EMBO/SourceData huggingface dataset:
f1a, Starvation (3 h)-induced autophagy results in a downregulation of H4K16ac in histone extracts of MEF cells.b, Upon rapamycin treatment (300 nM) LC3 conversion and downregulation of H4K16ac are observed in WT MEF cells but not in the autophagy-deficient atg5−/− and atg7−/− MEF cells.c, Rapamycin treatment increased the LC3-II/LC3-I ratio and promoted H4K16ac decrease in sirt1−/− and WT MEF cells.d, Rapamycin-induced autophagy led to downregulation of H4K16ac at 48 h in histone extracts of HeLa and U20S cells, and after 6 h in U1810 cells.e, Quantification of H4K16 acetylation by immunoblotting is depicted for rapamycin-treated cells. Data are expressed as mean ± s.e.m. (n = 3-5); *P 0.05; **P 0.01.
f3Rapamycin treatment (48 h) promoted the downregulation of the H4K16 histone acetyltransferase hMOF expression level in MEF (a)Rapamycin treatment (48 h) promoted the downregulation of the H4K16 histone acetyltransferase hMOF expression level in transfected HeLa cells (b).VPA (1 mM) treatment counteracted rapamycin-induced H4K16ac downregulation (c) and decreased the LC3 ratio (d).e, Co-treatment with chloroquine (CQ, 10 µM) showed that the decrease in LC3 ratio was a result of an increase in autophagic flux.f, Inhibition of autophagy by CQ after hMOF overexpression shows that hMOF does not inhibit autophagic flux.g, The yeast homologue of hMOF, Sas2, tagged with 3× HA showed a complete disappearance of HA signal upon autophagy induction after 3 h.h, Overexpression of Sas2 repressed the downregulation of H4K16ac upon rapamycin treatment.
f4a, VPA increases the autophagic flux in rapamycin-treated HeLa cells transfected with the mRFP-GFP-LC3 tandem reporter construct which allows distinction between autophagosomes (GFP+/RFP+ yellow puncta) and autolysosomes (GFP−/RFP+ red puncta).b, Confocal microscopy image of a cell treated with rapamycin and VPA depicting a high ratio of red to green LC3 puncta indicating an increase in autophagic flux.c, Co-treatment with VPA and rapamycin led to increased cell death.d, Co-treatment with CQ abrogated VPA + rapamycin-induced cell death.e, f, Increasing H4K16ac levels by either overexpression of hMOF, inhibition of SIRT1 by siRNA or the chemical inhibitor Ex527 promoted cell death upon rapamycin treatment.Co-treatment with the autophagy inhibitor bafilomycin A1 (BafA, 40 nM) (g) or 3-methyladenine (3MA, 5 mM) (h) abrogated VPA + rapamycin-induced cell death.Treatment of HeLa cells upon amino-acid starvation with VPA induced cell death (i), which was rescued when cells were co-treated with CQ (j). Data are expressed as mean ± s.e.m. (n = 3).
Figure 1A sWGS performed on sorted CD3+/Vβ+ malignant T-cells isolated from the blood of P1, shown as a representative example. Copy number profiles and ploidy estimates were calculated using ichorCNA. Blue represents balanced genomic regions, green indicates loss/µdeletion, and red indicates gain/amplification of regions on chromosome 17. Darker color shades indicate higher log-2 ratios.B Heatmap of extracted CNA [log2 ratios] of STAT genes on chr17 (i.e., STAT3, STAT5A and STAT5B), and other relevant tumor suppressors (i.e, STAT1, SOCS1 and TP53) and oncogenes (MYC). Red indicates gain/amplification, blue indicates loss/deletion. The balance threshold was set to +/- 0.1. P33 presented with two malignant clones in the blood and both were sequenced (P33_1 (Clone 1), P33_2 (Clone 2)). *Tetraploid malignant clone.
