EMBO/SourceData huggingface dataset:
Figure 6A, B Following treatment with 10 μM metformin for 3 days, the levels of HSF1 binding to endogenous HSP promoters were quantitated by chromatin IP (mean ± SD, n = 3, one-way ANOVA, **P < 0.01, ***P < 0.001). Normal rabbit IgG served as the negative control (A). Following the same treatment, HSP mRNA levels were quantitated by qRT-PCR (mean ± SD, n = 3, Student's t-test, ***P < 0.001) (B).C, D Following transfection with AMPKα1/2-targeting siRNAs for 2 days, WM115 cells were treated with 10 μM metformin for 3 days. HSP mRNA levels were quantitated by qRT-PCR (mean ± SD, n = 6, Student's t-test, n.s., not significant, *P < 0.05, **P < 0.01).C, D Following transfection with AMPKα1/2-targeting siRNAs for 2 days, WM115 cells were treated with 10 μM metformin for 3 days. HSP mRNA levels were quantitated by qRT-PCR (mean ± SD, n = 6, Student's t-test, n.s., not significant, *P < 0.05, **P < 0.01).E Following treatment with 10 μM metformin for 7 days, protein levels in WM115 and S462 cells were measured by immunoblotting.F, G Following treatment with 10 μM metformin for 7 days, levels of polyubiquitinated proteins were detected in both detergent-soluble and detergent-insoluble fractions in WM115 and A2058 cells using a Lys48-specific polyubiquitin antibody.H A2058 cells were treated with 10 μM metformin alone or co-treated with 2 μM compound C for 7 days. Individual proteins were detected by immunoblotting.I HEK293T cells stably expressing either scramble or HSF1-targeting shRNAs were treated with 10 μM metformin for 7 days. Individual proteins were detected by immunoblotting.J In HEK293T cells, a plasmid encoding HA-polyQ79-GFP was co-transfected with the indicated plasmids. Following treatment with and without 10 μM metformin for 5 days, the sizes of aggregates in detergent-insoluble fractions were quantitated by a Multisizer™ 3 Coulter Counter. Experimental details are described in Materials and Methods.K, L Immortalized and RAS-transformed MEFs were treated with either 10 μM metformin (K) or different concentrations of glucose (L) for 7 days. Individual proteins were detected by immunoblotting.M Following transfection with LacZ or polyQ79, HEK293T cells were grown under different concentrations of glucose for 5 days before measuring aggregate size.
Figure 7A, B A2058 cells were transduced with lentiviral scramble or HSF1-targeting (hA6 and hA9) shRNAs. (A) Cell numbers were quantitated by Hoechst 33342 DNA staining (mean ± SD, n = 4, two-way ANOVA, ***P < 0.001). (B) Protein levels were measured by immunoblotting.C A2058 cells stably expressing LacZ or HSF1 were grown in medium containing 4.5, 1.0, or 0.45 g/l glucose. Following treatment with 10 μM metformin, cell proliferation was measured by Hoechst 33342 DNA staining (mean ± SD, n = 5, two-way ANOVA, ***P < 0.001).D, E 1 × 106 LacZ- or HSF1-expressing A2058 cells were transplanted into female NOD/SCID mice. One day after transplantation, metformin was administered via drinking water at 1 mg/ml. Tumor incidence (log-rank test; D) and volumes (E) were measured (mean ± SEM, two-way ANOVA n.s., not significant, *P < 0.05, **P < 0.01, ***P < 0.001). Tumor growth curves were fitted to exponential growth models to calculate tumor doubling time (DT).F Individual proteins were measured by immunoblotting using lysates of three tumors from each treatment group.G Protein levels were measured by immunoblotting using lysates of mouse liver tissues.H A2058 cells stably expressing FLAG-HSF1WT or FLAG-HSF1S121A were treated with 10 μM metformin for 7 days. Protein levels were measured by immunoblotting.I A2058 cells stably expressing HSF1WT or HSF1S121A were grown in Corning®ultra-low attachment 96-well culture plates with 10 μM metformin and different concentrations of glucose for 7 days, 10,000 cells per well. Viable cells were counted by Guava flow cytometer using ViaCount® reagent (mean ± SEM, n = 8, Student's t-test, n.s., not significant, **P < 0.01, ***P < 0.001).J Tukey box plots showing the inverse correlation between AMPKα Thr172 phosphorylation and HSP mRNA expression in ccRCC. Patients were stratified on the median value of AMPKα Thr172 phosphorylation reverse-phase protein array (RPPA) scores (Student's t-test).
