Journal: Disease Models & Mechanisms

Article Title: Adenoviral TMBIM6 vector attenuates ER-stress-induced apoptosis in a neonatal hypoxic-ischemic rat model

doi: 10.1242/dmm.040352

Figure Lengend Snippet: Ad-TMBIM6 administered at 48 h pre-HI reduced percentage infarcted area and showed localization to neurons and microglia at 72 h post-HI. (A) Representative images of TTC-stained sections and quantification of percentage infarcted area at 72 h post-HI in brain tissue from neonatal rats expressing BI-1 through adenoviral transduction with Ad-TMBIM6. Data presented as mean±s.d.; * P <0.05 versus sham; # P <0.05 versus vehicle, n =6. One-way ANOVA followed by Tukey multiple-comparison post hoc analysis. (B–E) Representative microphotographs of double immunofluorescence staining of BI-1 (red) with neurons (NeuN, green) (B), IRE1α (red) with neurons (NeuN, green) (C), BI-1 (red) with astrocytes (GFAP, green) (D) and BI-1 (red) with microglia (Iba-1, green) (E) in the peri-infarcted area at 72 h post-HI. Blue, DAPI. n =1/group. Merged images show localization of BI-1 or IRE1α to neurons, astrocytes or microglia. There was minimal localization of BI-1 to astrocytes. Scale bars: 50 μm.

Article Snippet: Human adenoviral TMBIM6 vector (Ad-TMBIM6) (ADV-225645, Vector Biolabs) was injected intracerebroventricularly (icv) at 2 µl containing 1.6×10 11 PFU/ml per injection ( ) at 72 h, 48 h and 24 h pre-HI and 1 h post-HI.

Techniques: Staining, Expressing, Transduction, Comparison, Double Immunofluorescence Staining

Journal: Disease Models & Mechanisms

Article Title: Adenoviral TMBIM6 vector attenuates ER-stress-induced apoptosis in a neonatal hypoxic-ischemic rat model

doi: 10.1242/dmm.040352

Figure Lengend Snippet: Expression of BI-1 improved long-term neurological deficits at 4 weeks post-HI. (A,B) In rat pups expressing BI-1 through treatment with Ad-TMBIM6 , long-term motor, learning and memory were assessed via foot fault test (A) and water maze test (B) at 4 weeks post-HI. (C,D) Quantitative analysis of percentage of weight of ipsilateral and contralateral regions out of total brain weights (C) and percentage of tissue loss (D) at 4 weeks post-HI. Data presented as mean±s.d.; * P <0.05 versus sham; # P <0.05 versus vehicle; n =6–9. One-way ANOVA followed by Tukey multiple-comparison or Holm–Sidak post hoc analysis.

Article Snippet: Human adenoviral TMBIM6 vector (Ad-TMBIM6) (ADV-225645, Vector Biolabs) was injected intracerebroventricularly (icv) at 2 µl containing 1.6×10 11 PFU/ml per injection ( ) at 72 h, 48 h and 24 h pre-HI and 1 h post-HI.

Techniques: Expressing, Hi-C, Comparison

Journal: Disease Models & Mechanisms

Article Title: Adenoviral TMBIM6 vector attenuates ER-stress-induced apoptosis in a neonatal hypoxic-ischemic rat model

doi: 10.1242/dmm.040352

Figure Lengend Snippet: Effects of BI-1 siRNA and IRE1α CRISPR activation plasmid on infarct area at 72 h post-HI. (A,B) Representative images of infarcted tissue (A) and quantification of percentage infarcted area (out of total brain area) (B) at 72 h post-HI. Data presented as mean±s.d.; * P <0.05 versus sham; # P <0.05 versus vehicle; @ P <0.05 versus Ad-TMBIM6; $ P <0.05 versus BI-1 siRNA; + P <0.05 versus Ad-TMBIM6+scramble siRNA; & P <0.05 versus IRE1α CRISPR AP; % P <0.05 versus control CRISPR. n =6/group. One-way ANOVA followed by Tukey multiple-comparison post hoc analysis. (C) Representative schematic of BI-1/IRE1α/XBP1/CHOP signaling pathway using the intervention groups (BI-1 siRNA and IRE1α CRISPR activation plasmid) to study the mechanism. (D) Representative immunofluorescence staining of BI-1 (red) with neurons (NeuN, green), astrocytes (GFAP, green) and microglia (Iba-1, green) after siRNA administration. Representative picture demonstrating brain region from where immunofluorescence pictures were obtained is shown upper right. n =1/group. Scale bars: 50 μm.

Article Snippet: Human adenoviral TMBIM6 vector (Ad-TMBIM6) (ADV-225645, Vector Biolabs) was injected intracerebroventricularly (icv) at 2 µl containing 1.6×10 11 PFU/ml per injection ( ) at 72 h, 48 h and 24 h pre-HI and 1 h post-HI.

