Journal: Cell Death & Disease

Article Title: TMBIM6 enhances dopaminergic neuron survival by modulating the IRE1a pathway in Parkinson’s disease

doi: 10.1038/s41419-025-08391-5

Figure Lengend Snippet: A Representative images show red fluorescent dots of PLA assay to TMBIM6 HA /IRE1a in stable SN4741 TMBIM6 HA cells exposed to aSyn. B Quantification of PLA dots per cell. Kruskal-Wallis followed by Dunn’s multiple comparison test. C In SN4741 siRNA- mTMBIM6 cells, an RT-qPCR assay shows the effect of aSyn on mRNA levels of mouse XBP1s ( mXbp1s ), D mouse BLOCS1 (mBlocs1) , and E mouse BIP (mBip) . F In SN4741 TMBIM6 HA cells, RT-qPCR assay shows the effect of aSyn in mXbp1s , G mBlocs1 , and H mBip , mRNA levels. Results are expressed as fold change, and bars represent mean ± SEM. All bars represent mean ± SEM. In ( B ), a Kruskal–Wallis test was performed followed by Dunn’s multiple comparison test. For experiments involving more than two groups, a two-way ANOVA followed by Tukey’s multiple comparison test was performed ( C – H ). Statistical significance (p < 0.05) between samples is indicated in the figures as follows: *=p < 0.05; ***=p < 0.001; ****=p < 0.0001.

Article Snippet: The RNase activity of IRE1a was inhibited with 10 μM 4μ8C or 10 μM MKC (MedChemExpress, Monmouth Junction, NJ, USA).

Techniques: Comparison, Quantitative RT-PCR

Journal: Cell Death & Disease

Article Title: TMBIM6 enhances dopaminergic neuron survival by modulating the IRE1a pathway in Parkinson’s disease

doi: 10.1038/s41419-025-08391-5

Figure Lengend Snippet: A , B Cytotoxicity assay shows the effect of IRE1a inhibition using MKC or 4 μ 8c on cell death induced by aSyn in Tmbim6 KD cells after 24 h. Results are expressed as % of LDH release. C Cytotoxicity assay shows the effect of PERK inhibitor on cell death induced by aSyn in mTmbim6 KD cells after 24 h. D A RT-qPCR assay shows effective double knockdown of both mTmbim6 (left panel) and mIre1 a (right panel) in SN4741 cells. Results are expressed as fold change, and bars represent mean ± SEM. One way ANOVA, Dunnett´s multiple comparation test. E Cytotoxicity assay shows downregulation of mIRE1a over cell death induced by aSyn in mTmbim6 KD cells after 24 h. Results are expressed as % of LDH release. F Cytotoxicity assay shows the JNK inhibitor AS60125 over cell death induced by aSyn in mTmbim6 KD cells after 24 h. G Cytotoxicity assay shows the BAX inhibitor BAI-1 over cell death induced by aSyn in mTmbim6 KD cells after 24 h. H Cytotoxicity assay shows the pan-caspase inhibitor ZVAD-FMK (casp-inh) over cell death induced by aSyn in mTmbim6 KD cells after 24 h. All bars represent mean ± SEM. In ( D ), a one-way ANOVA followed by Dunnett’s multiple comparison test was performed, whereas in ( E , F , G , H ), a two-way ANOVA with Tukey’s multiple comparisons test was conducted. Statistical significance (p < 0.05) between samples is indicated in the figures as follows: *=p < 0.05; **=p < 0.01; ***=p < 0.001; ****=p < 0.0001.

Article Snippet: The RNase activity of IRE1a was inhibited with 10 μM 4μ8C or 10 μM MKC (MedChemExpress, Monmouth Junction, NJ, USA).

