Supplementary Figure 7: Oncogenic PI3K/AKT signaling regulates JNK signaling through SH3RF1.
From: Oncogenic activation of PI3K induces progenitor cell differentiation to suppress epidermal growth
(a), mRNA expression of Akt1-3 in keratinocytes transduced with control shScram or gene-targeting shRNAs. Two shRNAs for each gene that induced strongest transcript depletion (marked with red arrow) were used for functional studies in vivo. Experiment was repeated 3 times independently with similar results. Error bar: SD, center value: mean. (b-e), Depletion of Akt1-3 shows that Akt1 and Akt2 are required for epidermal differentiation and cell division phenotypes. EdU-BrdU pulse-chase assay shows that Akt1 and Akt2 are required for differentiation in Pik3ca 2 × (b) and WT (c) skin epithelium. Cell division rate is equally dependent on Akt1 and Akt2 in Pik3ca 2 × (d) and WT (e) epidermis. Statistics based on n = 3 animals of each condition. Two-tailed t test, P-value as indicated. Error bar: SD, center value: mean. (f), Sh3rf1 mRNA expression in keratinocytes transduced with gene-targeting shRNAs relative to control shScram. Two most efficient shRNAs (red arrows) were used in subsequent gene-depletion studies. Experiment was repeated 3 times independently with similar results. Error bar: SD, center value: mean. (g), SH3RF1 mutants do not affect cell division rate in both wild type and Pik3ca 2X epidermis. Statistics based on n = 3 animals of each condition. Two-tailed t test, P-value as indicated. Error bar: SD, center value: mean. (h), Immunofluorescence staining of phosphorylated JNK (red) in clonally activated Pik3ca 2X epidermis (green). Transduction with LV-Cre reduces p-JNK levels (top rows) that are rescued by overexpression of Sh3rf1 (bottom rows). Inset boundaries are marked by white rectangle in larger view images. Dotted line marks epidermal/dermal boundary. Dashed line marks the interface between Cre-transduced cells. Scale bar, 25 μm. Experiment was repeated 3 times independently with similar results. (i), MLK1 does not affect cell division rate in Pik3ca 2X epidermis. (j), Knockdown of Jnk1 does not affect cell division rate in Pik3ca 2X epidermis expressing SH3RF1SA. Statistics based on n = 3 animals of each condition. Two-tailed t test, P-value as indicated. Error bar: SD, center value: mean.
