Journal: Nature Communications

Article Title: ER-mitochondria contacts mediate lipid radical transfer via RMDN3/PTPIP51 phosphorylation to reduce mitochondrial oxidative stress

doi: 10.1038/s41467-025-56666-4

Figure Lengend Snippet: a Schematic of the construct and strategy for detection of MERCs. b Schematic of the MERBiT system. c Representative images of V5-TOMM20-SmBiT and LgBiT−3×HA-Sec61β localization in HeLa cells stably expressing V5-TOMM20-SmBiT and LgBiT−3×HA-Sec61β (MERBiT cells). Cells were stained with V5, HA, HSP60 and calnexin antibodies. HSP60 is used as a mitochondrial marker and calnexin is used as an ER marker. d , Representative immunoblots for each component of MERBiT cells. The lysates of MERBiT cells were analyzed by immunoblotting for V5 (V5-TOMM20-SmBiT), HA (LgBiT-3×HA-Sec61β), TOMM20, HSP60, calnexin, and α-tubulin. Black and white arrowheads indicate tagged and endogenous TOMM20, respectively. e Luminescence of MERBiT cells. Quantification of the luminescence of HeLa cells, MERBiT cells, and stably expressing V5-TOMM20-SmBiT HeLa cells. Data are mean ± s.e.m. ( n = 9). f Quantification of MERCs reduction during recovery from starvation in MERBiT cells. Cells were starved in HBSS for 1 h and then recovered in 10% FBS DMEM for the indicated times before luminescence was measured. Data are mean ± s.e.m. ( n = 3, triplicate). g, h Effects of knockdown of different MERCs tethering factors on MERBiT luminescence in MERBiT cells. Cells were transfected with the indicated siRNAs and then luminescence was measured or WB was performed with the indicated antibodies to confirm protein expression levels. Data are mean ± s.e.m. ( n = 3, triplicate). i MERCs linker increases luminescence. MERBiT cells were transfected with MERCs linker (pCAG-AKAP1(1-30 aa)-mTagBFP-V5-SACM1L (521-587 aa)) and luminescence was detected. Data are mean ± s.e.m. ( n = 3, triplicate). Statistical significance was analyzed by one-way analysis of variance (ANOVA) (e, f, g) or Student’s t -test, Two-tailed (i). P values are indicated as; ** p < 0.01; **** p < 0.0001.

Article Snippet: For immunoblotting: Mouse VAPB antibody (66191-1-Ig, 1:1000), rabbit PDZD8 antibody (25512-1-AP, 1:1000), rabbit polyclonal anti-PTPIP51 (RMDN3) antibody (20641-1-AP, 1:1000) and rabbit monoclonal anti-TOMM20 antibody (11802-1-AP, 1:2000) were purchased from Proteintech.

Techniques: Construct, Stable Transfection, Expressing, Staining, Marker, Western Blot, Knockdown, Transfection, Two Tailed Test

Journal: Nature Communications

Article Title: ER-mitochondria contacts mediate lipid radical transfer via RMDN3/PTPIP51 phosphorylation to reduce mitochondrial oxidative stress

