ptpn22 inhibitor  (MedChemExpress)

Journal: EMBO Reports

Article Title: Phosphatase PTPN22 functions as an adaptor in the mTORC2 complex

doi: 10.1038/s44319-025-00576-5

Figure Lengend Snippet: ( A ) Bar graphs showing the −log10 P values of the most enriched pathways in the interactome of PTPN22 derived from NCI-pathways (Nature 2016) using Enrichr. ( B ) HEK293T cells were co-transfected with SFB-PTPN22 along with Myc-tagged RICTOR, mSIN, mTOR, mLST8, RAPTOR and PRAS40. 24 h after transfection, cells were lysed in 0.3% CHAPS buffer and the lysates were immunoprecipitated with control IgG or anti-Flag antibody. The interactions were detected by immunoblotting with anti-Myc antibody. ( C ) Immunoprecipitation (IP) with control IgG or anti-PTPN22 antibody was performed with extracts derived from HeLa cells. Endogenous association of PTPN22 with mTOR complexes subunits (RICTOR, mTOR, and RAPTOR) were analysed by immunoblotting with specific antibodies. Due to low expression level of PTPN22 in HeLa cells, endogenous PTPN22 in input sample was shown by immunoprecipitating PTPN22 from cell extracts using its antibody. .

Article Snippet: Phorbol 12-myristate 13-acetate (PMA) (HY-18739) and PTPN22 inhibitor, PTPN22-IN-1 (HY-139693) were purchased from MedChemExpress.

Techniques: Derivative Assay, Transfection, Immunoprecipitation, Control, Western Blot, Expressing

Journal: EMBO Reports

Article Title: Phosphatase PTPN22 functions as an adaptor in the mTORC2 complex

doi: 10.1038/s44319-025-00576-5

Figure Lengend Snippet: ( A ) Partial interaction network of PTPN22 with its associated proteins derived from tandem affinity purification of SFB-PTPN22 followed by peptides identification by mass spectrometry analysis is shown. SFB-GFP purification was used as a control to filter out non-specific interactions. The list of SFB-PTPN22 and SFB-GFP associated proteins was derived from our earlier study (Kumar et al, ). mTOR complex components are highlighted in green colour. ( B ) Immunoprecipitation (IP) with control IgG or anti-PTPN22 antibody was performed with extracts derived from Jurkat cells lysed in 0.3% CHAPS buffer. Endogenous association of PTPN22 with mTOR complexes subunits (RICTOR, mSIN, mTOR, mLST8, and RAPTOR) were analysed by immunoblotting, immunoprecipitates with respective antibodies. ( C ) Jurkat cells were lysed in 0.3% CHAPS buffer and immunoprecipitates from control IgG or anti-mSIN antibody were analysed for the presence of PTPN22, and mTOR components by immunoblotting with their respective antibodies. ( D ) HEK293T cells were co-transfected with SFB-PTPN22 and Myc-mTOR constructs. At 24 h post-transfection, cells were lysed in 0.3% CHAPS buffer, followed by immunoprecipitation (IP) with control IgG or anti-Myc antibody and their interaction was detected by immunoblotting with anti-Flag antibody. ( E ) SFB vector, SFB-RICTOR, SFB-mSIN1, SFB-mLST8, SFB-PRAS40 or SFB-RAPTOR along with Myc-PTPN22 were transfected in HEK293T cells. At 24 h post-transfection, cells were lysed in 0.3% CHAPS buffer and were subjected to pulldown using S-protein agarose beads. Association of PTPN22 with components of mTORC1 and mTORC2 were analysed by immunoblotting with anti-Myc antibody. ( F ) Detection of PTPN22 expression by RT-PCR in different cell lines. GAPDH was used as a control. ( G ) Immunoprecipitation (IP) with anti-PTPN22 antibody was performed with extracts derived from indicated cell lines lysed in 0.5% NETN buffer. Endogenous levels of PTPN22 were analysed by immunoblotting, immunoprecipitates with anti-PTPN22 antibody. β-actin was used as a control. ( H ) HEK293T cells were lysed in 0.3% CHAPS buffer and immunoprecipitates from control IgG or anti-PTPN22 antibody were analysed for the presence of components of mTOR complexes by immunoblotting with their respective antibodies. As levels of endogenous PTPN22 are low in HEK293T cells, expression of PTPN22 in input sample was shown by immunoprecipitating PTPN22 from cell extracts using its antibody. ( I ) HCT116 cells were transfected with either SFB vector, SFB-PTPN22, SFB-PTPN5 or SFB-PRAS40. At 48 h post-transfection, cells were lysed in 0.3% CHAPS buffer and were subjected to pulldown using S-protein agarose beads. Interaction with subunits of mTOR complexes were detected by immunoblotting with their respective antibodies. ( J ) Jurkat cells were lysed in 0.3% CHAPS buffer and were fractionated using FPLC Superdex 200 column (gel-filtration chromatography). Molecular weights for fractions were estimated by running native molecular weight markers. Indicated fractions were resolved on SDS-PAGE followed by immunoblotting with respective antibodies as mentioned. .