Techniques: CRISPR, Activation Assay, Plasmid Preparation, Control, Comparison, Immunofluorescence, Staining

Journal: Disease Models & Mechanisms

Article Title: Adenoviral TMBIM6 vector attenuates ER-stress-induced apoptosis in a neonatal hypoxic-ischemic rat model

doi: 10.1242/dmm.040352

Figure Lengend Snippet: BI-1 exerted its protective effects via the IRE1α–XBP1–CHOP pathway at 72 h post-HI. (A) Representative immunoblots of protein expression levels in ipsilateral hemispheric tissue from 10-day-old rats at 72 h post-HI. (B–F) Quantitative analysis of BI-1 (B), pIRE1α (C), XBP1 (D), CHOP (E) and ROMO1 (F) expression levels (band density relative to actin). Data presented as mean±s.d.; * P <0.05 versus sham; # P <0.05 versus vehicle; @ P <0.05 versus Ad-TMBIM6; $ P <0.05 versus BI-1 siRNA; + P <0.05 versus Ad-TMBIM6+scramble siRNA; & P <0.05 versus IRE1α CRISPR AP; % P <0.05 versus control CRISPR; ^ P <0.05 versus Ad-TMBIM6+BI-1 siRNA; ε P <0.05 versus BI-1 siRNA-only group. n =6/group. One-way ANOVA followed by Tukey multiple-comparison post hoc analysis.

Article Snippet: Human adenoviral TMBIM6 vector (Ad-TMBIM6) (ADV-225645, Vector Biolabs) was injected intracerebroventricularly (icv) at 2 µl containing 1.6×10 11 PFU/ml per injection ( ) at 72 h, 48 h and 24 h pre-HI and 1 h post-HI.

Techniques: Western Blot, Expressing, CRISPR, Control, Comparison

Journal: Disease Models & Mechanisms

Article Title: Adenoviral TMBIM6 vector attenuates ER-stress-induced apoptosis in a neonatal hypoxic-ischemic rat model

doi: 10.1242/dmm.040352

Figure Lengend Snippet: Overexpression of BI-1 attenuated apoptosis at 72 h post-HI. (A) Representative immunoblots of protein expression levels in ipsilateral hemispheric tissue from 10-day-old rats at 72 h post-HI. (B–D) Quantification of Bcl-2 (B), BAX (C) and CC3 (D) expression level data (band density relative to actin). Data presented as mean±s.d.; * P <0.05 versus sham; # P <0.05 versus vehicle; @ P <0.05 versus Ad-TMBIM6; $ P <0.05 versus BI-1 siRNA; + P <0.05 versus Ad-TMBIM6+scramble siRNA; & P <0.05 versus IRE1α CRISPR AP; % P <0.05 versus control CRISPR. n =6/group. One-way ANOVA followed by Tukey multiple-comparison, Holm–Sidak or Dunnett's post hoc analysis. (E) Representative microphotographs of positively CC3-stained (red) neurons (NeuN, green) from 10-day-old rats at 72 h post-HI. (F) Representative microphotographs of Fluoro-Jade C (FJC)-positive degenerating neurons in the peri-infarcted area. n =1/group. Scale bars: 100 µm.

Article Snippet: Human adenoviral TMBIM6 vector (Ad-TMBIM6) (ADV-225645, Vector Biolabs) was injected intracerebroventricularly (icv) at 2 µl containing 1.6×10 11 PFU/ml per injection ( ) at 72 h, 48 h and 24 h pre-HI and 1 h post-HI.

Techniques: Over Expression, Western Blot, Expressing, CRISPR, Control, Comparison, Staining

Journal: Disease Models & Mechanisms

Article Title: Adenoviral TMBIM6 vector attenuates ER-stress-induced apoptosis in a neonatal hypoxic-ischemic rat model

doi: 10.1242/dmm.040352

Figure Lengend Snippet: Percentage cell viability significantly improved after transfection of PC-12 cells with Ad-TMBIM6 at MOI=100 in an in vitro OGD model. (A) Quantification of data shows time-dependent decrease in percentage cell viability for cultured PC12 cells when exposed to 1 h, 1.5, 3 h, 5 h and 6 h in OGD. (B) Quantification analysis of percentage cell viability of PC12 cells when exposed to varying concentrations (MOI) of viral vector. (C) Representative images of cell morphology and density of cells after OGD in all groups. Scale bar: 100 µm. (D) Quantification analysis of percentage cell viability with pathway interventions. Data presented as mean± s.d.; * P <0.05 versus control; # P <0.05 versus vehicle; @ P <0.05 versus Ad-TMBIM6+scramble siRNA (D) or versus MOI=100 (B); ε P <0.05 versus Ad-TMBIM6 only; $ P <0.05 versus BI-1 siRNA; & P <0.05 versus APY-29; + P <0.05 versus DMSO; % P <0.05 versus CCT020312; ^ P <0.05 versus STF-083010 and control CRISPR. n =5–6/group. One-way ANOVA followed by Tukey multiple-comparison post hoc analysis.

Article Snippet: Human adenoviral TMBIM6 vector (Ad-TMBIM6) (ADV-225645, Vector Biolabs) was injected intracerebroventricularly (icv) at 2 µl containing 1.6×10 11 PFU/ml per injection ( ) at 72 h, 48 h and 24 h pre-HI and 1 h post-HI.

