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

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: eLife

Article Title: A protein phosphatase network controls the temporal and spatial dynamics of differentiation commitment in human epidermis

doi: 10.7554/eLife.27356

Figure Lengend Snippet: ( a ) Heatmap showing differential expression at 4, 8 and 12 hr relative to 0 hr of phosphatases that are upregulated at 4 hr in the microarray dataset. ( b ) Effect of knocking down the 22 phosphatases identified in ( a ) on clonal growth of keratinocytes. Values plotted are average % clonal growth in n = 3 independent screens with n = 3 independent cultures per screen. Green: siSCR control. Red, blue: phosphatases with statistically significant effects on colony formation are shown (red: increase; blue: decrease). Grey: no statistically significant effect. ( c, d ) Effect of knockdowns on clonal growth after 0, 4, 8 or 12 hr in suspension. ( c ) Representative dishes. ( d ) Quantitation of mean % clonal growth ± SD ( n = 3 independent samples). p-values generated by unpaired T-test. ( e, f ) RT-qPCR quantification of TP63 ( e ) and TGM1 ( f ) mRNA levels (relative to 18 s expression) in the same conditions as in ( c ). n = 3 independent transfections. ( g ) Epidermal reconstitution assay following knockdown of DUSP6, PTPN1, PPP3CA or PTPN13. n = 2 independent transfections. Top row shows representative H and E images. Epidermal thickness was quantified in multiple fields from eight sections per replicate ± SD relative to scrambled control (siSCR). Middle row shows staining for TP63 (pink) with DAPI nuclear counterstain (blue). % DAPI-labelled nuclei that were TP63+ was quantified in n = 2–3 fields per replicate. Bottom row shows staining for Ki67 (brown) with haematoxylin counterstain (blue). % Ki67+ nuclei was quantified in n = 3–6 fields per replicate. Error bars represent mean ± s.d. p-values were calculated using one-way ANOVA with Dunnett's multiple comparisons test (*p<0.05; **p<0.01; ****p<0.0005; ****p<0.0001; ns = non significant).

Article Snippet: Antibodies against the following proteins were used: P-ERK (Cell Signaling # 9101; western blot – 1:1000 dilution), ERK (Cell Signaling # 9102; western blot – 1:1000), P-p38 (Cell Signaling # 9211; western blot – 1:1000), p38 (Cell Signaling # 9212; western blot – 1:1000), Cyclophilin B (R and D # MAB5410; western blot – 1:4000), MKP-3/DUSP6 (Abcam # ab76310; western blot - 1:1000 and R and D Systems # MAB3576-SP; immunostaining – 1:200), PPTC7 (Abcam # ab122548; western-blot 1:250 and Sigma # HPA039335; immunostaining – 1:200), PTPN1/PTP1B (Sigma # HPA012542; western-blot - 1:500; R and D Systems # AF1366-SP; immunostaining – 1:200), PTPN13 (R and D Systems # AF3577; western-blot - 1:300 and immunostaining – 1:200), PPP3CA/Calcineurin A (Sigma # HPA012778; western-blot 1/1000 and R and D Systems # MAB2839-SP; immunostaining – 1:200), DUSP10 (Abcam # 140123; western-blot - 1:1000 and immunostaining – 1:200), TP63 (SCBT # sc-8431 or Abcam # ab735; immunostaining – 1:100), Involucrin (SY5, in-house; immunostaining – 1:500, or DH1, in-house; immunostaining – 1:200), Ki67 (Abcam # ab16667; immunostaining – 1:100) and β1-Integrin (clone P5D2, in-house; immunostaining – 1:200).

Techniques: Quantitative Proteomics, Microarray, Control, Suspension, Quantitation Assay, Generated, Quantitative RT-PCR, Expressing, Transfection, Reconstitution Assay, Knockdown, Staining

Journal: eLife

Article Title: A protein phosphatase network controls the temporal and spatial dynamics of differentiation commitment in human epidermis