doi: 10.1038/s41467-025-56666-4

Figure Lengend Snippet: a RMDN3 and VAPB are critical tethering factors for MERCs formation induced by antimycin A and rotenone stimulation. MERBiT cells were transfected with the indicated siRNAs for 3 days and treated with or without rotenone (50 nM) and antimycin A (50 nM) for 1 h before luminescence measurements. Data are mean ± s.e.m. ( n = 3, triplicate). b Interaction between RMDN3 and VAPB increase in rotenone and antimycin A treatment. HeLa cells were transfected with the indicated vectors and treated with rotenone (50 nM) or antimycin A (50 nM) for 1 h. Cell lysates were subjected to IP assay (left). Ratio of RMDN3-VAPB interaction (right). Data are mean ± s.e.m. ( n = 3). c Schematic model of the RMDN3 domain. d The expression levels of RNAi-resistant RMDN3 vectors. The HeLa cells were transfected with RMDN3 siRNA for 2 days and then transfected with the indicated vectors such as RMDN3 RNAi-resistant vectors for 1 day. e FFAT but not TPR domain is important for mitochondrial ROS-induced MERCs formation. The MERBiT cells were transfected with the indicated siRNAs for 2 days. Then transfected with empty vectors or indicated RNAi-resistant vectors for 1 day. Before measuring luminescence, cells were treated with or without rotenone (50 nM) and antimycin A (50 nM) for 1 h. Data are mean ± s.e.m. ( n = 3, triplicate). f Threonine 160 mutant of RMDN3 decrease phosphorylation by antimycin A stimulation. HeLa cells were transfected with the indicated vectors and then treated with or without antimycin A (50 nM). Cell lysates were subjected to IP assay and then beads were incubated with or without lambda phosphatase (λPP). Pull-down lysates were subjected to Phos-tag-PAGE or SDS-PAGE. g Phosphorylation of RMDN3 T160 was important for interaction with VAPB by antimycin A treatment. HeLa cells were transfected with the indicated vectors and treated with antimycin A (50 nM) for 1 h. Cell lysates were subjected to IP assay and IB assay (left). Ratio of RMDN3-VAPB interaction (right). Data are mean ± s.e.m. ( n = 3). h Phosphorylation of RMDN3 T160 is important for MERCs formation induced by antimycin A stimulation. The MERBiT cells were transfected with the indicated siRNAs for 2 days and then transfected with vectors for 1 day before treatment with or without antimycin A (50 nM) for 1 h, and then the luminescence was measured. Data are mean ± s.e.m. ( n = 3, triplicate). Statistical significance was analyzed by one-way analysis of variance (ANOVA) ( a , b , e , g , h ). P values are indicated as * p < 0.05; ** p < 0.01; **** p < 0.0001; n.s., not significant.

Article Snippet: For immunoblotting: Mouse VAPB antibody (66191-1-Ig, 1:1000), rabbit PDZD8 antibody (25512-1-AP, 1:1000), rabbit polyclonal anti-PTPIP51 (RMDN3) antibody (20641-1-AP, 1:1000) and rabbit monoclonal anti-TOMM20 antibody (11802-1-AP, 1:2000) were purchased from Proteintech.

Techniques: Transfection, Expressing, Mutagenesis, Phospho-proteomics, Incubation, SDS Page

Journal: Nature Communications

Article Title: ER-mitochondria contacts mediate lipid radical transfer via RMDN3/PTPIP51 phosphorylation to reduce mitochondrial oxidative stress

doi: 10.1038/s41467-025-56666-4

Figure Lengend Snippet: a Rotenone or antimycin A treatment of RMDN3 and VAPB knockdown cells reduced cell viability. The HeLa cells were transfected with the indicated siRNAs for 5 days with or without rotenone (50 nM) and antimycin A (50 nM) for 2 days before cell viability was measured. Cell viability was determined and expressed as a fold change of si-NT. Viable cells were detected by cell viability assay using Cell Counting Kit-8. b The HeLa cells were transfected with the indicated siRNAs for 5 days with or without rotenone (50 nM) and antimycin A (50 nM) for 2 days before measuring cell viability. oxNAC (50 µM), NACS2 (50 µM) and mito-TEMPO (100 nM) were treated for 3 days before measuring cell viability. Cell viability was determined and expressed as a fold change of si-NT. Viable cells were detected by cell viability assay using Cell Counting Kit-8. c Lack of TPR domain does not rescue cell viability of RMDN3 knockdown with rotenone or antimycin A treatment. HeLa cells were transfected with RMDN3 siRNA for 5 days and with the indicated vectors for 3 days before measuring cell viability. Rotenone (50 nM) and antimycin A (50 nM) were treated for 2 days before cell viability was measured. Cell viability was determined and expressed as a fold change of si-NT. Viable cells were detected by cell viability assay using Cell Counting Kit-8. FLAG-resi-RMDN3 WT, FLAG-resi-RMDN3ΔFFAT, and FLAG-resi-RMDN3ΔTPR are RMDN3 RNAi-resistant vectors. d HeLa cells were transfected with RMDN3 siRNA for 5 days with or without rotenone (50 nM) and antimycin A (50 nM) for 2 days before measuring cell viability. The indicated inhibitors were treated for 2 days before measuring cell viability. Cell viability was determined and expressed as a fold change of si-NT. Viable cells were detected by cell viability assay using Cell Counting Kit-8. z-VAD-FMK (20 µM), necrostatin-1 (20 µM), ferrostatin-1 (Fer-1) (5 µM), and deferoxamine (DFO) (100 µM). Data are mean ± s.e.m. (n = 3, triplicate), and statistical significance was analyzed by one-way analysis of variance (ANOVA) (a-d). P values are indicated as **** p < 0.0001; n.s., not significant.

