Fig 2: PTPN14 is a negative regulator of YAP.A) An SF268 cell line stably expressing the YAP-responsive MCAT_Luc reporter was transfected with two independent siRNAs against YAP and analysed in a Resazurin and luciferase assay 72 h after transfection. mRNA levels of YAP were determined by QRTPCR. Luciferase results are normalized based on the Resazurin results. All results are the average of 5 experiments ± STDEV. Statistical analysis was carried out with a 2-tailed paired t-test for each siRNA; * p<0.0001. B) An SF268 cell line stably expressing the YAP-responsive MCAT_Luc reporter was transduced with lentivirus encoding for dox-inducible PTPN14 expression. After pharmacological selection, luciferase expression was measured for both cell lines in the presence and absence of dox (upper panel). A Resazurin assay was carried out in parallel for each sample and used to normalize the luciferase readings. Results are shown as the average of three independent experiments ± STDEV. Statistical analysis was carried out with a 2-tailed paired t-test; * p<0.0001. RLU: relative luciferase units. PTPN14 expression achieved by dox and YAP levels in each sample was analyzed by Western blot (lower panel). Tubulin serves as loading control C) 293A cells were transduced with lentivirus encoding dox-inducible PTPN14 expression. YAP localization in control and PTPN14-expressing cell lines was analysed 72 hours post dox induction by IF at low density using confocal miroscopy. D) 293A cells were transduced with lentivirus encoding two independent dox-inducible shRNAs targeting PTPN14. The nuclear/cytoplasmic ratio of YAP was quantified at high density 72 hours post dox induction using a Cellomics automated imager with a conventional microscope, and expressed relative to the control shGFP (left upper panel). Results are shown as the average of 3 independent experiments ± STDEV. For each experiment, the average ratio was calculated from three wells per sample (10 images per well). Statistical analysis was carried out with a 2-tailed paired t-test; * p<0.001, ** p<0.05. Western blot analysis of PTPN14 levels, indicating the efficiency of each shRNA (left lower panel). Tubulin serves as a loading control. Confocal microscopy images of cells 72 hours post dox induction (right panel).

Fig 3: YAP-PTPN14 binding is mediated through the WW domain-PPxY motif interaction.A) 293A cells were transfected with WT GST-PTPN14 and the indicated V5-YAP constructs (WT and mutants). A V5 IP was carried out and blotted for GST to study the interaction of the various YAP mutants with WT PTPN14. Input lanes were loaded with 10% of the amount of lysate used for each IP and used to compare the expression levels of each construct. B–C) 293A cells were transfected with WT V5-YAP and the indicated GST-PTPN14 constructs (WT and mutants). A V5 IP was carried out and blotted for GST to study the interaction of the various PTPN14 mutants with WT YAP. Input lanes were loaded with 10% of the amount of lysate used for each IP and used to compare the expression levels of each construct. All lanes in (C) are from a single blot and exposure. D) An SF268 cell line stably expressing the YAP-responsive MCAT_Luc reporter was transduced with lentivirus encoding for the indicated dox-inducible PTPN14 expression. Luciferase expression of each cell line was analysed 72 hours post dox induction (left panel). A Resazurin assay was carried out in parallel for each sample and used to normalize the luciferase readings. PTPN14 expression levels achieved for each construct were analysed by Western blot (right panel; arrows indicate the WT PTPN14 protein and the truncated ?PTP PTPN14 which migrates faster; all lanes from a single blot and exposure). Tubulin serves as loading control. Luciferase results are shown as the average of at least 3 independent experiments ± STDEV. Statistical analysis was carried out with a 2-tailed paired t-test; * p<0.05. E) The mRNA levels of the indicated YAP target genes were assessed in cells from (D) 72 hours post dox induction. Statistical analysis was carried out with a 2-tailed paired t-test; * p<0.001; **p<0.05, F) 293A cells were transduced with lentivirus encoding for the indicated dox-inducible PTPN14 expression. The nuclear/cytoplasmic YAP ratio was quantified at low density after 72 hours of dox induction using a Cellomics automated imager with a conventional microscope, and expressed relative to control (left panel). Results are shown as the average of three experiments ± STDEV. Statistical analysis was carried out with a 2-tailed paired t-test; * p<0.05. For each experiment, the average ratio was calculated from three wells per sample (10 images per well). PTPN14 expression levels were analysed by Western blot (right panel; arrows indicate the WT PTPN14 protein and the truncated ?PTP PTPN14 which migrates faster; all lanes from a single blot and exposure). Tubulin serves as a loading control G) Confocal microscopy images of 293A cells from (F) generated 72 hours post dox induction. H) The mRNA levels of the indicated YAP target genes were assessed in cells from (F) 72 hours post dox induction. Statistical analysis was carried out with a 2-tailed paired t-test; * p<0.05.

Fig 4: PTPN14 suppresses KRASG12V-induced transformation in Caco-2 cells. (A) Confocal images were captured for control and KRASG12V-transformed Caco-2 spheroids immunostained for PRKCI (green) and F-actin (magenta). (B) Quantification of the fold change of apical enrichment of PRKCI in control and KRASG12V-transformed Caco-2 cysts. (C) Western blot showing PRKCI and PTPN14 protein expression in control and KRASG12V-transformed Caco-2 spheroid lysates. (D) Immunoprecipitation of myc-EGFP or myc-PRKCI was performed with anti-myc as control and KRASG12V-transformed Caco-2 cysts. (E) Western blot was performed with anti-PRKCI and anti-PTPN14 in PRKCI knockdown Caco-2 cell lysates. (F) Western blot probed with anti-PRKCI and anti-PTPN14 antibodies in shScr, sh3- and sh5-PTPN14 knock-down Caco-2 cell lysates. PRKCI was up-regulated in PTPN14 knockdown Caco-2 cells. (G) Confocal images were captured for KRASG12V-transformed Caco-2 cysts with or without PTPN14 overexpression immunostained for PTPN14-V5 (yellow), PRKCI (green), and F-actin (blue). (H) Quantification of the fold change of apical enrichment of PRKCI in control, KRASG12V-transformed Caco-2 cysts, and KRASG12V-transformed Caco-2 cysts with PTPN14 overexpression. (I) Quantification of the fold change of cyst size of cultured KRASG12V-transformed Caco-2 cysts with or without PTPN14 overexpression. (J) Quantification of the percentage of single lumen formation of cultured KRASG12V-transformed Caco-2 cysts with or without PTPN14 overexpression. Scale Bars: A, 30 µm; G, 100 µm.

Fig 5: Down-regulation of PTPN14 enhances the oncogenic phenotype of YAP.A) MCF-10A cells were transduced with lentivirus encoding for YAP expression and after pharmacological selection transduced with lentivirus encoding for dox-inducible shRNAs targeting either PTPN14 or GFP (control) (two independent shRNAs targeting PTPN14). All six cell lines were subjected to an anoikis assay in low-attachment plates in the presence of dox for 48 hours. Results are shown as the average of three experiments ± STDEV. Statistical analysis was carried out with a 2-tailed paired t-test; * p<0.05, ** p = 0.05. B) Western blot analysis of cells from (A) 48 hours post dox induction. Tubulin serves as a loading control. C) Soft agar assay quantification of cells from (A). Results were obtained two weeks post dox induction and are the average of three experiments ± STDEV. D) Images of wells from (C) at the time of quantification. Scale: 1 mm.