doi: 10.7554/eLife.27356

Figure Lengend Snippet: ( a ) RT-qPCR quantification of phosphatase mRNA levels relative to 18 s RNA upon knockdown by lentiviral-mediated shRNA one week after infection and selection. p-values were generated by one-way ANOVA. ( b ) Epidermal reconstitution following knockdown of DUSP6, PPTC7, PTPN1, PPP3CA or PTPN13. n = 2 independent infections. Representative H and E images. ( c ) Epidermal thickness was quantified in eight non-consecutive sections per biological replicate as explained in and normalised to shSCR values. p-values were generated by one-way ANOVA. ( d ) Representative images of epidermis reconstituted by keratinocytes expressing shSCR or sh1DUSP6 co-stained for TP63 (yellow) and Ki67 (pink) with DAPI nuclear counterstain (blue). White asterisk shows a TP63+ Ki67 cell. ( e ) % Ki67-labelled nuclei that were also TP63+ was quantified in n = 5 fields per replicate. p-values generated by one-way ANOVA were >0.05. ( f ) Representative images of epidermis reconstituted by keratinocytes expressing shSCR or sh1DUSP6 co-stained for TP63 (yellow) and IVL (pink) with DAPI nuclear counterstain (blue). White asterisk shows a suprabasal cell co-stained for IVL and TP63. a, c, e. Error bars represent s.d. *p<0.05; **p<0.01; ***p<0.0005; ****p<0.0001; ns = non-significant.

Article Snippet: Antibodies against the following proteins were used: P-ERK (Cell Signaling # 9101; western blot – 1:1000 dilution), ERK (Cell Signaling # 9102; western blot – 1:1000), P-p38 (Cell Signaling # 9211; western blot – 1:1000), p38 (Cell Signaling # 9212; western blot – 1:1000), Cyclophilin B (R and D # MAB5410; western blot – 1:4000), MKP-3/DUSP6 (Abcam # ab76310; western blot - 1:1000 and R and D Systems # MAB3576-SP; immunostaining – 1:200), PPTC7 (Abcam # ab122548; western-blot 1:250 and Sigma # HPA039335; immunostaining – 1:200), PTPN1/PTP1B (Sigma # HPA012542; western-blot - 1:500; R and D Systems # AF1366-SP; immunostaining – 1:200), PTPN13 (R and D Systems # AF3577; western-blot - 1:300 and immunostaining – 1:200), PPP3CA/Calcineurin A (Sigma # HPA012778; western-blot 1/1000 and R and D Systems # MAB2839-SP; immunostaining – 1:200), DUSP10 (Abcam # 140123; western-blot - 1:1000 and immunostaining – 1:200), TP63 (SCBT # sc-8431 or Abcam # ab735; immunostaining – 1:100), Involucrin (SY5, in-house; immunostaining – 1:500, or DH1, in-house; immunostaining – 1:200), Ki67 (Abcam # ab16667; immunostaining – 1:100) and β1-Integrin (clone P5D2, in-house; immunostaining – 1:200).

Techniques: Quantitative RT-PCR, Knockdown, shRNA, Infection, Selection, Generated, Expressing, Staining

Journal: eLife

Article Title: A protein phosphatase network controls the temporal and spatial dynamics of differentiation commitment in human epidermis

doi: 10.7554/eLife.27356

Figure Lengend Snippet: ( a ) Heatmaps showing the effect of knocking down individual phosphatases on mRNA levels of other phosphatases with time in suspension (0, 4, 8, 12 hr). RT-qPCR is relative to 18 s mRNA ( n = 3 independent transfections; see for p-values generated by two-way ANOVA with Dunnett's multiple comparisons test). ( b ) Effects of TSA and PKCi on clonogenicity of keratinocytes recovered following suspension in methylcellulose for 12 hr. Representative dishes of n = 3. ( c ) mRNA levels of IVL, TGM, ITGα6, and TP63 in cells held in suspension for 12 hr. Cells were treated with TSA, PKCi or DMSO (vehicle control) ( n = 3 independent treated cultures with two technical replicates each). p-values for the comparisons were generated by one-way ANOVA. ( d ) Western blots showing phosphatase levels in primary keratinocytes upon knockdown of scrambled control (siSCR), DUSP6, PPTC7, PTPN1, PTPN13 or PPP3CA and suspension for 0, 4, 8 or 12 hr. Cyclophilin B: loading control. ( e ) RT qPCR quantification of phosphatase mRNA levels (relative to 18 s mRNA) following doxycycline-induced over-expression of DUSP6, mutant DUSP6 C293S and DUSP10. Cells were treated with 1 µg/ml doxycycline for 8, 24 or 48 hr ( n = 3 independent cultures; see for p-values generated by two-way ANOVA with Dunnett's multiple comparisons test).

