Fig. 1: Golgi stress transcriptionally upregulates SIRT2.
From: Golgi stress induces SIRT2 to counteract Shigella infection via defatty-acylation
A Immunoblots for SIRT2 in different cell lines treated with 5 μg/ml BFA for 24 h. HSP90 blots are shown as loading controls. Representative images from three independent experiments are shown. B Quantification of relative SIRT2 protein levels in different cell lines, normalized to HSP90. Statistical evaluation was done by a paired two-tailed Student’s t-test. n = 3 or 4 biological replicates. C In A549 cells, BFA treatment significantly increased SIRT2 protein level, while the ER stress inducers Tunicamycin (Tm) and Thapsigargin (Tg) only weakly induced SIRT2 expression. D RT-PCR analysis for SIRT2 and CREB3 mRNA levels in A549 cells treated with 5 μg/ml BFA for various incubation times. mRNA was normalized to 0 h. Statistical evaluation was done by an unpaired two-tailed Student’s t-test. n = 3 biological replicates. E ActD chase analysis showing SIRT2 upregulation is due to mRNA upregulation, which happens primarily during 12–24 h. A549 cells were treated with or without BFA at t = 0 h. During BFA treatment, ActD was added at t = 0, 12, 18, 23, 24 h. Cells were collected at t = 24 h and submit for western blot analysis. F CHX chase analysis showing SIRT2 upregulation is not due to protein stability. A549 cells were treated with or without BFA at t = 0 h. During BFA treatment, CHX was added at t = 0, 12, 24 h. At t = 24 h, cells were collected and submit for western blot analysis. G RT-PCR analysis for SIRT1-7 and HDAC11 mRNA level in A549 cells treated with 5 μg/ml BFA for 24 h. Fold change in mRNA was calculated by comparing samples with BFA treatment to control DMSO. n = 3 biological replicates. H Among all sirtuins and HDAC11 tested, SIRT2 protein level increase was the most drastic under Golgi stress. A549 cells were treated with BFA the indicated concentrations for 24 h before analyzed by western blot. α−tubulin blots were loading control. Data are represented as mean ± SEM. Statistical evaluation was done using unpaired two-tail Student’s t-test. ***p < 0.001.
