Journal: bioRxiv

Article Title: A CHCHD10 variant causing ALS elicits an unfolded protein response through the IRE1/XBP1 pathway

doi: 10.1101/2020.05.05.078881

Figure Lengend Snippet: (A) Dot plot representing the fold change of the transcripts of mitochondrial integrated protein response. Fold change is represented in log2 scale. Large circles represent significant values (FDR<0.05), small circles show non-significant values (FDR>0.05). ‘Glucose’ (green) represents the comparison of patient versus rescue cells in glucose. ‘Galactose’ (red) represents the comparison of patient versus rescue cells in galactose. (B) Immunoblot analysis of the proteins involved in mitochondrial integrated stress response. Whole cell extracts from patient and rescue fibroblasts grown in glucose or galactose for 2 days were separated by SDS-PAGE and probed with antibodies against indicated proteins. SDHA was used as a loading control. Bar plot indicates the quantification of three (CLPP, HSP10, CAT), 4 (HTRA2, HSP60) immunoblot analyses, normalized to SDHA, where patient is depicted in dark grey and rescue in light grey, *p<0.05, **p<0.01, ***p<0.001. (C) Immunoblot analysis of the proteins involved in autophagy. Whole cell extracts from patient and rescue fibroblasts grown in glucose or galactose for 2 days were separated by SDS-PAGE and probed with antibodies against indicated proteins. SDHA was used as a loading control. Bar plot indicates the quantification of three (LC3-A, LC3-B, p62) immunoblot analyses, normalized to SDHA, where patient is depicted in dark grey and rescue in light grey, *p<0.05, **p<0.01, ***p<0.001. (D) Immunoblot analysis of the proteins involved in apoptosis. Whole cell extracts from patient and rescue fibroblasts grown in glucose or galactose for 2 days were separated by SDS-PAGE and probed with antibodies against indicated proteins. SDHA was used as a loading control. Bar plot indicates the quantification of three (cleaved PARP) and four (cleaved CC3) immunoblot analyses, normalized to SDHA, where patient is depicted in dark grey and rescue in light grey, *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Antibodies directed against the following proteins were used in this study: SDHA (abcam, ab14715), AMPK (Cell Signaling, 2532), p-AMPK (Thr172) (Cell Signaling, 2535), AKT (Cell Signaling, 9272S), p-AKT (Ser473) (Cell Signaling, 4060S), mTOR (Cell Signaling, 2983P), p-mTOR (Ser2448) (Cell Signaling, 5536P), S6 (Cell Signaling, 2217), p-S6 (Ser235/236) (Cell Signaling, 4858), PERK (Cell Signaling, 5683), p-PERK (Thr980) (Cell Signaling, 3191), GCN2 (Cell Signaling, 3302), p-GCN2 (Thr899) (Abcam, ab75836), eIF2α (Cell Signaling, 2103), p-eIF2α (Ser 51) (SIGMA, SAB4504388), GADD34/PPP1R15A (Abclonal, A17117), IRE1/ERN1 (Cell Signaling, 3294), BIP (abcam, ab21685), XBP1 (Santa Cruz, sc-7160), CHOP/DDIT3 (Cell Signaling, 5554), JNK (Cell Signaling, 9252), p-JNK/SAPK (T183/Y185) (Cell Signaling, 9251), FGF21 (abcam, ab171941), GDF15 (abcam, ab106006), CLPP (Proteintech, 15698-1-1AP), HTRA2 (Proteintech, 15775-1-AP), HSP60 (Santa Cruz, sc-136291), CAT (Abclonal, A11777), HSP10 (Santa Cruz, SC-376313), LC3 (Cell Signaling, D3U4C), SQSTM1/p62 (Cell Signaling, 8025), PARP (Cell Signaling, 9542), cleaved PARP (Cell Signaling, 9541), cleaved caspase 3 (Cell Signaling, 9661S).

Techniques: Comparison, Western Blot, SDS Page, Control

Journal: Cell Death Discovery

Article Title: CTRP3 alleviates mitochondrial dysfunction and oxidative stress injury in pathological cardiac hypertrophy by activating UPRmt via the SIRT1/ATF5 axis

doi: 10.1038/s41420-024-01813-x

Figure Lengend Snippet: A – D Representative western blotting and quantification of ATF5, ClpP, Lonp1, HSP10, and HSP60 protein levels in heart tissues ( A and C ) and NRCMs ( B and D ) in response to different treatments. GAPDH served as the loading control ( n = 4). Data were analyzed by one-way ANOVA and presented as mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; ns not significant.

