C : H2O2 induces loss of TRIM28 SUMOylation in HeLa cells but not in U2OS cells . HeLa or U2OS cells were mock treated or treated with 500 µM H2O2 for 5 or 15 min . Cells were lysed with 1 % SDS in PBS buffer and 20 mM NEM . Upon immunoprecipitation of the SUMO1 proteome , samples were analyzed by immunoblotting with TRIM28 antibodies .

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C: H2O2 induces loss of TRIM28 SUMOylation in HeLa cells but not in U2OS cells. HeLa or U2OS cells were mock treated or treated with 500 µM H2O2 for 5 or 15 min. Cells were lysed with 1% SDS in PBS buffer and 20 mM NEM. Upon immunoprecipitation of the SUMO1 proteome, samples were analyzed by immunoblotting with TRIM28 antibodies.


D : Induction of the Uba2 ~ Ubc9 disulfide is a conserved mechanism . HeLa , MCF7 , U2OS or RPE cells were treated with increasing concentration of H2O2 for 15 min , lysed in non reducing buffer supplemented with 20 mM NEM , and analyzed by immunoblotting with Uba2 antibodies .

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D: Induction of the Uba2~Ubc9 disulfide is a conserved mechanism. HeLa, MCF7, U2OS or RPE cells were treated with increasing concentration of H2O2 for 15 min, lysed in non reducing buffer supplemented with 20 mM NEM, and analyzed by immunoblotting with Uba2 antibodies.


A : Upper panel : Strategy for the identification of oxidation resistant Ubc9 variants . Ubc9 was mutagenized by PCR under conditions that introduced 1 - 3 random mutations . Ubc9 variants were expressed in bacteria and the bacterial lysates of individual clones tested for E2 activity in the presence or absence of 1 mM H2O2 . For this purpose , we used a FRET - based in vitro SUMOylation assay with recombinant SUMO E1 , YFP - SUMO and CFP - RanGAPtail and ATP . Lower panel : Ubc9 W103R is H2O2 resistant . Bacterial lysates containing Ubc9 wt ( left panel ) or Ubc9 W103R ( right panel ) were tested as described .

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A: Upper panel: Strategy for the identification of oxidation resistant Ubc9 variants. Ubc9 was mutagenized by PCR under conditions that introduced 1 - 3 random mutations. Ubc9 variants were expressed in bacteria and the bacterial lysates of individual clones tested for E2 activity in the presence or absence of 1 mM H2O2. For this purpose, we used a FRET-based in vitro SUMOylation assay with recombinant SUMO E1, YFP-SUMO and CFP-RanGAPtail and ATP. Lower panel: Ubc9 W103R is H2O2 resistant. Bacterial lysates containing Ubc9 wt (left panel) or Ubc9 W103R (right panel) were tested as described.


B : Ubc9 W103 mutants are H2O2 resistant but not fully active . Left panel : Recombinant Ubc9 W103R , W103A and W103F were purified ( embedded panel ) . Resistance against oxidation was tested under conditions of limiting E1 enzyme : 21 nM Aos1 / Uba2 and 73 nM Ubc9 were incubated with H2O2 prior to the addition of 160 nM each of YFP - SUMO1 and CFP - RanGAPtail . Right panel : To compare specific activities of wt and mutants in the absence of H2O2 , sumoylation assays were carried out using limiting Ubc9 concentration . Reactions contained 35 nM Aos1 / Uba2 , 11 nM Ubc9 , 85 nM each of YFP - SUMO1 and CFP - RanGAPtail and 1 mM ATP .

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B: Ubc9 W103 mutants are H2O2 resistant but not fully active. Left panel: Recombinant Ubc9 W103R, W103A and W103F were purified (embedded panel). Resistance against oxidation was tested under conditions of limiting E1 enzyme: 21 nM Aos1/Uba2 and 73 nM Ubc9 were incubated with H2O2 prior to the addition of 160 nM each of YFP-SUMO1 and CFP-RanGAPtail. Right panel: To compare specific activities of wt and mutants in the absence of H2O2, sumoylation assays were carried out using limiting Ubc9 concentration. Reactions contained 35 nM Aos1/Uba2, 11 nM Ubc9, 85 nM each of YFP-SUMO1 and CFP-RanGAPtail and 1 mM ATP.


B : In FRET - based assay , Ubc9 D100A is active upon H2O2 treatment . Ubc9 D100A was purified and compared for activity in the presence of H2O2 as described in Fig . 2B .

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B: In FRET-based assay, Ubc9 D100A is active upon H2O2 treatment. Ubc9 D100A was purified and compared for activity in the presence of H2O2 as described in Fig. 2B.


C : Ubc9 wt and D100A are equally active in CFP - RanGAPtail SUMOylation . Assays were with 35 nM Aos1 / Uba2 and 15 nM Ubc9 .

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C: Ubc9 wt and D100A are equally active in CFP-RanGAPtail SUMOylation. Assays were with 35 nM Aos1/Uba2 and 15 nM Ubc9.


D : Ubc9 D100A is fully competent to form a thioester with SUMO . Single turnover reactions were performed using 630 nM E1 , 3 , 3 µM SUMO1 and 600 nM Ubc9 . Reactions were stopped at different time points by addition of non - reducing buffer . .

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D: Ubc9 D100A is fully competent to form a thioester with SUMO. Single turnover reactions were performed using 630 nM E1, 3,3 µM SUMO1 and 600 nM Ubc9. Reactions were stopped at different time points by addition of non-reducing buffer..


