Fig. 3: TUSC3 loss induces ER stress via PERK-eIF2α pathway, leading to impaired protein translation and cell death.

a Representative western blot of ER stress proteins in the hippocampus of 4-month-old female WT and TUSC3 KO mice. b–i Quantification of GRP78 (b), p-PERK (T982) (c), p-eIF2α (S51) (d), CHOP (e), XBP1s (f), p-AKT (S473) (g), p-CREB (S133) (h), and ATF6 (i) normalized to β-actin (n = 3 mice per group). j Representative western blot analysis of ER stress proteins in the striatum of 4-month-old female WT and TUSC3 KO mice (n = 3 per group). k–r Quantification of GRP78 (k), p-PERK (T982) (l), p-eIF2α (S51) (m), CHOP (n), XBP1s (o), p-AKT (S473) (p), p-CREB (S133) (q), and ATF6 (r) normalized to β-actin (n = 3 mice per group). s Non-ID and ID1/ID2 fibroblasts were chronically treated with MgT (400 μM) for 48 h and analyzed by western blotting (left). Quantification of p-eIF2α (S51) levels normalized to eIF2α (right) (n = 3 independent biological replicates). t Non-ID and ID-derived fibroblasts were pretreated with or without MgT (400 μM) for 24 h, followed by puromycin (5 μg/ml) treatment for 2 h. Representative western blot using an anti-puromycin antibody (left), and puromycin incorporation was quantified and normalized to β-actin (right) (n = 3 independent biological replicates). u SH-SY5Y shControl and shTUSC3#3-1 cells were pretreated with MgT (400 μM) for 12 h, then treated with either DMSO, thapsigargin (2 μM), tunicamycin (2 μg/ml), A23187 (2 μM), or etoposide (25 μM) for 24 h. Cells were stained with propidium iodide and Calcein-AM, and double-positive cells were quantified and normalized to Calcein-AM-positive cells. Data represent three independent biological experiments (n = 3). The exact number of cells analyzed per condition is provided in the Source Data file. v Representative western blot of primary cortical neurons (DIV7) treated with vehicle or 4-PBA (10 mM) for 24 h (left). Quantification of indicated protein levels normalized to TUBA (right) (n = 3 independent biological replicates). Two-tailed unpaired t-test (b–i, and k–r); one-way ANOVA followed by Tukey’s post hoc multiple comparison test (s, t, v); two-way ANOVA followed by Tukey’s multiple comparison test (u). Data are presented as mean ± S.E.M. or S.D. (t, u). Source data are provided as a Source data file.