Fig 2: TMBIM6 knockout induces senescence, nitro-oxidative stress and apoptosis during the aging process. (A) Animal groups and model of the study. (B) Hierarchical clustering of genes expressed in old WT and TMBIM6-/- mice. (C) Enrichment analysis results of up-and down-regulated differentially expressed genes in old-aged Tmbim6-/- mice compared to old-aged WT mice. (D) Representative immunohistochemistry images indicating the expression of ß-galactosidase of the indicated groups. The graph on the right is the measurement of ß-galactosidase positive staining. Yellow arrows indicate the predominant nuclear expression of ß-galactosidase. Scale bar: 100 µm. (E–F) ROS and RNS generation was assessed by fluorescent microscopy using DHE (10 µM) and DHR-123 (20 µM) staining in the liver sections of the indicated groups and genotypes. The bar graph (below) shows the relative fluorescence intensity to young WT (n = 6 per group). Scale bar: 25 µm. (G–H) Western blots showing the expression of caspase 12 and 7 (G) and immunohistochemistry of the cleaved caspase 3 (H) in the liver of WT and TMBIM6-/- mice of the indicated groups. The relative intensities are shown in graph (right) (n = 6 per group). Y: Young, M: Middle, O: Old age. All the data are represented as mean ± S.E.M. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 indicate significant differences indicated as specified in the bar graph. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig 3: Loss of TMBIM6 enhances impaired ER stress response during aging. (A) Immunoblot analysis of indicated proteins in young and aged liver. The bar graph (right) shows the relative band intensity of the indicated proteins (n = 6 per group). (B) The expression of several UPR elements/ER stress marker proteins in the liver of WT and TMBIM6-/- mice of different age groups. Bar graphs (right) show the band intensity of the indicated proteins to their loading control (n = 4 per group). Y: Young, M: Middle, O: Old age. (C) Representative confocal laser immunofluorescence photomicrography showing expressions of GRP78 (ER marker, green fluorescence) and CHOP (expressed in both cytoplasm and nucleus, red fluorescence) in the WT and TMBIM6-/- mice of indicated age groups. DAPI stain was used for nuclear localization. Red, green, and blue images were acquired using TRITC, FITC, and DAPI channels of the confocal microscope. The lowest panels show the merger views. Blue triangles indicate CHOP expression having no nuclear trans-localization and white triangles indicate the CHOP nuclear trans-localization. Magnification: 60×, Scale bar: 25 µm. TRITC, tetramethylrhodamine isothiocyanate; FITC, fluorescein isothiocyanate; DAPI, 4', 6-diamidino-2-phenylindole. The bar graph (below) shows the percentage of CHOP nuclear trans-localization of the indicated groups. (D) sXBP1 mRNA was analyzed by real-time PCR and a graph was plotted to the ratio with Gapdh (normalization). (E) The expression of sXBP1 was shown at the nuclear level from the liver of WT and TMBIM6-/- young and old mice (n = 3–4 per group). Histone was used as a nuclear marker. The intensity of the sXBP1 expression is shown in the bar graph (right). (F) The mRNA expression of XBP1 target genes such as Edem1 and Erdj4 were analyzed by real-time PCR and the graph was plotted to the ratio with Gapdh. (G) The mRNA expression of RIDD substrates such as Cyp2e1, Hgsnat, and Col6 was analyzed by real-time PCR and the graph was plotted to the ratio with Gapdh (n = 6 per group). Gapdh was used as a loading control. All the data are represented as mean ± S.E.M. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 indicate the significant differences among the groups. n.s; not significant. