Supplemental Figure 1: Densitometry analyses of western blots presented in figure 1. Protein bands were normalized to the β-actin content obtained from the same blots. The ratios of p- eIF-2α to eIF-2α, p-IRE-1α to IRE-1α, p-PERK to PERK were calculated directly using the results of densitometry. Data were compared by one-way analysis of variance (ANOVA) with Fisher’s test, with n = 8 in each group, and presented as means ±standard error (*, p<0.05; **, p<0.01; ***, p <0.001).
Supplemental Figure 2: Expression of three eIF-2αkinases, double-stranded RNA-activated protein kinase [PKR], general control non-repressed 2 [GCN2] and heme-regulated eIF2alpha kinase [HRI] in TO and OT cells. Expression of HRI is decreased in TO cells (p<0.001). Data were compared by one-way analysis of variance (ANOVA) with Fisher’s test, with n = 8 in each group, and presented as means ±standard error (*, p<0.05; **, p<0.01; ***, p <0.001).
Supplemental Figure 3: Impaired cellular availability of B12 induces ER stress. There is a net reduction of cellular proliferation marked by the reduction of Ki67. (3A) and a reduced level of molecular chaperons including HSP27 and HSP90 (3B). Confocal images of the co-immunostain of HSF1/ATF6a with apoptotic marker cleaved caspase3 and proliferation marker Ki67 (3C). Data were compared by one-way analysis of variance (ANOVA) with Fisher’s test, with n = 8 in each group, and presented as means ±standard error (*, p<0.05; **, p<0.01; ***, p <0.001).
Supplemental Figure 4: Over expression of the sirt1 construct in TO cell reduces the ER stress. (A)Over expression of the sirt1 construct in TO cell led not only to a higher SIRT1 protein level but also a higher HSF1 protein level (p<0.001). These appeared to increase the expression of heat shock proteins, including HSP27, HSP70 and HSP90 (all p<0.05), reduce the ER stress (p-IREα, p-PERK all <0.05) as well as the apoptosis (cleaved caspase3, p<0.05). In TO cell, these may explain the restoration of the cellular proliferation (marked by Ki67, p<0.05). (B) Data were compared by one-way analysis of variance (ANOVA) with Fisher’s test, with n = 8 in each group, and presented as means ±standard error (*, p<0.05; ***, p <0.001).
Supplemental Figure 5: Densitometry analyses of western blots from TO cells transfected either with siRNA against HSF1 (5A and 5B), with a constitutively activated Hsf1 (Hsf1-act), or with a dominant negative Hsf1 (Hsf1-inactive) construct (5C, 5D and 5E). Data were compared by one-way analysis of variance (ANOVA) with Fisher’s test, with n = 8 in each group, and presented as means ±standard error (*, p<0.05; **, p<0.01; ***, p <0.001).
Supplemental Figure 6A and 6B: Densitometry analysis of western blots from TO cells treated with celastrol, an activator of HSF1. Data were compared by one-way analysis of variance (ANOVA) with Fisher’s test, with n = 8 in each group, and presented as means ±standard error (*, p<0.05; **, p<0.01; ***, p <0.001).
Supplemental Figure 7: Thapsigargin[TG] increases the transcription of molecular chaperons of the HSP70 family. TG treatment (1µM for 5 hours) in TO cells increases significantly both the transcription of BIP (p<0.001) and HSP70 (p<0.01); however, it is not as effective in control OT cells. Data were compared by one-way analysis of variance (ANOVA) with Fisher’s test, with n = 8 in each group, and presented as means ±standard error (*, p<0.05; **, p<0.01; ***, p <0.001).
Supplemental Figure 8: Methylmalonic acid (MMA) has no effect on the ER stress of TO and OT cells. (A) Various doses of MMA were added to the culture media of the TO and OT cells for 7 days to determine if MMA can be neurotoxic to these cells. No significant difference were observed in ER stress markers, including pSer724-IRE1α and pThr980-PERK as well as SIRT1 and heat shock protein 70s (all p>0.05). (B) Data were compared by one-way analysis of variance (ANOVA) with Fisher’s test, with n = 8 in each group, and presented as means ±standard error.
Supplemental Figure 9: The proposed model for the influence of impaired cellular availability of B12 on N1E115 neuroblastoma cell proliferation through SIRT-1 dependent activation of ER stress.