Techniques: Transfection, In Vitro, Cell Culture, Plasmid Preparation, Control, CRISPR, Comparison

Journal: Disease Models & Mechanisms

Article Title: Adenoviral TMBIM6 vector attenuates ER-stress-induced apoptosis in a neonatal hypoxic-ischemic rat model

doi: 10.1242/dmm.040352

Figure Lengend Snippet: BI-1 exerted its anti-apoptotic effects via inhibition of IRE1α in an in vitro OGD model. (A) Representative immunoblots of protein expression levels in PC12 cultured cells. Quantification of BI-1 (B), pIRE1α (C), pPERK (D) and CC3 (E) expression levels (band density relative to actin). Data presented as mean±s.d.; * P <0.05 versus control; # P <0.05 versus vehicle; @ P <0.05 versus Ad-TMBIM6 and Ad-TMBIM6+scramble siRNA; $ P <0.05 versus BI-1 siRNA; & P <0.05 versus APY-29; + P <0.05 DMSO; % P <0.05 versus CCT020312; ^ P <0.05 versus STF-083010 and control CRISPR. n =5–6/group. One-way ANOVA followed by Tukey multiple-comparison post hoc analysis.

Article Snippet: Human adenoviral TMBIM6 vector (Ad-TMBIM6) (ADV-225645, Vector Biolabs) was injected intracerebroventricularly (icv) at 2 µl containing 1.6×10 11 PFU/ml per injection ( ) at 72 h, 48 h and 24 h pre-HI and 1 h post-HI.

Techniques: Inhibition, In Vitro, Western Blot, Expressing, Cell Culture, Control, CRISPR, Comparison

Journal: Nature Communications

Article Title: TMBIM6/BI-1 contributes to cancer progression through assembly with mTORC2 and AKT activation

doi: 10.1038/s41467-020-17802-4

Figure Lengend Snippet: a – e TMBIM6 expression was analyzed using the GEO database from NCBI. Fibrosarcoma (GSE2719; normal n = 3; tumor n = 7), cervix (GSE63678; cervical normal n = 5; tumor n = 5; endometrial normal n = 5; tumor n = 7; vulvar normal n = 7; tumor n = 6), breast (GSE31448; normal n = 31; basal n = 98; luminal A n = 89; luminal B n = 49; ERBB2 n = 25), lung (GSE19804; normal n = 60; tumor n = 60), and prostate (GSE69223; normal n = 15; tumor n = 15) datasets are presented. Center line of the box represents median; box bounds represent 25th and 75th percentiles; whiskers represent minimum and maximum values. f Representative immunohistochemical staining of TMBIM6 on tissue microarrays containing fibrosarcoma, cervix, breast, lung, and prostate tissue and adjacent normal tissues. TMBIM6 WT and KO HT1080 cells were used as a control for validation of the method. Right; quantification data of TMBIM6 expression (fibrosarcoma normal n = 9; tumor n = 8; cervix normal n = 20; tumor n = 80; breast normal n = 6; tumor n = 97; lung normal n = 15; tumor n = 75; prostate normal n = 32; tumor n = 160). Scale bar, 100 μm. (brown: positive antibody staining, blue: hematoxylin for nuclear staining). Data are presented as means ± SD. **** p < 0.0001, two-tailed unpaired t -test. g Kaplan–Meier curves showing the overall survival analysis in patients with high and low expression of TMBIM6 using GEPIA2 tool. P value with log-rank analysis. BRCA breast invasive carcinoma, CESC cervical squamous cell carcinoma and endocervical adenocarcinoma, SARC sarcoma, LUAD lung adenocarcinoma. h Differentially expressed genes by microarray analysis of mRNA expression levels in TMBIM6 KO and WT HT1080 cells. i Significant ratios in TMBIM6 KO and WT HT1080 cells determined by Gene Ontology analysis. j The graph indicates significant differences in downregulation and upregulation of the indicated category genes in the TMBIM6 KO cells compared with those in TMBIM6 WT cells.

Article Snippet: The plasmid containing sequences targeting human TMBIM6 were designed and constructed from the pRGEN_TMBIM6 expression vector by ToolGen (Seoul, Korea).

Techniques: Expressing, Immunohistochemical staining, Staining, Two Tailed Test, Microarray

Journal: Nature Communications

Article Title: TMBIM6/BI-1 contributes to cancer progression through assembly with mTORC2 and AKT activation