Figure 2A-C Spearman correlation analysis using CNA log2 ratios and the percentage of clonal CD3+ cells detected in patients for (A) STAT3, (B) STAT5A and (C) STAT5B.D-F RT-qPCR expression data of (C) STAT3, (D) STAT5A, and (E) STAT5B genes from whole PBMCs isolated from patient blood (n = 5) or healthy controls (n = 9). GAPDH expression was used for normalization. Statistical significance was calculated using two-tailed unpaired t-test. P-value: <0.01 (**), <0.0001 (****). One experiment was performed in technical triplicates. The bold dashed line in the middle of the violin plot denotes the median value, while the thin dotted lines denote the interquartile range.G Immunoblotting for total STAT3/5 protein isolated from the respective whole PBMCs from patient blood (n = 3), or from healthy controls (n = 3). HSC70 was used as loading control. One experiment was performed.H Representative image of H&E, CD3, pY-STAT5 single staining and CD3/pY-STAT5 double staining on skin biopsy tissue from P3 (n = 1), which demonstrates infiltration of CD3+ pY-STAT5+ (13.45%) cells into the dermal and subcutaneous layer of the skin. Arrows indicate pY-STAT5+ single and CD3+ pY-STAT5+ cells.I-M Quantification of IHC staining from skin-biopsy sections of L-CTCL patients (n = 6) and healthy individuals (n = 5) As the number of STAT5A/B positive cells was negligible (less than 0.03/per mm2) in healthy skin biopsy sections, healthy control samples were considered negative for pY-STAT5, and levels were set to 0. Statistical significance was calculated using an unpaired Welch's t-test. P-value: <0.05 (*).The bold dashed line in the middle of the violin plot denotes the median value, while the thin dotted lines denote the interquartile range.
Figure 3A-C Graphic depicts the aberration patterns on (A) chromosome 17 (STAT3/5), (B) chromosome 2 (STAT1) and (C) chromosome 16 (SOCS1) in the CTCL cell lines used in the study. Aberration patterns are represented as the log2 ratio of the fluorescence intensity of the tumor DNA vs. reference DNA and were generated by aCGH. Immunoblotting for total protein STAT1/3/5 and activation levels: phospo-Tyr (701)-STAT1/phospho-Tyr (705)-STAT3/phospho-Tyr (694/699)-STAT5, and SOCS1 protein levels. The cytokine-dependent cell line, SeAx, was collected 30 min after 5 ng/ml IL-2 and IL-4 cytokine addition. HSC70 was used as loading control. One representative out of three independent experiments is shown.
Figure 4A Heatmap of IC50 values calculated from drug response analysis using CellTiter-Glo viability assays upon 48h drug treatment. One representative, out of three independent experiments performed in triplicates, is shown.B, Immunoblot showing total protein STAT3/5 and activation levels: phospho-Tyr (705)-STAT3/phospho-Tyr (694/699)-STAT5A/B after 24h treatment with (B) JPX-0750 The cytokine-dependent cell line, SeAx, was collected 30 min after 5 ng/ml IL-2 and IL-4 cytokine addition. HSC70 was used as loading control. The normalized levels of phospho- and total STAT3/5 quantified by densitometry, are shown below the respective blots. One representative, out of three independent experiments is shown.C Immunoblot showing total protein STAT3/5 and activation levels: phospho-Tyr (705)-STAT3/phospho-Tyr (694/699)-STAT5A/B after 24h treatment with (C) IQDMA. The cytokine-dependent cell line, SeAx, was collected 30 min after 5 ng/ml IL-2 and IL-4 cytokine addition. HSC70 was used as loading control. The normalized levels of phospho- and total STAT3/5 quantified by densitometry, are shown below the respective blots. One representative, out of three independent experiments is shown.Real-Time apoptosis/necrosis assay in the (D, SeAx and (F, Myla cell lines treated with 2.5µM JPX-0750 RLU, Apoptosis [RLU] = relative luminescence; Necrosis [RFU] = relative fluorescence units. Error bars represent mean +/-SD. One experiment in technical triplicates was performed.Real-Time apoptosis/necrosis assay in the E) SeAx and G) Myla cell lines treated with 2.5µM IQDMA. RLU, Apoptosis [RLU] = relative luminescence; Necrosis [RFU] = relative fluorescence units. Error bars represent mean +/-SD. One experiment in technical triplicates was performed.H-I The effect of (H) JPX-0750 and (I) IQDMA on the viability of CTCL cells in comparison to viability of primary juvenile fibroblasts (JF) and keratinocytes (HaCat) at a low dose range (0.7-3µM). One experiment, in triplicates, was performed with JF and HaCat cells, and three experiments, in triplicates with the CTCL cells. Error bars represent mean +/-SD. Statistical significance was calculated by two-way ANOVA with Tukey's multiple comparisons test. P-value: <0.05 (*), <0.01 (**), <0.001 (***), <0.0001 (****).