E : Ubc9 wt and D100A are equally active in PIAS2b - dependent SUMOylation . Assays were with 300 nM GST - p53 , 170 nM E1 , 120 nM Ubc9 , 5 µM SUMO2 and 49 nM GST -

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E: Ubc9 wt and D100A are equally active in PIAS2b - dependent SUMOylation. Assays were with 300 nM GST-p53, 170 nM E1, 120 nM Ubc9, 5 µM SUMO2 and 49 nM GST-Pias2b


F : Ubc9 wt or D100A are equally active in RanBP2 - dependent SUMOylation . Assays were with 650 nM YFP - SP100 , 110 nM of E1 , 25 Ubc9 , 40 nM RanBP2ΔFG and 3 , 5 µM SUMO1 ( Left panel ) or SUMO2 ( Right panel ) .

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F: Ubc9 wt or D100A are equally active in RanBP2 - dependent SUMOylation. Assays were with 650 nM YFP-SP100, 110 nM of E1, 25 Ubc9, 40 nM RanBP2ΔFG and 3,5 µM SUMO1 (Left panel) or SUMO2 (Right panel).


A : Ubc9 D100A enzymatic activity can be recovered by limited amount of DTT . 21 nM Aos1 / Uba2 and 75 nM Ubc9 were incubated with H2O2 for 30 min prior addition of 65 nM each YFP - SUMO1 and CFP - RanGAPtail diluted in DTT - free buffer . The final DTT concentration in the reaction was 6 µM . Prior to addition of ATP , extra amount of DTT was added at indicated final concentration .

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A: Ubc9 D100A enzymatic activity can be recovered by limited amount of DTT. 21 nM Aos1/Uba2 and 75 nM Ubc9 were incubated with H2O2 for 30 min prior addition of 65 nM each YFP-SUMO1 and CFP-RanGAPtail diluted in DTT-free buffer. The final DTT concentration in the reaction was 6 µM. Prior to addition of ATP, extra amount of DTT was added at indicated final concentration.


B : Both Uba2 ~ Ubc9 disulfide formation and reduction are accelerated with Ubc9 D100A . 100 nM E1 and 1 µM E2 were incubated in presence of 1 mM H2O2 . One hour after H2O2 addition , 500 µM reduced glutathione was added . Samples were taken at indicated time points and the Uba2 ~ Ubc9 disulfide was monitored by immunoblotting against Uba2 . Lower panel : Quantification of the ratio between crosslinked Uba2 vs total

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B: Both Uba2~Ubc9 disulfide formation and reduction are accelerated with Ubc9 D100A.100 nM E1 and 1 µM E2 were incubated in presence of 1 mM H2O2. One hour after H2O2 addition, 500 µM reduced glutathione was added. Samples were taken at indicated time points and the Uba2~Ubc9 disulfide was monitored by immunoblotting against Uba2. Lower panel: Quantification of the ratio between crosslinked Uba2 vs total Uba2


A : Generation of polyclonal populations of stable cells . Top panel : Replacement strategy : U2OS cells transfected with pIRES - hrGFP II - based vectors were selected via addition of G418 . After 4 weeks , low level GFP expressing cells were sorted by FACS after 4 weeks and again after 8 weeks . Bottom panel : Stable cells express Ubc9 wt and D100A at levels comparable to endogenous Ubc9 : U2OS or U2OS stable cells were transfected with siRNA targeting human Ubc9 mRNA for 72h or mocked transfected . Cells were lysed in Laemmli buffer and Ubc9 and a - tubulin as loading control were detected by immunoblotting .

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A: Generation of polyclonal populations of stable cells. Top panel: Replacement strategy: U2OS cells transfected with pIRES-hrGFP II-based vectors were selected via addition of G418. After 4 weeks, low level GFP expressing cells were sorted by FACS after 4 weeks and again after 8 weeks. Bottom panel: Stable cells express Ubc9 wt and D100A at levels comparable to endogenous Ubc9: U2OS or U2OS stable cells were transfected with siRNA targeting human Ubc9 mRNA for 72h or mocked transfected. Cells were lysed in Laemmli buffer and Ubc9 and a-tubulin as loading control were detected by immunoblotting.


B : Ubc9 D100A is severely impaired for disulfide bond formation with Uba2 . Stable U2OS cells were transfected with siRNA against Ubc9 for 72h , treated with 250 µM H2O2 for different time points , from 2 min to 2 hours , lysed in non - reducing buffer and analyzed by immunoblotting against Ubc9 to detect the disulfide ( upper panel ) or free Uba2 as loading control ( see also Appendix Fig . S5 ) . Lower panel : relative intensities of Ubc9 bands were measured using imageJ . Highest intensities in both cell population were set to 1 .

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B: Ubc9 D100A is severely impaired for disulfide bond formation with Uba2. Stable U2OS cells were transfected with siRNA against Ubc9 for 72h, treated with 250 µM H2O2 for different time points, from 2 min to 2 hours, lysed in non-reducing buffer and analyzed by immunoblotting against Ubc9 to detect the disulfide (upper panel) or free Uba2 as loading control (see also Appendix Fig. S5). Lower panel: relative intensities of Ubc9 bands were measured using imageJ. Highest intensities in both cell population were set to 1.