doi: 10.1038/s41467-020-17802-4

Figure Lengend Snippet: a Proliferation of TMBIM6 KO and WT HT1080 cells, HeLa cells, and MEFs ( n = 3 independent experiments). Data are presented as means ± SD. ** p < 0.01, *** p < 0.001, two-way ANOVA followed by Bonferroni’s post hoc test. b Images of migrated cells in TMBIM6 KO and WT HT1080. Right, quantification of WT cells normalized to KO cells ( n = 6 independent experiments). Scale bars, 100 μm. Data are presented as means ± SD. **** p < 0.0001, two-tailed unpaired t -test. c Images of invasive cells in TMBIM6 KO and WT HT1080. Right, quantification of WT cells normalized to KO cells ( n = 5 independent experiments). Scale bars, 100 μm. Data are presented as means ± SD. **** p < 0.001, two-tailed unpaired t -test. d – f Tumor volume, weight, and size derived from TMBIM6 KO or WT HT1080 cells injected into the flank of 6-week-old nude mice ( n = 6 mice per group). Data are presented as means ± SD. * p < 0.05; ** p < 0.01; **** p < 0.0001, two-way ANOVA followed by Bonferroni’s post hoc test in D and two-tailed unpaired t -test in ( e ). g Histological images by immunohistochemical detection of Ki-67. Right, quantification of Ki-67-positive cells in xenograft tumors derived from TMBIM6 KO and WT HT1080 cells ( n = 6 mice per group). Scale bars, 100 μm. Data are presented as means ± SD. **** p < 0.0001, two-tailed unpaired t -test. h – j Tumor volume, weight, and size derived from TMBIM6 KO or WT HeLa cells injected into the flank of 6-week-old nude mice ( n = 6 mice per group). Data are presented as means ± SD. *** p < 0.001; **** p < 0.0001, two-way ANOVA followed by Bonferroni’s post hoc test in ( h ) and one-way ANOVA followed by Tukey’s post hoc test in ( i ). k Histological images by immunohistochemical detection of Ki-67. Right, quantification of Ki-67-positive cells in xenograft tumors derived from TMBIM6 KO and WT HeLa cells ( n = 6 mice per group). Scale bars, 100 μm. Data are presented as means ± SD. **** p < 0.0001, two-way ANOVA followed by Bonferroni’s post hoc test.

Article Snippet: The plasmid containing sequences targeting human TMBIM6 were designed and constructed from the pRGEN_TMBIM6 expression vector by ToolGen (Seoul, Korea).

Techniques: Two Tailed Test, Derivative Assay, Injection, Immunohistochemical staining

Journal: Nature Communications

Article Title: TMBIM6/BI-1 contributes to cancer progression through assembly with mTORC2 and AKT activation

doi: 10.1038/s41467-020-17802-4

Figure Lengend Snippet: a Expression and phosphorylation of 43 proteins were examined by Proteome Profile Human Phospho-Kinase Array in WT and TMBIM6 KO HT1080 cells. Right; the relative phosphorylations of indicated proteins were quantitated by ImagJ. Blots represent one of two experiments, with similar results obtained. b pAKT, pTSC2, and pNDRG1 were analyzed in TMBIM6 KO and WT HT1080 cells using Western blots (left) and normalized to total proteins of WT cells (right; n = 5 independent experiments). Data are presented as means ± SD. *** p < 0.001, **** p < 0.0001, two-way ANOVA followed by Bonferroni’s post hoc test. c Immunoblotting, RT-PCR, and its gene quantification analysis were performed in TMBIM6-HA-stably expressing or non-expressing TMBIM6 KO cells ( n = 3 independent experiments). Data are presented as means ± SD. * p < 0.05, ** p < 0.01, two-way ANOVA followed by Bonferroni’s post hoc test. d After serum starvation for 12 h, TMBIM6 KO and WT HT1080 cells were stimulated with insulin (100 ng/ml), IGF1 (100 ng/ml), or EGF (100 ng/ml), and immunoblotting was performed with indicated antibodies. e Gel filtration assay of extracts of TMBIM6 KO and WT MEFs. The red line represents the size marker. f PLA between indicated proteins (red dots) in TMBIM6 KO and WT HT1080 cells. For the PLA, the ribosomal protein S6 kinase beta-1 (S6K1) was also applied as a negative control. Scale bar, 15 μm. Right, quantification of red dots ( n = 5 independent experiments). Data are presented as means ± SD. * p < 0.05, ** p < 0.01, two-way ANOVA followed by Bonferroni’s post hoc test. g Immunoblot analysis of anti-RPL19 immunoprecipitate (IP) and whole cell lysate (WCL) of TMBIM6 KO and WT HT1080 cells.

Article Snippet: The plasmid containing sequences targeting human TMBIM6 were designed and constructed from the pRGEN_TMBIM6 expression vector by ToolGen (Seoul, Korea).

Techniques: Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Stable Transfection, Filtration, Marker, Negative Control

Journal: Nature Communications

Article Title: TMBIM6/BI-1 contributes to cancer progression through assembly with mTORC2 and AKT activation

doi: 10.1038/s41467-020-17802-4

Figure Lengend Snippet: a mRNA levels of glycolysis- and PPP-related genes in TMBIM6 KO and WT HT1080 cells, as determined by qRT-PCR. Quantification data represent the expression level of genes in the KO cells compared with those in normalized WT cells ( n = 3 independent experiments). Data are presented as means ± SD. **** p < 0.0001, two-way ANOVA followed by Bonferroni’s post hoc test. b , c Glucose consumption and lactate production in TMBIM6 KO and WT HT1080 cells ( n = 3 independent experiments). Data are presented as means ± SD. ** p < 0.01; *** p < 0.001, one-way ANOVA followed by Tukey’s post hoc test. d Metabolite analysis in TMBIM6 KO and WT HT1080 cells ( n = 2 independent experiments). e , f mRNA levels of GSH biosynthesis genes and de novo lipid biosynthesis genes in TMBIM6 KO and WT HT1080 cells, as determined by qRT-PCR. Quantification data represent the expression level of genes in the KO cells compared with those in normalized WT cells ( n = 3 independent experiments). Data are presented as means ± SD. * p < 0.05, ** p < 0.01, **** p < 0.0001, two-way ANOVA followed by Bonferroni’s post hoc test. g Immunofluorescence images of protein synthesis in TMBIM6 KO and WT HT1080 cells. Right, quantification data represent the expression intensity compared with that in normalized WT cells ( n = 3 independent experiments). Data are presented as means ± SD. * p < 0.05, two-tailed unpaired t -test. Scale bar, 15 μm. h Glycosylation-related gene lists in TMBIM6 KO and WT HT1080 cells by microarray. i Glycosylated protein levels in TMBIM6 KO and WT HT1080 cells ( n = 3 independent experiments). Data are presented as means ± SD. * p < 0.05, two-tailed unpaired t -test.

