Journal: Journal of Clinical Medicine

Article Title: Epithelial-Mesenchymal Transition-Related MicroRNAs and Their Target Genes in Colorectal Cancerogenesis

doi: 10.3390/jcm8101603

Figure Lengend Snippet: Probes used for miRNAs and mRNAs quantification using quantitative real-time PCR (qPCR).

Article Snippet: PTPN13 , Hs01106214_m1 , 65.

Techniques: Real-time Polymerase Chain Reaction

Journal: Journal of Clinical Medicine

Article Title: Epithelial-Mesenchymal Transition-Related MicroRNAs and Their Target Genes in Colorectal Cancerogenesis

doi: 10.3390/jcm8101603

Figure Lengend Snippet: Statistically significant correlations between expression of miR-200 family and their target genes and association with E-cadherin staining.

Article Snippet: PTPN13 , Hs01106214_m1 , 65.

Techniques: 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: Figure 2. Functional screen identifies candidate phosphatases that regulate commitment. (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). DOI: https://doi.org/10.7554/eLife.27356.004 The following figure supplements are available for figure 2:

Article Snippet: DOI: https://doi.org/10.7554/eLife.27356 15 of 20 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 # sc8431 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 b1-Integrin (clone P5D2, in-house; immunostaining – 1:200).

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

Journal: Cell Death & Disease

Article Title: Direct conversion of human umbilical cord mesenchymal stem cells into retinal pigment epithelial cells for treatment of retinal degeneration

doi: 10.1038/s41419-022-05199-5

Figure Lengend Snippet: The expression level of PTPN13 in shCont-iRPE and shPtpn13-iRPE cells were detected by A Western blotting and B quantitative analysis ( n = 3, P value measured by Student’s unpaired t test). C Representative images of shCont-iRPE and shPtpn13-iRPE cells. RPE-specific and EMT-associated markers in shCont-iRPE cells and shPtpn13-iRPE cells stimulated with TGF-β1 or TGF-β2 were detected by D immunostaining, E Western blotting, and F quantitative analysis ( n = 3, P value measured by one-way ANOVA and post hoc Bonferroni’s test). Phosphorylation of SMAD2/3, p38, and ERK1/2 in shCont-iRPE and shPtpn13-iRPE cells were analyzed by G immunostaining, H Western blotting, and I quantitative analysis ( n = 3, P value measured by one-way ANOVA and post hoc Bonferroni’s test). Scale bar = 50 μm. Results are expressed as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, shPtpn13-TGF-β1 compared with shCont-TGF-β1; # P < 0.01, ## P < 0.01, ### P < 0.001, shPtpn13-TGF-β2 compared with shCont-TGF-β2.

Article Snippet: After blocked with 3% BSA in PBS for 1 h, membranes were incubated with primary antibodies against RPE65 (1:1000, Novus Biologicals, Centennial, CO, USA); against TYRP1 (1:1000), MERTK (1:1000), FN1 (1:1000), α-SMA (1:2000), pSMAD2 (1:500), SMAD2 (1:1000), pSMAD3 (1:1000), SMAD3 (1:1000) TGF-β receptor1 (TGF-βR1) (1:1000), Abcam; against pERK1/2 (1:1000), ERK1/2 (1:1000), CST; against Claudin19 (1:1000, Invitrogen); against Flag (1:2000, MBL International, Woburn, MD, USA); against CRALBP (1:1000), syntenin1 (1:1000), DUSP4 (1:1000), DUSP10 (1:1000), PHLPP1 (1:1000), PPM1L (1:1000), PPP1CC (1:1000), PPP2R5A (1:1000), PTPN13 (1:1000), TGF-βR2 (1:1000), and β-Actin (1:5000), Proteintech; for 12 h at 4 °C, followed by incubation with corresponding secondary antibodies for 1 h at room temperature.

Techniques: Expressing, Western Blot, Immunostaining, Phospho-proteomics

Journal: Cell Death & Disease

Article Title: Direct conversion of human umbilical cord mesenchymal stem cells into retinal pigment epithelial cells for treatment of retinal degeneration

doi: 10.1038/s41419-022-05199-5

Figure Lengend Snippet: PTPN13 binding syntenin1 in iRPE cells were identified by A mass spectrometry and confirmed by B , C Western blotting. Phosphorylation of syntenin1 was detected by D anti-phosphorylated-Tyr antibody CO-IP assay and E quantitative analysis ( n = 3). The expression levels of TGF-βR1 and TGF-βR2 in shCont-iRPE and shPtpn13-iRPE cells were detected by F Western blotting, G quantitative analysis ( n = 3). The expression levels of TGF-βR1 and TGF-βR2 on cell membranes of shCont-iRPE and shPtpn13-iRPE cells were detected by H flow cytometry and I quantitative analysis of mean fluorescence density ( n = 3). Results are expressed as mean ± SD. P value measured by Student’s unpaired t test.

