Fig 1: SLC22A14 is localized at the inner membrane of spermatozoa mitochondria(A) SLC22A14 subcellular co-localization with mito-tracker red in HEK293T cells overexpressing the EGFP-SLC22A14 fusion protein.(B and C) Identification of hSLC22A14 (B) and mSlc22a14 (C) in mitochondria from HEK-hSLC22A14 and HEK-mSlc22a14 cells. The MS spectra of representative peptides are presented.(D) Identification of mSlc22a14 in mitochondria isolated from spermatozoa. The MS characteristics of representative peptides are presented.(E) Immunofluorescent staining of Slc22a14 in epididymal spermatozoa showed a mid-piece localization of Slc22a14. Blue, DAPI; red, Slc22a14 antibody.(F) Super resolution confocal images of mitochondria isolated from HEK293T cells overexpressing a FLAG-SLC22A14 fusion protein. The FLAG-SLC22A14 fusion protein was labeled with an anti-FLAG-tag antibody conjugated with Alexa Fluor 488 (green); inner membranes were labeled with an anti-COX-IV antibody and visualized with a secondary antibody conjugated to Alexa Fluor 594 (red); outer membranes were labeled with an anti-TOMM20 antibody and visualized with a secondary antibody conjugated to Alexa Fluor 647 (blue).(G) Plot profile of the COX-IV, FLAG-tag, and TOMM20 fluorescence intensity along the axis (line with arrow in F).(H) Super resolution confocal images of single mitochondria in HEK293T cells. FLAG-SLC22A14 fusion protein was labeled with an anti-FLAG-tag antibody conjugated with Alexa Fluor 488 (green); IMM was labeled with an anti-COX-IV antibody and visualized with a secondary antibody conjugated to Alexa Fluor 594 (red); outer mitochondrial membranes were labeled with anti-TOMM20 antibody and visualized with a secondary antibody conjugated to Alexa Fluor 647 (blue).(I) Plot profile of the COX-IV, FLAG-tag, and TOMM20 fluorescence intensity along the axis (line with arrow in H).See also Figures S5–S7.
Fig 2: Schematic representation showing SARS-CoV-2-induced disruption of mitochondrial homeostasis. Upon infection, SARS-CoV-2 releases single strand RNA known to replicate in DMV structures. Tom20 facilitates the entry process of viral RNA into mitochondria, resulting in mitochondrial dysfunction, including the loss of ΔΨm, MPTP opening and increased ROS release. Concomitantly, mitophagy is initiated through Pink1/Parkin pathway by host cell for mitochondrial quality control and virus clearance. However, SARS-CoV-2 hinders the binding of p62 to LC3 protein, thus inhibiting the p62-labeled mitochondria to be encapsulated by autophagosomes. Mitophagy stays in the early stage. This figure is created with BioRender.com.
Fig 3: NOX2-dependent accumulation of PTM forms of α-synuclein.(A) Confocal analysis of 4-HNE-α-syn adducts, detected as 4-HNE–α-syn PL signal, in pVMB neurons. Cells exposed to rotenone for 24 h showed significant increase of 4-HNE–α-syn, which was prevented by co-treatment with Nox2ds-tat but not Scr.Nox2ds-tat, indicating a role for NOX2 in the formation of this highly aggregable form of α-syn. (B) Quantification of PL 4-HNE–α-syn signal. Symbols represent the normalized means of the intensities (with vehicle set to 100) analyzed for each independent experiment (100–150 neurons/treatment group per experiment). Statistical analysis was performed as one-way ANOVA with Bonferroni’s correction (n = 3 independent experiments). ***denotes p < 0.0001 significance compared vehicle; ###denotes p < 0.0001 significance compared to rotenone. (C) Rotenone induced interaction of α-syn with TOM20 is prevented by NOX2 inhibition, consistent with the involvement of NOX2 in the cellular accumulation of posttranslational forms of α-syn able to interfere with TOM20- mediated protein import. (D) Quantification of the PL TOM20–α-syn signal. Symbols represent the normalized means of the intensities (with vehicle set to 100) analyzed for each independent experiment (100–150 neurons/treatment group per experiment). Statistical analysis was performed by one-way ANOVA with Bonferroni’s correction (n = 3 independent experiments). ***denotes p < 0.0001 significance compared to vehicle; ###denotes p < 0.0001 significance compared to rotenone.
Fig 4: CB2 is upregulated in aged kidneys. (A) Representative micrographs showing CB2 expression in kidneys from 2‐month‐old and 24‐month‐old mice. Cryosections were subjected to fluorescence in situ hybridization (FISH) staining for CB2. Arrow indicates positive staining. scale bar, 25 μm. (B‐E) Representative Western blot and quantitative data showing renal expression of CB2 from 2‐month‐old and 24‐month‐old mice (B and C) or mice which were administered subcutaneous injections of d‐gal at 150mg/kg/day for 6 weeks (D and E). Numbers (1–5) indicate each individual animal in a given group. **p < 0.01 versus 2‐month‐old mice group or the sham control group (n = 5). (F) Representative images showing renal expression of CB2 in d‐gal‐treated mice. Cryosections were subjected to fluorescence in situ hybridization (FISH) staining for CB2. Arrow indicates positive staining. scale bar, 25 μm. (G) Representative micrographs showing the colocalization of CB2 and various segment‐specific tubular markers in kidneys. Frozen kidney sections were stained for CB2 (red) using FISH and various segment‐specific tubular markers (green) by immunofluorescence. The following segment‐specific tubular markers were used: proximal tubule, lotus tetragonolobus lectin (LTL); distal tubule, peanut agglutinin (PNA); arrows indicate positive tubules with colocalization of CB2 and specific tubular markers. Scale bar, 25 μm. (H) Representative micrographs showing the expression of CB2 and TOMM20 in tubules in 2‐month‐old and 24‐month‐old mice. Cryosections were subjected to FISH staining of CB2 (red) and stained with TOMM20 (green) antibody by immunofluorescence. Arrows indicate positive staining. Scale bar, 25μm
Fig 5: CB2 deficiency protects renal mitochondrial homeostasis in the accelerated ageing mice. (A) Representative micrographs showing renal expression of PGC‐1α and TOMM20 in different groups. Paraffin‐embedded kidney sections were immunostained with an antibody against PGC‐1α or TOMM20. Arrows indicate positive staining. Scale bar, 50 μm. (B‐C) Quantitative data showing quantification of positive staining. *p < 0.05, ***p < 0.001 versus WT mice group alone; # p < 0.05, ### p < 0.001 versus the d‐gal‐treated WT mice group alone (n = 5–6). (D) Representative graph showing the production of adenosine triphosphate (ATP) in different groups. *p < 0.05 versus WT mice group alone; ## p < 0.01 versus the d‐gal‐treated WT mice group alone (n = 5–6). (E–H) Representative Western blot and quantitative data showing renal expression of PGC‐1α, TOMM20 and Cytb. Numbers (1–3) indicate each individual animal in a given group. *p < 0.05, **p < 0.01, ***p < 0.001 versus the WT mice group alone; # p < 0.05, ### p < 0.001 versus the d‐gal‐treated WT mice group alone (n = 5–6). (I) Representative transmission electron microscopy graphs showing mitochondrial ultrastructure of renal tubular cells in different groups. Arrows indicate damaged and abnormal‐shaped mitochondria. Scale bar, 1μm
Supplier Page from Abcam for Anti-TOMM20 antibody [EPR15581-54] - Mitochondrial Marker