Figure 5B Subcellular fractions of SeAx cells treated with IQDMA for 24h and immunoblotted for pY-STAT5 and total STAT5. The cells were collected 30 min after 1 ng/ml IL-2 cytokine addition. α-Tubulin and Histone H3 were used as loading controls for cytoplasmic and nuclear fractions, respectively. The normalized levels of phospho- and total STAT5 in the nucleus and cytoplasm, quantified by densitometry, are shown below the respective blots. One representative out of two independent experiments is shown.D-F Scatter plot depicting the profile of SeAx cells treated with (D) 1µM IQDMA, (E) 2.5µM IQDMA and (F) 10µM IQDMA. Protein abundances were determined using TMT-based quantification mass spectrometry Significant changes were assessed by a modified t-test as implemented in the limma package, with the negative log10 p values on the x-axis, and log2 fold change shown on the y-axis.G Heatmap of IC50 values calculated from drug response analysis using CellTiter-Blue or CellTiter-Glo viability assays upon 48h drug treatment. One representative out of three independent experiments performed in triplicates is shown. Grey represents the presence of STAT3/5 gain.
Figure 6A Inhibitor treatment was carried out with increased dose escalation for 24h. SeAx cells were collected 2h after 7,5 ng/ml IL-2 and IL-4 cytokine addition. STAT3/5 target gene products MYC, PIM1, MCL-1, and AKT1/2/3, as well as proteins regulating the cell cycle, such as CYCLIN D2 and D3, and CDK6 were probed by Western blotting. HSC70 served as loading control. The normalized protein levels, quantified by densitometry, are shown below the respective blots. One representative out of three independent experiments is shown.Synergy analysis of the indicated two-drug combinations in the SeAx and Myla cells after 48 h treatment. In each graph the most synergistic area (MSA) is highlighted, which represents the most synergistic 3-by-3 dose-window with the respective MSA score. The Zero interaction potency model was applied to quantify the degree of synergy, according to which an MSA score below -10 indicates that drugs are antagonistic (green), a score between -10 and 10 indicates that 2 drugs are additive (white), while a score above 10 indicates a synergistic effect (red).(D) Heatmap showing MSA scores of the combination of FRAx597 with the listed drugs. One representative out of two independent experiments is shown.E Heatmap showing the IC50 values upon treatment of primary PBMCs isolated from L-CTCL patients with JPX-0750, IQDMA and FRAx597. One experiment performed in triplicates using CellTiter-Glo viability assays upon 48h drug treatment is shown. The relative STAT3/5 log2 ratio value is depicted in grey.F-H Spearman correlation analysis using CNA log2 ratios and the IC50 values for the respective drug.
