Fig 2: Phosphatidylcholine depletion in IECs leads to ER stress and UPR activation. (A) Representative electron micrographs of the ER in colonic epithelial cells of control mice and CTαIKO mice. Arrow indicates ER. (B) The mRNA abundance of sXBP1, Ddit3, and Hspa5 in the colons of control mice and CTαIKO mice (n = 5/group). (C) Representative Western blots and (D) quantification of spliced XBP1, PERK, and ATF6 relative to loading controls in the colons of control mice and CTαIKO mice (n = 4–8/group). (E) The mRNA abundance of Atf4, Atf5, and Eif4ebp1 in the colons of control mice and CTαIKO mice (n = 5/group). (F) Representative Western blot and (G) quantification of P62 relative to tubulin in the colons of control mice and CTαIKO mice (n = 8/group). (H) Representative electron micrographs of autophagic vesicles in colonic epithelial cells of control mice and CTαIKO mice. Arrow indicates autophagosome. (I) Pathology scores for control mice and CTαIKO mice treated with and without PBA (n = 3–5/group). (J) Goblet cell depletion scores for control mice and CTαIKO mice treated with and without PBA (n = 3–5/group). (K) The mRNA abundance of sXBP1, Ddit3, and Hspa5 in the colons of control mice and CTαIKO mice treated with and without PBA (n = 3–5/group). (L) Colon weight of control mice and CTαIKO mice treated with and without PBA (n = 3–5/group). Values are means ± SEM. (I–L) Columns that do not share a letter (a or b) are significantly different (α = .05). ∗P < .05, ∗∗∗P < .001, and ∗∗∗∗P < .0001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; sXBP1, spliced XBP1.

Fig 3: SELENOK gene knockout induced increased ROS and ERS both in vitro and in vivo.(A) ROS levels were measured by flow cytometry after labeling with ROS assay kit (*p < 0.01 n = 3). (B) Intracellular Ca2+ levels were measured by flow cytometry after labeling with Fluo-3/AM. (**p < 0.01 n = 3). (C, D) Representative immunoblot analysis of BIP, ATF6, and CHOP in N2a cells and SELENOK-/- N2a cells (*p < 0.05, **p < 0.01, ***p < 0.001 n = 3).(E, F G, H) Representative immunoblot analysis of BIP, ATF6, and CHOP in cortical (E, F) and hippocampal (G, H) brain homogenates (*p < 0.05, **p < 0.01, ***p < 0.001 n = 7). The quantitative results were normalized to β-actin, GAPDH or α-tubulin expression.

Fig 4: The cell death involves an integrated stress response in the ER. a Kinetics of ER stress induction by 2DG, assessed by western blotting in NALM-6 cells treated with 1 mM 2DG for up to 6 h, in the presence or absence of mannose. Positive controls were ER stress inducers brefeldine A (BFA, 2.5μg/mL), tunicamycin (Tun, 1 μg/mL), and thapsigargin (Thps, 1 μM), analyzed at 6 h. Major markers of ER stress evaluated included p-eIF2α (ser51), ATF4, CHOP, the spliced form of XBP1 (XBP1s), ATF6, and BIP (GRP78). Actin served as a loading control. b RT-PCR data confirming that NALM-6 cells treated with 1 or 10 mM 2DG for 6 h exhibit unspliced XBP1 mRNA (XBP1u, 149 bp) but not the spliced mRNA (XBP1s, 109 bp). The spliced product is evident after treatment with thapsigargin and (faintly) BFA. HeLa cells yielded XBP1s with 10 mM 2DG and the other stimuli

Fig 5: Role of oxidative stress in PA-induced endoplasmic reticulum stress and apoptosis in podocytes. (A and C) Representative immunofluorescence images of intracellular ROS in podocytes (magnification, ×200) following treatment with different concentrations of PA, or PA (150 µmol/l) for 24 h after pretreatment with or without NAC (150 µmol/l) for 2 h. (B) Mean fluorescence intensities of ROS shown in (A). Data are presented as the mean ± SEM (n=3). *P<0.05 vs. PA (0 µmol/l) group, #P<0.05 vs. PA (50 µmol/l) group, ΔP<0.05 vs. PA (150 µmol/l) group. (D) Mean fluorescence intensities of ROS shown in (C). Data are presented as the mean ± SEM (n=3). *P<0.05 vs. control group, #P<0.05 vs. PA group, ΔP<0.05 vs. NAC group. (E and M) Representative images of BIP, PERK, ATF4, CHOP, ATF6, IRE1α, caspase 12 and cleaved-caspase 3 expression, as detected by western blotting after treatment with PA (150 µmol/l) or NAC (150 µmol/l). β-actin was used as a loading control. Densitometric analysis of the data shown in (E and M) for (F) BIP, (G) PERK, (H) ATF4, (I) CHOP, (J) ATF6, (K) IRE1α, (L) caspase 12 and (N) cleaved-caspase 3. Data are presented as the mean ± SEM (n=3). *P<0.05 vs. control group, #P<0.05 vs. PA group, ΔP<0.05 vs. NAC group. (O) Representative plots of podocyte apoptosis evaluated by flow cytometry after treatment with PA (150 µmol/l) and NAC (150 µmol/l) for 24 h. (P) Quantification of podocyte apoptosis shown in (O). Data are presented as the mean ± SEM (n=3). *P<0.05 vs. control group, #P<0.05 vs. PA group, ΔP<0.05 vs. NAC group. PA, palmitic acid; ROS, reactive oxygen species; NAC, N-acetylcysteine; BIP, binding immunoglobulin protein; PERK, protein kinase RNA-like endoplasmic reticulum kinase; ATF, activating transcription factor; CHOP, C/EBP homologous protein; IRE1α, inositol-requiring enzyme 1α; SEM, standard error of the mean.