Article Snippet: Antibodies against the following proteins were used: P-ERK (Cell Signaling # 9101; western blot – 1:1000 dilution), ERK (Cell Signaling # 9102; western blot – 1:1000), P-p38 (Cell Signaling # 9211; western blot – 1:1000), p38 (Cell Signaling # 9212; western blot – 1:1000), Cyclophilin B (R and D # MAB5410; western blot – 1:4000), MKP-3/DUSP6 (Abcam # ab76310; western blot - 1:1000 and R and D Systems # MAB3576-SP; immunostaining – 1:200), PPTC7 (Abcam # ab122548; western-blot 1:250 and Sigma # HPA039335; immunostaining – 1:200), PTPN1/PTP1B (Sigma # HPA012542; western-blot - 1:500; R and D Systems # AF1366-SP; immunostaining – 1:200), PTPN13 (R and D Systems # AF3577; western-blot - 1:300 and immunostaining – 1:200), PPP3CA/Calcineurin A (Sigma # HPA012778; western-blot 1/1000 and R and D Systems # MAB2839-SP; immunostaining – 1:200), DUSP10 (Abcam # 140123; western-blot - 1:1000 and immunostaining – 1:200), TP63 (SCBT # sc-8431 or Abcam # ab735; immunostaining – 1:100), Involucrin (SY5, in-house; immunostaining – 1:500, or DH1, in-house; immunostaining – 1:200), Ki67 (Abcam # ab16667; immunostaining – 1:100) and β1-Integrin (clone P5D2, in-house; immunostaining – 1:200).

Techniques: Suspension, Quantitative RT-PCR, Transfection, Generated, Control, Western Blot, Knockdown, Over Expression, Mutagenesis

Journal: Scientific Reports

Article Title: The tyrosine phosphatase PTPN13/FAP-1 links calpain-2, TBI and tau tyrosine phosphorylation

doi: 10.1038/s41598-017-12236-3

Figure Lengend Snippet: The C-terminus of calpain-2 binds to the PDZ domains of PTPN13. (A) Overlay of a PDZ domain array with the GST-tagged C-terminus of calpain-2 (800 nM). The GST-calpain-2 C-terminus exhibited specific binding to several PDZ domains, with PDZ domains 3, 4 and 5 of PTPN13 showing the strongest interactions (arrows). This overlay was repeated three times with similar results. ( B ) Schematic illustration of PTPN13 domains, including kinase non-catalytic C-lobe (KIND) domain and Four-point-one, ezrin, radixin, moesin (FERM) domain at the N-terminus, followed by 5 PDZ domains, and a Protein tyrosine phosphatase (PTP) domain at the C-terminus. ( C ) Co-immunostaining of calpain-2 and PTPN13 or calpain-1 and PTPN13 in the dendrites of CA1 pyramidal neurons. Scale bar = 20 µm. ( D ) Quantification of immunostaining results. Calpain-2 and PTPN13 IMF intensity were analyzed and exhibited a higher Pearson’s correlation coefficient, as compared to calpain-1 and PTPN13 IMF in CA1. N = 4 (animals). In each animal, 4–6 CA1 regions were assessed. ***P < 0.001. Two-tailed t -test. ( E ) Co-immunoprecipitation of PTPN13 with calpain-2, tau and c-Abl in P2 homogenates of mouse brain. Calpain-2 C-terminal peptide (C2CP) was added to the homogenates at a final concentration of 10 µM and incubated for 1 h before co-IP. Immunoprecipitation with rabbit IgG was performed as a negative control. Full-length blots are presented in Supplementary Figure . ( F ) Quantification of Western blots. Application of calpain-2 C-terminal peptide significantly reduced co-IP of PTPN13 with calpain-2. Data are means ± SEM of 3 independent experiments. **p < 0.01. Two-tailed t -test.

Article Snippet: The antibodies used for IP were PTPN13 (LS-C148268), normal rabbit IgG (2729, CST), GFP (12Ab hybridoma medium, gift from Dr. Steven Standley, Western University of Health Sciences), c-Abl (2862, CST), tau (AHB0042) and normal mouse IgG (sc-2025, Santa Cruz Biotechnology).

