Fig. 2.

CCN5 regulation by leptin in BC cells. a-b ~ 60–70% confluent MCF-7 cells were serum deprived for 24 h and then cells were treated with different doses of leptin or different times with a fixed dose of leptin (3.125 nM). Total RNAs from treated and untreated cells were extracted and were subjected to qRT-PCR. Values on the barograph represent CCN5 expression changes in treated and untreated groups. The data represents mean ± SEM of three independent experiments. c Serum deprived MCF-7 cells were grown in serum-deprived DMEM for 48 h in the presence or absence of Leptin (3.125 nM), and total RNAs from treated and untreated cells were extracted and were subjected to Northern blot analysis for CCN5 and GAPDH (loading control). Values on the barograph represent CCN5 expression changes in treated and untreated groups. The data represents mean ± SEM of three independent experiments. d Serum deprived MCF-7 and ZR-75-1 cells were treated with leptin for 48 h as indicated above, and total RNA extracts were subjected to qRT-PCR analysis for CCN5. Values on the bargraph represent CCN5 expression changes in treated and untreated groups. The data represents mean ± SEM of three independent experiments. e MCF-7 and ZR-75-1 cells treated with leptin (3.125 nM) for 48 h, and whole cell extracts were subjected to immunoblot analysis for CCN5 and β-actin (loading control). Values on the bargraph represent CCN5 expression changes in treated and untreated groups. The data represents mean ± SEM of three independent experiments. f MCF-7 cells were transiently transfected with CCN5/WISP-2 promoter. After 48 h, transfected cells were grown in treated with 3.125 nM leptin for 48 h or left untreated, and CAT assay was performed per the protocols indicated in Materials and Methods section. The results reflect the mean ± SEM of 3 independent experiments