Figure 1(A) Localization of endogenous SHP-1 in hematopoietic and non-hematopoietic cells was determined by confocal imaging after staining with SHP-1 antibody. DAPI was used for nuclear staining, Scale bar:5µm. Fluorescence intensities along the line drawn in a representative cell were obtained using Zen software and the co-localization plots were made using GraphPad (Red: SHP-1, Blue: DAPI).(C) SFB tagged PAF1, SFB-LEO1, SFB-CTR9, SFB-CDC73, SFB-RTF1, SFB-WDR61 along with Myc SHP-1 or Myc PTPN7 were transfected in 293T cells and interaction of SHP-1 with PAF complex components was detected by immunoblotting with Myc antibody after S protein beads pulldown in presence of TurboNuclease.(D) SFB empty vector, SFB SHP-1 or SFB PTPN7 was co-transfected with Myc tagged H2B in 293T cells. The interaction of H2B with SHP-1 was detected through immunoblotting using Myc antibody after pull down with streptavidin sepharose beads.(E) Endogenous association of SHP-1 with Paf1 complex subunits (CDC73, RTF1, WDR61, LEO1, Paf1, CTR9) was detected by immunoblotting after performing immunoprecipitation with either IgG or SHP-1 antibody.(F) Immunoprecipitation (IP) with control IgG or anti-SHP-1 antibody was performed with extracts prepared from MCF7 cells and endogenous association of H2B with SHP-1 was detected by immunoblotting.(G) Glutathione Sepharose beads immobilized with bacterially expressed recombinant GST or GST-H2B proteins were incubated with bacterially purified recombinant MBP-SHP-1. The association of SHP-1 with H2B was detected by immunoblotting with MBP antibody. Expression of GST, GST-H2B and MBP SHP-1 was shown by Coomassie staining.
Figure 2(A) Nuclear extracts of WT and SHP-1 knockout cells (derived from two independent guide RNAs) were made and the levels of H2BUb were detected.(B) Wild type (WT) 293T cells along with SHP-1 KO cells transfected with either Myc vector, Myc SHP-1 ∆SH2 or Myc SHP-1 NLS mutant were lysed. H2BUb levels were determined using specific antibody.(C) WT and SHP-1 KO 293T cells transfected with Myc vector, Myc SHP-1 or Myc SHP-1 C/S (C453S) mutant were lysed. H2BUb levels were determined using specific antibody.(D) H2B WT along with the indicated Y/F and Y/D mutants were transfected into 293T cells. Histone extract was prepared from cells transfected with each construct and subjected to western blotting with anti-H2B and anti-H2BUb antibodies.(E) In vitro ubiquitination assay was performed using bacterially purified GST, GST tagged H2B and its mutants as substrates. Recombinant E1, E2(UBE2A) and ubiquitin were included in each reaction. Ubiquitination was detected by H2BUb antibody.(F) In vitro ubiquitination was performed using biotin tagged non-phospho and phospho (pY121) peptide of H2B as substrates in the presence of E1 and E2. H2BUb was detected by immunoblotting with specific antibody.
