Journal: Nucleic Acids Research

Article Title: Ablation of Mto1 in zebrafish exhibited hypertrophic cardiomyopathy manifested by mitochondrion RNA maturation deficiency

doi: 10.1093/nar/gkab228

Figure Lengend Snippet: Western blotting analysis of mitochondrial proteins. ( A , C ) Twenty micrograms of total proteins from mutant and WT zebrafish were electrophoresed through a denaturing polyacrylamide gel, electroblotted and hybridized with antibodies for 5 subunits of OXPHOS (mitochondrion-encoding Nd1, Co2 and Atp8 and nucleus-encoding Sdha and Atp5a), Tufm, Tfam and Tom20 as a loading control. Quantification of levels of OXPHOS subunits ( B ) and other mitochondrial proteins ( D ). Average contents of Nd1, Co2, Atp8, Atp5a, Sdha, Tufm and Tfam were normalized to the average content of Tom20 in mutant and WT zebrafish. The values for the mutant zebrafish are expressed as percentages of the average values for the WT zebrafish. The calculations were based on three independent determinations. Graph details and symbols are explained in the legend to Figure .

Article Snippet: The antibodies used for this investigation were from Sigma [Mto1 (Sigma, HPA030232) and Gapdh (SAB2701826)], Abcam [Nd1 (ab74257), Sdha (ab151684), Atp5a (ab188107), Mtpap (ab154555) and Uqcrc2 (ab203832)] and Proteintech [Co2 (55070-1-AP), Atp8 (26723-1-AP), Ndufs1 (12444-1-AP), Cox5a (11448–1-AP), Atp5c (60284-1-Ig), Tfam (19998-1-AP), and Tufm (26730-1-AP) and Tom20 (1802-1-AP)].

Techniques: Western Blot, Mutagenesis

Journal: Nucleic Acids Research

Article Title: Ablation of Mto1 in zebrafish exhibited hypertrophic cardiomyopathy manifested by mitochondrion RNA maturation deficiency

doi: 10.1093/nar/gkab228

Figure Lengend Snippet: The synergic effects between Mto1 and Mtpap on the polyadenylation of mRNAs. ( A, B ) Immunoprecipitation analysis of MTO1 with MTPAP. HEK 293T cells transiently expressing with or without MTO1-FLAG were solubilized with a lysis buffer and lysate proteins were immuno-precipitated with immunocapture buffer (left) (input) and FLAG-antibody (right) (IP), respectively. Immunoprecipitates were analyzed by SDS-PAGE and Western blotting using anti-FLAG, anti-MTPAP and TOM20 antibodies, respectively. ( C ) Western blot analysis. Twenty micrograms of total proteins from mto1 −/− and WT zebrafish were electrophoresed through a denaturing polyacrylamide gel, electroblotted and hybridized with antibodies for Mtpap and Gapdh as a loading control. ( D ) Polyadenylation profiles of mitochondrial mRNAs upon mto1 −/− and WT zebrafish hearts. The 3′ termini of nd1, cytb , cox1 and cox3 mRNAs were assessed by RT-PCR amplifications from polyadenylated and oligoadenylated RNAs of mto1 −/− and WT zebrafish hearts and 3% agarose gel electrophoresis. Arrows indicated the positions of PCR products from the polyadenylated and oligoadenylated RNAs, respectively. ( E ) Quantification of poly(A) proportions of nd1, cytb , cox1 and cox3 transcripts in the WT and mto1 −/− zebrafish. ( F ) Poly(A) tail lengths from individually sequenced clones after 3’ end RACE analysis of nd1 transcripts in the mto1 −/− and WT zebrafish hearts. Graph details and symbols are explained in the legend to Figure .

Article Snippet: The antibodies used for this investigation were from Sigma [Mto1 (Sigma, HPA030232) and Gapdh (SAB2701826)], Abcam [Nd1 (ab74257), Sdha (ab151684), Atp5a (ab188107), Mtpap (ab154555) and Uqcrc2 (ab203832)] and Proteintech [Co2 (55070-1-AP), Atp8 (26723-1-AP), Ndufs1 (12444-1-AP), Cox5a (11448–1-AP), Atp5c (60284-1-Ig), Tfam (19998-1-AP), and Tufm (26730-1-AP) and Tom20 (1802-1-AP)].