Techniques: Binding Assay, Immunostaining, Two Tailed Test, Immunoprecipitation, Concentration Assay, Incubation, Co-Immunoprecipitation Assay, Negative Control, Western Blot

Journal: Scientific Reports

Article Title: The tyrosine phosphatase PTPN13/FAP-1 links calpain-2, TBI and tau tyrosine phosphorylation

doi: 10.1038/s41598-017-12236-3

Figure Lengend Snippet: Calpain-2 truncates PTPN13, generating stable breakdown products. ( A ) P2 fractions prepared from mouse brain homogenates were treated with 20 µM or 2 mM Ca 2+ and incubated for 20 or 60 min at 37 °C. In some groups, a calpain-2 selective inhibitor NA101 (200 nM) was added 10 min before Ca 2+ treatment. The truncation of PTPN13 and spectrin was analyzed using WB. Full-length blots are presented in Supplementary Figure . ( B ) Illustration of the two potential calpain cleavage sites on PTPN13 identified by calpain cleavage site predictor (SVM RBF prediction model). The cleavage at 1488 is predicted to generate a 170 kDa N-terminal fragment and the cleavage at 1965 to generate a 210 kDa N-terminal fragment. These two fragments contain the epitope of PTPN13 antibody (aa1279–1883, as indicated in the antibody manual) used in the WB of panel A and should be detected by this antibody. According to our result, the actual epitope of this antibody should be within aa1279–1488.

Article Snippet: The antibodies used for IP were PTPN13 (LS-C148268), normal rabbit IgG (2729, CST), GFP (12Ab hybridoma medium, gift from Dr. Steven Standley, Western University of Health Sciences), c-Abl (2862, CST), tau (AHB0042) and normal mouse IgG (sc-2025, Santa Cruz Biotechnology).

Techniques: Incubation

Journal: Scientific Reports

Article Title: The tyrosine phosphatase PTPN13/FAP-1 links calpain-2, TBI and tau tyrosine phosphorylation

doi: 10.1038/s41598-017-12236-3

Figure Lengend Snippet: Calpain-2 mediated cleavage of PTPN13 regulates tyrosine phosphorylation of c-Abl and tau. ( A ) Cos-1 cells were transfected with GFP-Tau and c-Fyn (lane 1), GFP-Tau and Flag-SYK (lane 2) or GFP-Tau plus Flag-His-c-Abl (lane 3). GFP-Tau was immunoprecipitated using anti-GFP antibody. Tyrosine phosphorylation of GFP-Tau was examined using anti-phosphotyrosine antibody. Full-length blots are presented in Supplementary Figure . ( B ) Cos-1 cells were transfected with GFP-Tau plus Flag-His-Abl (lane 1), GFP-Tau plus Flag-His-Abl plus Flag-PTPN13 WT (lane 2) and GFP-Tau plus Flag-His-Abl plus Flag-PTPN13 D2359A (lane 3). Flag-PTPN13 D2359A is an inactive mutant of PTPN13. GFP-Tau was immunoprecipitated by anti-GFP antibody and was precipitated GFP-Tau was analyzed with an anti-phosphotyrosine antibody. Full-length blots are presented in Supplementary Figure . ( C ) Cos-1 cells were transfected with GFP-Tau plus c-Fyn (lane 1), GFP-Tau plus c-Fyn plus Flag-PTPN13 WT (lane 2) and GFP-Tau plus c-Fyn plus Flag-PTPN13 D2359A (lane 3). Tyrosine phosphorylation of GFP-Tau was examined. Full-length blots are presented in Supplementary Figure . ( D ) Cos-1 cells were transfected with Flag-c-Abl (lane 1), Flag-c-Abl plus Flag-PTPN13 WT (lane 2) and Flag-c-Abl plus Flag-PTPN13 D2359A (lane 3). Flag-c-Abl was pulled-down using anti-Abl antibody. Tyrosine phosphorylation of Abl was examined using anti-phosphotyrosine, phospho-c-Abl Tyr245 and phospho-c-Abl Tyr412 antibodies. Full-length blots are presented in Supplementary Figure . ( E ) NMDA treatment of acute hippocampal slices from calpain-1 KO mice at postnatal day 10. Slices were treated with 10 µM NMDA at 37 °C for 30 min. In one group, slices were pre-treated with a calpain inhibitor (calpain inhibitor III, 10 µM) for 10 min before adding NMDA. Levels of indicated proteins in the collected slices were analyzed by WB. Lane 1 and 2 are two replicates. Full-length blots are presented in Supplementary Figure . ( F ) Quantification of WB. Ratios of P13BPs to full-length PTPN13 and phospho-c-Abl Tyr245 to total c-Abl were significantly increased with NMDA treatment. Treatment with CI-III significantly inhibited NMDA-mediated changes. Data are means ± SEM of 4 independent experiments. *p < 0.05, **p < 0.01. Two-way ANOVA followed by Bonferroni test.

