PTPN3 Acts as a Tumor Suppressor and Boosts TGF-β Signaling Independent of its Phosphatase Activity (2019)
Figure 1

Figure 1. PTPN3 enhances TGF-β-induced transcriptional responses

A, PTPN3 promotes TGF-β-induced CAGA-luc reporter gene activity. HaCaT cells were transfected with expression plasmids for CAGA-luc reporter gene, Renilla-luc reporter gene and PTPN3 as indicated and treated with TGF-β (2 ng/mL, 8 h). Relative luciferase activity was measured as described in the text. Data are shown as mean ± SEM; n = 3. Statistical analysis was performed using two-tailed Student's t-test. ***P < 0.001.

B, Knockdown of PTPN3 decreases TGF-β-induced CAGA-luc reporter gene activity. HaCaT cells were transfected with siRNA against PTPN3 or control siRNA. 24 h post-transfection, expression plasmids for CAGA-luc reporter and Renilla-luc reporter were transfected as indicated. Cells were harvested for relative luciferase assay after another 24 h with TGF-β treatment (2 ng/mL, 8 h). Data are shown as mean ± SEM; n = 3. Statistical analysis was performed using two-tailed Student's t-test. ***P < 0.001.

C, Knockdown of PTPN3 blocks TGF-β-induced p21 transcription. HaCaT cells were transfected with two independent siRNAs against PTPN3 and treated with TGF-β (2 ng/mL) for 8 h before harvested. Total mRNA was analyzed by qRT-PCR using primers specific to p21. Data are shown as mean ± SEM; n = 3. Statistical analysis was performed using two-tailed Student's t-test. **P<0.01, ***P < 0.001.

D, Knockdown of PTPN3 blocks TGF-β-induced PAI-1 gene transcription. Experiment was done as in (C) with qRT-PCR primers specific to PAI-1. Data are shown as mean ± SEM; n = 3. Statistical analysis was performed using two-tailed Student's t-test. ***P < 0.001.

E, Knockdown of PTPN3 abolishes TGF-β-induced p21 transcription. HaCaT cells were transfected with siRNA and then treated with TGF-β (2 ng/mL) for 0, 4, 8 or 24 h as indicated. qRT-PCR was carried out as in (C). Data are shown as mean ± SEM; n = 3.

F, Knockdown of PTPN3 abolishes TGF-β-induced transcription of PAI-1. Experiment was done as in (E). Data are shown as mean ± SEM; n = 3.

G, Depletion of PTPN3 abolishes TGF-β-induced target protein expression. HaCaT cells with stable knockdown of PTPN3 were treated with TGF-β (2 ng/mL) for 6 h before harvested. Cells were harvested for Western blotting with anti-p21, anti-PAI-1, anti-PTPN3 and anti-β-Actin antibodies.

H, PTPN3 enhances TGF-β-induced target protein expression. HaCaT cells stably expressing HA-PTPN3 were stimulated with 2 ng/mL of TGF-β for 6 h and then harvested for cell lysates. Protein levels were examined by Western blotting with appropriate antibodies as indicated.

I, Depletion of PTPN3 attenuates TGF-β global gene responses. Venn diagram shows the number of differential expression genes (DEGs) with TGF-β treatment in control or PTPN3-depleted HaCaT cells by RNA-Seq analysis. The overlap represents TGF-β target genes that are commonly regulated in both cell lines. HaCaT cells transfected with siControl or siPTPN3 were harvested for RNA-Seq after treated with or without TGF-β (2 ng/mL, 8 h).

J, Loss of PTPN3 markedly decreases TGF-β-dependent upregulation or downregulation of target genes. Heat map of DEGs in control HaCaT cells (siControl) or HaCaT cells depleted of PTPN3 (siPTPN3). Log2FC data represent fold change of TGF-β (2 ng/mL) treatment over vehicle for 8 h.

K, Heat map of Log2FC of select TGF-β target genes.