Journal: Cell Research

Article Title: Targeting PTPN13 with 11-amino-acid peptides of C-terminal APC prevents immune evasion of colorectal cancer

doi: 10.1038/s41422-025-01206-4

Figure Lengend Snippet: a Schematic illustration of the CT26-shApc in vivo CRISPR screening system. b Rank-ordered normalized robust rank aggregation (RRA) scores for genes negatively or positively enriched in WT Balb/c in the CT26-shApc in vivo CRISPR screen. Genes are highlighted in blue (immune-resistor genes) and red (immune-sensitizer genes). The top ten genes are indicated. Dot size is inversely scaled by FDR. FDR < 5%. c, d Ptpn13 knockout-GFP and control-mCherry CT26 cells were mixed in vitro and subcutaneously injected at a 1:1 ratio into WT Balb/c and nude mice or WT Balb/c mice pre-injected with anti-CD8α depletion antibody. The GFP/mCherry ratio in vitro and in vivo was determined by FACS. e Representative images and quantification of immunohistochemical staining against PTPN13 in paired CRC normal and tumor tissues. n = 50, paired t -test. f Survival analysis of PTPN13-high and PTPN13-low groups from the CRC patient cohort. Log-rank test. g Representative immunofluorescence staining of CK (purple), PTPN13 (green), and CD8 (yellow) in CRC primary tissues. h Scatterplot showing correlation between MFI of HLA-ABC and CD8 + cells. n = 80, Pearson’s r . i MFI of PTPN13 in APC WT and APC-mutated CRC primary tissues. n = 80 for each group, unpaired t -test. j The indicated cells were injected subcutaneously into Balb/c mice, and tumor growth was monitored. n = 8 per group, two-way ANOVA. k Ptpn13 was knocked out in Apc-silenced CT26 cells, and the cells were then intraperitoneally injected into Balb/c mice. The log-rank test was used to compare survival times. l The indicated CT26 cells were transplanted into Balb/c mice, and the tumor growth curve and weight were measured. n = 8 per group, two-way ANOVA. m Immmunofluorescent staining against CD8 in subcutaneous tumors of Balb/c mice. n = 8 per group, one-way ANOVA. n Numbers of IFN-γ + or TNF-α + CD8 + T cells in tumor-infiltrating lymphocytes (TILs) of Ptpn13-knockout or negative control sgRNA-transfected CT26-shAPC subcutaneous tumors were measured by flow cytometry. n = 3 per group, one-way ANOVA. o Optical colonoscopy and tumor scoring of orthotopic tumors of Ptpn13-knockout or negative control sgRNA-transfected AKP intestinal organoids injected into WT C57/B6 mice. n = 6 per group, unpaired t -test. p Representative immunofluorescence staining of CK (red) and CD8 (yellow) in tumor tissues and scatterplots showing numbers of CD8 + cells in three groups. n = 6 per group, one-way ANOVA. q Schematic illustration of the CRISPR/Cas9-based Ptpn13 conditional knockout system. Offspring of Ptpn13 conditional knockout mice were screened using PCR, and knockout efficiency was determined by immunoblotting. r Ptpn13 fl/fl mice were crossed with Apc Min/+ and Villin- CreER T2 mice (APV), and tamoxifen (TAM) or oil was injected intraperitoneally in APV mice aged 9 weeks. s Representative image of intestines of APV mice treated with TAM or oil. The entire small intestine was systematically divided into three segments, and one representative segment is shown. Red arrows, tumors stained with methylene blue. t Scatterplot showing the total number of intestinal tumors across the entire small intestine in APV mice treated with TAM or oil. n = 7, unpaired t -test. u Representative hematoxylin and eosin staining of intestines of APV mice treated with TAM or oil. The entire small intestine was systematically divided into three segments, and one representative segment is shown. Red arrows indicate tumors. v Representative immunofluorescence staining of CK (red) and CD8 (yellow) in tumor tissues. Scatterplots show numbers of CD8 positive cells in the tumor tissues of APV mice treated with TAM or oil. n = 7, unpaired t -test. Data are representative of three independent experiments. All data are mean ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Staining for (CK, CD3 and CD8) or (Epcam, HLA-ABC and CD8) or (APC, PTPN13 and p-STAT1) was performed using a sequential multiplexed immunofluorescence protocol with the isotype-specific primary antibodies anti-CK (1:800, #C2562, Sigma-Aldrich), Epcam (1:500, #93790S, Cell Signaling Technology), CD3E (1:500, #HPA043955, ATLAS), CD8 (1:500, #HPA037756, ATLAS), HLA-ABC (1:200, #565292, BD Biosciences), APC (1:100, #ab15270, abcam), PTPN13 (1:500, #NB100-56139, Novus) and p-STAT1 (1:50, #9167S, Cell Signaling Technology).

