Journal: Frontiers in Cell and Developmental Biology

Article Title: Mitofusins are required for specialized mitochondrial morphology and function of rod photoreceptor cells

doi: 10.3389/fcell.2026.1724328

Figure Lengend Snippet: Abnormal mitochondrial morphologies in rod photoreceptor inner segments and synapses due to ablation of Mfn1 and Mfn2 at 1 month of age. (A) Representative electron micrographs of rod photoreceptor inner segments. Magnification = ×8,800. Scale bar = 1 micron. (B) Higher-magnification images of the regions outlined by red rectangles in panel (A) . (C) Representative electron micrographs of rod photoreceptor synapses. Magnification = ×8,800. (D) Quantification of the number of mitochondria in rod photoreceptor inner segments. (E) Quantification of the number of mitochondria in the rod photoreceptor synapse. (F) Quantification of the size of mitochondria in the rod photoreceptor synapse. Dots represent individual data points. Number in the parenthesis denotes the number of mice used in the experiment. Data is presented as mean±SD. *P < 0.05, ****P < 0.0001 by one-way ANOVA with post hoc Tukey’s test. (G,H) Photoreceptor cell degeneration in mice with rod-specific ablation of Mfn1 and Mfn2. Representative images of H&E-stained retinal sections of 1-, 2- and 3-month-old mice. Magnification = ×40. Scale bar = 20 microns (G) . Outer nuclear layer thickness (ONLT) ratios (H) . Note that Rho-Cre/Mfn1 flx/flx /Mfn2 flx/flx mice exhibit significant reduction of ONLT at 2 and 3 months of age. Number in the parenthesis denotes the number of mice used in the study. Data is presented as mean±SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-way ANOVA with post hoc Tukey’s test.

Article Snippet: Rho-Cre mice (B6.Cg- Pde6b + Tg [Rho-icre]1Ck/Boc [JAX stock #015850, RRID: IMSR_JAX:015850]) , Mfn1 floxed mice ( Mfn1 flx/flx ; B6.129 [Cg]- Mfn1 tm2Dcc /J [JAX stock #026401, RRID: IMSR_JAX:026401]) , and Mfn2 floxed mice ( Mfn2 flx/flx ; B6.129 [Cg]- Mfn2 tm3Dcc /J [JAX stock #026525, RRID: IMSR_JAX:026525]) ( ) were purchased from The Jackson Laboratory.

Techniques: Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: Mitofusins are required for specialized mitochondrial morphology and function of rod photoreceptor cells

doi: 10.3389/fcell.2026.1724328

Figure Lengend Snippet: ERG analysis of Rho-Cre/Mfn1 flx/flx /Mfn2 flx/flx mice and age-matched WT controls. (A,B) Representative scotopic (rod-driven) ERG traces showing a- and b-waves (A) and c-waves (B) from 1-month-old WT (black) and Rho-Cre/Mfn1 flx/flx /Mfn2 flx/flx (blue) mice, and 2-month-old WT (yellow) and Rho-Cre/Mfn1 flx/flx /Mfn2 flx/flx (green) mice. (C–E) Quantification of scotopic ERG responses. Compared to WT controls, Rho-Cre/Mfn1 flx/flx /Mfn2 flx/flx mice exhibit a reduction in a-wave amplitude as early as 1 month of age (C) followed by a significant reduction in both a-wave and b-wave amplitude by 2 months (D) , and an age-dependent decline in c-wave amplitude (E) , consistent with progressive impairment of rod photoreceptor function and photoreceptor–RPE coupling. Number in the parenthesis denotes the number of mice used in the study. Data are presented as mean ± SEM, and statistical significance (*P < 0.05, **P < 0.01, ***P < 0.001) was determined using two-tailed Student’s t -tests.