Figure 7B Growth rate of Hut78, Myla and H9-derived tumors measured on the respective day post-injection. The mean of the respective tumor volumes for H9 group and for the vehicle-treated groups for Hut78 and Myla are shown. Tumor volume = (length x width2)/2. Error bars represent mean +/-SEM. n represents the total number of tumors that engrafted per group.C-D Myla-derived tumor weight upon experiment termination, after 20 days of treatment with 5 mg/kg of (C) JPX-0750 (n = 5) and IQDMA (n = 6), or vehicle (n = 7), and (D) FRAx597 (n = 5) or vehicle (n = 4), with n representing the number of analyzed tumors per group. Statistical significance was calculated using two-tailed paired t-test. P- value: <0.05 (*). Error bars represent mean +/-SEM.H&E analysis of Myla-derived tumors treated with JPX-0750 (n = 6), IQDMA (n = 6) or vehicle (n = 6), and FRAx597 (n = 5) or vehicle (n = 4), stained with (E, F) H&E with n representing the number of analyzed tumors per group. Pictures shown are from contiguous sections. Dotted rectangles indicate magnified areas. Scale bars, 200, 100 and 25 µm. Violin plots show the perimeter of the annotated tumor cell-infiltration/expansion region into the intradermal and subcutaneous region of the skin Statistical significance was calculated using two-tailed paired t-test with Welch´s correction. P-value: <0.05 (*), <0.01 (**), <0.001 (***). The bold dashed line in the middle of the violin plot denotes the median value, while the thin dotted lines denote the interquartile range.IHC analysis of Myla-derived tumors treated with JPX-0750 (n = 6), IQDMA (n = 6) or vehicle (n = 6), and FRAx597 (n = 5) or vehicle (n = 4), stained with (G, H) Ki67, as a marker for proliferation with n representing the number of analyzed tumors per group. Pictures shown are from contiguous sections. Dotted rectangles indicate magnified areas. Scale bars, 200, 100 and 25 µm. Violin plots show the quantification of the percentage of Ki67+ cells in the tissue. Statistical significance was calculated using two-tailed paired t-test with Welch´s correction. P-value: <0.05 (*), <0.01 (**), <0.001 (***). The bold dashed line in the middle of the violin plot denotes the median value, while the thin dotted lines denote the interquartile range.IHC analysis of Myla-derived tumors treated with JPX-0750 (n = 6), IQDMA (n = 6) or vehicle (n = 6), and FRAx597 (n = 5) or vehicle (n = 4), stained with (I, J) CD31, as a tumor vessel marker, with n representing the number of analyzed tumors per group. Pictures shown are from contiguous sections. Dotted rectangles indicate magnified areas. Scale bars, 200, 100 and 25 µm. CD31 staining was quantified as raw counts of vessels with lumen. Statistical significance was calculated using two-tailed paired t-test with Welch´s correction. P-value: <0.05 (*), <0.01 (**), <0.001 (***). The bold dashed line in the middle of the violin plot denotes the median value, while the thin dotted lines denote the interquartile range.
Figure 2(B) Histogram displaying the SILAC ratios (log2) of stress-induced phosphorylation sites in setup cdc55Δ (upper panel) and setup rts1Δ (lower panel) (Hollenstein et al., 2020), stratified by their Hog1-dependence (grey area versus thick black line).(D) Line plot monitoring growth rate (y-axis) of indicated strains (colored lines) under different osmolarity conditions (x-axis).
Figure 4B) Western-blot illustrating the depletion of AID-Cdc55 at various time points after addition of auxin (upper panel); Pgk2 serves as a negative control (lower panel).(D) Transcriptional analysis of stress-induced expression of ESR genes CTT1 (upper panel) and PGM2 (lower panel) under different strain conditions (i.e. presence/absence of IGO1/2 and induction of iAID-Cdc55 depletion). Measured time points after stress application are depicted on the x-axis, target gene mRNA levels are illustrated on the y-axis as fold over reference gene IPP1, values are normalized relative to wild type without induction of iAID-Cdc55 depletion at 30 minutes post-stress.E) Transcriptional analysis of stress-induced expression of ESR genes CTT1 (upper panel) and PGM2 (lower panel) at 30 minutes post-stress. Target gene mRNA levels are illustrated on the y-axis as fold over reference gene IPP1, values are normalized relative to wild type without induction of iAID-Cdc55 depletion. dep.: Cdc55 depleted, n.d.: Cdc55 non-depleted.
Figure 5(C) Mobility shift assays monitoring phosphorylation-induced mobility changes of Gis1 (two upper rows) and Rph1 (two lower rows) upon hyperosmotic stress in wild type, igo1∆igo2∆ and Cdc55-depleted cells (right-hand side).(E) Western blot phosphorylation shift assays of wild type Gis1-myc (upper panel) and Rph1-Flag (lower panel), and mutated forms Gis1-5A-myc (upper panel) and Rph1-5A-Flag (lower panel). The measurement was done using Phos-tag (Kinoshita et al., 2006) and at the indicated time points after exposure to hyperosmotic stress (0.5 M NaCl) using Pgk1 protein levels as a negative control. The location of the mutated residues is shown in the respective schematics. Red boxes: demethylase associated domains (JmjN and JmjC), blue box: zinc finger domain.(F) Transcriptional analysis of stress-induced expression of ESR genes CTT1 (upper panel) and PGM2 (lower panel) at 30 minutes post-stress across indicated strains expressing either wild type or point-mutated forms of Gis1 and Rph1 in a gis1∆rph1∆ deletion background, with the empty vector as control.