Techniques: Immunoprecipitation, Expressing, Lysis, SDS Page, Western Blot, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Clone Assay

Journal: bioRxiv

Article Title: Lipid Dysregulation Unveil the Intricate Interplay of Lysosomal and Mitochondrial Changes in Frontotemporal Dementia with GRN Haploinsufficiency

doi: 10.1101/2024.01.22.576606

Figure Lengend Snippet: A. Protein Levels of LC3B-II and P62 Assessed by Western Blotting in Primary Human Fibroblasts. Primary human fibroblasts were cultured with or without a 6-hour STV pre-assay period and treated with CLQ at 30 µM for 5 hours. Western blotting was performed to assess the protein levels of LC3B-II and P62, and quantification are shown as bar plot graphs. B. Representative Images of LC3B Puncta (green) and DAPI (blue) Staining in Primary Human Fibroblasts. Primary human fibroblasts were treated with or without a 5-hour pre-assay period with CLQ at 30 µM. LC3B puncta (green) staining and DAPI (blue) staining were used to visualize autophagosomes and nuclei, respectively. Scale bar = 100 µm. C. Representative TEM images of primary human fibroblasts, showing autophagosomes (purple). Insets: Autophagosome. Scale bar is indicated in the figure. D. Protein Levels of LC3B-II and PINK1 Analyzed by Western Blotting in Cytoplasmic and Mitochondrial Fractions of Primary Human Fibroblasts. Primary human fibroblasts were treated with or without a 5-hour pre-assay period with CLQ at 30 µM. Western blotting was performed to analyze the protein levels of LC3B-II and PINK1 in both cytoplasmic and mitochondrial fractions. E. Representative Images of LC3B (green) and TOM20 (red) Staining in Primary Human Fibroblasts. Bar plot shows the quantification of the relative area of LC3B colocalized with TOM20. Scale bar = 10 µm. Healthy control fibroblasts(CTL), FTD-GRN patient fibroblast (GRN). Starving (STV), Chloroquine (CLQ), non treated (NT). Data are presented as means ± SEMs (n = 3). Statistical significance represented by *p < 0.05 and **p < 0.01, determined using Student’s t test.

Article Snippet: Primary Antibodies: PGRN (1μg/mL, AF2420, R&D systems), LAMP-1(1:10 000, ab25630, Abcam), LAMP-2 (1:1 000, #49067, Cell Signaling), LC3Bxp (1:1 000, #3868, Cell Signaling), SQSTM1/P62 (1:1 000, #5114, Cell Signaling), β-Tubulin (1:5 000, MA5-16308, Thermo Fisher) PINK1 (1:1 000, BC100-494, Novus Bio) TOM20 (1:1 000, 11802-1-AP, Proteintech), GAPDH (1:50 000, 60004-1-Ig, Proteintech), Lamin A+C (1:10 000, ab108595, Abcam), TDP-43 (1:1 000, ab104223, Abcam).

Techniques: Western Blot, Cell Culture, Staining

Journal: Theranostics

Article Title: TRPM2 protects against cisplatin-induced acute kidney injury and mitochondrial dysfunction via modulating autophagy