Article Snippet: Rho-Cre mice (B6.Cg- Pde6b + Tg [Rho-icre]1Ck/Boc [JAX stock #015850, RRID: IMSR_JAX:015850]) , Mfn1 floxed mice ( Mfn1 flx/flx ; B6.129 [Cg]- Mfn1 tm2Dcc /J [JAX stock #026401, RRID: IMSR_JAX:026401]) , and Mfn2 floxed mice ( Mfn2 flx/flx ; B6.129 [Cg]- Mfn2 tm3Dcc /J [JAX stock #026525, RRID: IMSR_JAX:026525]) ( ) were purchased from The Jackson Laboratory.

Techniques: Two Tailed Test

Journal: Frontiers in Cell and Developmental Biology

Article Title: Mitofusins are required for specialized mitochondrial morphology and function of rod photoreceptor cells

doi: 10.3389/fcell.2026.1724328

Figure Lengend Snippet: Molecular pathways altered in the neural retina by rod-specific ablation of Mfn1 and Mfn2 . (A) Top10 enriched pathways were identified by Gene Set Enrichment Analysis (GSEA) and ranked by P value (pval) and adjusted P value (padj). NES stands for normalized enrichment score. (B) Overrepresentation analysis of the CEBPG_TARGET_GENES gene sets with the highest expression variation ratio. (C) Heatmap showing genes involved in mTORC1 signaling, amino acid (AA) metabolism, unfolded protein response (UPR), and C/EBPγ that are significantly enriched, and glycolysis-related genes that are significantly downregulated in the neural retina by rod-specific ablation of Mfn1 and Mfn2 . False discovery rate (FDR), logFC, and average expression (AvgExpr) are shown in the left column for each gene.

Article Snippet: Rho-Cre mice (B6.Cg- Pde6b + Tg [Rho-icre]1Ck/Boc [JAX stock #015850, RRID: IMSR_JAX:015850]) , Mfn1 floxed mice ( Mfn1 flx/flx ; B6.129 [Cg]- Mfn1 tm2Dcc /J [JAX stock #026401, RRID: IMSR_JAX:026401]) , and Mfn2 floxed mice ( Mfn2 flx/flx ; B6.129 [Cg]- Mfn2 tm3Dcc /J [JAX stock #026525, RRID: IMSR_JAX:026525]) ( ) were purchased from The Jackson Laboratory.

Techniques: Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Mitofusins are required for specialized mitochondrial morphology and function of rod photoreceptor cells

doi: 10.3389/fcell.2026.1724328

Figure Lengend Snippet: Metabolic changes in the neural retina resulting from rod-specific ablation of Mfn1 and Mfn2 . (A) Volcano plot showing differentially changed metabolites in the neural retina of Rho-Cre/Mfn1 flx/flx /Mfn2 flx/flx mice versus WT mice. (B) Heatmap showing significantly changed metabolites in the neural retina of Rho-Cre/Mfn1 flx/flx /Mfn2 flx/flx mice versus WT mice (P < 0.05). (C) Schematic diagram of pyrimidine and purine synthesis, and relative metabolite levels associated with nucleotide synthesis in Rho-Cre/Mfn1 flx/flx /Mfn2 flx/flx neural retina compared to WT neural retina. (D) Schematic diagram of glycolysis and TCA cycle, and relative metabolite levels associated with these pathways in Rho-Cre/Mfn1 flx/flx /Mfn2 flx/flx neural retina compared to WT neural retina. Data are presented as mean ± SD. Asterisks (*) indicate P < 0.05 significance by t-test. Five mice were used for each group in the study. Dots represent individual data points.

Article Snippet: Rho-Cre mice (B6.Cg- Pde6b + Tg [Rho-icre]1Ck/Boc [JAX stock #015850, RRID: IMSR_JAX:015850]) , Mfn1 floxed mice ( Mfn1 flx/flx ; B6.129 [Cg]- Mfn1 tm2Dcc /J [JAX stock #026401, RRID: IMSR_JAX:026401]) , and Mfn2 floxed mice ( Mfn2 flx/flx ; B6.129 [Cg]- Mfn2 tm3Dcc /J [JAX stock #026525, RRID: IMSR_JAX:026525]) ( ) were purchased from The Jackson Laboratory.