Figure 1(A) Heat map showing a subset of mRNAs of genes immunoprecipitated by anti-EZH2 antibody in LNCaP and C4-2 prostate cancer cell lines, which was generated based on the distinct RIP-seq reads on specific gene exons. The units of the heatmap values were reads per kilobase million (RPKM).(B) Screen shots from the UCSC genome browser showing signal profiles of p53 mRNA immunoprecipitated by anti-EZH2 antibody in LNCaP and C4-2 cells.(C) C4-2 cells were subjected to UV-RIP assay. RT-qPCR measurement of p53 mRNA immunoprecipitated by IgG or anti-EZH2 antibody. Data shown as means ± SD (n=3). Statistical significance was determined by two-tailed Student's t-test. ** P<0.01.(E) Top, RT-qPCR analysis of p53 mRNA in C4-2 cell lysate pulled down by GST or GST-EZH2 recombinant proteins EZ1-EZ4. Bottom, western blotting analysis of GST or GST-EZH2 proteins used for GST pull-down assay. Asterisks indicate the protein bands at expected molecular weight. Data shown as means ± SD (n=3). Statistical significance was determined by two-tailed Student's t-test.** P<0.01.(F) C4-2 cells were transfected with Myc-tagged EZH2-WT or EZH2-∆336-554 for 24 h and cells were harvested for RIP with IgG or anti-Myc tag antibody. Transfected proteins and pull-down p53 mRNAs were analyzed by western blot and RT-qPCR, respectively. Data shown are means ± SD (n=3). * P<0.01. ERK2, a loading control.(G) GST pull-down assay using in vitro transcribed different fragments of p53 mRNA and indicated GST proteins followed by RT-qPCR analysis of pull-down p53 mRNA. FL, full length; ORF, open reading frame; UTR, untranslated region.(H, I) RNA EMSA evaluation of EZh2 binding of p53 mRNA. GST-EZH2 recombinant proteins (EZ1 to EZ4) were incubated with biotin-labeled in vitro transcribed p53 5'UTR (biotin-labeled probe) in the presence or absence of 100-fold unlabeled p53 5'UTR (unlabeled probe), followed by PAGE and immune blotting with HPR-conjugated streptavidin.
Figure 2(A) Biotin pull down assay by incubating biotin-labelled different fragments of p53 mRNA and HOTAIR (positive control) with C4-2 cell lysate followed by western blot with EZH2 antibody.(B) Biotin pull down assay as in (A) using unmutated and various internally deleted mutants of 5'UTR of p53 mRNA. Top, diagram of different p53 5'UTR deletion mutants.(C) Upper, the linear map of the pRF bicistronic report plasmid. The SV40 promoter (purple box) was used to drive firefly luciferase (Fluc) and Renilla luciferase (Rluc) gene transcription. Different p53 5'UTR fragments were inserted between the Fluc and Rluc genes. Lower, at 24 h after transfection, cells were lysed and luciferase activities were measured using a dual-luciferase kit and the ratio of Fluc/Rluc was calculated. Data shown as means±SD (n=3). Statistical significance was determined by two-tailed Student's t-test. * P<0.01.(D) EZH2 knockdown C4-2 cells were transfected with the bicistronic reporter vector in combination with empty vector, Myc-tagged EZH2 WT or deletion mutants followed by western blot analysis with indicated antibodies (bottom) and luciferase assay (top). Data shown as means±SD (n=3). * P<0.01.(E) C4-2 and U2OS cell lines were transfected with non-specific control (siC) or two independent EZH2-specific siRNAs and harvested for western blot analysis with indicated antibodies. ERK2 and β-TUBULIN, loading controls.(F) U2OS cells were transfected with control (siC) or EZH2-specific siRNAs and treated with 200 nM of CPT followed by western blot analysis for indicated proteins.(G) C4-2 cells were transfected with control (siC) or EZH2-specific siRNAs and plasmids for empty vector, EZH2 WT or deletion mutants followed by western blot analysis for indicated proteins.