Article Snippet: Phorbol 12-myristate 13-acetate (PMA) (HY-18739) and PTPN22 inhibitor, PTPN22-IN-1 (HY-139693) were purchased from MedChemExpress.

Techniques: Derivative Assay, Affinity Purification, Mass Spectrometry, Purification, Control, Immunoprecipitation, Western Blot, Transfection, Construct, Plasmid Preparation, Expressing, Reverse Transcription Polymerase Chain Reaction, Filtration, Chromatography, Molecular Weight, SDS Page

Journal: EMBO Reports

Article Title: Phosphatase PTPN22 functions as an adaptor in the mTORC2 complex

doi: 10.1038/s44319-025-00576-5

Figure Lengend Snippet: ( A ) Representative immunoblots showing analysis of whole cell lysates derived from Jurkat cells depleted of endogenous PTPN22 by two independent shRNAs (shRNA-Scramble used as a negative control) via lentiviral mediated infection, for examine the total and phosphorylated states of AKT, p70S6K, GSK-3β and FoxO with their respective antibodies. ( B–F ) Quantification of immunoblotting data from ( A ). The pixel intensity of phosphorylated protein bands were normalized by the pixel intensity of corresponding total protein bands. Individual data points from three independent experiments are shown in the graph. ( G–I ) Representative immunoblots showing analysis of whole cell lysates derived from HCT116 cells depleted of endogenous PTPN22 by two independent shRNAs (shRNA-Scramble used as a negative control) via lentiviral mediated infection, for examine the total and phosphorylated states of SGK1 and PKCα with their respective antibodies ( G ). Quantification of SGK1 phosphorylation ( H ) and PKCα phosphorylation ( I ) from ( G ). Individual data points from three independent experiments are shown in the graph. ( J ) Cell extracts of control and PTPN22 knockout cells (derived from two independent guide RNAs) were analysed by immunoblotting to determine the total and phosphorylated states of AKT and GSK-3β with specific antibodies. ( K ) Immunoblots showing analysis of whole cell lysates derived from Jurkat cells transfected with Myc vector, Myc-tagged PTPN22 (WT) or PTPN22 (D/A-C/S) constructs, for determine the total and phosphorylated states of AKT and FoxO with specific antibodies. .

Article Snippet: Phorbol 12-myristate 13-acetate (PMA) (HY-18739) and PTPN22 inhibitor, PTPN22-IN-1 (HY-139693) were purchased from MedChemExpress.

Techniques: Western Blot, Derivative Assay, shRNA, Negative Control, Infection, Phospho-proteomics, Control, Knock-Out, Transfection, Plasmid Preparation, Construct