Techniques:

Journal: Frontiers in Cell and Developmental Biology

Article Title: Mitofusins are required for specialized mitochondrial morphology and function of rod photoreceptor cells

doi: 10.3389/fcell.2026.1724328

Figure Lengend Snippet: Identified changes in protein levels associated with pathways presumed to respond to mitochondrial fusion defects. (A) Schematic diagram of the glycolysis pathway of lactate synthesis from glucose through pyruvate in the cytosol of cells. (B) Western blot analysis of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), pyruvate kinase M2 (PKM2), and lactate dehydrogenase (LDH), which are related to glycolysis pathway in the neural retina of Rho-Cre/Mfn1 flx/flx /Mfn2 flx/flx mice versus WT mice. (C) Western blot analysis of each subunit comprising the complexes (Complex I (CI): NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 8 (NDUFB8), Complex II (CII): succinate dehydrogenase B (SDHB), Complex III (CIII): ubiquinol-cytochrome c reductase core protein 2 (UQCRC2), Complex IV (CIV): mitochondrially encoded cytochrome c oxidase I (MTCO1), Complex V (CV): ATP synthase F1 subunit alpha (ATP5A)) responsible for oxidative phosphorylation (OXPHOS). (D) Western blot analysis of another CII subunit, succinate dehydrogenase A (SDHA). (E) Schematic diagram of the substrate uptake and pathway toward mitochondrial β-oxidation. (F) Western blot analysis of carnitine-acylcarnitine translocase (CACT) and carnitine palmitoyl transferase II (CPT2), involved in mitochondrial β-oxidation. (G) Western blot analysis of mammalian target of rapamycin (mTOR) and phosphorylated-mTOR-S2448 (p-mTOR). Protein levels of p-mTOR were normalized by that of mTOR. Alpha-tubulin (TUB) served as the loading control for this Western blot experiments except for the result of p-mTOR. Data are presented as mean ± SD. Asterisks (*) indicates P < 0.05 significance following a significant difference detected by t-test. Six one-month-old mice were used in both groups in study. Dots represent individual data points. The protein size next to the immunoblot images denotes the size of the immunobands measured for this analysis.

Article Snippet: Rho-Cre mice (B6.Cg- Pde6b + Tg [Rho-icre]1Ck/Boc [JAX stock #015850, RRID: IMSR_JAX:015850]) , Mfn1 floxed mice ( Mfn1 flx/flx ; B6.129 [Cg]- Mfn1 tm2Dcc /J [JAX stock #026401, RRID: IMSR_JAX:026401]) , and Mfn2 floxed mice ( Mfn2 flx/flx ; B6.129 [Cg]- Mfn2 tm3Dcc /J [JAX stock #026525, RRID: IMSR_JAX:026525]) ( ) were purchased from The Jackson Laboratory.

Techniques: Western Blot, Phospho-proteomics, Control

Journal: Materials Today Bio

Article Title: A dual-responsive CO-releasing nanogel ameliorates retinal ischemia–reperfusion injury by restoring mitochondrial homeostasis and attenuating cGAS-STING pathway activation

