: 100μm. Data are presented as mean ± SEM. Two-tailed Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001.Expression of marker genes for each cell subtype of myofibroblast-like cells (n=6 samples).
Figure 2Flow cytometric analysis of the proportion of CD14+ cells, CD301+ macrophages, CD80+ macrophages and CD163+ macrophages in the endometrium of normal controls and patients with IUA. Macro, macrophage (n=20 samples for each group). Data are presented as mean ± SEM. Two-tailed Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001. NS, not significant.Representative immunostaining images of CD301 and CD68 from samples of normal controls and patients with IUA (n=5 samples for each group). Representative immunostaining images of CD301 and α-SMA from samples of normal controls and patients with IUA (n=5 samples for each group). Scale bar: 100μm. Data are presented as mean ± SEM. Two-tailed Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001. NS, not significant.The protein levels of CD14 and CD301 in CD301- macrophages and CD301+ macrophages examined by western blot (n=3 technical replicates for each group). Data are presented as mean ± SEM. Two-tailed Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001. NS, not significant.The protein levels of Fibronectin, Collagen 1, α-SMA and CTGF expression in primary human endometrial stromal cells (hESCs) treated by the supernatants of CD301- and CD301+ macrophages for 48h, respectively examined by western blot (n=3 technical replicates for each group). Data are presented as mean ± SEM. Two-tailed Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001. NS, not significant.
Figure 3The protein levels of GAS6 and CCL8 expression in sorted human endometrial CD301- and CD301+ macrophages (n=3 technical replicates for each group). Data are presented as mean ± SEM. Two-tailed Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001. NS, not significant.Localization of GAS6 and CD301 in endometrium of normal controls and patients with IUA examined by immunofluorescence (n=5 samples for each group). Localization of AXL and α-SMA in endometrium of normal controls and patients with IUA examined by immunofluorescence (n=5 samples for each group). Scale bar: 100μm. Data are presented as mean ± SEM. Two-tailed Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001. NS, not significant.Western blot analysis of phospho-AXL (p-AXL) and AXL in hESCs treated with GAS6 at dose of 10, 20, 50, 100 and 200 ng/mL for 72h (n=3 technical replicates for each group). The protein levels of Collagen 1 and α-SMA expression in hESCs after treatment with GAS6 at dose of 10, 20, 50, 100 and 200 ng/mL for 72h examined by western blot (n=3 technical replicates for each group). Data are presented as mean ± SEM. One-way ANOVA with Tukey's post hoc analysis. *, P<0.05; **, P<0.01; ***, P<0.001. NS, not significant.Western blot analysis of α-SMA and Collagen 1 in hESCs treated with Bemcentinib (1μM) for 1 hour, followed by GAS6 (50ng/mL) for 72h (n=3 technical replicates for each group). Western blot analysis of α-SMA and Collagen 1 in hESCs treated with the supernatants of CD301- and CD301+ macrophages and GAS6 neutralizing antibody (2.5μg/mL) for 72h (n=3 technical replicates for each group). Data are presented as mean ± SEM. One-way ANOVA with Tukey's post hoc analysis. *, P<0.05; **, P<0.01; ***, P<0.001. NS, not significant.
Figure 4Western blot analysis of phospho-p65 (p-p65) and p65 expression in hESCs treated with GAS6 (50ng/mL) at time of 3, 6, 12, 24 and 48h (n=3 technical replicates for each group). Data are presented as mean ± SEM. One-way ANOVA with Tukey's post hoc analysis. *, P<0.05; **, P<0.01; ***, P<0.001.Western blot analysis of p-p65, p65, Collagen 1 and α-SMA expression in hESCs treated with or without GAS6 neutralizing antibody (2.5μg/mL) or Bemcentinib (1μM) for 1 hour, followed by GAS6 protein (50ng/mL) for 72h (n=3 technical replicates for each group). Data are presented as mean ± SEM. One-way ANOVA with Tukey's post hoc analysis. *, P<0.05; **, P<0.01; ***, P<0.001.Representative immunostaining images of p-p65 in endometrium of normal controls and patients with IUA (n=5 samples for each group). Scale bar: 100μm. Data are presented as mean ± SEM. (D) Two-tailed Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001.qRT-PCR analysis of ACTA2 and COL1A1 expression in hESCs treated with GAS6 (50ng/mL) or JSH23 (10μM) for 72h (n=3 biological replicates for each group). Data are presented as mean ± SEM. One-way ANOVA with Tukey's post hoc analysis. *, P<0.05; **, P<0.01; ***, P<0.001.Western blot analysis of Collagen 1 and α-SMA expression in hESCs treated with GAS6 (50ng/mL) or JSH23 (10μM) for 72h (n=3 technical replicates for each group). Data are presented as mean ± SEM. One-way ANOVA with Tukey's post hoc analysis. *, P<0.05; **, P<0.01; ***, P<0.001.
Figure 5Representative images of Masson trichrome staining and immunostaining for Collagen 1 between endometrium of Sham and IUA mice (n=5 mice for each group). Scale bar: 100μm. Data are presented as mean ± SEM. Two-tailed Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001.Flow cytometric analysis of the proportions of CD11b+F4/80+ cells (pan-macrophages) and CD301b+ macrophages in the uterus of Sham and IUA mice (n=5 mice for each group). Macro, macrophage. Data are presented as mean ± SEM. Two-tailed Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001.Representative images of immunostaining for GAS6 (red) and CD301b (green) in the endometrium of Sham and IUA mice (n=5 mice for each group). Scale bar: 100μm. Data are presented as mean ± SEM. Two-tailed Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001.Representative images of immunostaining for AXL in the endometrium of Sham and IUA mice (n=5 mice for each group). Scale bar: 100μm. Data are presented as mean ± SEM. (F) Mann-Whitney U test. *, P<0.05; **, P<0.01; ***, P<0.001.
Figure 6qRT-PCR analysis of Acta2 and Col1a1 expression in the uterus of Sham, IUA and IUA+DT mice (n=5 mice for each group). Data are presented as mean ± SEM. One-way ANOVA with Tukey's post hoc analysis. *, P<0.05; **, P<0.01; ***, P<0.001.Representative images of immunostaining for AXL, Collagen 1 and p-p65 and Masson trichrome staining in the endometria of Sham, IUA and IUA+DT mice (n=5 mice for each group). Scale bar: 100μm. Data are presented as mean ± SEM. One-way ANOVA with Tukey's post hoc analysis. *, P<0.05; **, P<0.01; ***, P<0.001.
Figure 7qRT-PCR analysis of Acta2 and Col1a1 expression in the uterus of IUA+PBS and IUA+BEM mice (n=5 mice for each group). Data are presented as mean ± SEM. (B, Two-tailed Student's t-test.Representative images of immunostaining for AXL, Collagen 1, p-p65 and Masson trichrome staining in the endometrium of IUA+PBS group and IUA+BEM group (n=5 mice for each group). Scale bar, 100μm. Data are presented as mean ± SEM. C for AXL and Collagen 1 Two-tailed Student's t-test. (C for p-p65) Mann-Whitney U test. *, P<0.05; **, P<0.01; ***, P<0.001.
Figure 1Primary wild type MEF were treated with DMSO or Aurora kinase inhibitor ZM447439 (ZM, 2μM) for 72h, followed by RNA isolation and mRNA-sequencing. (A) Volcano plot representation of differentially expressed genes (DEGs) between ZM and DMSO treatment. Adjusted p value ≤ 0.01 and log2 FC ≥ 1 or log2 FC ≤ -1.