Figure 4: Altered HO-1 expression levels by recombinant expression or RNAi also influence Stat3 activation in keratinocytes.

(a) Altered HO-1 expression by RNAi increased Stat3 phosphorylation and downstream genes expression in HaCaT cells. (b) Altered HO-1 expression by RNAi increased Stat3 phosphorylation and downstream genes expression in A431 cells. In the RNAi experiments, HaCaT or A431 cells were cultured to 80% confluency in six-well plates. These cells were transfected with 100 nM specific HO-1 siRNA or control siRNA and treated with 25 μM Hemin for 24 h before exposure to 25 ng/mL IL-6 for 30 min. Stat3 phosphorylation levels and downstream proteins were determined by Western blot. (c) Enhanced HO-1 expression using the transfected recombinant plasmids decreased Stat3 activation in the presence or absence of IL-6 stimulus. (d) Enhanced HO-1 expression decreased the expression of Stat3 downstream proteins in the presence or absence of IL-6 stimulus. In gene overexpression experiments, HaCaT cells were transfected with GV230-HO-1 recombinant plasmid for 24 h before exposure to 25 ng/mL IL-6 for 30 min or 24 h, and the Stat3 phosphorylation levels and the expression of Stat3 downstream genes were determined by Western blot. Blots shown are derived from multiple gels. The gels were run under the same experimental conditions. The membrane was cut based on molecular weight and probed with antibody of interest. Band of interest is indicated with an arrow. All full-length blots are presented in Supplementary Fig. 2.