Article Snippet: The plasmid containing sequences targeting human TMBIM6 were designed and constructed from the pRGEN_TMBIM6 expression vector by ToolGen (Seoul, Korea).

Techniques: Quantitative RT-PCR, Expressing, Immunofluorescence, Two Tailed Test, Microarray

Journal: Nature Communications

Article Title: TMBIM6/BI-1 contributes to cancer progression through assembly with mTORC2 and AKT activation

doi: 10.1038/s41467-020-17802-4

Figure Lengend Snippet: a Gel filtration assay of lysates of TMBIM6 KO HT1080 cells transiently overexpressing TMBIM6-HA. b Anti-RICTOR immunoprecipitate (IP) of pooled fractions of TMBIM6 KO HT1080 cells transiently overexpressing TMBIM6-HA. The inputs were analyzed by immunoblotting. c Immunoblot analysis of anti-HA IP and whole cell lysate (WCL) of TMBIM6-HA overexpressing HeLa cells. d PLA between TMBIM6-HA and mTORC2 components (red dots) in TMBIM6 stably overexpressing HT1080 cells. Right, quantification of red dots ( n = 5 independent experiments). Scale bar, 15 μm. Data are presented as means ± SD. *** p < 0.001, **** p < 0.0001, two-way ANOVA followed by Bonferroni’s post hoc test. e GST pull-down assay between GST-TMBIM6 and myc-RICTOR. f GST pull-down assay in the presence or absence of HA-TMBIM6. g Immunoblot analysis of WCL of HT1080 cells stably expressing TMBIM6 and transfected with scrambled, mTOR, RICTOR, or SIN1 siRNA and immunoprecipitated with anti-HA antibody. h Immunoblot analysis of the immunoprecipitates with anti-RICTOR antibody and whole cell lysates of HT1080 cells transiently transfected with TMBIM6 and TMBIM6 mutant constructs. i Immunoblot analysis of the immunoprecipitates with anti-HA antibody and input of HT1080 cells transiently transfected with TMBIM6 and TMBIM6 mutant constructs.

Article Snippet: The plasmid containing sequences targeting human TMBIM6 were designed and constructed from the pRGEN_TMBIM6 expression vector by ToolGen (Seoul, Korea).

Techniques: Filtration, Western Blot, Stable Transfection, Pull Down Assay, Expressing, Transfection, Immunoprecipitation, Mutagenesis, Construct

Journal: Nature Communications

Article Title: TMBIM6/BI-1 contributes to cancer progression through assembly with mTORC2 and AKT activation

doi: 10.1038/s41467-020-17802-4

Figure Lengend Snippet: a Immunofluorescence images and fluorescence intensity (right) of TMBIM6-GCaMP3 and TMBIM6 D213A-GCaMP3 in the presence or absence of 10 µM BAPTA-AM. ( n = 5 independent experiments). Scale bar, 15 μm. Data are presented as means ± SD. ** p < 0.01, *** p < 0.001, two-way ANOVA followed by Bonferroni’s post hoc test. b PLA between Rictor and mTOR or between Rictor and RPL19 (red dots) in empty vector, TMBIM6, or D213A-transfected HT108 cells. ( n = 3 independent experiments). Scale bar, 15 μm. Data are presented as means ± SD. **** p < 0.0001, two-way ANOVA followed by Bonferroni’s post hoc test. c Immunoblot analysis of the immunoprecipitates with Anti-HA antibody and input of cell lysates with the indicated antibodies. d Immunoblotting and quantification of phosphorylation of AKT in TMBIM6 KO HT1080 cells transfected with empty vector, TMBIM6 WT, and TMBIM6 D213A. ( n = 3 independent experiments). Data are presented as means ± SD. ** p < 0.01, *** p < 0.001, one-way ANOVA followed by Tukey’s p ost hoc test. e Immunofluorescence images and quantification of AKT phosphorylation ( n = 3 independent experiments, total of nine images). Scale bar, 15 μm. Data are presented as means ± SD. **** p < 0.0001, one-way ANOVA followed by Tukey’s post hoc test. f Proliferation analysis of empty vector, TMBIM6, and TMBIM6 D213A-expressed HT1080 cells ( n = 3 independent experiments). Data are presented as means ± SD. ** p < 0.01, **** p < 0.0001, two-way ANOVA followed by Bonferroni’s p ost hoc test. g The quantification analysis of mRNA levels of glycolysis- and PPP-related genes in empty vector, TMBIM6, and TMBIM6 D213A-rescued TMBIM6 KO HT1080 cells, as determined by qRT-PCR. Quantification data represent the expression level of genes compared with those in normalized WT HT1080 cells (red line, n = 3 independent experiments). Data are presented as means ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, two-way ANOVA followed by Bonferroni’s post hoc test. h , i Glucose consumption and lactate production in empty vector, TMBIM6, and TMBIM6 D213A-rescued TMBIM6 KO HT1080 cells ( n = 3 independent experiments). Data are presented as means ± SD. * p < 0.05, ** p < 0.01, one-way ANOVA followed by Tukey’s post hoc test. j Metabolite analysis in empty vector, TMBIM6, and TMBIM6 D213A-rescued TMBIM6 KO HT1080 cells. Quantification data represent the metabolite level compared with those in empty vector-rescued TMBIM6 KO HT1080 cells ( n = 2 independent experiments).