doi: 10.7150/thno.84655

Figure Lengend Snippet: TRPM2 deficiency aggravates cisplatin-induced acute kidney injury in mice. (A) Representative confocal images showing TRPM2 expression in the renal cortex of WT mice without cisplatin treatment. LTL, Lotus Tetragonolobus Lectin ; G, glomeruli. Scale bars, 100 μm. (B) Representative confocal images of tubular epithelial cells double-labeled TRPM2 and mitochondrial marker TOM20 (colocalization indicated by white arrows) . Scale bars, 10 μm. (C) Kidney function assessed by the serum creatinine level in Trpm2 -/- and WT mice following treatment with cisplatin (CP, 18 mg/kg) or normal saline (NS) for 72 h (n = 7). (D) Kidney function assessed by the serum creatinine level in WT mice pretreated with vehicle or 2-APB. (E, F) Representative PAS staining images and quantification of tubular injury score (n = 7). Scale bars, 100 μm. (G, H) Representative images of TUNEL staining and quantification of apoptotic cells (n = 7). Scale bars, 100 μm. (I) Immunoblotting analysis and quantification of cleaved and total caspase-3 (CASP3) and BAX. (J, K) Representative confocal images of Kim-1 staining and quantification of Kim-1 positive cells (n = 7). Scale bars, 100 μm. Data are presented as mean ± SEM. Statistical analysis was performed using one-way ANOVA with Tukey post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: For immunofluorescence, PFA-fixed frozen kidney sections or cells were permeabilized with 0.3% Triton X-100 for 20 min and blocked with 5% bovine serum albumin for 1 h. After washed with PBS, the slides were incubated with primary antibodies targeting Calnexin (1:100, Santa Cruz, sc-23954), kidney injury marker-1 (Kim-1, 1:500, R&D Systems, AF1817), LC3B (1:200, Sigma-Aldrich, L7543), lysosomal associated membrane protein 1 (LAMP1, 1:100, Santa Cruz, sc-19992), LAMP2 (1:500, Proteintech, 66301-1-Ig), TPRM2 (1:100, Abcam, ab240540; 1:100, Bethyl Laboratories, A300-414A), TOM20 (1:500, Proteintech, 11802-1-AP), Vimentin (1:200, Huabio, HA500437) or AQP-1 (1:100, Huabio, ET1703-34), respectively or in combination at 4 °C overnight, followed by incubation with corresponding secondary antibodies for 1 h. Brush borders of proximal tubules were labelled by Lotus tetragonolobus lectin (LTL, 1:200, Vector Laboratories, B-1325), in combination with Cy3-conjugated streptavidin (1:500, Vector Laboratories, SA-1300).

Techniques: Expressing, Labeling, Marker, Saline, Staining, TUNEL Assay, Western Blot

Journal: Theranostics

Article Title: TRPM2 protects against cisplatin-induced acute kidney injury and mitochondrial dysfunction via modulating autophagy

doi: 10.7150/thno.84655

Figure Lengend Snippet: TRPM2 deficiency exacerbates tubular mitochondrial fragmentation and oxidative damage in the kidneys of cisplatin-treated mice. (A-C) Representative TEM images showing tubular mitochondrial morphology in the kidneys of Trpm2 -/- and WT mice following treatment with cisplatin (CP, 18 mg/kg) or normal saline (NS) for 72 h and related analysis on mitochondrial area and aspect ratio. Scale bars, 1 μm. (D, E) Representative confocal images of DHE staining and quantification of DHE positive cells (n = 6). Scale bars, 100 μm. (F, G) Representative immunohistochemistry images of 8-OHdG staining and quantification of 8-OHdG positive cells (n = 6). Scale bars, 100 μm. (H) Relative mitochondrial DNA content calculated by the ratio of mitochondrial DNA (mtDNA) to nuclear DNA (nDNA) (n = 4). (I) Immunoblotting analysis and quantification of DRP1 and TOM20 in kidneys. (J) Immunoblotting analysis and quantification of cytochrome-c (Cyt-c) in the fractions of mitochondria and cytoplasm deprived of mitochondria. Cyto, cytoplasm; Mito, mitochondria. Data are presented as mean ± SEM. Statistical analysis was performed using one-way ANOVA with Tukey post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: For immunofluorescence, PFA-fixed frozen kidney sections or cells were permeabilized with 0.3% Triton X-100 for 20 min and blocked with 5% bovine serum albumin for 1 h. After washed with PBS, the slides were incubated with primary antibodies targeting Calnexin (1:100, Santa Cruz, sc-23954), kidney injury marker-1 (Kim-1, 1:500, R&D Systems, AF1817), LC3B (1:200, Sigma-Aldrich, L7543), lysosomal associated membrane protein 1 (LAMP1, 1:100, Santa Cruz, sc-19992), LAMP2 (1:500, Proteintech, 66301-1-Ig), TPRM2 (1:100, Abcam, ab240540; 1:100, Bethyl Laboratories, A300-414A), TOM20 (1:500, Proteintech, 11802-1-AP), Vimentin (1:200, Huabio, HA500437) or AQP-1 (1:100, Huabio, ET1703-34), respectively or in combination at 4 °C overnight, followed by incubation with corresponding secondary antibodies for 1 h. Brush borders of proximal tubules were labelled by Lotus tetragonolobus lectin (LTL, 1:200, Vector Laboratories, B-1325), in combination with Cy3-conjugated streptavidin (1:500, Vector Laboratories, SA-1300).