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig 4: TMBIM6 deficiency in aging increases redox-mediated post-translational modifications of IRE1a in vivo. (A) Representative images for S-nitrosylated IRE1a in the livers from young and old WT and TMBIM6-/- mice (magnification 60×, scale bar 25 µm). Green images, IRE1a; red, S-nitrosylation; blue, DAPI; SNO-IRE1, merged images; -AS, without ascorbic acid (used as a negative control for S-nitrosylation). The bar graph shows the colocalization between IRE1a and nitrosylated proteins (right) (n = 6–7 per group). (B) IRE1a and cysteine sulfonate proximity ligation assay (PLA) in the liver of young and old WT and TMBIM6-/- mice. Red blobs are the indication of PLA-positive cells. Red images, IRE1a sulfonation; blue, DAPI. Magnification 40×, scale bar 25 µm. NC indicates negative control, where only a single antibody (anti-IRE1a) was used. The bar graph shows the graphical representation of the number of dots per cell of IRE1 sulfonation (below). (C) Urea polyacrylamide gel electrophoresis of sXBP1 and SPARC (as RIDD) substrate with FAM cleaved by IRE1c with GSNO (nitrosylation) and H2O2 (sulfonation) donor. RNase A was used as a control. (D) Sequence motif of XBP1 and TMBIM6 with their potential IRE1a cleavage sites (G/C sites marked with red color). (E) In vitro IRE1a-mediated TMBIM6 cleavage assay. In vitro–transcribed TMBIM6 and XBP1 RNA were incubated with or without recombinant IRE1a protein in the reaction buffer. The cleavage reaction products were resolved on an agarose gel. (F) Predicted secondary structures of TMBIM6 with their potential IRE1a cleavage sites (upper) and urea polyacrylamide gel electrophoresis of TMBIM6 substrate with FAM cleaved by IRE1c. (G) Urea polyacrylamide gel electrophoresis of TMBIM6 substrate with FAM cleaved by IRE1c with GSNO and H2O2 donor. The bar graph (below) shows the relative band intensity to control (n = 3 independent experiments). (H) Sequence motif of miR-338-3p binding sites to PTP1B. (I–J) The mRNA expression of Ptp1b (I) and miR-338 (J) were analyzed by real-time PCR in the liver of mice (n = 6 mice per group). (K) Sequence motif for the IRE1a cleavage sites (upper) and predicted secondary structures of miR-338b with their potential IRE1a cleavage sites (G/C sites marked with red arrows). (L) In vitro IRE1a-mediated miR-338b cleavage assay was performed. All the data are represented as mean ± S.E.M. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 indicate the significant differences among the groups. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig 5: TMBIM6 is downregulated in fatty degenerated and in aged human and mice livers. The liver was isolated from young and old WT mice and subjected to RNA-sequencing. (A) Hierarchical clustering of genes expressed in young and old WT mice. (B) Enrichment analysis results of up/down-regulated differentially expressed genes (DEGs) in old-aged mice compared to young mice. (C–D) Human normal and fat-degenerated liver sections were selected from tissue microarray (LV1201a and BC03116a) and analyzed for H&E and immunohistochemistry using TMBIM6 antibody. H&E images were adopted from Biomax. The bar graph (right) shows the average TMBIM6 expression score (LV1201a: normal = 14, fatty = 15; BC03116a: normal = 21, fatty = 9). Scale bar: 50 µm. (E) Normal tissues and fat-degenerated tissues were selected from young (<50 years) and old patients (>50 years) (selected as explained in the method section), and TMBIM6 expression was analyzed by immunohistochemistry using a specific antibody. Bar graph (below) shows the average TMBIM6 expression (total young normal = 81, total old normal = 21, young fatty = 14, and old fatty = 10). Scale bar: 50 µm. (F) TMBIM6 mRNA expression was analyzed by real-time PCR in different age groups of wild-type mice. (G) Immunohistochemistry of the expression of TMBIM6 in liver sections of young and old WT and TMBIM6-/- mice. The graph (right) shows the average TMBIM6 expression score (n = 6 per group). Note: TMBIM6-/- mice liver sections were used to validate the antibody and expressed none. Scale bar: 100 µm. (H) TMBIM6 association with various proteins of ER stress sensors (human ERN1/IRE1, EIF2AK3/PERK, and ATF6). The figure was plotted using STRING, a functional protein association network. The detail is in the legends of Supplementary Fig. 1. All the data are represented as mean ± S.E.M. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 indicate significant differences indicated as specified in the bar graph.