Article Snippet: For immunoblotting: Mouse VAPB antibody (66191-1-Ig, 1:1000), rabbit PDZD8 antibody (25512-1-AP, 1:1000), rabbit polyclonal anti-PTPIP51 (RMDN3) antibody (20641-1-AP, 1:1000) and rabbit monoclonal anti-TOMM20 antibody (11802-1-AP, 1:2000) were purchased from Proteintech.

Techniques: Knockdown, Transfection, Viability Assay, Cell Counting

Journal: Nature Communications

Article Title: ER-mitochondria contacts mediate lipid radical transfer via RMDN3/PTPIP51 phosphorylation to reduce mitochondrial oxidative stress

doi: 10.1038/s41467-025-56666-4

Figure Lengend Snippet: a, b Suppression of RMDN3 does not affect the induction of thermogenic genes. Cells were transfected with the indicated siRNAs and harvested at 0 or 4 days after differentiation. The cell lysates were analyzed by immunoblotting with the indicated antibodies ( a ). mRNA levels of differentiation markers were measured by qRT-PCR. Data were normalized to s18 mRNA and expressed relative to si-NT on day 4 ( b ). c Representative images of lipid droplets (LDs) in cells treated with the indicated siRNAs. Cells were fixed on day 4. The LDs and mitochondria were labeled with LipidTOX and anti-TOMM20 antibodies. The LDs and mitochondria were quantified for total LD area in ( d ), average LD size in ( e ), total mitochondrial area in ( f ), and the ratio of total LD area to total mitochondrial area in ( g ) from the ROI of ( c ). 2−3 cells from three independent experiments for the control and si-RMDN3#1 cells, respectively. Data are mean ± s.e.m. ( n = 3) h Lipid peroxide production increases with NE stimulation. MitoPeDPP (10 µM) was stained 30 min after stimulation with or without NE (1 µM) for 1 h and MitoPeDPP signals were detected in brown adipocytes (day 6). i, j RMDN3 and VAPB binding is increased by mitochondrial ROS generated under NE stimulation. Cell lysates were subjected to IP assay with anti-RMDN3 antibody and IB assay with the indicated antibodies ( j ). Ratio of RMDN3-VAPB interaction, plotted data for NE (1 µM) with or without Mito-TEMPO (10 µM) treatment versus control. Data are mean ± s.e.m. (n means three independent experiments). k Phosphorylation of RMDN3 by mitochondrial ROS and binding with VAPB is necessary for the suppression of lipid peroxide production. Cells were transfected with the indicated siRNAs and expressed human RMDN3 or human RMDN3 T160A before measuring MitoPeDPP fluorescence. NE was treated for 1 h. Data are mean ± s.e.m. (n means three independent experiments). Statistical significance was analyzed by one-way analysis of variance (ANOVA) ( b , j , k ) or Student’s t-test, Two-tailed ( d – h ). P values are indicated as * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; n.s., not significant.

Article Snippet: For immunoblotting: Mouse VAPB antibody (66191-1-Ig, 1:1000), rabbit PDZD8 antibody (25512-1-AP, 1:1000), rabbit polyclonal anti-PTPIP51 (RMDN3) antibody (20641-1-AP, 1:1000) and rabbit monoclonal anti-TOMM20 antibody (11802-1-AP, 1:2000) were purchased from Proteintech.

Techniques: Transfection, Western Blot, Quantitative RT-PCR, Labeling, Control, Staining, Binding Assay, Generated, Phospho-proteomics, Fluorescence, Two Tailed Test