C : Ubc9 D100A impairs cell survival upon H2O2 treatment . Ubc9 wt or Ubc9 D100A cells were transfected with siRNA against endogenous Ubc9 for 72h , transferred into 6 well plates ( 20 000 cells / well ) and treated with increasing H2O2 concentration for 1h . Ten days later , cells were washed with PBS , fixed with 4 % formaldehyde and stained with crystal violet . The area occupied by the cells was evaluated using ImageJ and the ColonyArea plugin . An arbitrary value of 1 was given to the area occupied by non - treated cells . The graph is showing the mean of 4 independent experiments . Error bars are S . E . M . * : p < 0 .

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C: Ubc9 D100A impairs cell survival upon H2O2 treatment. Ubc9 wt or Ubc9 D100A cells were transfected with siRNA against endogenous Ubc9 for 72h, transferred into 6 well plates (20 000 cells / well) and treated with increasing H2O2 concentration for 1h. Ten days later, cells were washed with PBS, fixed with 4% formaldehyde and stained with crystal violet. The area occupied by the cells was evaluated using ImageJ and the ColonyArea plugin. An arbitrary value of 1 was given to the area occupied by non-treated cells. The graph is showing the mean of 4 independent experiments. Error bars are S.E.M. *: p < 0.05


D : Constitutive expression of Ubc9 D100A affects cellgrowth . 103 cells were plated per 10 cm plate and cultivated for 10 days in 20 % or 5 % O2 incubators . Subsequently , cells were colored using crystal violet . The area of each clone was measured using ImageJ . Representative plates are shown in the upper panel . Lower left panel : Distribution of colony sizes , shown for one of the three biological replicates . Lower right panel : Comparison of the colonies size between " Ubc9 wt " and " Ubc9 DA " cells . The average size of " Ubc9 wt " was set to 1 . Shown is the mean of 3 independent experiments , each of which was performed in technical triplicates . Error bars are S . E . M .

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D: Constitutive expression of Ubc9 D100A affects cellgrowth. 103 cells were plated per 10 cm plate and cultivated for 10 days in 20% or 5% O2 incubators. Subsequently, cells were colored using crystal violet. The area of each clone was measured using ImageJ. Representative plates are shown in the upper panel. Lower left panel: Distribution of colony sizes, shown for one of the three biological replicates. Lower right panel: Comparison of the colonies size between "Ubc9 wt"and "Ubc9 DA" cells. The average size of "Ubc9 wt" was set to 1. Shown is the mean of 3 independent experiments, each of which was performed in technical triplicates. Error bars are S.E.M.


A : Phosphorylation of ATM is impaired in Ubc9 D100A cells . Stable U2OS cell populations were depleted from endogenous Ubc9 by siRNA for 72 h before addition of 250 µM H2O2 . At the indicated times , cells were lysed in Laemmli buffer and analysed by immunoblotting with the indicated antibodies .

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A: Phosphorylation of ATM is impaired in Ubc9 D100A cells. Stable U2OS cell populations were depleted from endogenous Ubc9 by siRNA for 72 h before addition of 250 µM H2O2. At the indicated times, cells were lysed in Laemmli buffer and analysed by immunoblotting with the indicated antibodies.


B : Phosphorylation of gamma - H2AX is impaired in Ubc9 D100A . Experiment was performed as in A . Stable U2OS cell populations were depleted from endogenous Ubc9 by siRNA for 72 h before addition of 250 µM H2O2 . At the indicated times , cells were lysed in Laemmli buffer and analysed by immunoblotting with the indicated antibodies .

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B: Phosphorylation of gamma-H2AX is impaired in Ubc9 D100A. Experiment was performed as in A. Stable U2OS cell populations were depleted from endogenous Ubc9 by siRNA for 72 h before addition of 250 µM H2O2. At the indicated times, cells were lysed in Laemmli buffer and analysed by immunoblotting with the indicated antibodies.


C : Phosphorylation of Chk2 is impaired in Ubc9 D100A . Experiment was performed as described in A . Stable U2OS cell populations were depleted from endogenous Ubc9 by siRNA for 72 h before addition of 250 µM H2O2 . At the indicated times , cells were lysed in Laemmli buffer and analysed by immunoblotting with the indicated antibodies .

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C: Phosphorylation of Chk2 is impaired in Ubc9 D100A. Experiment was performed as described in A. Stable U2OS cell populations were depleted from endogenous Ubc9 by siRNA for 72 h before addition of 250 µM H2O2. At the indicated times, cells were lysed in Laemmli buffer and analysed by immunoblotting with the indicated antibodies.


D : Ubc9 D100A cells are fully competent for phosphorylation of ATM upon hydroxyurea exposure . Stable U2OS cell populations were exposed to 2 . 5 mM hydroxyurea or 250 µM H2O2 . Cells were lysed at indicated time points and analysed by immunoblotting with the indicated antibodies .

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D: Ubc9 D100A cells are fully competent for phosphorylation of ATM upon hydroxyurea exposure. Stable U2OS cell populations were exposed to 2.5 mM hydroxyurea or 250 µM H2O2. Cells were lysed at indicated time points and analysed by immunoblotting with the indicated antibodies.


E : Phosphorylation of Chk1 is not impaired in Ubc9 D100A cells . Stable U2OS cells were depleted from endogenous Ubc9 by siRNA for 72 h before addition of 2 . 5 mM hydroxyurea . At the indicated times , cells were lysed in Laemmli buffer and analysed by immunoblotting with the indicated antibodies .