Techniques: In Vivo, CRISPR, Knock-Out, Control, In Vitro, Injection, Immunohistochemical staining, Staining, Immunofluorescence, Negative Control, Transfection, Flow Cytometry, Western Blot

Journal: Cell Research

Article Title: Targeting PTPN13 with 11-amino-acid peptides of C-terminal APC prevents immune evasion of colorectal cancer

doi: 10.1038/s41422-025-01206-4

Figure Lengend Snippet: a Apc-silenced CT26 cells transfected with negative control (N.C) or two single guide RNAs (sgRNAs) targeting Ptpn13 were stimulated with different concentrations of IFNγ. Total cell lysates were subjected to immunoblot analysis with antibodies to the indicated proteins. Data are representative of three independent experiments. b Irf1, Lmp2, Tap1, Tap2, MHC-I , and B2m mRNA expression was determined by RT-qPCR in Ptpn13-knockout or negative control sgRNA-transfected CT26-shAPC cells with 12 h exposure to IFNγ (50 ng/mL). n = 6 per group, one-way ANOVA. c Flow cytometry histogram and levels of the MHC-I complex on the surfaces of the indicated cells pretreated for 24 h with IFNγ (100 ng/mL) or BSA and stained with anti-H-2Kd/2Dd antibody. Data were calculated from three independent experiments. One-way ANOVA. d Ptpn13-knockout or negative control sgRNA-transfected MC38-OVA-shAPC cells were stimulated with IFNγ (100 ng/mL) or BSA for 24 h, and the numbers of H-2Kb-OVA 257-264 positive cells and MFI were detected by flow cytometry. Data were calculated from three independent experiments. One-way ANOVA. e Numbers of OVA-tetramer positive CD8 + T cells in TILs of Ptpn13-knockout or negative control sgRNA-transfected MC38-OVA-shAPC subcutaneous tumors, as detected by flow cytometry. n = 3 for each group, one-way ANOVA. f Irf1, Lmp2, Tap1, Tap2, MHC-I , and B2m mRNA expression was determined by RT-qPCR in intestinal tumors of TAM- or oil-treated APV mice. n = 6 per group, one-way ANOVA. g MFI of H-2Kb/2Db positive cells in intestinal tumors of TAM- or oil-treated APV mice as detected by flow cytometry. n = 6 per group, unpaired t -test. h Scatterplot showing correlation between MFI of HLA-ABC and Ptpn13 IF staining in CRC primary tissues. n = 80, Pearson’s r . i, j CRC-patient-derived organoids (PDO) were cultivated and transfected with Ptpn13-knockout or negative control sgRNA. i Representative immunofluorescence staining of Epcam (green), HLA-ABC (red) and CD8 (cyan). j MFI of HLA-ABC IF staining in Ptpn13-knockout or negative control sgRNA-transfected PDOs. One-way ANOVA. Data were calculated from three independent experiments. All data are mean ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Staining for (CK, CD3 and CD8) or (Epcam, HLA-ABC and CD8) or (APC, PTPN13 and p-STAT1) was performed using a sequential multiplexed immunofluorescence protocol with the isotype-specific primary antibodies anti-CK (1:800, #C2562, Sigma-Aldrich), Epcam (1:500, #93790S, Cell Signaling Technology), CD3E (1:500, #HPA043955, ATLAS), CD8 (1:500, #HPA037756, ATLAS), HLA-ABC (1:200, #565292, BD Biosciences), APC (1:100, #ab15270, abcam), PTPN13 (1:500, #NB100-56139, Novus) and p-STAT1 (1:50, #9167S, Cell Signaling Technology).