Figure 3(A) List of top hits of EZH2-bound proteins identified by TAP-MS. The number of peptides and coverage of each protein are indicated.(B) Reciprocal co-IP of endogenous EZH2 and eIF4G2 or PABP1 in C4-2 cells.(C) Co-IP of endogenous EZH2 with eIF4G2 or PABP1 from C4-2 cell lysate pre-treated with RNaseA in 37oC for 30 min. The effectiveness of RNaseA treatment was monitored by RT-PCR analysis of the presence of GAPDH mRNA.(D) C4-2 cells were transfected control (siC) or EZH2-specific siRNA and lysed for polysome fractionation. RNA was extracted from even-number fractions followed by RT-qPCR analysis for p53 and GAPDH mRNA. The β-ACTIN mRNA was used as an internal control. Data shown as means±SD (n=3). Statistical significance was determined by two-tailed Student's t-test. * P<0.01; NS, no significance.(E) C4-2 cells were transfected with control (siC) or EZH2-specific siRNAs in combination with empty vector, EZH2 WT or different deletion mutants followed by polysome fractionation and RT-qPCR. β-ACTIN mRNA was used as an internal control. Data shown as means±SD (n=3). * P<0.01.
Figure 4(A) IHC analysis of Pten, Ezh2 and p53 proteins in prostatic tissues of 4-month-old mice with different genotypes: Ptenpc-/- (n=11); Ezh2pc-/- (n=8); Ptenpc-/-;Ezh2pc-/- (n=10) and 'wild-type' littermate controls (n=10). Scale bars, 50(B) Western blot analysis of expression of indicated proteins in four different groups of mice as described (n=2 mice/group).(C) Analysis of correlation of Ezh2 and p53 protein levels determined by IHC in Pten knockout (Ptenpc-/-) mice (n=11).(D) RT-qPCR analysis of p53 mRNA expression in four different genotypes of mice as described Statistical significance was determined by two-tailed Student's t-test. * P<0.01.(E) RT-qPCR analysis of mRNA expression of p53 downstream target gene Bax in four different genotypes of mice as indicated. Statistical significance was determined by two-tailed Student's t-test. * P< 0.01.(F) H&E analysis of ventral prostate (VP) of 4-month-old mice with indicated genotypes. The inset shows a high magnification image of the representative (framed) area in each panel. Scale bars, 50 µm. Scale bars in insets, 10 µm.(G) Quantification of nonmalignant, low-grade PIN (LGPIN) and high-grade PIN (HGPIN) or cancerous acini in the prostates (including anterior prostate (AP), ventral prostate (VP) and dorsolateral prostate (DLP)) of mice with the indicated genotypes and number as in (A).
Figure 5(A) Correlation analysis of EZH2 and p53 mRNA expression measured by RT-qPCR in primary and metastatic prostate cancer specimens of a cohort of patients from University of Washington.(B) IHC analysis of EZH2 and p53 protein expression in human prostate cancer tissues of a cohort of patients from Mayo Clinic. Case 1 and 2 show low and high expression in primary tissues, respectively; case 3 shows high expression in lymph node metastasis. Scale bar, 50 µm. Scale bars in insets, 10 µm.(C) Correlation analysis of EZH2 and p53 protein expression in primary and metastatic prostate cancer specimens of the Mayo Clinic cohort.(D) IHC analysis of EZH2 and p53 protein expression in commercially purchased TMA with patient specimens of brain, colorectal, pancreatic cancer and sarcoma. Scale bars in low magnification images, 200 µm; Scale bars in high magnification images, 50 µm. Arrows, indicated the zoom out-box. (E) IHC data as shown in (D) were quantified and used for correlation analysis of EZH2 and p53 protein expression in patient specimens of brain, colorectal, pancreatic cancer and sarcoma. Pearson correlation r and P values in each cancer type are indicated.