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E: Phosphorylation of Chk1 is not impaired in Ubc9 D100A cells. Stable U2OS cells were depleted from endogenous Ubc9 by siRNA for 72 h before addition of 2.5 mM hydroxyurea. At the indicated times, cells were lysed in Laemmli buffer and analysed by immunoblotting with the indicated antibodies.


A : Impaired y - H2AX and 53BP1 accumulation in Ubc9 D100A cells . Stable U2OS cells were transfected with siRNA against endogenous Ubc9 for 72 h . Cells were treated with 125 µM H2O2 and immunofluorescence microscopy was performed . Left panel : representative immunofluorescence images . Right panel : quantification of cells with γ - H2AX and 53BP1 foci . Error bars represent S . E . M , n = 3 independent experiments * p < 0 . 05 .

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A: Impaired y-H2AX and 53BP1 accumulation in Ubc9 D100A cells. Stable U2OS cells were transfected with siRNA against endogenous Ubc9 for 72 h. Cells were treated with 125 µM H2O2 and immunofluorescence microscopy was performed. Left panel: representative immunofluorescence images. Right panel: quantification of cells with γ-H2AX and 53BP1 foci. Error bars represent S.E.M, n=3 independent experiments * p < 0.05.


B : H2O2 exposure leads to prolonged DNA damage in Ubc9 wt and Ubc9 D100A cells . Cells were treated as described in A . Stable U2OS cells were transfected with siRNA against endogenous Ubc9 for 72 h . Cells were treated with 125 µM H2O2 and immunofluorescence microscopy was performed . An alkaline comet assay was performed to evaluate the DNA damage . Representative images are shown . Error intervals represent S . E . M , n = 4 independent experiments .

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B: H2O2 exposure leads to prolonged DNA damage in Ubc9 wt and Ubc9 D100A cells. Cells were treated as described in A. Stable U2OS cells were transfected with siRNA against endogenous Ubc9 for 72 h. Cells were treated with 125 µM H2O2 and immunofluorescence microscopy was performed. An alkaline comet assay was performed to evaluate the DNA damage. Representative images are shown. Error intervals represent S.E.M, n=4 independent experiments.


A : Diamide is inducing the SUMO E1 ~ E2 disulfide but not ATM phosphorylation . U2OS stable cell populations were depleted from endogenous Ubc9 by siRNA for 72 h before addition of 500 µM diamide or 500 µM H2O2 . Cells were subsequently washed in PBS with 20 mM NEM and lysed in Laemmli buffer . The lysates were resolved by SDS - PAGE .

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A: Diamide is inducing the SUMO E1~E2 disulfide but not ATM phosphorylation. U2OS stable cell populations were depleted from endogenous Ubc9 by siRNA for 72 h before addition of 500 µM diamide or 500 µM H2O2. Cells were subsequently washed in PBS with 20 mM NEM and lysed in Laemmli buffer. The lysates were resolved by SDS-PAGE.


B : Left panel : The PP2A inhibitor okadaic acid rescues the y - H2AX defect of the " Ubc9 DA " cells . " Ubc9 wt " or " Ubc9 DA " cells were treated with 0 . 25 µM okadaic acid for 30 min . After addition of 250 µM H2O2 in fresh medium for 3 hours , immunofluorescence microscopy was performed as described in Fig . 7A . Error bars represent S . E . M , n = 3 independent experiments . Right panel : Representative immunofluorescence images .

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B: Left panel: The PP2A inhibitor okadaic acid rescues the y-H2AX defect of the "Ubc9 DA" cells. "Ubc9 wt" or "Ubc9 DA" cells were treated with 0.25 µM okadaic acid for 30 min. After addition of 250 µM H2O2 in fresh medium for 3 hours, immunofluorescence microscopy was performed as described in Fig. 7A. Error bars represent S.E.M, n=3 independent experiments. Right panel: Representative immunofluorescence images.


C : Left panel : Ubc9 D100A enhances sensitivity to Ara - C or etoposide . Cell survival assays were performed as described in Fig . 5C . Cells were exposed to Ara - C or etoposide ( VP16 ) for 1 h and evaluated 10 days later . Right panel : N - acetylcysteine treatment rescues the higher toxicity of Ara - C or etoposide on " Ubc9 DA " cells . Cells were treated with 0 . 5 mM NAC 16 h prior to addition of etoposide or Ara - C . Graphs represent means of three to four independent experiments , error bars represent S . E . M . * p < 0 . 05 .

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C: Left panel: Ubc9 D100A enhances sensitivity to Ara-C or etoposide. Cell survival assays were performed as described in Fig. 5C. Cells were exposed to Ara-C or etoposide (VP16) for 1 h and evaluated 10 days later. Right panel: N-acetylcysteine treatment rescues the higher toxicity of Ara-C or etoposide on "Ubc9 DA" cells. Cells were treated with 0.5 mM NAC 16 h prior to addition of etoposide or Ara-C. Graphs represent means of three to four independent experiments, error bars represent S.E.M. * p < 0.05.


B Following treatment with 10 mM metformin overnight , NIH3T3 reporter cells were heat - shocked at 43 ° C for 30 min and recovered at 37 ° C for 5 h . GFP induction was quantitated by flow cytometry . HS : heat shock .

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B Following treatment with 10 mM metformin overnight, NIH3T3 reporter cells were heat-shocked at 43°C for 30 min and recovered at 37°C for 5 h. GFP induction was quantitated by flow cytometry. HS: heat shock.


C Individual proteins in reporter cells treated with and without 10 mM metformin overnight were detected by immunoblotting .