doi: 10.1016/j.mtbio.2026.102974

Figure Lengend Snippet: COPN restores mitochondrial quality control and interrupts the ROS‒cGAS‒inflammation axis in OGD/R-treated R28 cells. a , TEM images showing changes in the mitochondrial ultrastructure of R28 cells subjected to various treatments (PBS, PDNs, COPN-L, and COPN-H) after OGD/R. Scale bar: 500 nm b , Quantitative analyses of mitochondrial length and number in R28 cells as indicated in a . c , Representative fluorescence images showing JC-1 staining of the mitochondrial Δψm: aggregates (red) indicate healthy mitochondria, whereas monomers (green) represent depolarized mitochondria. Nuclei were stained with DAPI (blue). Scale bar: 50 μm d , Intracellular ATP content assay revealing improved energy production after COPN treatment under OGD/R stress conditions. e , mRNA expression analysis of mitochondrial dynamic regulatory genes (Opa1, Mfn2, Drp1, and Fis1) and mitochondrial DNA transcription levels (mt-ND1 and mt-COX1). f , Quantitative analysis of the JC-1 fluorescence ratio (aggregates/monomers) and mitochondrial ROS levels (MitoSOX staining). g–h , Western blot analysis of proteins involved in mitochondrial autophagy (Pink1, Parkin, and P62) (g) and mitochondrial fusion/fission (Opa1, Mfn2, Mfn1, Drp1, and Fis1) (h) . i , Confocal fluorescence microscopy images of mitochondria (green, MitoTracker) and lysosomes (red, LysoTracker) in R28 cells after different treatments. Nuclei were stained with Hoechst (blue). Scale bar: 50 μm ∗ ∗∗∗P < 0.0001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The following primary antibodies were used: Drp1 (CST, #8570, 1:1000, ∼80 kDa), Fis1 (Proteintech, 10956-1-AP, 1:1000, ∼17 kDa), Mfn1 (Abcam, ab104274, 1:1000, ∼84 kDa), Mfn2 (CST, #9482, 1:1000, ∼86 kDa), Opa1 (CST, #80471, 1:1000, ∼100–120 kDa), Pink1 (CST, #6946, 1:1000, ∼63 kDa), cGAS (CST, #15102, 1:1000, ∼60 kDa), STING (CST, #13647, 1:1000, ∼42–45 kDa), TBK1 (CST, #3013, 1:1000, ∼84 kDa), p-TBK1 (CST, #5483, 1:1000, ∼84 kDa), COX IV (Abcam, ab14744, 1:2000, ∼17 kDa), β-actin (Proteintech, 66009-1-Ig, 1:5000, ∼43 kDa), and GAPDH (Proteintech, 60004-1-Ig, 1:5000, After washing, the membranes were incubated with HRP-conjugated secondary antibodies (goat anti-rabbit or goat anti-mouse IgG, CST, 1:5000) for 1 h at room temperature.

Techniques: Control, Fluorescence, Staining, Expressing, Western Blot, Microscopy

Journal: Cell Discovery

Article Title: Complement 5a receptor 2 attenuates diabetic kidney disease by promoting mitochondria-associated endoplasmic reticulum membrane formation mediated by PSS-MFN2 interaction