Journal: EMBO Reports

Article Title: Phosphatase PTPN22 functions as an adaptor in the mTORC2 complex

doi: 10.1038/s44319-025-00576-5

Figure Lengend Snippet: ( A , B ) HCT116 cells were transduced with either control or two independent PTPN22 shRNAs, and stable cell lines were generated. Knockdown of PTPN22 was confirmed either by ( A ) examining the expression levels of PTPN22 and GAPDH mRNAs by reverse transcription polymerase chain reaction (RT-PCR), ( B ) or by immunoprecipitating PTPN22 from cell lysates using endogenous antibody, followed by immunoblot analysis of whole cell lysates and anti-PTPN22 immunoprecipitates with antibodies as indicated. ( C , D ) Immunoblot analysis of whole cell lysates derived from stable HCT116 cell lines expressing the control shRNA or two independent PTPN22 shRNAs, with indicated antibodies was done to determine the activation of AKT and its downstream substrates ( C ). Quantification of immunoblotting data, the pixel intensity of AKT-pS473 protein bands were normalized by the pixel intensity of total AKT protein bands. Individual data points from three independent experiments are shown in the graph ( D ), and the phospho-protein/total protein levels of pAKT-T308, pGSK-3β-pS9 and pFoxO are indicated as mean ± SD from three independent experiments are shown below their respective blots. ( E , F ) Control or PTPN22 depleted HCT116 cells were serum starved for 16 h, followed by stimulation with EGF (100 ng/ml) at indicated time points. Whole cell lysates were immunoblotted with indicated antibodies to determine the activation of AKT ( E ). Quantification of immunoblotting data, the pixel intensity of AKT-pS473 protein bands were normalized by the pixel intensity of total AKT protein bands. Individual data points from three independent experiments are shown in the graph ( F ). ( G , H ) Whole cell lysates of control shRNA or PTPN22 shRNA transfected with SFB-vector, SFB-PTPN22 (WT) or SFB-PTPN22 (D/A-C/S) shRNA-resistant plasmids were examined to determine the total and phosphorylated states of AKT with specific antibodies ( G ). Quantification of immunoblotting data, the pixel intensity of AKT-pS473 protein bands were normalized by the pixel intensity of total AKT protein bands. Individual data points from three independent experiments are shown in the graph ( H ). ( I , J ) PTPN22 depleted HCT116 cells expressing Myc-PTPN22 (WT) shRNA-resistant construct were treated with torin (250 nM) for 2 h. Cell lysates were analysed for total and phosphorylated states of AKT with specific antibodies. Untreated cells or PTPN22 depleted HCT116 cells expressing Myc-vector were used as controls ( I ). Quantification of immunoblotting data, the pixel intensity of AKT-pS473 protein bands were normalized by the pixel intensity of total AKT protein bands. Individual data points from three independent experiments are shown in ( J ). ( K ) Schematic showing experimental workflow of in vitro mTORC2 kinase assays using bacterially purified GST-AKT (K179D) as a substrate. ( L , M ) mTORC2 purified from control or PTPN22 depleted HCT116 cells were incubated with bacterially purified GST-AKT (K179D) for in vitro kinase reactions, and substrate phosphorylation was assessed with AKT-pS473 antibody. Cell lysates and pulldowns were immunoblotted with the indicated antibodies ( L ). Quantification of immunoblotting data, the pixel intensity of AKT-pS473 protein bands were normalized by the pixel intensity of total AKT protein bands. Individual data points from three independent experiments are shown in the graph ( M ). .

Article Snippet: Phorbol 12-myristate 13-acetate (PMA) (HY-18739) and PTPN22 inhibitor, PTPN22-IN-1 (HY-139693) were purchased from MedChemExpress.

Techniques: Transduction, Control, Stable Transfection, Generated, Knockdown, Expressing, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Western Blot, Derivative Assay, shRNA, Activation Assay, Transfection, Plasmid Preparation, Construct, In Vitro, Purification, Incubation, Phospho-proteomics