Techniques: Transfection, Negative Control, Western Blot, Expressing, Quantitative RT-PCR, Knock-Out, Flow Cytometry, Staining, Derivative Assay, Immunofluorescence

Journal: Cell Research

Article Title: Targeting PTPN13 with 11-amino-acid peptides of C-terminal APC prevents immune evasion of colorectal cancer

doi: 10.1038/s41422-025-01206-4

Figure Lengend Snippet: a Flag-tagged Stat1 expression vector (0.5 μg) was transfected into CT26 cells. Total cell lysates were immunoprecipitated with anti-Flag and immunoblotted with anti-PTPN13. b Schematic diagram showing the GST-fused PTPN13 motifs and His-tagged STAT1 used in the GST pull-down assays. c GST pull-down assays examining the interactions between GST-fused PTPN13 fragments and His-tagged STAT1 protein. Data are representative of three independent experiments. d Phosphorylated STAT1 was immunoprecipitated with anti-Flag from IFNγ-stimulated 293T transfectants and incubated with 0.2 mg/mL recombinant GST-PTPase domain of PTPN13. Immunoprecipitates were immunoblotted with anti-phospho-STAT1. Equal loading was verified by reprobing with anti-STAT1. e Total cell lysates of CT26 cells were immunoprecipitated with anti-APC and immunoblotted with anti-PTPN13. Data are representative of three independent experiments. f CT26 cells were treated with the indicated concentrations of IFNγ, and cell lysates were immunoprecipitated with anti-APC and immunoblotted with anti-PTPN13. g A Flag-tagged Stat1 vector (0.5 μg) was transfected into CT26-shApc cells or their control cells. Total cell lysates from the indicated cells were immunoprecipitated with anti-Flag and immunoblotted with anti-PTPN13. Data are representative of three independent experiments. h Alphafold3-predicted binding pattern of human APC (brown) and the PDZ2a domain of PTPN13 (cyan). The APC V2843 residue is labeled, and APC Q2829–V2843 residues are shown as sticks and colored in yellow. Hydrogen bonds are shown as yellow dotted lines. i CT26 cells were transfected with HA-tagged Apc-WT or Apc V2860A mutant plasmids, and total cell lysates were immunoprecipitated with anti-HA and immunoblotted with anti-PTPN13 and anti-CTNNB1. Data are representative of three independent experiments. j IFNγ (50 ng/mL) was administered to CT26 cells transfected with Apc-WT or Apc V2860A mutant plasmids for 12 h, and Apc, Lgr5, Axin2, Irf1, Lmp2, Tap1, Tap2, H2-D1, H2K1 , and B2m mRNA expression was detected by RT-qPCR. Data are representative of three independent experiments. One-way ANOVA. k CRISPR/Cas9-based establishment of APC V2860A point mutation. l CT26 cells transfected with APC-WT or APC V2860A mutant plasmids (CT26-APC V2860A -1/2) were incubated with or without IFNγ at the indicated concentrations for 2 h, and total cell lysates were subjected to immunoblot analysis. m IFNγ (50 ng/mL) was administered to CT26 cells transfected with Apc-WT or Apc V2860A mutant plasmids (CT26-APC V2860A -1/2) for 12 h, and Apc, Lgr5, Axin2, Irf1, Lmp2, Tap1, Tap2, H2-D1, H2K1 , and B2m mRNA expression was detected by RT-qPCR. Data are representative of three independent experiments. One-way ANOVA. n CT26 cells harboring gRNA-induced mutant APC V2860A (CT26-APC V2860A -1/2) and the WT control were subcutaneously injected (2 × 10 6 cells) into Balb/c mice, and tumor growth was monitored. n = 8 for each group, two-way ANOVA. o Immunofluorescence of CD8 + cell infiltration in subcutaneous tumors of CT26-APC V2860A and the control group. n = 8 for each group, one-way ANOVA. Data are representative of three independent experiments. All data are mean ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Staining for (CK, CD3 and CD8) or (Epcam, HLA-ABC and CD8) or (APC, PTPN13 and p-STAT1) was performed using a sequential multiplexed immunofluorescence protocol with the isotype-specific primary antibodies anti-CK (1:800, #C2562, Sigma-Aldrich), Epcam (1:500, #93790S, Cell Signaling Technology), CD3E (1:500, #HPA043955, ATLAS), CD8 (1:500, #HPA037756, ATLAS), HLA-ABC (1:200, #565292, BD Biosciences), APC (1:100, #ab15270, abcam), PTPN13 (1:500, #NB100-56139, Novus) and p-STAT1 (1:50, #9167S, Cell Signaling Technology).

