Fig 1: ER stress and unfolded protein response by CerS downregulationactivates ATF6α in MDR cells. Cells were transiently transfected with CerS-specific siRNA for 72 h or treated with SERCA inhibitor thapsigargin (TG) (3 μM for 6 h) as positive control. A, analysis of GRP78 by immunoblotting in A498 cells. A′, densitometry analysis of CerS-specific siRNA transfected A498 cells after 72 h (n = 5–6). B, quantitative PCR analysis of ABCB1 and CHOP mRNA in A498 cells (n = 6). Immunoblots and densitometry analyses for full length (FL, 90 kDa) and cleaved (50 kDa) ATF6α in CerS siRNA (C, C′) and CerS6 overexpressing (D) cells after 72 h (representative immunoblots of n = 7–9). Values indicate densitometry analysis with correction to GAPDH. Immunoblots in C for CerS2, CerS6, and GAPDH are reused from Figure 2D because target proteins were analyzed from the same set of samples. E, E′, immunofluorescence staining and quantification of nuclear ATF6α using threshold analysis in CerS siRNA cells after 72 h in fixed A498 cells. A minimum of 500 nuclei per treatment were analyzed from thresholded images using Fiji/ImageJ from two independent experiments. The scale bar represents 10 μm. ATF6α, activating transcription factor 6α; CerS, ceramide synthase; CHOP, C/EBP homologous protein; ER, endoplasmic reticulum; GRP78, glucose-regulated protein 78 kDa; MDR, multidrug resistance; SERCA, sarco-/endoplasmic reticulum calcium ATPase.