Techniques: Saline, Staining, Immunohistochemistry, Western Blot

Journal: Theranostics

Article Title: TRPM2 protects against cisplatin-induced acute kidney injury and mitochondrial dysfunction via modulating autophagy

doi: 10.7150/thno.84655

Figure Lengend Snippet: TRPM2 is required for cisplatin-evoked autophagy in vitro and in vivo . (A) Immunoblotting analysis and quantification of LC3B II and p62 in the kidneys of Trpm2 -/- and WT mice following treatment with cisplatin (CP, 18 mg/kg) or normal saline (NS) for 72 h. (B) Formation of autolysosomes in the kidneys evaluated by immunofluorescence double-labeled LC3B and LAMP1. Scale bars, 25 μm. Formation of mitolysosomes in the kidneys evaluated by immunofluorescence double-labeled TOM20 and LAMP1. Scale bars, 20 μm. Quantification of (C) autolysosomes and (D) mitolysosomes in the renal tubules (n = 4). (E) Immunoblotting analysis and quantification of LC3B II in Trpm2 -/- and WT MEFs treated with the indicated concentrations of CP for 24 h. (F) Immunoblotting analysis and quantification of LC3B II and p62 in Trpm2 -/- and WT MEFs incubated with 10 μM chloroquine (CQ) or vehicle for 1 h before treatment with 20 μM CP for 24 h. (G) Representative confocal images of LC3 puncta in Trpm2 -/- and WT MEFs expressing mRFP-GFP-LC3. Scale bars, 20 μm. (H) Quantification of autophagosomes (yellow dots, RFP + GFP + ) and autolysosomes (red dots, RFP + GFP - ) (n = 4). (I) Formation of mitolysosomes in Trpm2 -/- and WT MEFs evaluated by immunofluorescence double-labeled TOM20 and LAMP2. Scale bars, 20 μm. (J) Quantification of mitolysosomes in MEFs (n = 4). Data are presented as mean±SEM. Statistical analysis was performed using one-way ANOVA with Tukey post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: For immunofluorescence, PFA-fixed frozen kidney sections or cells were permeabilized with 0.3% Triton X-100 for 20 min and blocked with 5% bovine serum albumin for 1 h. After washed with PBS, the slides were incubated with primary antibodies targeting Calnexin (1:100, Santa Cruz, sc-23954), kidney injury marker-1 (Kim-1, 1:500, R&D Systems, AF1817), LC3B (1:200, Sigma-Aldrich, L7543), lysosomal associated membrane protein 1 (LAMP1, 1:100, Santa Cruz, sc-19992), LAMP2 (1:500, Proteintech, 66301-1-Ig), TPRM2 (1:100, Abcam, ab240540; 1:100, Bethyl Laboratories, A300-414A), TOM20 (1:500, Proteintech, 11802-1-AP), Vimentin (1:200, Huabio, HA500437) or AQP-1 (1:100, Huabio, ET1703-34), respectively or in combination at 4 °C overnight, followed by incubation with corresponding secondary antibodies for 1 h. Brush borders of proximal tubules were labelled by Lotus tetragonolobus lectin (LTL, 1:200, Vector Laboratories, B-1325), in combination with Cy3-conjugated streptavidin (1:500, Vector Laboratories, SA-1300).