Journal: EMBO Reports

Article Title: Phosphatase PTPN22 functions as an adaptor in the mTORC2 complex

doi: 10.1038/s44319-025-00576-5

Figure Lengend Snippet: ( A ) A schematic showing sensitivity of mTORC2 towards different detergents during isolation of mTORC2. Cell lysis with 0.3% CHAPS buffer preserves mTORC2 integrity, while 1% Triton X-100 containing buffer cause dissociation of mTORC2 into two modules; one having mTOR and mLST8, and other having RICTOR and mSIN. ( B ) Immunoprecipitation (IP) with control IgG or anti-PTPN22 antibody was performed with extracts derived from Jurkat cells lysed in a buffer containing either 0.3% CHAPS or 1% Triton X-100. Endogenous association of PTPN22 with mTORC2 components were analysed by immunoblotting with respective antibodies. ( C ) Bacterially expressed recombinant MBP or MBP-PTPN22 proteins immobilized to dextran Sepharose beads were incubated with concentrated bacterial cell lysates expressing GST-RICTOR. The MBP-pulldowns were resolved by SDS-PAGE and the interactions were analyzed by immunoblotting with anti-RICTOR antibody. Expression of MBP and MBP-PTPN22 was shown by Coomassie staining. ( D ) Bacterially expressed recombinant GST, GST-mSIN and MBP-PTPN22 were purified using glutathione sepharose and dextran sepharose beads, respectively. 2 µg of purified MBP-PTPN22 was incubated with glutathione sepharose beads bound GST or GST-mSIN. The GST-pulldowns were resolved on SDS-PAGE and analysed by immunoblotting with anti-MBP antibody to check for the interaction. Recombinant protein expression was shown by Coomassie staining. ( E ) Lentiviral transduction with control shRNA or PTPN22 shRNA was performed in HCT116 cells. Knockdown of PTPN22 was verified by examining the expression levels of PTPN22 and GAPDH mRNAs by reverse transcription polymerase chain reaction (RT-PCR). ( F ) Control or PTPN22 depleted HCT116 cells were co-transfected with SFB-mSIN and Myc-RICTOR. At 48 h post-transfection, cells were lysed in 0.3% CHAPS buffer and lysates were pulldown using S-protein agarose beads. The interactions were detected by immunoblotting with anti-Myc antibody. ( G ) Immunoprecipitation (IP) with control IgG or anti-RICTOR antibody was performed with extracts derived from either control sgRNA or PTPN22 knockout HCT116 cells. Endogenous association of RICTOR with mSIN were analysed by immunoblotting with anti-mSIN antibodies. Due to low expression level of PTPN22 in HCT116 cells, endogenous PTPN22 in input sample was shown by immunoprecipitating PTPN22 from cell extracts using its antibody. ( H , I ) Bacterially purified GST-mSIN immobilized on glutathione sepharose beads were incubated with the purified SFB-RICTOR, either in the presence of recombinant PTPN22 or equal volume of corresponding buffer. The interaction of mSIN-RICTOR was assessed by immunoblotting with anti-Flag antibody. GST-protein was used as a negative control ( H ). Individual data points for relative RICTOR bound to mSIN were plotted as graph from three independent experiments ( I ). ( J ) Jurkat cells treated either with phorbol 12-myristate 13-acetate (PMA, 1 µM) or Dimethylsulphoxide (DMSO) were lysed in 0.3% CHAPS buffer and immunoprecipitates from control IgG or anti-RICTOR antibody were analysed for the presence of PTPN22, and mTOR components by immunoblotting with their respective antibodies. ( K ) Immunoblot (IB) analysis of whole cell lysates derived from Jurkat cells treated with DMSO, PMA (1 µM), PTPN22-IN-1 (1.4 µM) or PMA treatment followed by PTPN22-IN-1 treatment, with indicated antibodies to determine the activation of AKT. .

Article Snippet: Phorbol 12-myristate 13-acetate (PMA) (HY-18739) and PTPN22 inhibitor, PTPN22-IN-1 (HY-139693) were purchased from MedChemExpress.

Techniques: Isolation, Lysis, Immunoprecipitation, Control, Derivative Assay, Western Blot, Recombinant, Incubation, Expressing, SDS Page, Staining, Purification, Transduction, shRNA, Knockdown, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Transfection, Knock-Out, Negative Control, Activation Assay

Journal: EMBO Reports

Article Title: Phosphatase PTPN22 functions as an adaptor in the mTORC2 complex

doi: 10.1038/s44319-025-00576-5

Figure Lengend Snippet: ( A–C ) Control or PTPN22 depleted HCT116 cells were co-transfected either with ( A ) SFB-mSIN and Myc-mLST8, ( B ) SFB-RICTOR and Myc-mTOR, or ( C ) SFB-mLST8 and Myc-mTOR. At 48 h post-transfection, cells were lysed in 0.3% CHAPS buffer and lysates were pulldown using S-protein agarose beads. The interactions were detected by immunoblotting with anti-Myc antibody. ( D ) HEK293T cells were transfected with either SFB vector or SFB-PTPN22. At 24 h post-transfection, cells were serum starved for 16 h, followed by stimulation with EGF (100 ng/ml) for 15 min and lysed in 0.3% CHAPS buffer and were subjected to pulldown using S-protein agarose beads. Association of PTPN22 with RICTOR and mSIN were detected by immunoblotting with their respective antibodies. ( E ) Control or PTPN22 depleted HCT116 cells were co-transfected either with SFB vector or SFB-RICTOR along with Myc-mSIN. At 48 h post-transfection, cells were serum starved for 16 h, followed by stimulation with EGF (100 ng/ml) for 15 min. Cells were lysed in 1% Triton X-100 buffer and lysates were pulldown using S-protein agarose beads. The interactions were detected by immunoblotting with anti-Myc antibody. .

