Fig. 2.

Knockdown of WNK4 impairs lipid accumulation and expression of adipogenic genes in 3T3-L1 cells. (a) Oil-Red O staining of 3T3-L1 cells expressing si-WNK4 showed decreased lipid accumulation at day 6 of differentiation. (b) Lipids extracted from Oil-Red O stained cells were quantified (n = 6 per group). Cells expressing si-WNK4 showed impaired lipid accumulation. (c) Real-time PCR analysis of 3T3-L1 adipocytes expressing si-Nega or si-WNK4 at day 4 of differentiation. The mRNA levels of PPARγ, C/EBPα, and PPARγ targeting genes were reduced in si-WNK4-transfected 3T3-L1 cells (n = 6 per group). (d) Real-time PCR analysis of 3T3-L1 adipocytes expressing si-Nega or si-WNK4 within 8 h of differentiation (n = 5 per group). The level of C/EBPβ mRNA in the early phase of differentiation was not affected by si-WNK4. (e) WNK4 knockdown in 3T3-L1 cells prevented PPARγ and C/EBPα protein expression at day 2 of differentiation (n = 6 per group). Whole course of C/EBPβ, PPARγ, and C/EBPα proteins during adipogenesis under si-WNK4 are shown in Fig. S3.