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C Individual proteins in reporter cells treated with and without 10 mM metformin overnight were detected by immunoblotting.


D , E Following heat shock and recovery for 4 h , mRNA levels in reporter cells treated with and without 10 mM metformin overnight were quantitated by qRT - PCR ( mean ± SD , n = 3 , Student ' s t - test , * * P < 0 . 01 , * * * P < 0 . 001 ) . β - actin was used as the internal control .

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D, E Following heat shock and recovery for 4 h, mRNA levels in reporter cells treated with and without 10 mM metformin overnight were quantitated by qRT-PCR (mean ± SD, n = 3, Student's t-test, **P < 0.01, ***P < 0.001). β-actin was used as the internal control.


F Reporter cells were treated with 10 mM metformin overnight , and viability was measured using Guava ViaCount ® reagents ( mean ± SD , n = 5 , n . s . , not significant , Student ' s t - test ) .

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F Reporter cells were treated with 10 mM metformin overnight, and viability was measured using Guava ViaCount® reagents (mean ± SD, n = 5, n.s., not significant, Student's t-test).


G Left panel , graphic depiction of the ELISA - based DNA binding assay . HSE : heat shock element . Right panel : Immediately following heat shock at 43 ° C for 30 min , nuclear proteins of the reporter cells treated with and without 10 mM metformin were extracted for assay ( mean ± SD , n = 3 , Student ' s t - test , * * P < 0 . 01 , * * * P < 0 . 001 ) . Experimental details are described in Materials and Methods .

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G Left panel, graphic depiction of the ELISA-based DNA binding assay. HSE: heat shock element. Right panel: Immediately following heat shock at 43°C for 30 min, nuclear proteins of the reporter cells treated with and without 10 mM metformin were extracted for assay (mean ± SD, n = 3, Student's t-test, **P < 0.01, ***P < 0.001). Experimental details are described in Materials and Methods.


I HEK293T cells stably expressing a scramble or HSF1 - targeting shRNA were treated with 10 mM metformin overnight and heat - shocked at 43 ° C for 30 min . Cells were fixed for PLA to detect HSF1 - DNA interactions . Experimental details are described in Materials and Methods . Scale bars : 50 μm for low magnification and 10 μm for high magnification .

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I HEK293T cells stably expressing a scramble or HSF1-targeting shRNA were treated with 10 mM metformin overnight and heat-shocked at 43°C for 30 min. Cells were fixed for PLA to detect HSF1-DNA interactions. Experimental details are described in Materials and Methods. Scale bars: 50 μm for low magnification and 10 μm for high magnification.


J Immediately following heat shock , cytoplasmic and nuclear proteins of the reporter cells treated with and without 10 mM metformin were extracted for immunoblotting . LDH and Lamin A / C were used as cytoplasmic and nuclear markers , respectively . C : cytoplasm ; N : nucleus .

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J Immediately following heat shock, cytoplasmic and nuclear proteins of the reporter cells treated with and without 10 mM metformin were extracted for immunoblotting. LDH and Lamin A/C were used as cytoplasmic and nuclear markers, respectively. C: cytoplasm; N: nucleus.


K Reporter mice ( ventral side shown ) were pre - treated with metformin for 3 days , 2 mg / mouse / day , via i . p . injection . Following a single i . p . injection of velcade ( 5 mg / kg ) and recovery for 6 h , whole - body luciferase activities were quantitated by bioluminescence imaging . The photon flux of each mouse was expressed as photon / s / cm2 / steradian ( right panel , mean ± SD , n = 3 , one - way ANOVA , * P < 0 . 05 ; * * P < 0 . 01 ) .

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K Reporter mice (ventral side shown) were pre-treated with metformin for 3 days, 2 mg/mouse/day, via i.p. injection. Following a single i.p. injection of velcade (5 mg/kg) and recovery for 6 h, whole-body luciferase activities were quantitated by bioluminescence imaging. The photon flux of each mouse was expressed as photon/s/cm2/steradian (right panel, mean ± SD, n = 3, one-way ANOVA, *P < 0.05; **P < 0.01).


A - C Reporter cells were treated with and without 1 μM A - 769662 for 3 h followed by heat shock at 43 ° C for 30 min and recovery at 37 ° C for 5 h . Hspa1a and Hspb1 mRNA levels were quantitated by qRT - PCR ( mean ± SD , n = 3 , Student ' s t - test , * * * P < 0 . 001 ) . ACC phosphorylation was detected by immunoblotting ( C ) .

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A-C Reporter cells were treated with and without 1 μM A-769662 for 3 h followed by heat shock at 43°C for 30 min and recovery at 37°C for 5 h. Hspa1a and Hspb1 mRNA levels were quantitated by qRT-PCR (mean ± SD, n = 3, Student's t-test, ***P < 0.001). ACC phosphorylation was detected by immunoblotting (C).


D - F Primary MEFs were derived from Ampkα1fl / fl ; Ampkα2fl / fl embryos and transduced with either adenoviral GFP or Cre to delete Ampkα1 / 2 in vitro . AMPKα levels were detected 4 days after transductions ( D )

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D-F Primary MEFs were derived from Ampkα1fl/fl; Ampkα2fl/fl embryos and transduced with either adenoviral GFP or Cre to delete Ampkα1/2 in vitro. AMPKα levels were detected 4 days after transductions (D)


Following viral transductions , Hsp mRNA levels were quantitated by qRT - PCR in MEFs with and without 10 mM metformin treatment overnight followed by 43 ° C heat shock for 30 min and recovery for 4 h ( mean ± SD , n = 3 , one - way ANOVA , n . s . , not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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Following viral transductions, Hsp mRNA levels were quantitated by qRT-PCR in MEFs with and without 10 mM metformin treatment overnight followed by 43°C heat shock for 30 min and recovery for 4 h (mean ± SD, n = 3, one-way ANOVA, n.s., not significant, *P < 0.05, **P < 0.01, ***P < 0.001).