Article Snippet: The antibodies used for IP were PTPN13 (LS-C148268), normal rabbit IgG (2729, CST), GFP (12Ab hybridoma medium, gift from Dr. Steven Standley, Western University of Health Sciences), c-Abl (2862, CST), tau (AHB0042) and normal mouse IgG (sc-2025, Santa Cruz Biotechnology).

Techniques: Phospho-proteomics, Transfection, Immunoprecipitation, Mutagenesis

Journal: Scientific Reports

Article Title: The tyrosine phosphatase PTPN13/FAP-1 links calpain-2, TBI and tau tyrosine phosphorylation

doi: 10.1038/s41598-017-12236-3

Figure Lengend Snippet: TBI triggers calpain-2 mediated PTPN13 cleavage and tyrosine phosphorylation of c-Abl and tau. ( A ) A calpain-2 selective inhibitor inhibits TBI-triggered PTPN13 cleavage and tyrosine phosphorylation of c-Abl and tau. Ipsilateral cortex of WT mice was collected and homogenized 6 h after surgery. C-Abl or tau was then immunoprecipitated from homogenates to test its tyrosine phosphorylation. Lane 1, Sham surgery. Immunoprecipitation was performed with tau or c-Abl antibody. Lane 2, Controlled cortical impact (CCI). Immunoprecipitation was performed with tau or c-Abl antibody. Lane 3, NA101 was injected i.p. 1 h after CCI. Immunoprecipitation was performed with tau or c-Abl antibody. Lane 4, Sham surgery. Immunoprecipitation was performed with mouse or rabbit IgG as a negative control. Full-length blots are presented in Supplementary Figure . ( B ) Ipsilateral cortex of calpain-1 KO mice was collected and homogenized 6 h after CCI or sham surgery. PTPN13 and spectrin cleavage and tau tyrosine phosphorylation were analyzed with WB. Lane 1, Sham surgery. Immunoprecipitation was performed with tau antibody. Lane 2, CCI. Immunoprecipitation was performed with tau antibody. Lane 3, Sham surgery. Immunoprecipitation was performed with mouse IgG as a negative control. Full-length blots are presented in Supplementary Figure . ( C ) Quantification of WB results similar to panel A. The ratios of P13BPs to PTPN13, phospho-tyrosine tau to total tau, and phospho-Tyr245 of c-Abl to total c-Abl were significantly increased following CCI. Injection with NA101 significantly inhibited those changes following CCI. Data are means ± SEM of 3–5 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. Two-way ANOVA followed by Bonferroni test. ( D ) Quantification of WB results similar to panel B. The ratios of P13BPs to PTPN13, and phospho-tyrosine tau to total tau were significantly increased following CCI. Data are means ± SEM of 3 independent experiments. *p < 0.05, **p < 0.01. Two-way ANOVA followed by Bonferroni test. ( E – G ) IHC with a phospho-c-Abl (Tyr245) antibody in coronal brain sections (Bregma −0.58 mm) 6 h after TBI. Vehicle (5% DMSO in PBS) or NA101 (0.3 mg/kg) was injected i.p. 1 h after TBI. Scale bar in the DAPI images under low-magnification, 500 µm. Scale bar in the zoomed-in images, 100 µm. ( H ) High-magnification images of phospho-c-Abl (Tyr245) staining of cortical neurons in ipsilateral cortex 6 h after TBI. Scale bar, 10 µm. ( I ) Quantification of IHC results. Three coronal brain sections (Bregma −0.58 mm, −1.58 mm and −1.94 mm) from each brain were immunostained and IMF intensity analyzed. Changes in phospho-Tyr245 level were calculated as the mean fluorescence intensity (MFI) of p-Tyr245 staining in the ipsilateral side of the insult subtracted by the MFI of p-Tyr245 staining in the contralateral side. Changes in p-Tyr245 in all 3 sections from the same brain were averaged. N = 3–5 (animals). *p < 0.05, **p < 0.01. One-way ANOVA followed by Bonferroni test.

Article Snippet: The antibodies used for IP were PTPN13 (LS-C148268), normal rabbit IgG (2729, CST), GFP (12Ab hybridoma medium, gift from Dr. Steven Standley, Western University of Health Sciences), c-Abl (2862, CST), tau (AHB0042) and normal mouse IgG (sc-2025, Santa Cruz Biotechnology).