Figure 3(A) In vitro phosphorylated GST H2B by Syk kinase was incubated with bacterially purified recombinant wild type SHP-1 or elution buffer and the amount of released phosphate was assayed colorimetrically using the malachite green reagent (A620 nm). Data represent mean absorbance from four independent experiments. Error bar indicates SD; *p<0.05, by Student's t test.(B) Bacterially purified GST H2B was subjected to in vitro kinase assay using HEK293T cell lysate, and then phosphorylated proteins were incubated with either bacterially purified MBP tagged constitutively active (SHP-1 ΔSH2), or catalytically inactive SHP-1 C/S mutant. The phosphorylation status of recombinant H2B was detected by immunoblotting with phospho-tyrosine antibody.(C) Phospho (pH2B) Y121 and non-phospho peptide of H2B was incubated with constitutively active (SHP-1 ΔSH2) or catalytically inactive mutant (C/S) of SHP-1 and the released phosphate was assayed colorimetrically using the malachite green reagent (A620nm). n=3 independent experiments, Error bars indicate SD; ***p<0.001 (one-way ANOVA, Tukey's multiple comparison test).(D) 293T cells were transfected with vector, Myc tagged SHP-1 WT or C/S mutant. The phosphorylation status of endogenous H2B was detected by immunoblotting with pY121-H2B antibody.(E) Nuclear extracts of 293T WT and SHP-1 KO cells were made and the levels of H2B phosphorylation were detected using pY121-H2B antibody.(F) Wild type (WT) along with SHP-1 KO 293T cells transfected with either Myc vector, Myc SHP-1 or Myc SHP-1 C/S mutant were lysed. Levels of pY121-H2B and H2BUb were determined using specific antibodies.(G) 293T Cells were treated with SHP-1 inhibitor TPI (50nm) for 1hr and levels of pY121-H2B was determined using pY121H2B antibody.MCF7 cells were transduced with (H) CTR9 shRNA along with control shRNA. Post 72 h, cells were collected and lysed to isolate soluble and nuclear fractions. Levels of phosphorylated and ubiquitinated H2B were detected by immunoblotting with indicated specific antibodies.MCF7 cells were transduced with (I) Paf1 shRNA, along with control shRNA. Post 72 h, cells were collected and lysed to isolate soluble and nuclear fractions. Levels of phosphorylated and ubiquitinated H2B were detected by immunoblotting with indicated specific antibodies.MCF7 cells were transduced with (J) RTF1 shRNA along with control shRNA. Post 72 h, cells were collected and lysed to isolate soluble and nuclear fractions. Levels of phosphorylated and ubiquitinated H2B were detected by immunoblotting with indicated specific antibodies.
Figure 4(A) Cell lysates derived from 293T cells transfected with empty vector, SFB-tagged H2B, H2B Y121F or H2B Y121D constructs were pulled down with S-protein agarose beads. RNF20 interaction with H2B was detected by immunoblotting with specific antibody.(B) HEK293T cells were co-transfected with the SFB-tagged H2B, H2B Y121F or H2B Y121D constructs along with Myc-tagged UBE2A. Cell lysates were pulled down with S-protein beads, and interaction was detected by immunoblotting with Myc antibody.(C) Glutathione Sepharose beads bound with bacterially expressed recombinant GST-H2B, GST H2B Y121F and GST-H2B Y121D proteins were incubated with bacterially purified recombinant His UBE2A, and interaction of UBE2A with H2B was detected by immunoblotting with His antibody. Recombinant protein expression and pulldown was shown by Coomassie staining.(D) Bacterially purified GST, GST H2B and GST H2B-p (phosphorylated using Syk) was incubated with recombinant His UBE2A, and interaction of UBE2A with H2B was detected by immunoblotting with His antibody. Recombinant protein expression and pulldown was shown by Coomassie staining.(E) WT 293T cells along with SHP-1 KO were co-transfected with SFB vector or SFB H2B along with Myc UbE2A. Cell lysates were pulldown using S-protein beads, and interaction of UBE2A with H2B was determined by Western blotting with the indicated antibodies.(F) HEK293T cells were transfected with SFB vector or SFB H2B along with Myc UBE2A.Cells were treated with SHP-1 inhibitor TPI (50nm) for 1hr after 24hrs of transfection. Cells lysates were subjected to pull-down with S-protein beads, and western blotting was performed using specific antibodies.