G Transcriptional activities of HSF1 were measured by a dual reporter system consisting of two plasmids : the HSF1 - dependent reporter pHSE - SEAP , in which ideal HSEs drive the expression of secreted alkaline phosphatase ( SEAP ) , and the control reporter pCMV - Gaussia Luc , in which a CMV promoter drives the expression of secreted Gaussia luciferase . In HEK293T cells , either LacZ or GST - AMPKα1CA plasmid was co - transfected with the two reporter plasmids . After 24 h , the transfected cells were heat - shocked at 44 ° C for 45 min . The culture supernatants were collected to measure SEAP and luciferase activities 24 h after heat shock . SEAP signals were normalized to Gaussia luciferase signals ( mean ± SD , n = 5 , Student ' s t - test , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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G Transcriptional activities of HSF1 were measured by a dual reporter system consisting of two plasmids: the HSF1-dependent reporter pHSE-SEAP, in which ideal HSEs drive the expression of secreted alkaline phosphatase (SEAP), and the control reporter pCMV-Gaussia Luc, in which a CMV promoter drives the expression of secreted Gaussia luciferase. In HEK293T cells, either LacZ or GST-AMPKα1CA plasmid was co-transfected with the two reporter plasmids. After 24 h, the transfected cells were heat-shocked at 44°C for 45 min. The culture supernatants were collected to measure SEAP and luciferase activities 24 h after heat shock. SEAP signals were normalized to Gaussia luciferase signals (mean ± SD, n = 5, Student's t-test, **P < 0.01, ***P < 0.001).


H , I Following treatment with 10 mM metformin overnight , endogenous AMPKα and HSF1 proteins were co - precipitated using anti - AMPKα agarose conjugates from lysates of immortalized Hsf1 + / + MEFs ( H ) .

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H, I Following treatment with 10 mM metformin overnight, endogenous AMPKα and HSF1 proteins were co-precipitated using anti-AMPKα agarose conjugates from lysates of immortalized Hsf1+/+MEFs (H).


HC : heavy chain . Endogenous HSF1 - AMPKα interactions in immortalized Hsf1 + / + and Hsf1 − / − MEFs were visualized in situ by PLA ( I ) . Experimental details are described in Materials and Methods . Scale bars : 50 μm .

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HC: heavy chain. Endogenous HSF1-AMPKα interactions in immortalized Hsf1+/+ and Hsf1−/− MEFs were visualized in situ by PLA (I). Experimental details are described in Materials and Methods. Scale bars: 50 μm.


A In HEK293T cells that stably express an shRNA targeting the 3 ′ UTR of human HSF1 , HSF1WT or HSF1S121A was expressed . Following treatment with 10 mM metformin overnight , the levels of HSF1 phosphorylation at Ser121 were detected by immunoblotting using a specific phospho - HSF1 Ser121 antibody ( A8041 , Assay Biotechnology ) .

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A In HEK293T cells that stably express an shRNA targeting the 3′ UTR of human HSF1, HSF1WT or HSF1S121A was expressed. Following treatment with 10 mM metformin overnight, the levels of HSF1 phosphorylation at Ser121 were detected by immunoblotting using a specific phospho-HSF1 Ser121 antibody (A8041, Assay Biotechnology).


B Primary Ampkα1fl / fl ; Ampkα2fl / fl MEFs were transduced with adenoviral GFP or Cre . Following treatment with 10 mM metformin overnight or treatment with 10 μM A - 769662 for 3 h , HSF1 Ser121 phosphorylation was examined by immunoblotting .

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B Primary Ampkα1fl/fl; Ampkα2fl/fl MEFs were transduced with adenoviral GFP or Cre. Following treatment with 10 mM metformin overnight or treatment with 10 μM A-769662 for 3 h, HSF1 Ser121 phosphorylation was examined by immunoblotting.


C HSF1WT or HSF1S121A was co - expressed with either GFP or GST - AMPKαCA in HSF1 - deficient HEK293T cells . HSF1 Ser121 phosphorylation was examined by immunoblotting .

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C HSF1WT or HSF1S121A was co-expressed with either GFP or GST-AMPKαCA in HSF1-deficient HEK293T cells. HSF1 Ser121 phosphorylation was examined by immunoblotting.


D AMPK complexes were immunoprecipitated from HEK293T cells treated with and without 10 mM metformin overnight . Aliquots of AMPK complexes were incubated with 400 ng purified recombinant His - tagged HSF1 proteins with and without 100 μM AMP or 20 μM compound C ( CC ) . HSF1 Ser121 phosphorylation was detected by immunoblotting .

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D AMPK complexes were immunoprecipitated from HEK293T cells treated with and without 10 mM metformin overnight. Aliquots of AMPK complexes were incubated with 400 ng purified recombinant His-tagged HSF1 proteins with and without 100 μM AMP or 20 μM compound C (CC). HSF1 Ser121 phosphorylation was detected by immunoblotting.


