SB202190-induced transcriptional changes are not necessary for vacuole formation in HT29 cells.

(A) HT29 cells were treated in the presence of DMSO or SB202190 (5 µM) for 24 hours and the levels of BNIP3L and GABARAP mRNAs were analyzed by RT- PCR. The relative mRNA levels were normalized to actin mRNA and plotted (* denotes p-value<0.04). (B) HT29 cells stably transfected with FHRE-luc reporter were treated with 10 µM SB202190 for indicated time points and luciferase activity measured and normalized to protein content. (C) Cells seeded in 12 well plates were treated with Act-D (5 µg/mL), SB202190 (5 µM) or both together and phase contrast images were taken at indicated time points (original magnification 600×). (D) Percentage of vacuolated cells was calculated using Image J software. Percentage vacuolated-cells plotted is the average of three measurements from three independent images. (E) HT29 cells treated as indicated were analyzed by RT- PCR for the expression of GABARAP, BNIP3L and LC3.