Figure 5(A) Representative super-resolution images of MCF7 cells immunostained with SHP-1 in combination with CDK9, total RNA Polymerse II, RNA polymerase II Ser2P, and RNA Polymerase II Ser5P. Scale bar: 5µm. Fluorescence intensities along the line drawn in a representative section of cell were obtained using Zen software and the co-localization plots were made using GraphPad.(B) Co-localization of SHP-1 with CDK9, total RNA Polymerase II, RNA Polymerase II S2P, and RNA Polymerase II S5P was measured by Pearson correlation coefficient using ImageJ. Values presented as the mean ± SD, n=10 (from three independent experiments). ***p value < 0.001. (One-way ANOVA).(C) Endogenous interaction of SHP-1 with RNA Polymerase II, RNA polymerase II Ser2P, and RNA Polymerase II Ser5P and CDK9 was detected by immunoblotting after performing immunoprecipitation with either IgG or SHP-1 antibody using TurboNuclease treated cell lysate.(D) Nuclear extracts of MCF-7 WT and SHP-1 knockout cells were made and the levels of RNA Polymerase II, RNA Polymerase II Ser2P and RNA Polymerase II Ser5P were detected by immunoblotting using specific antibodies.(E) Nuclear extracts of cells treated with either DMSO or SHP-1 inhibitor TPI (50nm) were used to detect total RNA Polymerase II and its modifications by immunoblotting using specific antibodies.(F) Nuclear extracts of MCF-7 WT and SHP-1 knockout cells transfected with Myc vector, H2B Y121F or H2B Y121D constructs were made and the levels of RNA Polymerase II, RNA Polymerase II Ser2P, RNA Polymerase II Ser5P and Lamin B1 were detected by immunoblotting using specific antibodies.(G) Immunofluorescence of WT or SHP-1 knockout MCF7 cells labelled with the 5-Ethynyl Uridine (EU) for 60 minutes to capture nascent RNA synthesis. Scale bars 10μm. (H) The EU incorporation in MCF-7 WT and SHP-1 KO cells was quantified in three independent experiments. Values presented as the mean ± SD, ***indicates p<0.001 (One-way ANOVA).(I) Representative images of immunofluorescence of DMSO or TPI treated (50nm) MCF7 cells labelled with EU for 60 minutes was shown. Scale bar: 40µm. (J) The EU incorporation in MCF7 cells treated with DMSO or TPI was quantified in three independent experiments. Values presented as the mean ± SD, ***indicates p<0.001 (One-way ANOVA).
Figure 7(C, D) MCF-7 WT cells along with SHP-1 KO cells transfected with either vector, H2B Y121F or H2B Y121D mutant were used for ChIP q-PCR analysis of the H2BUb at indicated genes. The data shown is derived from two independent experiments. Error bars indicate the mean ± SD.
Figure 8(A) Lysates of WT and SHP-1 knockout MCF7 cells were made and the levels of LC3-I and II were detected by immunoblotting. (B) Levels of LC3 II were normalized against total LC3 (LC3 I+LC3 II). The data shown is derived from three independent experiments. Error bars indicate the mean ± SD. *p value < 0.05, Student's t-test.(C) Representative super- resolution images of WT and SHP-1 KO MCF7 cells immunostained with LC3 antibody, (Scale bar: 5µm). (D) Quantification of LC3 puncta accumulation per cell done by using ImageJ was shown. Bars represents mean ± SD.(N=60) ***p value < 0.001, Student's t-test.(E) WT along with SHP-1 KO MCF7 cells transfected with either Myc vector, Myc SHP-1 or Myc SHP-1 NLS mutant were lysed. Levels of LC3 and actin were determined using specific antibodies.(F) WT and SHP-1 knockout MCF7 cells transfected with the Myc vector or Myc-tagged H2B, H2B Y121F or H2B Y121D constructs were immunostained with endogenous p62 and Myc antibody. Image acquisition was done using LSM 700, (Scale bar: 20µm). (G) Accumulation of p62 was quantified by counting p62 positive puncta per cell. (N= 25); values presented as the mean ± SD. ***p value < 0.001 (One-way ANOVA).(H) Representative super-resolution images of LC3 and LAMP2 immunostaining in WT and SHP-1 KO MCF7 cells. (Scale bar: 20 µm for merge & 5 µm for Zoom). (I) LC3 puncta co-localizing with LAMP2 per cell were quantified using ImageJ. (N=45); values presented as the mean ± SD. ***p value < 0.001, Student's t-test.
Figure 1B. UTP6 in U-2 OS cells exemplify proteins localized to whole nucleoli (HPA025936).C. Fibrillar center localization shown by a UBTF IF staining in U-2 OS cells (HPA006385).E. Dual localization of LEO1 to both the fibrillar center and nucleoplasm in GFP-tagged HeLa cells (magenta), also supported by IF antibody staining using HPA040741 (green).F. The multilocalizing ribosomal protein RPL13 detected in the nucleoli, cytosol and ER in MCF-7 cells (HPA051702).
