Fig 2: EerI administration promoted-ERAD and stimulated-UPR pathway in fibrotic kidney. (a) Representative western blotting images of GRP78, PDI, ATF4 and Derlin-1 expressions. The fold expression of GRP78 (b), PDI (c), ATF4(d), Derlin 1 (e). Data are presented as the mean ± SD (n = 6).

Fig 3: Gestational regulation of ER stress signaling in the pregnant mouse uterus from E6–E19.A) Analysis of protein expression of DDIT3 and XBP1(S) in the nuclear fraction of a gestational series of mouse uteri from E6–19. NCOA3 was used as a loading control. Graphs of ROD of B) DDIT3 and C) XBP1(S) normalized to NCOA3. D) Representative western blots of cleaved (CL) and full length (FL) CASP12 in the cytoplasmic fraction across gestation. PDI was used as a loading control. ROD of E) CL CASP12 and F) FL CASP12 normalized to PDI. Data are represented as Mean ROD ± SE. N = 3 for each time point. Data labeled with different letters are significantly different from each other (P<0.05).

Fig 4: Effect of goniothalamin-MFE2 interaction. Knockdown MFE2 expression using siMFE2 transfection into HeLa cell line resulting in decreased MFE2 expression and CHOP induction after three days, mock-transfection into HeLa cell line was used as control (A). Goniothalamin treatment in other cell line showed similar results in CHOP induction (B), but no effect on MFE2 expression (C). The ER stress sensor transmembrane proteins activation by goniothalamin was observed via detection of XBP1 splicing, for IRE1a activation (D), and detection of phospho-eIF2a, for PERK activation (E). The mediators of protein folding process, Ero1-La and PDI were observed, but not altered in the expression. These results suggest goniothalamin - MFE2 interaction may cause indirect ER stress induction. The results of immunoblotting analysis (B,C,E) and reverse transcription-PCR (D) use 0.5% DMSO treatment as control.

Fig 5: Intracellular Localization of SEC61A1 in Affected Kidney(A and D) In affected kidney, SEC61A1 is present in coarsely granular structures.(B, C, E, and F) Co-staining of SEC61A1 with Protein disulphide isomerase (PDI) (B), a marker of endoplasmic reticulum (ER), and with 58k Golgi-protein (E) demonstrate localization of the SEC61A1 in the ER (C) and in the Golgi (F).(G and J) In control kidney, SEC6A1A is present in finely granular structures.(H, I, K, and L) Co-staining with (H) PDI and (K) GM130 demonstrating localization of SEC61A1 in ER (I) but not in the Golgi (L). The degree of SEC6A1A colocalization with selected markers is demonstrated by the fluorescent signal overlap coefficient values that ranging from 0 to 1. The resulting overlap coefficient values are presented as the pseudo color which scale is shown in corresponding lookup table.