Fig 2: Western blots of secreted (a) and intracellular (b) protein fractions of Expi293F cells expressing WT and B.1.351 RBDmfc together with increasing amounts of PDIA2. Secreted and intracellular protein fractions described in Figure 3 were analysed by Western blot using antibodies directed against the recombinant RBDmfc proteins (anti-RBD, anti-Fc), against chaperones (anti-PDIA2, anti-HSPA5, anti-HSP90B1), and against ßactin as loading control. M, protein ladder (apparent MW in kDa indicated on the left); *, endogenous PDIA2 protein (anti-PDIA2 blot); arrow, RBDmfc proteins (anti-RBD and anti-Fc blots in panel (b)) likely lacking disulphide bonds and mature glycosylations, and thus running in SDS-PAGE with an apparent MW of 50 kDa (compare to 59 kDa reduced, mature proteins shown in Figure 2, lanes 3 and 4). Quantification of signal intensity of chaperones is shown in Figure S2. Data are from one representative experiment (out of three independent experiments). Uncropped blot images are shown in Appendix A Figure A3 and Figure A4.

Fig 3: Role of BiP in nuclear NRF1 and ABCA1 expression under high cholesterol condition. a–e UCB-MSCs were pretreated BiP inhibitor, Ver155008 (10 µM) prior to cholesterol treatment (200 µM, 24 h). a Immunocytochemistry was conducted with NRF1 (green), BiP (red) specific antibodies and DAPI (blue), and then visualized by SRRF imaging system. Scale bar was set as 8 µm (magnification × 1000). The co-localization of BiP and NRF1, nuclear translocation of NRF1 were analyzed with Fiji software. (n = 7). b The expression level of ABCA1 was quantified with western blotting (n = 5). c The concentration of the cellular cholesterol was measured with cholesterol quantification kit (n = 5). d Cells were incubated with CM-H2DCFDA and relative fluorescence units (RFU) were measured by luminometer (n = 5). e Cell viability was measured by trypan blue exclusion assay (n = 5). All blot and images are representative and quantitative data are presented as a mean ± standard error of the mean. *p < 0.05 vs control, #p < 0.05 vs cholesterol

Fig 4: Involvement of melatonin-activated Sp1/miR-597 pathway in BiP suppression. a UCB-MSCs were treated with melatonin (1 µM, 24 h) and total RNAs were extracted. MicroRNA microarray was conducted and the expression levels of microRNAs were analyzed using hierarchical clustering with heatmap. b A cluster of expression-increased microRNAs was selected and their expression levels under high cholesterol and melatonin condition were analyzed by qPCR (n = 5, *p < 0.05 vs control). c, d miR-597 mimic was transfected to UCB-MSCs for 24 h prior to cholesterol treatment (200 µM, 12 h). The mRNA expressions of HSPA5 and ABCA1 and protein expression levels of BiP and ABCA1 were quantified with qPCR and western blotting (n = 5, *p < 0.05 vs NT mimic, #p < 0.05 vs NT mimic + cholesterol). e, f MT2 inhibitor 4-P-PDOT (10 µM) was pretreated prior to melatonin treatment (1 µM, 12 h). e The phosphorylation of Sp1 (Thr453) were measured by western blotting (n = 5, *p < 0.05 vs control, #p < 0.05 vs melatonin). f Immunocytochemistry was conducted with Sp1 (green) specific antibody and DAPI (blue). Scale bar was set as 8 µm (magnification × 1000, n = 5, *p < 0.05 vs control, #p < 0.05 vs melatonin). g Sp1 inhibitor Mithramycin A (5 nM) was pretreated prior to melatonin treatment (1 µM, 12 h), and the change of expression level of miR-597-5p was assessed through qPCR (n = 5, *p < 0.05 vs control, #p < 0.05 vs melatonin). All images are representative and quantitative data are presented as a mean ± standard error of the mean