Techniques: Cytotoxicity Assay, Inhibition, Quantitative RT-PCR, Knockdown, Comparison

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Autophagy and the unfolded protein response promote profibrotic effects of TGF-β 1 in human lung fibroblasts

doi: 10.1152/ajplung.00372.2017

Figure Lengend Snippet: XBP splicing is required for TGF-β1-induced fibrosis in primary human IPF, but not non-IPF, fibroblasts. A: primary human non-IPF fibroblasts were stimulated with TGF-β1 (2.5 ng/ml) in the presence or absence of the IRE1α inhibitor MKC8866 (10 μM) for the indicated time points. The representative immunoblot shows the abundance of collagen 1α2, fibronectin, and sXBP in 4 different primary non-IPF fibroblasts. B and C: densitometry analysis (n = 4) confirmed that MKC8866 did not significantly reduce TGF-β1-induced collagen 1α2 or fibronectin biosynthesis in non-IPF human fibroblasts. Data are shown in dot plots of from 4 individual experiments using different cell lines. The horizontal line in each column represents the mean, with error bars showing SE. NSP > 0.05, no significant difference. D: primary human IPF fibroblasts were stimulated with TGF-β1 (2.5 ng/ml) in the presence or absence of IRE1 inhibitor (10 μM) for the indicated time points. Immunoblots are arranged to show expression of collagen 1α2, fibronectin and sXBP. The immunoblot is representative of experiments performed on 4 different primary IPF fibroblasts (n = 4). E and F: densitometry analysis confirmed that treatment of IPF fibroblasts with IRE1 inhibitor significantly reduced TGF-β1-induced collagen 1α2 and fibronectin biosynthesis. Data are shown in dot plots of from 4 individual experiments using different cell lines. The horizontal line in each column represents the mean, with error bars showing SE. ***P < 0.001; **P < 0.01. G: primary human IPF fibroblasts were stimulated with TGF-β1 (2.5 ng/ml) in the presence or absence of IRE1 inhibitor (10 μM) for 96 h. For a baseline control, cells were maintained in serum- and TGF-β1-deficient media. Quantitative PCR showed that MKC8866 significantly inhibited TGF-β1-induced fibronectin mRNA accumulation in IPF fibroblasts. The experiments were performed in duplicate on 2 different primary IPF fibroblast cell lines. Data are shown in dot plots from each experiment. The horizontal line in each column represents the mean, with error bars showing SE. ***P < 0.001.

Article Snippet: Bafilomycin-A1 (Baf-A1), rabbit anti-human/mouse/rat LC3βII (L8918, 1:3,000), anti-mouse IgG (A8924, 1:3,000), and anti-rabbit IgG (A6154, 1:5,000) were obtained from Sigma-Aldrich (Oakville, CA).The IRE1-specific inhibitor (MKC8866), which inhibits both basal and thapsigargin induced splicing of XBP1 mRNA ( 47 ), was provided by Mannkind (Westlake Village, CA).

Techniques: Western Blot, Expressing, Real-time Polymerase Chain Reaction

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Autophagy and the unfolded protein response promote profibrotic effects of TGF-β 1 in human lung fibroblasts

doi: 10.1152/ajplung.00372.2017

Figure Lengend Snippet: Autophagy flux inhibition and IRE1α inhibition modulates UPR and autophagy in IPF fibroblasts. Primary human IPF fibroblasts were stimulated with TGF-β1 (2.5 ng/ml) in the presence or absence of bafilomycin-A1 (10 nM) or IRE1 inhibitor MKC8866 (10 μM) for 48 or 96 h. Immunoblots show abundance of autophagy (LC3βII, p62) and UPR (BIP) markers. The blot shown is representative of experiments performed on 2 different primary IPF fibroblast cell lines.

Article Snippet: Bafilomycin-A1 (Baf-A1), rabbit anti-human/mouse/rat LC3βII (L8918, 1:3,000), anti-mouse IgG (A8924, 1:3,000), and anti-rabbit IgG (A6154, 1:5,000) were obtained from Sigma-Aldrich (Oakville, CA).The IRE1-specific inhibitor (MKC8866), which inhibits both basal and thapsigargin induced splicing of XBP1 mRNA ( 47 ), was provided by Mannkind (Westlake Village, CA).

Techniques: Inhibition, Western Blot