Techniques: Expressing, Plasmid Preparation, Transfection, Immunoprecipitation, Incubation, Recombinant, Control, Binding Assay, Residue, Labeling, Mutagenesis, Quantitative RT-PCR, CRISPR, Western Blot, Injection, Immunofluorescence

Journal: Cell Research

Article Title: Targeting PTPN13 with 11-amino-acid peptides of C-terminal APC prevents immune evasion of colorectal cancer

doi: 10.1038/s41422-025-01206-4

Figure Lengend Snippet: a Schematic diagram showing the indicated residues at the APC C terminus. b Kinetics of the interaction between PDZ-2a and the indicated residues of APC were explored by surface plasmon resonance-based binding assays. c Binding affinities of PDZ2a to APC C-terminal peptides of different lengths, as measured by an FP assay. d The 2.1-Å complex structure of the PDZ2a domain (1364–1446 aa) and the APC11 peptide. PDZ2a is shown in cyan and presented as a surface diagram, whereas the peptide is shown in yellow and presented as a stick diagram. e Detailed interactions between the APC11 peptide and PDZ2a within the complex. The PDZ2a residues involved are labeled and shown as magenta sticks, and the peptide-interacting water molecules are shown as green balls. Hydrogen bonds are shown as yellow dotted lines. f Binding affinity of PDZ2a to the WT APC11 peptide and the APC11M mutant (V2843A) as measured by an FP assay. g GST-fused STAT1 was incubated with HA-tagged PDZ2a and with TAT-APC11 or TAT-APC11M peptides, immunoprecipitated with GST beads, and immunoblotted with anti-GST and anti-HA antibodies. Data are representative of three independent experiments. h CT26 cells transfected with a Flag-tagged Stat1 vector were incubated with 25 μM TAT-HA2, with or without 50 μM TAT-APC11 or TAT-APC11M, for 4 h. Total cell lysates were immunoprecipitated with anti-Flag and immunoblotted with anti-PTPN13. Data are representative of three independent experiments. i Apc-silenced CT26 cells were incubated with 25 μM TAT-HA2, with or without 50 μM TAT-APC11 or TAT-APC11M, for 2 h and then treated with or without IFNγ at the indicated concentrations for 2 h. Total cell lysates were subjected to immunoblot analysis. Data are representative of three independent experiments. j Irf1, Lmp2, Tap1, Tap2, H2-D1, H2K1 , and B2m mRNA expression (RT-qPCR) in Apc-silenced CT26 cells exposed to IFNγ (50 ng/mL) for 12 h before collection from three independent experiments. One-way ANOVA. k Apc-silenced CT26 cells were incubated with 25 μM TAT-HA2, with or without 50 μM TAT-APC11 or TAT-APC11M, for 2 h and then treated with or without IFNγ (100 ng/mL) for 24 h. FACS histogram and quantification of the MHC-I complex on the surfaces of the indicated cells stained with anti-H-2Kd/2Dd antibody or isotype control antibodies. Data were calculated from three independent experiments. One-way ANOVA. l Apc-silenced MC38-OVA 257-264 cells were incubated with 25 μM TAT-HA2, with or without 50 μM TAT-APC11 or TAT-APC11M, for 2 h and then treated with or without IFNγ (100 ng/mL) for 24 h. FACS histogram and quantification of OVA 257-264 -specific MHC-I complex on the surfaces of the indicated cells stained with anti-H-2Kb/SIINFEKL antibody or isotype control antibodies. Data represent three independent experiments. One-way ANOVA. m DLD1 cells were incubated with 25 μM TAT-HA2, with or without 50 μM TAT-APC11 or TAT-APC11M, for 2 h and then treated with or without IFNγ at the indicated concentrations for 2 h. Total cell lysates were subjected to immunoblot analysis. Data are representative of three independent experiments. n IRF1, LMP2, TAP1, TAP2, HLA-A, HLA-B, HLA-C , and B2M mRNA expression (RT-qPCR) in the indicated cells after exposure to IFNγ (50 ng/mL) for 12 h before collection from three independent experiments. One-way ANOVA. All data are mean ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Staining for (CK, CD3 and CD8) or (Epcam, HLA-ABC and CD8) or (APC, PTPN13 and p-STAT1) was performed using a sequential multiplexed immunofluorescence protocol with the isotype-specific primary antibodies anti-CK (1:800, #C2562, Sigma-Aldrich), Epcam (1:500, #93790S, Cell Signaling Technology), CD3E (1:500, #HPA043955, ATLAS), CD8 (1:500, #HPA037756, ATLAS), HLA-ABC (1:200, #565292, BD Biosciences), APC (1:100, #ab15270, abcam), PTPN13 (1:500, #NB100-56139, Novus) and p-STAT1 (1:50, #9167S, Cell Signaling Technology).

