Journal: bioRxiv

Article Title: The ER Thioredoxin-Related Transmembrane Protein TMX2 Controls Redox-Mediated Tethering of ER-Mitochondria Contacts (ERMCS)

doi: 10.1101/2024.04.12.589228

Figure Lengend Snippet: (A) Glycosylation of endogenous TMX2. U251 MG cell lysates were treated with Endo H and PNGase F for 60 min at 37°C. Samples were then analyzed with Ero1ɑ and TMX2 antibodies. (B) Glycosylation scanning analysis. HeLa cells were transfected with the indicated artificial glycosylation mutants. Then, the glycosidase digestion assay was performed and the samples were analyzed with the Ero1ɑ and Flag antibodies. (C) 3D reconstruction of transfected Flag-tagged TMX2. HeLa cells were transfected with Flag-TMX2-wild-type for 24 h. Then immunofluorescence microscopy was performed, and TMX2 localization was observed after three-dimensional reconstruction (white arrow). Scale bar, 20 µm. (D) Percoll membrane fractionation of TMX2 in HeLa cells. Protein components of subcellular fractions prepared were analyzed by immunoblotting. Equal amounts were loaded and analyzed for the indicated ERMCS-regulatory proteins (H: homogenate; ER: endoplasmic reticulum; MAM: mitochondria-associated membrane; Mc: crude mitochondria; Mp: purified mitochondria; C: cytosol). (E) Redox dependence of TMX2 localization. After transfecting with Flag-TMX2-WT, HeLa cells were treated with 200 µM H 2 O 2 for 20 min or 5 mM NAC for 1h. Mander’s coefficient analysis was utilized to measure the co-localization between TMX2 and MitoTracker TM Red or Erp57 [n = 23-36 of 2 technical replicates for each group, statistical analysis was performed with two-way ANOVA (Sidak’s multiple comparisons test)]. Scale bar, 20 µm. (F, G) TMX2 expression levels after knockout and knock-down. TMX2-KO cells were generated with the CRISPR/Cas9 method using U251 MG cells. For TMX2 knock-down, U251 MG cells were transfected with RNAi (siTMX2) using Oligofectamine. Experiments were performed 48 h after transfection. The lysates were probed with a TMX2 antibody, and γ-tubulin served as a loading control. (H) ERMCS analysis in U251 MG TMX2 control and knock-down cells. Transmission electronic microscopy was performed with TMX2-KD cells. Mitochondria were labeled in red, and ER was labeled in green. Mitochondria circumference, average ER-mitochondria distance, and total ERMCS length were measured and analyzed [n = 69-166 of 3 technical replicates for each group, *p < 0.05, **p < 0.01, and ***p < 0.001 by Kruskal-Wallis test (Dunn’s multiple comparisons test)]. The ERMCS were classified and quantified by the mean distance. Scale bar, 0.5 µm.

Article Snippet: On the experiment day (24 or 48 h after transfection), cells were rinsed 3 times with PBS++ and fixed with 2% paraformaldehyde (PFA; 15710, Electron Microscopy Sciences) in PBS++ for 15 min at R.T. Then the cells were rinsed 3 times with PBS++ to quickly remove most of the fixative and quenched with 50 mM NH 4 Cl for 10 min. Next, cells were washed with PBS++, permeabilized with 0.1% Triton X-100 in PBS++ for 10 min, and rinsed with PBS++ before blocking with 2% BSA in PBS++ for 1 h. Cells were then incubated with primary antibodies [rabbit anti-TMX2 (1:100, 19838-1-AP, Proteintech); mouse anti-TOM70 (1:150, H00009868-B01P, Abnova); mouse anti-Erp57 (1:100, SMC-168B, StressMarq); mouse anti-Flag (1:200, 200-301-B13, Rockland); rabbit anti-Flag (1:400, 14793, Cell Signaling); mouse anti-HA (1:200, 901513, BioLegend)] for 1-2 h at R.T. in a humidified, light-protected chamber.

Techniques: Transfection, Immunofluorescence, Microscopy, Membrane, Fractionation, Western Blot, Purification, Expressing, Knock-Out, Knockdown, Generated, CRISPR, Control, Transmission Assay, Labeling