E , F HSF1 activities were measured by the dual reporter system in HSF1 - deficient HEK293T cells . Either FLAG - HSF1WT or FLAG - HSF1S121A was co - expressed with LacZ orAMPKα1CA ( E ) .

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E, F HSF1 activities were measured by the dual reporter system in HSF1-deficient HEK293T cells. Either FLAG-HSF1WT or FLAG-HSF1S121A was co-expressed with LacZ orAMPKα1CA (E).


E , F HSF1 activities were measured by the dual reporter system in HSF1 - deficient HEK293T cells . Either FLAG - HSF1WT or FLAG - HSF1S121A was co - expressed with LacZ or AMPKα1CA ( E ) . Following expression of HSF1WT or HSF1S121A , cells were treated with and without 10 mM metformin overnight ( F ) ( mean ± SD , n = 5 , one - way ANOVA , n . s . , not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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E, F HSF1 activities were measured by the dual reporter system in HSF1-deficient HEK293T cells. Either FLAG-HSF1WT or FLAG-HSF1S121A was co-expressed with LacZ or AMPKα1CA (E). Following expression of HSF1WT or HSF1S121A, cells were treated with and without 10 mM metformin overnight (F) (mean ± SD, n = 5, one-way ANOVA, n.s., not significant, *P < 0.05, **P < 0.01, ***P < 0.001).


G , H FLAG - HSF1WT or FLAG - HSF1S121A plasmids were co - transfected with LacZ or AMPKα1CA plasmids into HSF1 - deficient HEK293T cells for 3 days ( G ) . Following transfection with FLAG - HSF1WT or FLAG - HSF1S121A plasmids for 3 days , HSF1 - deficient HEK293T cells were treated with and without 10 mM metformin overnight ( H ) . Nuclear proteins were extracted to measure HSF1 - DNA binding by the ELISA - based DNA binding assay using anti - FLAG antibodies ( mean ± SD , n = 3 , one - way ANOVA , n . s . , not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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G, H FLAG-HSF1WT or FLAG-HSF1S121A plasmids were co-transfected with LacZ or AMPKα1CA plasmids into HSF1-deficient HEK293T cells for 3 days (G). Following transfection with FLAG-HSF1WT or FLAG-HSF1S121A plasmids for 3 days, HSF1-deficient HEK293T cells were treated with and without 10 mM metformin overnight (H). Nuclear proteins were extracted to measure HSF1-DNA binding by the ELISA-based DNA binding assay using anti-FLAG antibodies (mean ± SD, n = 3, one-way ANOVA, n.s., not significant, *P < 0.05, **P < 0.01, ***P < 0.001).


I , J Following transfections and metformin treatment as described in ( G ) and ( H ) , cytoplasmic and nuclear proteins were extracted for immunoblotting using anti - FLAG antibodies .

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I, J Following transfections and metformin treatment as described in (G) and (H), cytoplasmic and nuclear proteins were extracted for immunoblotting using anti-FLAG antibodies.


A , B NIH3T3 reporter cells were cultured overnight in DMEM with and without leucine ( A ) or 4 . 5 g / l glucose ( B ) . Following heat shock at 43 ° C for 30 min and recovery for 5 h , GFP induction was quantitated by flow cytometry .

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A, B NIH3T3 reporter cells were cultured overnight in DMEM with and without leucine (A) or 4.5 g/l glucose (B). Following heat shock at 43°C for 30 min and recovery for 5 h, GFP induction was quantitated by flow cytometry.


C , D Nuclear proteins were extracted from reporter cells with and without nutrient deprivations overnight for the ELISA - based DNA binding assay ( mean ± SD , n = 3 , Student ' s t - test , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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C, D Nuclear proteins were extracted from reporter cells with and without nutrient deprivations overnight for the ELISA-based DNA binding assay (mean ± SD, n = 3, Student's t-test, *P < 0.05, **P < 0.01, ***P < 0.001).


E , F Following nutrient deprivations overnight , the reporter cells were heat - shocked at 43 ° C for 30 min and recovered overnight . Individual proteins were detected by immunoblotting .

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E, F Following nutrient deprivations overnight, the reporter cells were heat-shocked at 43°C for 30 min and recovered overnight. Individual proteins were detected by immunoblotting.


G , H Reporter cells were subjected to nutrient starvation for the indicated time . AMPKα Thr172 and HSF1 Ser121 phosphorylation was measured by immunoblotting .

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G, H Reporter cells were subjected to nutrient starvation for the indicated time. AMPKα Thr172 and HSF1 Ser121 phosphorylation was measured by immunoblotting.


I , J HEK293T cells were sequentially transfected with control or AMPKα1 / 2 - targeting siRNAs and dual HSF1 reporter plasmids . 24 h later , transfected cells were starved overnight before reporter activities were measured ( mean ± SD , n = 6 , Student ' s t - test , n . s . , not significant , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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I, J HEK293T cells were sequentially transfected with control or AMPKα1/2-targeting siRNAs and dual HSF1 reporter plasmids. 24 h later, transfected cells were starved overnight before reporter activities were measured (mean ± SD, n = 6, Student's t-test, n.s., not significant, **P < 0.01, ***P < 0.001).


K , L FLAG - HSF1WT or FLAG - HSF1S121A plasmids were co - transfected with the dual HSF1 reporter plasmids into HSF1 - deficient HEK293T cells for 2 days , followed by overnight starvation ( mean ± SD , n = 5 , Student ' s t - test , n . s . , not significant , * P < 0 . 05 , * * P < 0 . 01 ) .