Article Snippet: After blocked with 3% BSA in PBS for 1 h, membranes were incubated with primary antibodies against RPE65 (1:1000, Novus Biologicals, Centennial, CO, USA); against TYRP1 (1:1000), MERTK (1:1000), FN1 (1:1000), α-SMA (1:2000), pSMAD2 (1:500), SMAD2 (1:1000), pSMAD3 (1:1000), SMAD3 (1:1000) TGF-β receptor1 (TGF-βR1) (1:1000), Abcam; against pERK1/2 (1:1000), ERK1/2 (1:1000), CST; against Claudin19 (1:1000, Invitrogen); against Flag (1:2000, MBL International, Woburn, MD, USA); against CRALBP (1:1000), syntenin1 (1:1000), DUSP4 (1:1000), DUSP10 (1:1000), PHLPP1 (1:1000), PPM1L (1:1000), PPP1CC (1:1000), PPP2R5A (1:1000), PTPN13 (1:1000), TGF-βR2 (1:1000), and β-Actin (1:5000), Proteintech; for 12 h at 4 °C, followed by incubation with corresponding secondary antibodies for 1 h at room temperature.

Techniques: Binding Assay, Mass Spectrometry, Western Blot, Phospho-proteomics, Co-Immunoprecipitation Assay, Expressing, Flow Cytometry, Fluorescence

Journal: Cell Death & Disease

Article Title: Direct conversion of human umbilical cord mesenchymal stem cells into retinal pigment epithelial cells for treatment of retinal degeneration

doi: 10.1038/s41419-022-05199-5

Figure Lengend Snippet: Knockdown efficiency of syntenin1 was determined by A qRT-PCR ( n = 3, P value measured by one-way ANOVA and post hoc Bonferroni’s test), B Western blotting, and C quantitative analysis ( n = 3, P value measured by one-way ANOVA and post hoc Bonferroni’s test). RPE-specific and EMT-associated markers in shCont-shPtpn13-iRPE and shSyntenin1-shPtpn13-iRPE cells were analyzed by D Western blotting and E quantitative analysis ( n = 3, P value measured by one-way ANOVA and post hoc Bonferroni’s test). Phosphorylation of SMAD2/3, p38, and ERK1/2 in shCont-shPtpn13-iRPE and shSyntenin1-shPtpn13-iRPE cells were analyzed by F Western blotting and G quantitative analysis ( n = 3, P value measured by one-way ANOVA and post hoc Bonferroni’s test). The expression levels of TGF-βR1 and TGF-βR2 on the cell membranes of shCont-shPtpn13-iRPE and shSyntenin1-shPtpn13-iRPE cells were detected by H flow cytometry and I quantitative analysis of mean fluorescence density ( n = 3, P value measured by Student’s unpaired t test). J Schematic model for the PTPN13 dephosphorylating syntenin1 mediates TGF-β receptor internalization and degradation. Results are expressed as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, shCont-shPtpn13-TGF-β1 compared with shSyntenin1-shPtpn13-TGF-β1; # P < 0.01, ## P < 0.01, ### P < 0.001, shCont-shPtpn13-TGF -β 2 compared with shSyntenin1-shPtpn13-TGF-β2.

Article Snippet: After blocked with 3% BSA in PBS for 1 h, membranes were incubated with primary antibodies against RPE65 (1:1000, Novus Biologicals, Centennial, CO, USA); against TYRP1 (1:1000), MERTK (1:1000), FN1 (1:1000), α-SMA (1:2000), pSMAD2 (1:500), SMAD2 (1:1000), pSMAD3 (1:1000), SMAD3 (1:1000) TGF-β receptor1 (TGF-βR1) (1:1000), Abcam; against pERK1/2 (1:1000), ERK1/2 (1:1000), CST; against Claudin19 (1:1000, Invitrogen); against Flag (1:2000, MBL International, Woburn, MD, USA); against CRALBP (1:1000), syntenin1 (1:1000), DUSP4 (1:1000), DUSP10 (1:1000), PHLPP1 (1:1000), PPM1L (1:1000), PPP1CC (1:1000), PPP2R5A (1:1000), PTPN13 (1:1000), TGF-βR2 (1:1000), and β-Actin (1:5000), Proteintech; for 12 h at 4 °C, followed by incubation with corresponding secondary antibodies for 1 h at room temperature.

