Fig. 2

ERS is aggravated by dual celecoxib plus orlistat drug therapy in mucinous colon cancer and correlates with MUC2 expression levels. a Western blot assay of ERS protein GRP78 (BiP) following treatment of LS174T cells with celecoxib (0–100 μM) or Orlistat (0–100 μM) for 24 and 48 h. qPCR assay of mRNA expression for ERS markers following treatment with celecoxib, orlistat, or combination for 24 h in LS174T cells b and MCC explant tissue c; representative pictures of IF assay with bar graph demonstrating mean intensity difference. d Western blot assay of ERS markers comparing high MUC2 expressing cells (dnTCF4-LS174T cells exposed to doxycycline for 48 h) versus low MUC2 expressing cells (dnTCF4-LS174T cells without doxycycline exposure) and comparing stable MUC2 KD in LS174T cells compared to LV control cells following treatment with celecoxib, orlistat, or combination for 24 h. e Ratio of ER:cytoplasmic calcium concentration at 30 s in LS174T cells following combination treatment (celecoxib + orlistat). f ELISA assay of MUC2 secretion from COS-7 cells transfected with pSNMUC2-MG vector expressing MUC2 N-terminal following treatment with celecoxib, orlistat, or combination for 24 h. g FASN enzymatic activity assay in LS174T cells following combination treatment (celecoxib + orlistat) for 24 h; and h western blot analysis of FASN expression levels. i N-terminal MUC2-palmitoylation in COS-7 cells stably expressing MUC2 N-terminal following single or dual drug therapy for 24 h was determined by ABE assay and quantified by western blot assay; hydroxylamine (HAM), a strong reducing agent that cleaves palmitate from cysteine residues, is necessary for biotinylation, the omission of HAM cleavage (HAM -) serves as negative control. Error bars represents standard deviation (S.D.) from triplicate experiments (* p < 0.05, ** p < 0.01, *** p < 0.001)