Article Snippet: Phorbol 12-myristate 13-acetate (PMA) (HY-18739) and PTPN22 inhibitor, PTPN22-IN-1 (HY-139693) were purchased from MedChemExpress.

Techniques: Control, Transfection, Western Blot, Plasmid Preparation

Journal: EMBO Reports

Article Title: Phosphatase PTPN22 functions as an adaptor in the mTORC2 complex

doi: 10.1038/s44319-025-00576-5

Figure Lengend Snippet: ( A–C ) The predicted Alphafold model of RICTOR-mSIN-PTPN22 complex is shown and the proteins are highlighted in indicated colours: mSIN ( blue ), RICTOR ( red ), and PTPN22 ( green ). The N-terminus and Interdomian along with C-terminal domain of PTPN22 are denoted by the labels. ipTM = 0.49 and pTM = 0.48 represents the confidence score of the predicted structure ( A ). ( B ) 3D model depicting PTPN22 and mSIN interaction, from the predicted structure. N-terminus and the CRIM domain of mSIN are denoted by labels. ( C ) 3D model depicting PTPN22 and RICTOR interaction, from the predicted structure. The cyan colour indicates the ARM4 region of RICTOR. ( D ) HEK293T cells were transduced with either control shRNA or pool of multiple mSIN shRNAs, via lentiviral mediated infection. Stable cell lines were generated and knockdown efficiency was verified by immunoblotting with anti-mSIN antibody. ( E ) HEK293T cells were transduced with either control shRNA or pool of multiple RICTOR shRNAs, via lentiviral mediated infection, and stable cell lines were generated. The knockdown efficiency was verified by immunoblotting with anti-RICTOR antibody. ( F , G ) MBP or MBP-mSIN bound on dextran sepharose beads were incubated with purified GST-AKT either in the presence of recombinant PTPN22 or equal volume of corresponding buffer, and its effect on the AKT-mSIN interaction was assessed by immunoblotting MBP-pulldowns with AKT antibody ( F ), and individuals data points for relative AKT bound to mSIN were plotted from three independent experiments ( G ). ( H ) SFB-mSIN along with increasing concentrations (0–4 µg plasmid) of Myc-PTPN22 were transfected in HEK293T cells. At 24 h post-transfection, cells were lysed and subjected to pulldown using S-protein agarose beads, and the effect of PTPN22 on the interaction of AKT and mSIN was assessed by immunoblotting pulldowns with AKT specific antibody. SFB vector was used as a negative control. β-actin was used as a loading control. .

Article Snippet: Phorbol 12-myristate 13-acetate (PMA) (HY-18739) and PTPN22 inhibitor, PTPN22-IN-1 (HY-139693) were purchased from MedChemExpress.

Techniques: Transduction, Control, shRNA, Infection, Stable Transfection, Generated, Knockdown, Western Blot, Incubation, Purification, Recombinant, Plasmid Preparation, Transfection, Negative Control

Journal: EMBO Reports

Article Title: Phosphatase PTPN22 functions as an adaptor in the mTORC2 complex

doi: 10.1038/s44319-025-00576-5

Figure Lengend Snippet: ( A ) A schematic showing the full length PTPN22 along with its various deletion mutants lacking indicated domains. ( B ) SFB vector, SFB-PTPN22 full length (FL) or SFB-tagged deletion constructs of PTPN22 (shown in ( A )) along with Myc-mSIN were co-transfected in HEK293T cells having stable knockdown (KD) of RICTOR. At 24 h post-transfection, cells were lysed in 1% Triton X-100 buffer and were subjected to pulldown using S-protein agarose beads, and their interactions were analysed by immunoblotting with anti-Myc antibody. ( C ) SFB vector, SFB-PTPN22 full length (FL) or SFB-tagged deletion constructs of PTPN22 (shown in ( A )) along with Myc-tagged RICTOR were co-transfected in HEK293T cells having stable knockdown (KD) of mSIN. 24 h after transfection, cells were lysed in 1% Triton X-100 buffer and lysates were pulldown with S-protein agarose beads, and their interactions were analysed by immunoblotting with anti-Myc antibody. ( D ) HCT116 cells stably expressing PTPN22 shRNA were transfected either with SFB-vector, SFB-PTPN22 (WT) shRNA-resistant plasmids or SFB-tagged D1 and D3 deletion constructs of PTPN22. Whole cell lysates were immunoblotted to determine the activation of AKT by examined the total and phosphorylated states of AKT with specific antibodies. HCT116 cells expressing scrambled shRNA were used as a control. ( E ) Schematic representation of mSIN full length, and its various truncation mutants lacking indicated domains. ( F ) Schematic representation of RICTOR full length (FL), and its various deletion mutants lacking indicated domains. ( G ) RICTOR depleted HEK293T cells were transfected with SFB vector or SFB-tagged mSIN constructs (shown in ( E )) along with Myc-PTPN22. Cell lysates were pulldown with S-protein agarose beads, and their interactions were detected by immunoblotting with anti-Myc antibody. ( H ) mSIN depleted HEK293T cells expressing either SFB vector or SFB-tagged RICTOR constructs (shown in ( F )) along with Myc-PTPN22 were lysed in 1% Triton X-100 buffer and pulldown with S-protein agarose beads. Interaction of PTPN22 with various domains was detected by immunoblotting with anti-Myc antibody. .