Techniques: In Vitro, In Vivo, Western Blot, Saline, Immunofluorescence, Labeling, Incubation, Expressing

Journal: Theranostics

Article Title: TRPM2 protects against cisplatin-induced acute kidney injury and mitochondrial dysfunction via modulating autophagy

doi: 10.7150/thno.84655

Figure Lengend Snippet: TRPM2 regulates cisplatin-induced autophagy in kidneys via suppressing the AKT-mTOR signaling pathway. (A) PCA analysis on the RNA-sequencing data from cisplatin (CP)-treated Trpm2 -/- and WT mice (n = 3). (B, C) Heatmap and volcano plot of differentially expressed genes (|log2(FoldChange)| > 1, P value < 0.05). (D) Top 10 upregulated and downregulated Kyoto Encyclopedia of Genes and Genomes pathway enrichments. ARVC, Arrhythmogenic Right Ventricular Cardiomyopathy; TNF, tumor necrosis factor. (E) Gene Set Enrichment Analysis of PI3K-AKT-mTOR pathway generated from Trpm2 -/- and WT mice subjected to cisplatin. (F) Immunoblotting analysis and quantification of p-mTOR, mTOR, p-AKT, AKT in the kidneys of Trpm2 -/- and WT mice. (G) Immunoblotting analysis and quantification of LC3B II in the kidneys of CP-treated mice. The mice were either injected i.p. with 1 mg/kg rapamycin (Rapa) or vehicle 1 hour prior to and 1 day after CP treatment. (H) Immunoblotting analysis and quantification of LC3B II in the kidneys of CP-treated mice. The mice were injected i.p. with either 50 mg/kg VIII or vehicle 1 h prior to cisplatin injection. (I) Formation of autolysosomes evaluated by immunofluorescence double-labeled LC3B and LAMP1. Scale bars, 25 μm. Formation of mitolysosomes evaluated by immunofluorescence double-labeled TOM20 and LAMP1. Scale bars, 20 μm. Quantification of (J) autolysosomes and (K) mitolysosomes in the renal tubules (n = 6). Data are presented as mean±SEM. Statistical analysis was performed using one-way ANOVA with Tukey post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: For immunofluorescence, PFA-fixed frozen kidney sections or cells were permeabilized with 0.3% Triton X-100 for 20 min and blocked with 5% bovine serum albumin for 1 h. After washed with PBS, the slides were incubated with primary antibodies targeting Calnexin (1:100, Santa Cruz, sc-23954), kidney injury marker-1 (Kim-1, 1:500, R&D Systems, AF1817), LC3B (1:200, Sigma-Aldrich, L7543), lysosomal associated membrane protein 1 (LAMP1, 1:100, Santa Cruz, sc-19992), LAMP2 (1:500, Proteintech, 66301-1-Ig), TPRM2 (1:100, Abcam, ab240540; 1:100, Bethyl Laboratories, A300-414A), TOM20 (1:500, Proteintech, 11802-1-AP), Vimentin (1:200, Huabio, HA500437) or AQP-1 (1:100, Huabio, ET1703-34), respectively or in combination at 4 °C overnight, followed by incubation with corresponding secondary antibodies for 1 h. Brush borders of proximal tubules were labelled by Lotus tetragonolobus lectin (LTL, 1:200, Vector Laboratories, B-1325), in combination with Cy3-conjugated streptavidin (1:500, Vector Laboratories, SA-1300).

Techniques: RNA Sequencing, Generated, Western Blot, Injection, Immunofluorescence, Labeling

Journal: Theranostics

Article Title: TRPM2 protects against cisplatin-induced acute kidney injury and mitochondrial dysfunction via modulating autophagy