Techniques: SPR Assay, Binding Assay, FP Assay, Labeling, Mutagenesis, Incubation, Immunoprecipitation, Transfection, Plasmid Preparation, Western Blot, Expressing, Quantitative RT-PCR, Staining, Control

Journal: Biomedicines

Article Title: Reversal of Myofibroblast Apoptosis Resistance and Collagen Deposition by Phaseoloidin-Induced Autophagy Attenuates Pulmonary Fibrosis

doi: 10.3390/biomedicines13112679

Figure Lengend Snippet: PTPN13 mediated by phaseoloidin links autophagy and apoptosis. ( A ) Western blot analysis of PTPN13 levels in FasL and different dose phaseoloidin co-treated myofibroblasts ( left ) and quantification of the PTPN13 band intensity ( right ). ( B ) Western blotting reflects the CO-IP assay of PTPN13 binding to p62 under co-treatment with FasL and phaseoloidin or CQ. ( C ) Western blot analysis of PTPN13 levels in FasL and 100 μM phaseoloidin co-treated primary mouse myofibroblasts in the presence or absence of CQ ( top ) and quantification of the PTPN13 band intensity ( bottom ). ( D ) Flow cytometry was used to detect the percentage of apoptosis in the above experimental groups. ( E ) Flow cytometry was used to detect the percentage of apoptosis in groups with or without vector-PTPN13. Data in this figure represent the means ± S.D. (* p < 0.05, ** p < 0.01, **** p < 0.0001).

Article Snippet: A total of 10% of the proteins electrophoresed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels were transferred to PVDF membranes (Bio-Rad) and 5% ( w / v ) skim milk with TBS buffer containing 0.1% ( v / v ) Tween 20 (TBST) TBS buffer for blocking. β-ACTIN monoclonal antibody (1:5000, Proteintech, Wuhan, China), α-SMA monoclonal antibody (1:1000, CST, USA), Caspase3 polyclonal antibody (1:1000, Proteintech, China), collagen I polyclonal antibody (1:1000, Millipore, USA), Bcl-2 polyclonal antibody (1:1000, Proteintech, China), BAX polyclonal antibody (1:1000, Proteintech, China), PTPN13 polyclonal antibody (1:1000, RD, Minneapolis, MN, USA), p70S6K polyclonal antibody (1:1000, CST, USA), elf-4B polyclonal antibody (1:500, ABclonal, Wuhan, China), mTOR monoclonal antibody (1:1000, CST, USA), Pho-mTOR monoclonal antibody (1:1000, CST, USA), AMPK monoclonal antibody (1:1000, CST, USA) and Pho-AMPK monoclonal antibody (1:1000, CST, USA) were incubated overnight at 4 °C.

Techniques: Western Blot, Co-Immunoprecipitation Assay, Binding Assay, Flow Cytometry, Plasmid Preparation