Figure 2A. KRI1 localized to the nucleoli in MCF-7 cells (HPA043574). Data information: Protein of interest is shown in green, nuclear marker DAPI in blue and the reference marker of microtubules in red. Scale bar 10 µm.B. GPATCH4 localized to the nucleoli in U-2 OS cells (HPA054319). Data information: Protein of interest is shown in green, nuclear marker DAPI in blue and the reference marker of microtubules in red. Scale bar 10 µm.C. FOXL2NB localized to the fibrillar center in SiHa cells (HPA061017). Data information: Protein of interest is shown in green, nuclear marker DAPI in blue and the reference marker of microtubules in red. Scale bar 10 µm.D. FAM71E1 localized to the fibrillar center in U-2 OS cells (HPA048111). Data information: Protein of interest is shown in green, nuclear marker DAPI in blue and the reference marker of microtubules in red. Scale bar 10 µm.E. LRRC23 localized to the nucleoli in U-2 OS cells (HPA057533). Data information: Protein of interest is shown in green, nuclear marker DAPI in blue and the reference marker of microtubules in red. Scale bar 10 µm.F. ZNF853 localized to the nucleoli in U-2 OS cells (HPA067690). Data information: Protein of interest is shown in green, nuclear marker DAPI in blue and the reference marker of microtubules in red. Scale bar 10 µm.G. METTL5 localized to the nucleoli in U-2 OS cells (HPA038223). Data information: Protein of interest is shown in green, nuclear marker DAPI in blue and the reference marker of microtubules in red. Scale bar 10 µm.
Figure 3B. IF staining of MKI67 shows localization to nucleoli rim in U-251 cells (CAB000058). Data information: Protein of interest is shown in green, nuclear marker DAPI/Hoechst in blue and microtubules reference marker in red. Scale bar 10 µm.C. IF staining of GNL3 shows localization to nucleoli rim in U-2 OS cells (HPA036743). Data information: Protein of interest is shown in green, nuclear marker DAPI/Hoechst in blue and microtubules reference marker in red. Scale bar 10 µm.D. HEK 293T cells expressing endogenous levels of N-terminus mNG-MKI67 show a faint nucleoli rim localization, although still visible. White arrows indicate cells where the rim could be seen. Data information: Protein of interest is shown in green, nuclear marker DAPI/Hoechst in blue and microtubules reference marker in red. Scale bar 10 µm.E. Fixed HEK 293T cells expressing mNG-tagged MKI67 show a clear nucleoli rim localization. Data information: Protein of interest is shown in green, nuclear marker DAPI/Hoechst in blue and microtubules reference marker in red. Scale bar 10 µm.F. HEK 293T cells expressing endogenous levels of C-terminus tagged GNL3 show nucleoli rim localization. White arrows indicate cells where the rim could be seen. Data information: Protein of interest is shown in green, nuclear marker DAPI/Hoechst in blue and microtubules reference marker in red. Scale bar 10 µm.
Figure 4A. MKI67 (HPA000451). B. NOC2L (HPA044258). C. EMG1 (HPA039304). D. BMS1 (HPA043081). E. BRIX1 (HPA039614). F. RSL1D1 (HPA043483). Data information: Protein of interest is shown in green, microtubules in red and DAPI in blue. Images of interphase cells were acquired from a different experiment and staining intensities cannot be compared between interphase and mitotic cells. Scale bar 10 μm.
Figure 5C-E. IF stainings of previously uncharacterized mitotic chromosome proteins predicted to be highly disordered. Protein of interest shown in green and DAPI in blue. Scale bar 10 µm. C. CCDC86 (HPA041117), 100% predicted disorder level. D. RRP15 (HPA024639), 87% predicted disorder level. E. PRR19 (HPA070350), 67% predicted disorder level.