Article Snippet: The plasmid containing sequences targeting human TMBIM6 were designed and constructed from the pRGEN_TMBIM6 expression vector by ToolGen (Seoul, Korea).

Techniques: Immunofluorescence, Fluorescence, Plasmid Preparation, Transfection, Western Blot, Quantitative RT-PCR, Expressing

Journal: Nature Communications

Article Title: TMBIM6/BI-1 contributes to cancer progression through assembly with mTORC2 and AKT activation

doi: 10.1038/s41467-020-17802-4

Figure Lengend Snippet: a Proliferation of TMBIM6 WT HT1080, TMBIM6 KO HT1080, MCF7, MDA-MB-231, and SKBR3 cells treated with BIA ( n = 3 independent experiments). Data are presented as means ± SD. b Gel filtration assay of HT1080 cells treated with 1.0 μM BIA. The red line represents the size marker. c Immunoblot analysis of anti-HA immunoprecipitate (IP) and whole cell lysate (WCL) of HT1080 cells transiently overexpressing TMBIM6-HA and treated with BIA. d Immunoblotting of p-AKT, AKT, and actin in the indicated cell lines following treatment with BIA. e PLA between TMBIM6-HA and mTORC2 components or between TMBIM6-HA and RPL19 (red dots) in BIA-treated or non-treated TMBIM6 stably overexpressing HT1080 cells. Bottom, quantification of red dots ( n = 5 independent experiments). Scale bar, 20 μm. Data are presented as means ± SD. ** p < 0.01, **** p < 0.0001, two-way ANOVA followed by Bonferroni’s post hoc test. f , g Real-time lapse images of HT1080 cells stably expressing TMBIM6-GCaMP3 and G-CEPIAer treated with BIA. Right, mean green intensity of every cells normalized to untreated cells ( n = 5 independent experiments, total of 20 cells for TMBIM6-GCaMP3; total of 16 cells for G-CEPIAer). Scale bar, 15 μm. Data are presented as means ± SD.

Article Snippet: The plasmid containing sequences targeting human TMBIM6 were designed and constructed from the pRGEN_TMBIM6 expression vector by ToolGen (Seoul, Korea).

Techniques: Filtration, Marker, Western Blot, Stable Transfection, Expressing

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 In silico TMBIM6 mRNA expression in human CNS from THPA database. B Tmbim6 mRNA expression on N2a cells after 18 h of exposure to 25 μM 6-OHDA or 50 μM rotenone. C Tmbim6 mRNA levels over time in PCNs exposed to aSyn for 96 h. D Changes of expression of Tmbim6 , BcL2 , and Bax over time in PCNs exposed to aSyn for 96 h. E Representative Western blots of total protein extracts from postmortem human SN from neurologically healthy controls and PD patients, probed for TMBIM6 and GAPDH (loading control). Full, uncropped blots are provided in Supplementary Material. F Densitometric quantification of TMBIM6 from blots in ( E ). Band intensities were normalized to GAPDH for each lane; individual data points are shown with mean ± SEM (n = 9–10 per group). For experiments involving more than two groups, a two-way ANOVA followed by Tukey’s multiple comparison test was performed. Pairwise comparisons between two groups were analyzed using unpaired t-test ( B ) or the Mann–Whitney U test ( F ). All bars represent mean ± SEM. 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: Using data on TMBIM6 mRNA expression levels from The Human Protein Atlas database (THPA) [ ], we confirmed through in silico analysis that TMBIM6 is expressed in various cortical regions of the human brain, with notably high expression in the midbrain and retina, two areas rich in DAergic cells [ , ] (Fig. ).