doi: 10.1038/s41421-026-00873-w

Figure Lengend Snippet: a MAM formation in TECs was analyzed via TEM imaging. Scale bars = 2 µm. The ER and mitochondria in the TEM images were graphically reconstructed to visualize MAM formation. Mito: mitochondria (orange), ER (red). b Quantification of the MAM length to the mitochondrial perimeter ratio in different groups of WT mice, n = 15 (150 mitochondria); C5ar2 −/− mice, n = 15 (193 mitochondria); diabetic WT mice, n = 15 (232 mitochondria); and diabetic C5ar2 −/− mice, n = 15 (175 mitochondria) microscopic fields from 5 mice/group. c Representative western blotting images of the specified proteins in the cytoplasm, mitochondrial fractions, ER fractions, and MAM fractions in renal cortex tissue from WT mice. d – f Representative western blotting images and quantitative analysis of PSS1 and PSS2 protein levels in the MAM fraction of renal cortex tissue from different groups of mice ( n = 3 per group). Total proteins stained with Ponceau S served as an internal reference for quantifying MAM proteins. d , g Representative western blotting images and quantitative analysis of calnexin and VDAC1 protein levels in the MAM fraction in renal cortex tissues from different groups of mice ( n = 3 per group). h Representative live-cell confocal microscopy images of ER–mitochondria contacts in TCMK-1 cells under different treatments. The ER was labeled with sec61β-GFP (green), the mitochondria were labeled with Mito Orange (red), and the nuclei were labeled with DAPI (blue). Z-stack images with orthogonal projections of the boxed regions are shown (scale bars = 10 µm). i Quantification of ER–mitochondria colocalization using Pearson’s correlation coefficient ( n = 15 microscopic fields from three independent experiments). j Representative in situ proximity ligation assay (PLA) images of TCMK-1 cells probed with IP3R1 and VDAC1 antibodies. The red dots indicate colocalization sites between IP3R1 and VDAC1. The cells were counterstained with DAPI (blue) (scale bars = 100 μm). k Quantitative analysis of PLA red spot counts in TCMK-1 cells subjected to different treatments. l Surface representation of the molecular docking between truncated MFN2 (Protein Data Bank code 6JFK) and PSS1. MFN2 (orange), PSS1 (blue). m Surface representation of the molecular docking between truncated MFN2 (6JFK) and PSS2. MFN2 (orange), PSS2 (red). n Schematic diagram of the structure of the MFN2 domain and truncation mutants. FL full-length, GTPase GTPase domain, HR1 heptad repeat 1, PR proline-rich domain, TM1/TM2 transmembrane domains 1 and 2, HR2 heptad repeat 2, Δ1/Δ2/Δ3 truncation mutants with the indicated deletions. o , p Co-IP analysis of PSS1–MFN2 and PSS2–MFN2 interactions using full-length and truncation mutants of MFN2 in HEK293T cells. q Representative confocal microscopy images of ER–mitochondria contacts in human kidney cortical proximal tubule epithelial cells (HK-2 cells) transfected with full-length or truncation mutants of MFN2 under HG/HF conditions (scale bars = 10 µm). r Quantification of ER–mitochondria colocalization in HK-2 cells ( n = 15 microscopic fields from three independent experiments). s Representative PLA images of Lv Pss2 ( Flag )-TCMK-1 cells probed with FLAG and MFN2 antibodies. Red spots indicate colocalization sites between FLAG and MFN2. The cells were counterstained with DAPI (blue) (scale bars = 100 μm). t Quantitative analysis of PLA red spot counts in Lv Pss2 -TCMK-1 cells subjected to different treatments. The data in the graphs are presented as the means ± SD. The data were analyzed by two-sided one-way ANOVA with Tukey’s test. ns not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: The membranes were blocked with 5% skim milk and then incubated with primary antibodies against C5aR2 (sc-515734; Santa Cruz Biotechnology), PSS1 (ab157222; Abcam), PSS2 (ARP49961_P050; Aviva Systems Biology Corporation), MFN2 (12186-1-AP; Proteintech), XBP-1s (143F; BioLegend), p-EIF2α (28740-1-AP; Proteintech), EIF2α (11170-1-AP; Proteintech), CHOP (15204-1-AP; Proteintech), COX IV (11242-1-AP; Proteintech), calnexin (10427-2-AP; Proteintech), ERK1/2 (4695; Cell Signaling Technology), p-ERK1/2 (4370; Cell Signaling Technology), HA (ab9110, Abcam), FLAG (ab213519, Abcam), α-tubulin (HRP-80762; Proteintech), and β-actin (Ac028; ABclonal).

Techniques: Imaging, Western Blot, Staining, Confocal Microscopy, Labeling, In Situ, Proximity Ligation Assay, Co-Immunoprecipitation Assay, Transfection