Figure 6A. MKI67 is detected on the chromosomes throughout all mitotic phases and exemplifies proteins in the early recruitment category. B-F. IF stainings of proteins where localization to mitotic chromosomes is seen after metaphase, showing a typical late recruitment expression pattern. B. NOL12 (HPA003547). C. TIGD1 (HPA041717). D. BYSL (HPA031219). E. ACTL6B (HPA045126). F. ZNF202 (HPA059229). Data information: Protein of interest is shown in green, microtubules in red and DAPI in blue. Images of interphase cells were acquired from a different experiment and staining intensities cannot be compared between interphase and mitotic cells. Scale bar 10 μm.
Figure 7A-B. GNL3 and PES1 are localizing to protein aggregates around the chromosomes in the MKI67 KO cells. A. GNL3 (HPA036743). Median overlapKO = 0.539, median overlapWT = 0.795 (P = 9.34x10-9) and median Pearson correlationKO = -0.335, median Pearson correlationWT = 0.134 (P = 3.58x10-6). B. PES1 (HPA040210). Median overlapKO = 0.516, median overlapWT = 0.755 (P = 1.77x10-13) and median Pearson correlationKO = -0.293, median Pearson correlationWT = 0.129 (P = 4.06x10-13). Data information: Protein of interest is shown in green and DAPI in blue. Scale bar 10 μm. For the colocalization analysis data; biological replicate 1 highlighted in grey and biological replicate 2 in green. Both the overlap and Pearson correlation between DAPI/Protein staining was measured and a two-tailed unpaired Wilcoxon test was used.C. No protein aggregates were observed in MKI67 KO cells for PWP1 (HPA038708), despite being localized to mitotic chromosomes in WT cells. Median overlapKO = 0.523, median overlapWT = 0.861 (P = 1.05x10-13) and median Pearson correlationKO = -0.190, median Pearson correlationWT = 0.515 (P = 8.71x10-15). Data information: Protein of interest is shown in green and DAPI in blue. Scale bar 10 μm. For the colocalization analysis data; biological replicate 1 highlighted in grey and biological replicate 2 in green. Both the overlap and Pearson correlation between DAPI/Protein staining was measured and a two-tailed unpaired Wilcoxon test was used. Box limits mark 1st and 3rd quantile, whiskers the 1.5x interquartile range and center line the median.D-E. Late recruited proteins do not follow the same recruitment pattern in the MKI67 KO cells compared to WT HeLa cells. White arrows indicate where protein aggregates can be seen. D. DENND4A (HPA065343). Median overlapKO = 0.671, median overlapWT = 0.803 (P = 1.02x10-4) and median Pearson correlationKO = -0.224, median Pearson correlationWT = 0.236 (P = 1.91x10-5). E. NOL12 (HPA003547). Median overlapKO = 0.468, median overlapWT = 0.820 (P = 1.01x10-17) and median Pearson correlationKO = -0.318, median Pearson correlationWT = 0.381 (P = 1.81x10-10). White arrow indicates metaphase cell. Data information: Protein of interest is shown in green and DAPI in blue. Scale bar 10 μm. For the colocalization analysis data; biological replicate 1 highlighted in grey and biological replicate 2 in green. Both the overlap and Pearson correlation between DAPI/Protein staining was measured and a two-tailed unpaired Wilcoxon test was used. Box limits mark 1st and 3rd quantile, whiskers the 1.5x interquartile range and center line the median.
Figure 1c, Log normalised expression of markers for epithelial (EPCAM), mature luminal epithelial (ESR1), myoepithelial (KRT5, KRT14 and ACTA2) and proliferating cancer cells (MKI67).e, Log normalised expression of markers for fibroblasts (PDGFRB, THY1, COL1A1, ITGB1 and S100A4), endothelial cells (PECAM1), T-cells (CD3D), CD8 T cells (CD8A), T-regulatory cells (FOXP3), B-cells (MS4A1), myeloid cells (CD68) and plasma cells (JCHAIN).
Figure 2c, Log expression of parenchymal gene markers commonly associated with CAFs and perivascular cells.
Figure 3b, Candidate transcriptional drivers of each CAF and PVL subset. Violin plots showing the log normalised gene expression (left) of the TF and its respective AUC regulon activity (right). TFs ZEB1 and FOXP1 enriched in myofibroblast-like CAFs, EGR2 and TCF7L2 enriched in inflammatory-like CAFs, MEF2C enriched in PVL cells and NR2F2 enriched in immature-PVL cells.