Techniques: In Silico, Expressing, Western Blot, Control, Comparison, MANN-WHITNEY

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 Graph shows validation of decreased Tmbim6 mRNA levels after 48 h of siRNA transfection in SN4741. B , C Representative immunoblots and quantification show mTmbim6 KD cells after 48 h. D Cytotoxicity assay shows cell death induced by 10 μM Tunicamycin after 24 h in KD cells. Results are expressed as % of LDH release. E Cytotoxicity assay shows cell death induced by 50 μM 6-OHDA after 24 h in KD cells. F Retention of DiOC6(3) assay shows the effect of aSyn on ΔΨm in KD cells after 18 h. Results are expressed as % of DiOC6(3) retention. G MTT assay shows mitochondrial-dependent cell death induced by aSyn in KD cells after 24 h. Results are expressed as % of MTT. H DEVD-AMC fluorescent assay shows the effect of aSyn on Caspase-3 activity in Tmbim6 KD cells after 24 h. Results are expressed as fold change of DEVD-AMC fluorescence intensity relative to vehicle. I Cytotoxicity assay shows the KD cell death induced by aSyn after 24 h. J Representative immunoblot of high–molecular-weight (HMW) aSyn species in SN4741 cells transfected with si Cntrl or si Tmbim6 and treated with 10 µM aSyn PFFs for 24 h; Tubulin was used as a loading control. K Densitometric quantification of HMW aSyn bands from the experiment described in ( J ) (integrated density normalized to Tubulin). All bars represent mean ± SEM. For experiments involving more than two groups, a two-way ANOVA followed by Tukey’s multiple comparison test was performed. Pairwise comparisons between two groups were analyzed using unpaired t-test ( A , C ) or the Mann–Whitney U test ( K ). Statistical significance (p < 0.05) between samples is indicated in the figures as follows: *=p < 0.05; **=p < 0.01; ***=p < 0.001.

Article Snippet: Using data on TMBIM6 mRNA expression levels from The Human Protein Atlas database (THPA) [ ], we confirmed through in silico analysis that TMBIM6 is expressed in various cortical regions of the human brain, with notably high expression in the midbrain and retina, two areas rich in DAergic cells [ , ] (Fig. ).

Techniques: Biomarker Discovery, Transfection, Western Blot, Cytotoxicity Assay, MTT Assay, Fluorescence, Activity Assay, High Molecular Weight, Control, Comparison, MANN-WHITNEY

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 d Tmbim6 mRNA expression on homogenized flies’ heads. Results are expressed as fold change of mRNA expression. The bars represent mean ± SEM. B Optic image of eye integrity in RNAi-dTmbim6 flies incubated at 25 °C. C Quantification of eye integrity score in RNAi- dTmbim6 flies incubated at 25 °C (n = 25 per group). D Immunostaining of TH+ neurons in the lamina of RNAi-dTmbim6 flies incubated at 25 °C (n = 6 per group). E Quantification of the number of TH+ neurons in the lamina of RNAi-dTmbim6 flies incubated at 25 °C (n = 6 per group). F Schematic representation of rotenone-induced PD model in D. mel . G Spontaneous activity in DAergic RNAi-dTmbim6 flies after exposition to rotenone 300 μM for 7 days (n = 6 populations of 12 flies). H Climbing assay showed the motor ability of DAergic RNAi-dTmbim6 flies exposed to rotenone 300 μM for 7 days (n = 12 per group). I Representative confocal images of IF assay showed TH+ cells from DAergic RNAi-dTmbim6 flies exposed to rotenone 300 μM for 7 days. J The numbers of TH+ cells were quantified in each DAergic cluster, and K the somal size was analyzed (n = 7 per group). All bars represent mean ± SEM. For experiments involving more than two groups, a two-way ANOVA followed by Tukey’s multiple comparison test was performed. Pairwise comparisons between two groups were analyzed using unpaired t-test ( A , C ) or the Mann–Whitney U test ( E ). 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: Using data on TMBIM6 mRNA expression levels from The Human Protein Atlas database (THPA) [ ], we confirmed through in silico analysis that TMBIM6 is expressed in various cortical regions of the human brain, with notably high expression in the midbrain and retina, two areas rich in DAergic cells [ , ] (Fig. ).

Techniques: Expressing, Incubation, Immunostaining, Activity Assay, Climbing Assay, Comparison, MANN-WHITNEY

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 immunoblot shows a stable expression of TMBIM6 HA in SN4741 cells. B Cytotoxicity assay shows the cell death induced by Tunicamycin in SN4741 TMBIM6 HA cells after 24 h. C , D MTT assay shows cell death induced by 6-OHDA or rotenone in SN4741 TMBIM6 HA cells after 24 h. Results are expressed as % of MTT. E Retention of DiOC6(3) assay shows the effect of aSyn on ΔΨm in SN4741 hTMBIM6 HA cells after 18 h. Results are expressed as % of DiOC6(3) retention. F MTT assay shows cell death induced by aSyn in SN4741 TMBIM6 HA cells after 24 h. G DEVD-AMC fluorescent assay shows the effect of aSyn on Caspase-3 activity in SN4741 TMBIM6 HA cells after 24 h. H Cytotoxicity assay shows the cell death induced by aSyn in SN4741 TMBIM6 HA cells after 24 h. I Representative immunoblot of HMW aSyn species in Mock or TMBIM6 HA cells treated with aSyn PFFs for 24 h; TCE staining was used as a loading control. J Densitometric quantification of HMW aSyn bands from the experiment described in J (integrated density normalized to TCE). K Representative immunoblot shows expression of TMBIM6 HA and TMBIM6 D213A/HA in SN4741 cells. L MTT assay shows cell death induced by Tunicamycin and Thapsigargin in SN4741 Mock, TMBIM6 HA , and TMBIM6 D213A/HA cells after 24 h. Results are expressed as % of MTT. M Cytotoxicity assay shows the cell death induced by aSyn in SN4741 TMBIM6 HA and TMBIM6 D213A/HA cells after 24 h. N Cytotoxicity assay shows the cell death induced by aSyn after 10 days in PCNs transfected with Mock, TMBIM6 HA and TMBIM6 D213A/HA constructs. All bars represent mean ± SEM. For experiments involving more than two groups, a two-way ANOVA followed by Tukey’s multiple comparison test was performed. Pairwise comparisons between two groups were analyzed using the Mann–Whitney U test. 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: Using data on TMBIM6 mRNA expression levels from The Human Protein Atlas database (THPA) [ ], we confirmed through in silico analysis that TMBIM6 is expressed in various cortical regions of the human brain, with notably high expression in the midbrain and retina, two areas rich in DAergic cells [ , ] (Fig. ).

