Fig. 4: s-HBEGF stabilizes SIRT1 in keratinocytes by increasing its phosphorylation at Ser 27 via JNK2.
a HaCaT cells or human primary keratinocytes were treated with or without sorafenib, CdMCTRL or CdMSORA. The expression level of SIRT1 was detected by western blot. b HaCaT cells or human primary keratinocytes were treated with s-HBEGF for 24 h. The expression level of SIRT1 was analyzed by western blot. c Representative western blot indicated the expression of SIRT1 in the stratum corneum of mice in control or sorafenib-treated group (n = 5/group). d Representative western blot indicated the expression of SIRT1 in the stratum corneum of mice in control or s-HBEGF-treated group (n = 5/group). e Representative immunohistochemistry images showing SIRT1-stained paws of mice in control, sorafenib, HBEGF neutralizing antibody or combination group (left panel). Representative immunohistochemistry images showing SIRT1-stained paws of mice with or without s-HBEGF treatment (right panel). Scale bar, 50 µm. f HaCaT cells were transfected with non-targeting siRNA or siRNA targeting JNK2, followed by treatment with or without s-HBEGF (2.5 ng/mL) for 24 h. The expression levels of p-JNK1/2, JNK2 and SIRT1 were determined by western blot. g HaCaT cells were treated with s-HBEGF for 24 h. The transcription level of SIRT1 was measured by RT-qPCR (n = 3). h HaCaT cells were treated with CHX with or without s-HBEGF at different time points, and SIRT1 protein level was measured by western blot. i HaCaT cells were treated with s-HBEGF for 24 h. The expression levels of p-SIRT1 and SIRT1 were analyzed by western blot. j Human primary keratinocytes were treated with s-HBEGF for 24 h. The expression levels of p-SIRT1 and SIRT1 were analyzed by western blot. k HaCaT cells were treated with s-HBEGF for 24 h. Cell lysates were immunoprecipitated with anti-SIRT1 antibody and probed with anti-Ub antibody or with anti-SIRT1 antibody. Protein expression levels of endogenous p-SIRT1 and SIRT1 are displayed. l HaCaT cells were transfected with non-targeting siRNA or siRNA targeting JNK2, followed by treatment with or without s-HBEGF (2.5 ng/mL) for 24 h. Cell lysates were immunoprecipitated with anti-SIRT1 antibody and probed with anti-Ub antibody or with anti-SIRT1 antibody. Protein expression levels of endogenous p-SIRT1, SIRT1, p-JNK1/2, JNK2 are displayed. Densitometric values are shown as optical density after ACTB or GAPDH normalization using Image J. The results in (g) are presented as the mean ± SD. Statistical analyses were performed using one-way ANOVA with LSD post hoc test in (g). n.s. no significance. SORA sorafenib, CHX cycloheximide, CdM HUVECs conditional medium, CTRL control, IP immunoprecipitant, WCL whole cell lysate.