Journal: Cell Discovery

Article Title: Complement 5a receptor 2 attenuates diabetic kidney disease by promoting mitochondria-associated endoplasmic reticulum membrane formation mediated by PSS-MFN2 interaction

doi: 10.1038/s41421-026-00873-w

Figure Lengend Snippet: a Experimental scheme for assessing PSS2 overexpression. AAV9-Ksp- Pss2 was administered via tail vein (i.v.) injection at week 5, and the mice were sacrificed at week 20 for analysis ( n = 6 per group). b uACRs in different groups of mice ( n = 6 per group). c Representative images of PAS staining of the glomeruli and tubulointerstitium (scale bars = 50 µm); representative TEM micrographs (scale bars = 2 µm). MAM formation was analyzed via TEM imaging in TECs (scale bars = 5 µm). The ER and mitochondria in the TEM images were graphically reconstructed to visualize MAM formation. Mito (orange) and ER (red). d , e Quantitative analysis of mesangial matrix expansion ( d ) and the tubulointerstitial injury index ( e ) in different groups of mice. f , g Quantification of the GBM thickness ( f ) and foot process width ( g ) in different groups of mice ( n = 6 per group). h Quantification of the MAM length to mitochondrial perimeter ratio in different groups of mice: STZ/HFD-WT + OE Ctrl mice, n = 15 (with 167 mitochondria); STZ/HFD- C5ar2 −/− + OE Ctrl mice, n = 15 (with 272 mitochondria) microscopic fields from 5 mice/group; STZ/HFD-WT + OE PSS2 mice, n = 15 (with 193 mitochondria); STZ/HFD- C5ar2 −/− + OE PSS2 mice, n = 15 (with 220 mitochondria) microscopic fields from 6 mice/group. i The mitochondrial OCRs of LvNC and Lv Pss2 -TCMK-1 cells were analyzed in the presence or absence of HG/HF or C5ar2 knockdown. j , k Representative OCR tracings (from three independent experiments) and quantified OCR values are presented. l Representative PLA images of TCMK-1 cells probed with IP3R1 and VDAC1 antibodies. The red spots indicate sites of IP3R1 and VDAC1 colocalization. The cells were counterstained with DAPI (blue) (scale bars = 50 μm). m Quantitative analysis of PLA red spot counts in TCMK-1 cells subjected to different treatments. n Representative live-cell confocal microscopy images of ER–mitochondria contacts in TCMK-1 cells under different treatments. The ER was labeled with sec61β-GFP (green), the mitochondria were labeled with Mito Orange (red), and the nuclei were labeled with DAPI (blue). Z-stack images with orthogonal projections of the boxed regions are shown (scale bars = 5 µm). o Working model of how PSS interacts with MFN2 at the mitochondria–ER interface to maintain MAM formation and PS homeostasis. The data in the graphs are presented as the means ± SDs. The data were analyzed by two-sided one-way ANOVA with Tukey’s test. ns not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: The membranes were blocked with 5% skim milk and then incubated with primary antibodies against C5aR2 (sc-515734; Santa Cruz Biotechnology), PSS1 (ab157222; Abcam), PSS2 (ARP49961_P050; Aviva Systems Biology Corporation), MFN2 (12186-1-AP; Proteintech), XBP-1s (143F; BioLegend), p-EIF2α (28740-1-AP; Proteintech), EIF2α (11170-1-AP; Proteintech), CHOP (15204-1-AP; Proteintech), COX IV (11242-1-AP; Proteintech), calnexin (10427-2-AP; Proteintech), ERK1/2 (4695; Cell Signaling Technology), p-ERK1/2 (4370; Cell Signaling Technology), HA (ab9110, Abcam), FLAG (ab213519, Abcam), α-tubulin (HRP-80762; Proteintech), and β-actin (Ac028; ABclonal).

Techniques: Over Expression, Injection, Staining, Imaging, Knockdown, Confocal Microscopy, Labeling