Techniques: Western Blot, Expressing, Cytotoxicity Assay, MTT Assay, Fluorescence, Activity Assay, Staining, Control, Transfection, Construct, Comparison, MANN-WHITNEY

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 An in-silico assay using Ingenuity Pathway Analysis (IPA) software shows the canonical pathways significantly associated with TMBIM6 interactors. B , C UMAP visualizations of the snRNA-seq dataset (GEO: GSE178265 ) from human postmortem substantia nigra. B shows TMBIM6 expression across all nuclei, while C distinguishes nuclei from healthy and PD donors. D Dot plot comparing the expression of TMBIM6 and UPR-related genes ( HSPA5, ERN1, XBP1, BLOC1S1 ) between healthy and PD conditions across all nuclei. E UMAP plot identifying resistant and vulnerable DAergic neuron populations within the dataset. F Dot plot comparing gene expression between resistant and vulnerable DAergic neurons within the PD cohort. For dot plots ( D , F ), dot size represents the percentage of cells expressing the gene, and color intensity indicates the mean expression level. Statistical significance for the differential expression shown in ( D , F ) was determined using the Model-based Analysis of Single-cell Transcriptomics (MAST) test. Full statistical details, including FDR-adjusted p-values, are provided in Supplementary Fig. .

Article Snippet: Using data on TMBIM6 mRNA expression levels from The Human Protein Atlas database (THPA) [ ], we confirmed through in silico analysis that TMBIM6 is expressed in various cortical regions of the human brain, with notably high expression in the midbrain and retina, two areas rich in DAergic cells [ , ] (Fig. ).

Techniques: In Silico, Software, Expressing, Gene Expression, Quantitative Proteomics, Single-cell Transcriptomics

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: Using data on TMBIM6 mRNA expression levels from The Human Protein Atlas database (THPA) [ ], we confirmed through in silico analysis that TMBIM6 is expressed in various cortical regions of the human brain, with notably high expression in the midbrain and retina, two areas rich in DAergic cells [ , ] (Fig. ).

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: Using data on TMBIM6 mRNA expression levels from The Human Protein Atlas database (THPA) [ ], we confirmed through in silico analysis that TMBIM6 is expressed in various cortical regions of the human brain, with notably high expression in the midbrain and retina, two areas rich in DAergic cells [ , ] (Fig. ).

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

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 Effect of two 6-OHDA doses on motor performance of mice, as a pharmacologic in vivo PD model (n = 4 per condition). Results are expressed as a percentage of contralateral forelimb use. B Timeline of in vivo transduction of the AAV-TMBIM6 HA/GFP and AAV-Mock GFP in SN and motor performance measurements in mice wild-type lesioned with 6-OHDA in CPu. C Cylinder test shows the effect of AAV-TMBIM6 HA/GFP and AAV-Mock GFP expression in SN over forelimb use after 2-, 3-, 4-, and 5-weeks post-injection (wpi). The colored area shows treatment with 6-OHDA injuries in the CPu. Results are expressed as a percentage of contralateral forelimb use (n Mockl = 4 and n TMBIM6 = 5). D Beam test shows the effect of the AAV-TMBIM6 HA/GFP and AAV-Mock GFP expression in SN on balance and coordination after 2, 3, 4, and 5 weeks after injection. The colored area shows treatment with 6-OHDA injury in the CPu. Results are expressed as the number of paws slips (n Mockl = 4 and n TMBIM6 = 5). E Effect of the AAV-TMBIM6 HA/GFP and AAV-Mock GFP expression over time, animals used to complete the Beam test during 2-, 3-, 4-, and 5-wpi. The colored area shows treatment with 6-OHDA injury in the CPu. Results are expressed as the time in seconds to complete the test (n Mockl = 4 and n TMBIM6 = 5). All bars represent mean ± SEM. In all tests, two-way ANOVA followed by Tukey’s multiple comparison test was performed. 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: Using data on TMBIM6 mRNA expression levels from The Human Protein Atlas database (THPA) [ ], we confirmed through in silico analysis that TMBIM6 is expressed in various cortical regions of the human brain, with notably high expression in the midbrain and retina, two areas rich in DAergic cells [ , ] (Fig. ).

Techniques: In Vivo, Transduction, Expressing, Injection, Comparison