Techniques: Knockdown, Quantitative RT-PCR, Western Blot, Phospho-proteomics, Expressing, Flow Cytometry, Fluorescence

Journal: PLoS ONE

Article Title: Pair-Wise Regulation of Convergence and Extension Cell Movements by Four Phosphatases via RhoA

doi: 10.1371/journal.pone.0035913

Figure Lengend Snippet: (a) Protein structures are shown encoded by ptpn20 homologue and the immediately 5′ upstream FRMPD2 , as currently annotated in five fish genomes, the human genome and the mouse genome. In some cases like Fugu and Tetraodon a single known coding transcript exists besides separate transcripts encoding the PTP domain and the “FRMPD" part. For comparison the protein structure encoded by human ptpn13 (PTPBL) is added below. (b) Primers were designed as indicated, leaving approximately 100 bp known coding sequence for the purpose of alignment of generated sequences. PCR products with forward primers on the second to last known exon of human and zebrafish FRMPD2 and reverse oligos on the second exon of PTPN20 . A schematic representation of retrieved sequences blasted to the genome are indicated in green (not to scale). (c) Generated PCR products on human (top) and zebrafish (bottom) cDNA libraries using the described primer sets. Generated band sizes are consistent with expected values based on homology with the ptpn13 gene.

Article Snippet: Ptpn13 RNA was transcribed from full length mouse cDNA kindly provided by Wiljan Hendriks (Department of Cell Biology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands).

Techniques: Comparison, Sequencing, Generated

Journal: PLoS ONE

Article Title: Pair-Wise Regulation of Convergence and Extension Cell Movements by Four Phosphatases via RhoA

doi: 10.1371/journal.pone.0035913

Figure Lengend Snippet: Morpholinos targeting ptpn13 and ptpn20 were injected in the zebrafish at the one cell stage, and concentrations were titrated down until no phenotype was observed. Normal (red), low (green) concentrations and combined low concentrations of ptpn13 and ptpn20 morpholino were micro-injected and embryos were grown to 3dpf under normal conditions. Pictures were taken from all embryos and tails were measured using ImageJ imaging software, from the yolk to the tip of the tail, and compared to non-injected control. Average tail length compared to non-injected control is plotted as a percentage deviating from 100% in (a) and representative fish are shown for each condition in (b). Zebrafish embryos were microinjected as described above, using low concentration combined knockdown of ptpra with either ptpn13 or ptpn20 , or ptpre with either ptpn13 or ptpn20 and tail lengths are plotted in (c) and (d). (e) Shown are representative fish from the experiments depicted in (c) and (d). All error bars are standard error of the mean. Student t-test was performed where indicated; no asterisk indicates P>0.05, * indicates 0.05>P>0.001 and ** indicates P<0.001. Morpholino concentrations are color coded: red for “full" knockdown, giving full phenotype without being toxic and green for “low" concentration, giving no observable phenotype.

Article Snippet: Ptpn13 RNA was transcribed from full length mouse cDNA kindly provided by Wiljan Hendriks (Department of Cell Biology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands).

Techniques: Injection, Imaging, Software, Control, Concentration Assay, Knockdown

Journal: PLoS ONE

Article Title: Pair-Wise Regulation of Convergence and Extension Cell Movements by Four Phosphatases via RhoA

doi: 10.1371/journal.pone.0035913

Figure Lengend Snippet: (a) Low dose combined knockdowns of ptpn13 or ptpn20 and arhgap29b were performed by injecting indicated amounts of morpholino at the one cell stage. Tail lengths were measured at 3dpf and plotted. Co-knockdowns with arhgap5 were included as a control. (b) Similar co-knockdowns as in (a) but with ptpra and ptpre knockdown instead of ptpn13 and ptpn20 knockdown. (c) Zebrafish embryos were micro-injected with morpholinos targeting the different phosphatases in low concentrations together with low dose arhgef27 ( ngef ) morpholino. Embryos were grown to 3 dpf and tail lengths were determined and plotted as a percentage of non-injected control. All error bars are standard error of the mean. Student t-test was performed where indicated; no asterisk indicates P>0.05, * indicates 0.05>P>0.001 and ** indicates P<0.001.

Article Snippet: Ptpn13 RNA was transcribed from full length mouse cDNA kindly provided by Wiljan Hendriks (Department of Cell Biology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands).

Techniques: Control, Knockdown, Injection