Techniques: Phospho-proteomics, Immunoprecipitation, Injection, Negative Control, Staining, Fluorescence

Journal: Scientific Reports

Article Title: The tyrosine phosphatase PTPN13/FAP-1 links calpain-2, TBI and tau tyrosine phosphorylation

doi: 10.1038/s41598-017-12236-3

Figure Lengend Snippet: Inhibition of calpain-2 or c-Abl attenuates the formation of tau oligomers in brain following TBI. (A) IHC with an anti-tau oligomer antibody (T22) in cortical and hippocampal CA1 areas of coronal brain sections 24 h after TBI or sham surgery. Vehicle (5% DMSO in PBS), NA101 (0.3 mg/kg) or Nilotinib (25 mg/kg) was injected i.p. 1 h after TBI. Scale bar = 100 µm. ( B ) Quantification of IHC results. Three coronal brain sections (Bregma −1.58 mm, −1.94 mm and −2.30 mm) from each brain were imaged. MFIs in cortical and CA1 areas from each section were measured in ImageJ. N = 3 for Sham, C2I and Nilotinib. N = 5 for Vehicle. **p < 0.01, ****p < 0.0001 Sham vs. Vehicle, # p < 0.05 C2I vs. Vehicle, ## p < 0.01 Nilotinib vs. Vehicle. Two-way ANOVA followed by Bonferroni test. ( C ) ELISA analysis of brain homogenates using Tau-5 and T22 antibodies 24 h after TBI or sham surgery. Vehicle (5% DMSO in PBS), NA101 (0.3 mg/kg) or Nilotinib (25 mg/kg) was injected i.p. 1 h after TBI. The ratios of tau oligomers to total tau were analyzed. N = 4 (animals). ***p < 0.001 sham vs. vehicle, # p < 0.05 vehicle vs. C2I. ## p < 0.01 vehicle vs. Nilotinib. One-way ANOVA followed by Bonferroni test. ( D ) Levels of Tau monomers and oligomers and Serine/Threonine phosphorylation of tau at S202, T205, T231 and S416 in ipsilateral cortical homogenates collected 24 h after sham (lane 1) or TBI (lane 2) or TBI plus NA101 injection (lane 3) were analyzed with WB of non-reducing SDS-PAGE (NuPAGE 4–12% Bis-Tris gel). Full-length blots are presented in Supplementary Figure . ( E ) Quantification of tau oligomers (boxed area) as detected by WB. N = 3 (animals). ****p < 0.0001 sham vs. TBI. ### p < 0.001 TBI vs. TBI + NA101. Two-way ANOVA followed by Bonferroni test. ( F ) Post-mortem hippocampal samples from 4 AD patients and 4 age-matched controls (N) were probed with antibodies against PTPN13 and actin in WB. Full-length PTPN13 at 275 kDa and a P13BP at approximately 210 kDa were detected. ( G ) Quantification of the Western blots. The ratio of P13BP to full-length PTPN13 was significantly higher in AD patients than that in controls. N = 4. *p < 0.05. Two-tailed t -test.

Article Snippet: The antibodies used for IP were PTPN13 (LS-C148268), normal rabbit IgG (2729, CST), GFP (12Ab hybridoma medium, gift from Dr. Steven Standley, Western University of Health Sciences), c-Abl (2862, CST), tau (AHB0042) and normal mouse IgG (sc-2025, Santa Cruz Biotechnology).

Techniques: Inhibition, Injection, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, SDS Page, Western Blot, Two Tailed Test

Journal: Scientific Reports

Article Title: The tyrosine phosphatase PTPN13/FAP-1 links calpain-2, TBI and tau tyrosine phosphorylation

doi: 10.1038/s41598-017-12236-3

Figure Lengend Snippet: A new link between TBI, calpain-2, tauopathy and AD pathogenesis. TBI triggers calpain-2 activation. PTPN13 is cleaved and inactivated. This results in increased tyrosine phosphorylation of c-Abl at tyr245, which increases its kinase activity and phosphorylates tau at tyr394. Elevation of tau tyrosine phosphorylation contributes to tau oligomer accumulation, which may lead to increased risk of AD or AD-related disease. Administration of a calpain-2 selective inhibitor post-TBI reduces TBI-induced calpain-2 activation, tau tyrosine phosphorylation and tau oligomer formation, thus may inhibit tauopathy and AD pathogenesis.

Article Snippet: The antibodies used for IP were PTPN13 (LS-C148268), normal rabbit IgG (2729, CST), GFP (12Ab hybridoma medium, gift from Dr. Steven Standley, Western University of Health Sciences), c-Abl (2862, CST), tau (AHB0042) and normal mouse IgG (sc-2025, Santa Cruz Biotechnology).

Techniques: Activation Assay, Phospho-proteomics, Activity Assay