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K, L FLAG-HSF1WT or FLAG-HSF1S121A plasmids were co-transfected with the dual HSF1 reporter plasmids into HSF1-deficient HEK293T cells for 2 days, followed by overnight starvation (mean ± SD, n = 5, Student's t-test, n.s., not significant, *P < 0.05, **P < 0.01).


A Reporter cells were heat - shocked at 43 ° C for the indicated time . AMPKα Thr172 and HSF1 Ser121 phosphorylation was measured by immunoblotting .

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A Reporter cells were heat-shocked at 43°C for the indicated time. AMPKα Thr172 and HSF1 Ser121 phosphorylation was measured by immunoblotting.


B Immediately following heat shock at 43 ° C for 40 min , AMPK complexes were immunoprecipitated from HEK293 cells and in vitro kinase assays were performed as described in Figure 3D using recombinant human ACC1 proteins as substrates .

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B Immediately following heat shock at 43°C for 40 min, AMPK complexes were immunoprecipitated from HEK293 cells and in vitro kinase assays were performed as described in Figure 3D using recombinant human ACC1 proteins as substrates.


C Following heat shock at 43 ° C for 40 min , both detergent - soluble and detergent - insoluble fractions of cellular proteins were extracted from HEK293 cells as described in Materials and Methods for immunoblotting .

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C Following heat shock at 43°C for 40 min, both detergent-soluble and detergent-insoluble fractions of cellular proteins were extracted from HEK293 cells as described in Materials and Methods for immunoblotting.


D , E Immediately following heat shock at 43 ° C for 40 min , HEK293 cells were subjected to nutrient deprivations for 4 h . AMPKα Thr172 and ACC Ser79 phosphorylation was measured by immunoblotting .

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D, E Immediately following heat shock at 43°C for 40 min, HEK293 cells were subjected to nutrient deprivations for 4 h. AMPKα Thr172 and ACC Ser79 phosphorylation was measured by immunoblotting.


F - H HSF1WT or HSF1S121A plasmids were transfected into HSF1 - deficient HEK293T cells . Transfected cells were heat - shocked at 43 ° C for 40 min . HSP mRNAs were quantitated by qRT - PCR following overnight recovery . HSF1 - DNA binding was measured immediately after heat shock ( mean ± SD , n = 3 , Student ' s t - test , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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F-H HSF1WT or HSF1S121A plasmids were transfected into HSF1-deficient HEK293T cells. Transfected cells were heat-shocked at 43°C for 40 min. HSP mRNAs were quantitated by qRT-PCR following overnight recovery. HSF1-DNA binding was measured immediately after heat shock (mean ± SD, n = 3, Student's t-test, **P < 0.01, ***P < 0.001).


A , 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 ) .

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A, 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 ) .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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M Following transfection with LacZ or polyQ79, HEK293T cells were grown under different concentrations of glucose for 5 days before measuring aggregate size.


A , 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 .

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A, 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 ) .

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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 ) .

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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 .

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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 .

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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 .

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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 ) .

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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 ) .

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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).


( 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 ) .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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.


( 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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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.


( 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 .

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(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 .

O O O O O B-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O

(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 ) .

O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE O O B-EXP_ASSAY O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 .

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(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 .

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(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 .

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(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 .

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(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 .

B-CELL_LINE O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O B-EXP_ASSAY O B-EXP_ASSAY B-GENEPROD O O O B-EXP_ASSAY O O O O O

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 .

B-CELL_LINE O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O B-EXP_ASSAY O B-EXP_ASSAY B-GENEPROD O O O B-EXP_ASSAY O O O O O

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 .

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.


( 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 .

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(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 .

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(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 .

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(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 .

O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O

(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 .

O O O O B-CELL_LINE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O

(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 .

O O O B-CELL_LINE O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O

(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.


( 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 .

O O O O O O O O O B-CELL_LINE O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O

(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 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 .

O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-GENEPROD O O O B-EXP_ASSAY O O B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O

(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 .

O O O B-SUBCELLULAR O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O B-EXP_ASSAY O O O O

(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 .

O O O B-SUBCELLULAR O O O O O O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O B-EXP_ASSAY O O O O

(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 .

O O O B-SUBCELLULAR O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O O O

(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 ) .

O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 ) .

O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-CELL_LINE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O B-CELL_LINE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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).


( 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 .

O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

(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.


( 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 .

O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 .

O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 .

O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O

(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 ) .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 .

O O O O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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.


B . UTP6 in U - 2 OS cells exemplify proteins localized to whole nucleoli ( HPA025936 ) .

O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-EXP_ASSAY O O B-SUBCELLULAR O O O O

B. 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 ) .

O O B-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O

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 ) .

O O O B-EXP_ASSAY O B-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O B-GENEPROD O O B-CELL_LINE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O

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 ) .

O O O B-EXP_ASSAY B-SUBCELLULAR O B-GENEPROD O O O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O

F. The multilocalizing ribosomal protein RPL13 detected in the nucleoli, cytosol and ER in MCF-7 cells (HPA051702).


A . 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 .

O O B-GENEPROD B-EXP_ASSAY O O B-SUBCELLULAR O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O B-SUBCELLULAR O O O O O O O O

A. 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 .

O O B-GENEPROD B-EXP_ASSAY O O B-SUBCELLULAR O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O B-SUBCELLULAR O O O O O O O O

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 .

O O B-GENEPROD B-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR O B-CELL_LINE O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O B-SUBCELLULAR O O O O O O O O

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.