Journal: Cell Discovery

Article Title: Complement 5a receptor 2 attenuates diabetic kidney disease by promoting mitochondria-associated endoplasmic reticulum membrane formation mediated by PSS-MFN2 interaction

doi: 10.1038/s41421-026-00873-w

Figure Lengend Snippet: a UMAP plot showing five subpopulations of PT cells in vehicle-treated m/m mice, vehicle-treated db/db mice, and P59-treated db/db mice ( n = 3 per group). Each dot corresponds to a single cell and is colored according to the cell type. b , c Violin plots showing Pss1 and Pss2 transcript expression levels of the indicated genes in PT cells from vehicle-treated m/m mice, vehicle-treated db/db mice, and P59-treated db/db mice. d Expression levels of Pss2 in each PT cluster revealed by scRNA-seq. e Representative western blotting images and quantitative analysis of PSS1 and PSS2 expression in renal cortex tissues from different groups of mice ( n = 6 per group). f MAM formation in TECs was analyzed via TEM imaging (scale bars = 2 µm). The ER and mitochondria in TEM images were graphically reconstructed to visualize MAM formation. Mito (orange) and ER (yellow). g Quantification of the MAM length to mitochondrial perimeter ratio in different groups of mice; m/m + vehicle group, n = 15 (with 261 mitochondria); m/m + P59 (3 mg/kg) group, n = 15 (with 270 mitochondria); db/db + vehicle group, n = 15 (with 192 mitochondria); db/db + P59 (3 mg/kg) group, n = 15 (with 235 mitochondria) microscopic fields from 6 mice/group. h – j Representative western blotting images and quantitative analysis of PSS1 and PSS2 protein levels in the MAM fraction in renal cortex tissues from different groups of mice ( n = 3 per group). Total proteins stained with Ponceau S served as an internal reference for quantifying MAM proteins. h , k Representative western blotting images and quantitative analysis of calnexin and VDAC1 protein levels in the MAM fraction in renal cortex tissues from different groups of mice ( n = 4 in the db/db + P59-3 mg/kg group; n = 3 in the other groups). l ELISA analysis of PS levels in the MAM fraction of renal tubules in different groups of mice ( n = 6 per group). m Representative live-cell confocal microscopy images of ER–mitochondria contacts in TCMK-1 cells under different treatments. The ER was labeled with sec61β-GFP (green), the mitochondria were labeled with Mito Orange (red), and the nuclei were labeled with DAPI (blue). Z-stack images with orthogonal projections of the boxed regions are shown (scale bars = 10 µm). n Representative PLA images of TCMK-1 cells probed with IP3R1 and VDAC1 antibodies. The red spots indicate sites of IP3R1 and VDAC1 colocalization. The cells were counterstained with DAPI (blue) (scale bars = 100 μm). o Quantitative analysis of PLA red spot counts in TCMK-1 cells subjected to different treatments. p Representative PLA images of Lv Pss2 ( Flag )-TCMK-1 cells probed with FLAG and MFN2 antibodies. Red spots indicate colocalization sites between FLAG and MFN2. The cells were counterstained with DAPI (blue) (scale bars = 100 μm). q Quantitative analysis of PLA red spot counts in Lv Pss2 ( Flag )-TCMK-1 cells subjected to different treatments. The data in the graphs are presented as the means ± SD. The data were analyzed by two-sided one-way ANOVA with Tukey’s test. ns not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: The membranes were blocked with 5% skim milk and then incubated with primary antibodies against C5aR2 (sc-515734; Santa Cruz Biotechnology), PSS1 (ab157222; Abcam), PSS2 (ARP49961_P050; Aviva Systems Biology Corporation), MFN2 (12186-1-AP; Proteintech), XBP-1s (143F; BioLegend), p-EIF2α (28740-1-AP; Proteintech), EIF2α (11170-1-AP; Proteintech), CHOP (15204-1-AP; Proteintech), COX IV (11242-1-AP; Proteintech), calnexin (10427-2-AP; Proteintech), ERK1/2 (4695; Cell Signaling Technology), p-ERK1/2 (4370; Cell Signaling Technology), HA (ab9110, Abcam), FLAG (ab213519, Abcam), α-tubulin (HRP-80762; Proteintech), and β-actin (Ac028; ABclonal).

Techniques: Single Cell, Expressing, Western Blot, Imaging, Staining, Enzyme-linked Immunosorbent Assay, Confocal Microscopy, Labeling