doi: 10.7150/thno.84655

Figure Lengend Snippet: mTOR inhibitor rapamycin alleviates cisplatin-induced kidney injury, cell apoptosis and mitochondrial dysfunction in Trpm2 -/- mice. (A) Kidney function assessed by serum creatinine level in cisplatin (CP, 18 mg/kg)-treated Trpm2 -/- and WT mice (n = 6). The mice were either injected i.p. with 1 mg/kg rapamycin (Rapa) or vehicle 1 hour prior to and 1 day after CP treatment. (B, C) Representative PAS staining images and quantification of tubular injury score (n = 6). Scale bars, 100 μm. (D, E) Representative images of TUNEL staining and quantification of apoptotic cells (n = 6). Scale bars, 100 μm. (F, G) Representative confocal images of Kim-1 staining and quantification of Kim-1 positive cells (n = 6). Scale bars, 100 μm. (H) Immunoblotting analysis and quantification of cleaved and total caspase-3 (CASP3). (I) Representative TEM images showing the mitochondrial morphology in the renal tubules. Scale bars, 1 μm. (J, K) Representative confocal images of DHE staining and quantification of DHE positive cells (n = 6). Scale bars, 100 μm. (L, M) Representative immunohistochemistry images of 8-OHdG staining and quantification of 8-OHdG positive cells (n = 6) . Scale bars, 100 μm. (N) Relative mitochondrial DNA content calculated by the ratio of mitochondrial DNA (mtDNA) to nuclear DNA (nDNA) (n = 4). (O) Immunoblotting analysis and quantification of DRP1 and TOM20. (P) Immunoblotting analysis and quantification of cytochrome-c (Cyt-c) in the fractions of mitochondria and cytoplasm deprived of mitochondria. Cyto, cytoplasm; Mito, mitochondria. Data are presented as mean±SEM. Statistical analysis was performed using one-way ANOVA with Tukey post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: For immunofluorescence, PFA-fixed frozen kidney sections or cells were permeabilized with 0.3% Triton X-100 for 20 min and blocked with 5% bovine serum albumin for 1 h. After washed with PBS, the slides were incubated with primary antibodies targeting Calnexin (1:100, Santa Cruz, sc-23954), kidney injury marker-1 (Kim-1, 1:500, R&D Systems, AF1817), LC3B (1:200, Sigma-Aldrich, L7543), lysosomal associated membrane protein 1 (LAMP1, 1:100, Santa Cruz, sc-19992), LAMP2 (1:500, Proteintech, 66301-1-Ig), TPRM2 (1:100, Abcam, ab240540; 1:100, Bethyl Laboratories, A300-414A), TOM20 (1:500, Proteintech, 11802-1-AP), Vimentin (1:200, Huabio, HA500437) or AQP-1 (1:100, Huabio, ET1703-34), respectively or in combination at 4 °C overnight, followed by incubation with corresponding secondary antibodies for 1 h. Brush borders of proximal tubules were labelled by Lotus tetragonolobus lectin (LTL, 1:200, Vector Laboratories, B-1325), in combination with Cy3-conjugated streptavidin (1:500, Vector Laboratories, SA-1300).

Techniques: Injection, Staining, TUNEL Assay, Western Blot, Immunohistochemistry

Journal: Theranostics

Article Title: TRPM2 protects against cisplatin-induced acute kidney injury and mitochondrial dysfunction via modulating autophagy

