Fig. 4: MBOAT7 deficiency induces ER stress.

a Western blot analysis of MBOAT7 expression in subcellular fractions of THP-1 cells. Phosphorylation of ER stress marker, IRE1A, analyzed by b immunoblot and c immunofluorescence in MDMs transfected with MBOAT7 siRNA or control with and without LPS treatment (n = 3). d Induction of Xbp-1 splicing analyzed by PCR and normalized to GAPDH in MDMs transfected with MBOAT7 siRNA or control with and without LPS treatment (n = 4). Total cellular ROS production with and without LPS stimulation in e knockdown and f overexpression MBOAT7 in MDMs (n = 16 and 8 includes 3 and 2 biological replicates from independent individuals and 4–5 technical replicates, respectively). g Real-time PCR for the relative mRNA expression of UPR branches in MDMs transfected with MBOAT7 siRNA or scramble as control. Effect of inhibition of UPR branches on h TNF-α secretion assessed by ELISA and i sXBP1 mRNA expression. j Real-time PCR of NADPH complex and antioxidant enzymes in MDMs transfected with MBOAT7 siRNA or scramble (n = 3). k Effect of 4µ8C (IREA1 inhibitor) on TNF-α secretion assessed by ELISA in MDMs knocked-down for MBOAT7 and challenged with LPS (n = 4). l Intracelluar Ca2⁺ levels in in MDMs transfected with MBOAT7 siRNA or scramble after stimulation with LPS. m LPS-induced TNF-α secretion from MBOAT7 deficient cells was suppressed by the intracellular Ca2⁺-chelating agent BAPTA-AM and o high extracellular K⁺ (n = 3). n Expression levels of TRPC1, Orail1 and STIM1 in control and MBOAT7 depleted cells (n = 3). MDMs were transfected with scramble, MBOAT7 siRNA, STIM1siRNA, Orail1 siRNA, or a combination of the three siRNAs for 48 h and treated with LPS for 4 h. Shown is p total cellular ROS (n = 9 includes three biological replicates from independent individuals and three technical replicates) and q TNF-α secretion in MDMs. (n = 6 includes three biological replicates from independent individuals and two technical replicates in g–i, l, and q). P-value <0.04 for all comparisons. Statistical differences between groups were assessed by two-tailed Student’s t test and the error bars indicate the mean ± sem; *P < 0.05, ** P < 0.01, ****P < 0.001. Source data are provided as a Source Data file.