Fig 2: Aging decreases TMBIM6 expression, induces aberrant UPR expression, S-nitrosylation, and sulfonation of IRE1a in human liver. (A) TMBIM6 mRNA expression and (B) protein expression were analyzed by real-time PCR and immunohistochemistry respectively in young (n = 7) and old human liver tissues (n = 15). (C) Western blotting was performed to check the expression of ER stress response elements p-IRE1, IRE1, GRP78, and CHOP. GAPDH was used as a loading control. Densitometry analysis is shown in the graph (right). (D) The mRNA expression of sXBP1 was analyzed by real-time PCR. (E) The mRNA expression of XBP1 target genes such as EDEM1 and ERDJ4 were measured by real-time PCR. (F) The mRNA expression of the IRE1a-dependent decay (RIDD) target genes such as CYP2E1 and GEMIN5 were examined in the human patient liver samples and analyzed by real-time PCR. (G) Western blotting was performed to check the expression of ER stress-apoptosis-related proteins such as caspase 12, caspase 7, p-JNK, and JNK. GAPDH was used as a loading control. (H) Dihydroethidium (DHE) and Dihydrorhodamine-123 (DHR-123) fluorescence staining were performed in liver sections of young and old patients using confocal microscopy. Red fluorescence indicates DHE positive cells, green fluorescence indicates DHR-123 positive cells, and blue fluorescence indicates DAPI for nuclear staining. The bar graph (right) shows the intensity (n = 7–9 for DHE, n = 5–11 for DHR-123). Magnification: 40×, Scale bar: 25 µm. (I) Representative images for S-nitrosylated IRE1a in the livers from young and old patients (magnification 40×, scale bar 25 µm). S-nitrosylation staining was performed by a modified in situ biotin switch method. 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 (bottom) shows the colocalization between IRE1a and nitrosylated proteins (n = 7 per group). (J) IRE1a and cysteine sulfonate proximity ligation assay (PLA) in the liver of young and old patients' livers. Red blots are the indication of PLA-positive cells. Red images, IRE1a sulfonation; blue, DAPI. Magnification 60×, scale bar 25 µm. The bar graph (right) shows the graphical representation of the number of dots per cell of IRE1 sulfonation (n = 7 per group). All the data are represented as mean ± S.E.M. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, indicates the significant differences among the groups (n = 7/young, and n = 15/old patients in all experiments). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig 3: A putative D domain within WDR62 is responsible for WDR62–JNK association(A) Alignment of the amino acid sequences of the D domains derived from WDR62, JIP1 and c-Jun. The consensus D domain sequence identified in numerous proteins is shown. (B) Schematic representation of the WDR62 deletion constructs used in the experiments. Amino acid positions are numbered. The light green rectangle represents the position of the GST tag. (C) HEK-293T cells were transfected with plasmids encoding GST, 1018-C–GST or 1018-C?D–GST together with HA–JNK1/2 as indicated. GST-containing complexes were isolated from cell lysates with glutathione–agarose beads, washed extensively and eluted with reduced glutathione. The protein complexes were subjected to Western blotting (WB) with anti-pJNK (top panel), anti-HA (second panel from the top) or anti-GST (bottom panel) antibodies. The expression level of HA–JNK1/2 was determined by blotting the total cell lysate with anti-HA antibody (middle panel). The migration of the relevant proteins is indicated by arrows. (D) Top panel: schematic representation of the full-length WDR62 wild-type and deletion mutant constructs used in the HeLa cell immunofluorescence staining experiments. Amino acid positions are numbered. The black square represents the position of the Myc epitope tag. Bottom panels: HeLa cells were co-transfected with an EGFP–JNK1 expression plasmid together with plasmids encoding either wild-type WDR62 (Wt) or the D domain deletion mutant (?D). Cells were fixed and stained with anti-Myc antibody (red). EGFP–JNK1 is shown in green. Co-localization appears in yellow in the merged image. Nuclei were stained with DAPI (blue). Representative confocal microscopy images are shown. Scale bar, 10 µm. GPD, GST pull-down.

Fig 4: Activation of mitogen activated protein kinase (MAPK) in ZIKV infected trophoblasts.a Immunoblot analysis of JNK and p38 activation via phosphorylation were observed 8–24 h after infection with 0.1 MOI r-MRV in JEG-3 cells showed increase in phosphorylation of p54 and p46 components of JNK and phosphorylation of p38 MAPK as well compared to uninfected vehicle cells. Similarly, JNK and p38 activation via phosphorylation was also observed in JAR cells (b) and HTR-8 (c) 24 h of postinfection with 0.1 MOI r-MRV compared to uninfected vehicle cells. Total JNK and Total p38 levels were mostly unaltered between ZIKV infected and vehicle uninfected cells. d Activation of JNK was also seen with 0.1 MOI PRV 24–48 h of postinfection in JEG-3 cells compared to uninfected vehicle cells.

Fig 5: Neurotoxicity of primary cultured SN dopaminergic neurons caused by miR-204-5p is mediated by the increased level of DYRK1A protein. (A) Cotransfection of shRNAs of DYRK1A or treatment of DYRK1A inhibitor harmine (1µM) attenuated miR-204-5p mimic-induced upregulation of a-Syn, phospho-a-Syn, tau, or phospho-tau in primary cultured dopaminergic neurons. (B) In the presence of DYRK1A shRNAs and DYRK1A inhibitor harmine, miR-204-5p mimic failed to upregulate the expression of ER stress-related proteins, including Grp78, IRE1a, CHOP, and active caspase-12, in primary cultured dopaminergic neurons. (C) The miR-204-5p mimic-induced decrease in protein expression of autophagy markers, including Beclin-1, Atg7, Atg16L1, and LC3-II/I ratio, in primary cultured dopaminergic neurons was prevented by cotransfection of shRNA of DYRK1A or treatment of DYRK1A inhibitor harmine. (D) Knockdown of DYRK1A expression or treatment of harmine in primary cultured dopaminergic neurons attenuated miR-204-5p mimic-induced activation of JNK, c-Jun, caspase-9, and caspase-3 in primary cultured SN dopaminergic neurons. (E) In the presence of DYRK1A shRNA or DYRK1A inhibitor harmine, the miR-204-5p mimic failed to significantly upregulate the mRNA level of Bim in primary cultured dopaminergic neurons. Each qRT-PCR experiment was performed in triplicate. (F) Cotransfection of DYRK1A shRNA or treatment of DYRK1A inhibitor harmine reversed miR-204-5p mimic-induced cell death of primary cultured dopaminergic neurons. For the assays of cell viability, each experiment was performed in triplicate. Each bar indicates the mean ± SEM value of four independent experiments. *p < 0.05, **p < 0.01 compared to scramble miR control (SC)-transfected primary cultured dopaminergic neurons.