doi: 10.7150/thno.84655

Figure Lengend Snippet: TRPM2 protects against cisplatin-induced cell apoptosis and mitochondrial dysfunction in vitro by the regulation of autophagy via suppressing the AKT-mTOR signaling pathway. Trpm2 -/- MEFs or mRTECs were pre-incubated with AKT inhibitor VIII (5 μM) or mTOR inhibitor rapamycin (Rapa, 50 nM) for 1 h before cisplatin (CP) intervention. (A) Immunoblotting analysis and quantification of LC3B II, p-AKT and p-p70S6K in Trpm2 -/- and WT mRTECs following treatment with 5 μM CP for 24 h. (B) Representative confocal images of LC3 puncta in Trpm2 -/- and WT MEFs expressing mRFP-GFP-LC3 f ollowing treatment with 20 μM CP for 24 h . Scale bars, 20 μm. (C) Quantification of autophagosomes (yellow dots, RFP + GFP + ) and autolysosomes (red dots, RFP + GFP - ) (n = 4). (D, E) Representative confocal images and quantification of mitolysosomes evaluated by immunofluorescence double-labeled TOM20 and LAMP2 in mRTECs (n = 4). Scale bars, 20 μm. Then, Trpm2 -/- mRTECs and MEFs were pre-incubated with rapamycin (50 nM), Mito-TEMPO (Mito-T, 200 nM) or Rapamycin (50 nM) plus 3-MA (5 mM) for 1 h followed by intervention of 5 and 20 μM CP for 24 h, respectively. (F) CP-induced apoptosis in mRTECs determined by flow cytometry (n = 3). (G) Level of mitochondrial ROS in mRTECs determined by the mean fluorescent intensity (MFI) of Mito-SOX (n = 3). (H) Mitochondrial membrane potential of mRTECs indicated by the ratio of red to green fluorescence intensity of JC-1 (n = 3). (I) Mitochondrial morphology of MEFs detected by the fluorescence of MitoTracker Red. Scale bars, 20 μm. Data are presented as mean±SEM. Statistical analysis was performed using one-way ANOVA with Tukey post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: For immunofluorescence, PFA-fixed frozen kidney sections or cells were permeabilized with 0.3% Triton X-100 for 20 min and blocked with 5% bovine serum albumin for 1 h. After washed with PBS, the slides were incubated with primary antibodies targeting Calnexin (1:100, Santa Cruz, sc-23954), kidney injury marker-1 (Kim-1, 1:500, R&D Systems, AF1817), LC3B (1:200, Sigma-Aldrich, L7543), lysosomal associated membrane protein 1 (LAMP1, 1:100, Santa Cruz, sc-19992), LAMP2 (1:500, Proteintech, 66301-1-Ig), TPRM2 (1:100, Abcam, ab240540; 1:100, Bethyl Laboratories, A300-414A), TOM20 (1:500, Proteintech, 11802-1-AP), Vimentin (1:200, Huabio, HA500437) or AQP-1 (1:100, Huabio, ET1703-34), respectively or in combination at 4 °C overnight, followed by incubation with corresponding secondary antibodies for 1 h. Brush borders of proximal tubules were labelled by Lotus tetragonolobus lectin (LTL, 1:200, Vector Laboratories, B-1325), in combination with Cy3-conjugated streptavidin (1:500, Vector Laboratories, SA-1300).

Techniques: In Vitro, Incubation, Western Blot, Expressing, Immunofluorescence, Labeling, Flow Cytometry, Membrane, Fluorescence

Journal: medRxiv

Article Title: Atrial fibrillation is associated with decreased claudin-5 in cardiomyocyte

doi: 10.1101/2023.07.11.23292531

Figure Lengend Snippet: (A) Exemplar images of electrocardiogram (ECG) and apical four- chamber view of the heart in AF patient and control. Red box indicates left atrium (LA). (B) Left appendages of the LA was excised during the modified radiofrequency ablation maze procedure indicated in the right panel. Left panel indicates the traditional percutaneous radiofrequency ablation for AF. (C) Representative western blot of Cldn5 in left appendages of AF patients. Human lung tissue is used as positive control. (D) Exemplar left atrial appendage section stained with anti-Cldn5, CD31 and TOMM20 antibodies. Anti-Cldn5 primary antibody is Alexa Fluor 488 conjugated (Green) while CD31 and TOMM20 antibodies is visualized by Fluorescein donkey anti-rabbit IgG Alexa Fluor 594 (Red). DAPI is used to stain nucleus. Scale bar, 20 µm. (E) Quantification of immunofluorescent staining of Cldn5 in cardiomyocytes. (F) Exemplar left atrial appendage section stained with WGA for assessing cardiomyocyte’s cross section. Scale bar, 20 µm. (G) Quantification of cross-section of cardiomyocytes. * P < 0.05 vs. Non-AF; n = 5.

Article Snippet: Briefly, the human and mice heart tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sections (3–5μm thickness) were stained with primary antibodies for Cldn5-Alexa Fluor 488 (Invitrogen, Catalog#352588), CD31 (Proteintech, Catalog#11265-1-AP) or Tomm20 (Abcam, ab186734) for 1h at room temperature.

Techniques: Modification, Western Blot, Positive Control, Staining