Article Snippet: Phorbol 12-myristate 13-acetate (PMA) (HY-18739) and PTPN22 inhibitor, PTPN22-IN-1 (HY-139693) were purchased from MedChemExpress.

Techniques: Plasmid Preparation, Construct, Transfection, Knockdown, Western Blot, Stable Transfection, Expressing, shRNA, Activation Assay, Control

Journal: EMBO Reports

Article Title: Phosphatase PTPN22 functions as an adaptor in the mTORC2 complex

doi: 10.1038/s44319-025-00576-5

Figure Lengend Snippet: ( A ) HCT116 cells were transduced with either control shRNA or two independent PTPN22 shRNAs via lentiviral mediated infection, and stable cell lines were generated. Knockdown efficiency was shown by immunoprecipitating PTPN22 from cell lysates using PTPN22 antibody, followed by immunoblot analysis of anti-PTPN22 immunoprecipitates and whole cell lysates with specific antibodies. ( B , C ) Colony formation assays were performed in HCT116 cells expressing control shRNA or PTPN22 shRNA constructs. ( B ) Images were captured after staining with crystal violet, and ( C ) quantification data for number of colonies from three independent experiments are shown. Error bars indicate mean ± standard deviation ( n = 3), *** P = 0.000024 for control shRNA v/s PTPN22 shRNA-1, *** P = 0.000018 for control shRNA v/s PTPN22 shRNA-2 (one-way ANOVA with Bonferroni’s multiple comparisons test). ( D ) The expression of shRNA-resistant PTPN22 wild-type (WT) or PTPN22 catalytic mutant (D/A-C/S) constructs transfected in PTPN22 depleted cells was shown by immunoblotting with anti-Flag antibody. ( E ) HCT116 cells expressing either control shRNA or PTPN22 shRNAs were transfected with indicated constructs. After day 1, cell number was measured every 2 days for the indicated durations. Quantification of cell number was calculated from three independent experiments. Error bars indicate mean ± SD ( n = 3), *** P = 0.0003, ns: not significant (two-way ANOVA with Tukey’s multiple comparisons test). ( F , G ) Representative images from sub-G1 population measurement by flow cytometric analysis in HCT116 cells expressing indicated constructs are shown ( F ), and ( G ) quantified data for percentage of sub-G1 population from three independent experiments were plotted. Error bars represent mean ± standard deviation ( n = 3), *** P = 0.00000616 for control shRNA v/s PTPN22 shRNA, *** P = 0.00000803 for PTPN22 shRNA v/s PTPN22 shRNA + PTPN22 (WT) shRNA Res., *** P = 0.0000135 for PTPN22 shRNA v/s PTPN22 shRNA + PTPN22 (D195A/C227S) shRNA Res., ns: not significant for control shRNA v/s PTPN22 shRNA + PTPN22 (WT) shRNA Res., and for PTPN22 shRNA + PTPN22 (WT) shRNA Res. v/s PTPN22 shRNA + PTPN22 (D195A/C227S) shRNA Res. (one-way ANOVA with Bonferroni’s multiple comparisons test). ( H , I ) Representative images from sub-G1 population measurement by flow cytometric analysis in HCT116 cells expressing indicated constructs, and were treated either with torin (250 nM) or with DMSO control for 2 h are shown ( H ), and scatter plot for percentage of sub-G1 population from two independent experiments were plotted ( I ). ( J ) Quantification data for the percentage of mitotic index (number of pH3 positive cells/total number of cells (DAPI) per field) in PTPN22 depleted cells as compared to the control cells were plotted. The plotted data points were from three biological replicates and represent mean ± SD, *** P = 0.0002 (unpaired two-tailed Student’s t test). ( K ) Immunoblot showing PTPN22 knockdown efficiency in cell line used for nude mice xenograft experiments. .

