Fig. 2: CREB3 promotes SIRT2 transcription under Golgi stress.

A RT-PCR analysis for Sirt2 mRNA in A549 CREB3 KD cells treated with or without BFA. mRNA is normalized to DMSO treated control (shLuc) cells. Statistical evaluation was done by two-way ANOVA. B Immunoblots for SIRT2 protein levels in A549 control (shLuc) and CREB3 KD cells treated with or without BFA. HSP90 blot was used as the loading control. The quantification of SIRT2 protein level, normalized to HSP90, is shown on the right. Statistical evaluation was done by two-way ANOVA. C Immunoblots for SIRT2 in control and CREB3 KD cells treated with BFA and rescued with CREB3 transfection. SIRT2 level relative to actin loading indicated below. D A schematic representation of SIRT2 promoter. The annotated regions are on Chromatin 19 complement [39,389,400-39,391,600], reference: GRCh37. E SIRT2-promoter driven firefly transcription in cells under Flag-tagged CREB3 N-terminal bZIP domain (CREB3-N) overexpression. The Renilla luciferase construct was used as an internal control. F Firefly luciferase transcription in cells treated with BFA. Cells were transfected with an empty pGL3-basic vector (ctrl) or pGL3-basic vector with 994 base pairs of SIRT2 5' UTR (Sirt2). G CREB3 chromatin IP followed by qPCR of the SIRT2 promoter using primers surrounding the putative CREB3 binding site. Results are from three separate experiments with three technical replicates each. Data are represented as mean ± SEM. Statistical evaluation was done using unpaired two-tail Student’s t-test. ***p < 0.001.