Figure 3.
PTPN23 is required for PI3KC2α-mediated production of PI(3,4)P2 and AKT activation. (A) Interaction between exogenous HA-PI3KC2α and Flag-PTPN23 was detected by immunoprecipitation in A375 and SK-MEL-28 cells. (B) Endogenous PI3KC2α and PTPN23 interaction was detected by immunoprecipitation in A375 and SK-MEL-28 cells. (C) Immunoblot showing the phosphorylation of serine, threonine, and tyrosine residues of PI3KC2α after PTPN23 depletion in A375 cells. (D) Immunofluorescence showing the level of PI(3,4)P2 in A375 cells with pcDNA3.1-HA, PI3KC2αWT, PI3KC2αS329A, and PI3KC2αS329D overexpression. Quantification of PI(3,4)P2 signal intensity was obtained from three independent images (per group). Scale bar, 20 μm. One-way ANOVA, Dunnett’s test. (E) Immunofluorescence showing the level of PI(3,4)P2 after PTPN23 depletion in A375 and SK-MEL-28. Quantification of PI(3,4)P2 signal intensity was obtained from three independent images (per group). Scale bar, 20 μm. Two-way ANOVA, Dunnett’s test. (F) Relative cell number of A375 cells after PTPN23 depletion with or without exogenous soluble PI(3,4)P2 treatment, measured by the CCK8 assay after 72 h (n = 3). One-way ANOVA, Bonferroni’s test. (G) Relative cell number of PTPN23-depleted A375 cells overexpressing PI3KC2α WT, S329A, or S329D, measured by the CCK8 assay after 72 h (n = 3). One-way ANOVA, Bonferroni’s test. (H) Colony formation assay of PTPN23-depleted A375 cells overexpressing PI3KC2α WT, S329A, or S329D. Colonies were stained with crystal violet after 14 days. (I) Immunoblot analysis of indicated proteins in A375 cells after endogenous PTPN23 depletion with or without the overexpression of HA-PI3KC2α WT, S329A, and S329D. (J) Immunoblot showing protein expression in A375 cells after PTPN23 depletion with the overexpression of AKT2 WT or S474D mutant. (K) Relative cell number of PTPN23-depleted A375 cells with the overexpression of AKT2 WT and S474D. Cell viability was measured by the CCK8 assay after 72 h (n = 3). One-way ANOVA, Bonferroni’s test. (L) Colony formation assay of PTPN23-depleted A375 cells with the overexpression of AKT2 WT or S474D mutant. Colonies were stained with crystal violet after 14 days. ****P < 0.0001; **P < 0.01; *P < 0.05; P > 0.05; not significant (ns). Source data are available for this figure: SourceData F3. Refer to the image caption for details.

PTPN23 is required for PI3KC2α-mediated production of PI(3,4)P2 and AKT activation. (A) Interaction between exogenous HA-PI3KC2α and Flag-PTPN23 was detected by immunoprecipitation in A375 and SK-MEL-28 cells. (B) Endogenous PI3KC2α and PTPN23 interaction was detected by immunoprecipitation in A375 and SK-MEL-28 cells. (C) Immunoblot showing the phosphorylation of serine, threonine, and tyrosine residues of PI3KC2α after PTPN23 depletion in A375 cells. (D) Immunofluorescence showing the level of PI(3,4)P2 in A375 cells with pcDNA3.1-HA, PI3KC2αWT, PI3KC2αS329A, and PI3KC2αS329D overexpression. Quantification of PI(3,4)P2 signal intensity was obtained from three independent images (per group). Scale bar, 20 μm. One-way ANOVA, Dunnett’s test. (E) Immunofluorescence showing the level of PI(3,4)P2 after PTPN23 depletion in A375 and SK-MEL-28. Quantification of PI(3,4)P2 signal intensity was obtained from three independent images (per group). Scale bar, 20 μm. Two-way ANOVA, Dunnett’s test. (F) Relative cell number of A375 cells after PTPN23 depletion with or without exogenous soluble PI(3,4)P2 treatment, measured by the CCK8 assay after 72 h (n = 3). One-way ANOVA, Bonferroni’s test. (G) Relative cell number of PTPN23-depleted A375 cells overexpressing PI3KC2α WT, S329A, or S329D, measured by the CCK8 assay after 72 h (n = 3). One-way ANOVA, Bonferroni’s test. (H) Colony formation assay of PTPN23-depleted A375 cells overexpressing PI3KC2α WT, S329A, or S329D. Colonies were stained with crystal violet after 14 days. (I) Immunoblot analysis of indicated proteins in A375 cells after endogenous PTPN23 depletion with or without the overexpression of HA-PI3KC2α WT, S329A, and S329D. (J) Immunoblot showing protein expression in A375 cells after PTPN23 depletion with the overexpression of AKT2 WT or S474D mutant. (K) Relative cell number of PTPN23-depleted A375 cells with the overexpression of AKT2 WT and S474D. Cell viability was measured by the CCK8 assay after 72 h (n = 3). One-way ANOVA, Bonferroni’s test. (L) Colony formation assay of PTPN23-depleted A375 cells with the overexpression of AKT2 WT or S474D mutant. Colonies were stained with crystal violet after 14 days. ****P < 0.0001; **P < 0.01; *P < 0.05; P > 0.05; not significant (ns). Source data are available for this figure: SourceData F3.

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