Article Snippet: Phorbol 12-myristate 13-acetate (PMA) (HY-18739) and PTPN22 inhibitor, PTPN22-IN-1 (HY-139693) were purchased from MedChemExpress.

Techniques: Transduction, Control, shRNA, Infection, Stable Transfection, Generated, Knockdown, Western Blot, Expressing, Construct, Staining, Standard Deviation, Mutagenesis, Transfection, Two Tailed Test

Journal: EMBO Reports

Article Title: Phosphatase PTPN22 functions as an adaptor in the mTORC2 complex

doi: 10.1038/s44319-025-00576-5

Figure Lengend Snippet: ( A ) Equal numbers of HCT116 cells stably expressing either control shRNA or PTPN22 shRNAs were seeded, and after day 1, cell number was measured every 2 days for the indicated durations. Quantification of cell number was calculated from three independent experiments. Error bars indicate mean ± SD ( n = 3), * P = 0.0151, ** P = 0.0026, *** P < 0.0001, ns: not significant (two-way ANOVA with Tukey’s multiple comparisons test). ( B , C ) HCT116 cells expressing either control shRNA or PTPN22 shRNA were transfected with indicated constructs, and colony formation assay was performed. Images were captured after staining with crystal violet ( B ), and quantification data for number of colonies from three independent experiments are shown ( C ). Error bars indicate mean ± standard deviation ( n = 3), *** P = 0.00001566 for control shRNA + empty vector v/s PTPN22 shRNA-2 + empty vector, *** P = 0.00001134 for PTPN22 shRNA-2 + empty vector v/s PTPN22 shRNA-2 + PTPN22 (WT) shRNA Res., *** P = 0.00003107 for PTPN22 shRNA-2 + empty vector v/s PTPN22 shRNA-2 + PTPN22 (D/A-C/S) shRNA Res., ns: not significant for control shRNA + empty vector v/s PTPN22 shRNA-2 + PTPN22 (WT) shRNA Res., for control shRNA + empty vector v/s PTPN22 shRNA-2 + PTPN22 (D/A-C/S) shRNA Res., and PTPN22 shRNA-2 + PTPN22 (WT) shRNA Res. v/s PTPN22 shRNA-2 + PTPN22 (D/A-C/S) shRNA Res. (one-way ANOVA with Bonferroni’s multiple comparisons test). ( D , E ) Representative images of transwell migration assays showed the migration potential of HCT116 cells expressing indicated PTPN22 constructs, or a control vector. Phase-contrast microscopic images were taken after staining the migrated cells with crystal violet ( D ). Scatter plot showing the relative migration of cells/field (derived from average of four different fields) from two independent experiments are shown ( E ). ( F – H ) Knockdown of PTPN22 inhibits subcutaneous tumor growth in nude mice xenograft model. HCT116 cells stably expressing either control shRNA or PTPN22 shRNA were injected subcutaneously into athymic nude mice (Foxn1 −/− ) for xenograft tumor growth. Tumor images ( F ), tumor weights ( G ), and tumor volumes ( H ) are shown. Error bars indicate mean ± standard deviation (for tumor weights), and mean ± standard error of mean (for tumor volumes) ( n = 7), * P = 0.0379, ** P = 0.0054 (unpaired two-tailed Student’s t test). ( I ) A proposed model to depict the role of PTPN22 in mTORC2-AKT axis activation. PTPN22 acts as a molecular bridge for mSIN-RICTOR association, thereby facilitating the proper assembly, and enhancing the kinase activity of mTORC2 towards AKT. .

Article Snippet: Phorbol 12-myristate 13-acetate (PMA) (HY-18739) and PTPN22 inhibitor, PTPN22-IN-1 (HY-139693) were purchased from MedChemExpress.

Techniques: Stable Transfection, Expressing, Control, shRNA, Transfection, Construct, Colony Assay, Staining, Standard Deviation, Plasmid Preparation, Migration, Derivative Assay, Knockdown, Injection, Two Tailed Test, Activation Assay, Activity Assay