Article Snippet: Membranes were then incubated with the following diluted primary antibodies: anti-SIRT1 (1:1000, 21535-1-AP), anti-β-MHC (1:1000, 22280-1-AP), anti-ANP (1:1000, 27426-1-AP), anti-collagen-3 (1:1000, 22734-1-AP), anti-Cyt-b (1:1000, 55090-1-AP), anti-TOMM20 (1:1000, 11802-1-AP), anti-ClpP (1:1000, 15698-1-AP), and anti-Lonp1 (1:1000, 15440-1-AP) purchased from Proteintech (Wuhan, China); anti-α-SMA (1:5000, ab7817), anti-MTCO1 (1:1000, ab14705), and anti-ATF5 (1:2000, ab184923) purchased from ABCAM (Cambridge, UK); anti-GAPDH (1:5000, AT0002) purchased from Engibody (WI, USA); anti-HSP10 (1:1000, A5580) purchased from ABclonal (Wuhan, China); anti-HSP60 (1:1000, 4870s) purchased from CST (MA, USA); and anti-OPA1(1:1000, 612606) purchased from BD Biosciences (NY, USA) at 4 °C overnight.

Techniques: Western Blot

Journal: Cell Death Discovery

Article Title: CTRP3 alleviates mitochondrial dysfunction and oxidative stress injury in pathological cardiac hypertrophy by activating UPRmt via the SIRT1/ATF5 axis

doi: 10.1038/s41420-024-01813-x

Figure Lengend Snippet: A Representative western blotting of ATF5 protein level in the heart tissues of ATF5-KO mice. α-Tubulin served as a loading control. B Top panel, representative echocardiographic images 4 weeks after TAC surgery. Bottom two panels, LVEF and LVFS determined by analyzing echocardiographic images ( n = 10). C Left panel, representative images of heart sections stained with HE and Masson’s trichrome. Right panel, quantification of LV cross-sectional area and left ventricular collagen volume ( n = 5, 10–15 random fields were analyzed per sample). D Representative western blotting and quantification of β-MHC, ANP, collagen-1, and α-SAM protein levels in heart tissues. GAPDH served as a loading control ( n = 4). E Representative western blotting and quantification of ATF5, ClpP, Lonp1, HSP10, and HSP60 protein levels in heart tissues. GAPDH served as a loading control ( n = 4). F Top panel, representative electron microscope images of cardiomyocyte mitochondria in heart tissues. Bottom two panels, quantification of mitochondria density, cristae/mitochondrial area in heart tissues ( n = 5, 10–15 random fields were analyzed per sample). G Quantification of ATP levels in heart tissues ( n = 5). H Left panel, representative fluorescence images of cardiac sections stained with DHE (red) to indicate ROS levels; nuclei were stained with DAPI (blue). Right panel, relative quantification of ROS fluorescence intensity in heart tissues ( n = 6, 10–15 random fields were analyzed per sample). I Quantification of MDA levels in heart tissues ( n = 10). J Quantification of SOD activities in heart tissues ( n = 10). Data were analyzed by one-way ANOVA and presented as mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; ns not significant, HE hematoxylin and eosin, MDA malondialdehyde, SOD superoxide dismutase, TAC transverse aortic constriction.

Article Snippet: Membranes were then incubated with the following diluted primary antibodies: anti-SIRT1 (1:1000, 21535-1-AP), anti-β-MHC (1:1000, 22280-1-AP), anti-ANP (1:1000, 27426-1-AP), anti-collagen-3 (1:1000, 22734-1-AP), anti-Cyt-b (1:1000, 55090-1-AP), anti-TOMM20 (1:1000, 11802-1-AP), anti-ClpP (1:1000, 15698-1-AP), and anti-Lonp1 (1:1000, 15440-1-AP) purchased from Proteintech (Wuhan, China); anti-α-SMA (1:5000, ab7817), anti-MTCO1 (1:1000, ab14705), and anti-ATF5 (1:2000, ab184923) purchased from ABCAM (Cambridge, UK); anti-GAPDH (1:5000, AT0002) purchased from Engibody (WI, USA); anti-HSP10 (1:1000, A5580) purchased from ABclonal (Wuhan, China); anti-HSP60 (1:1000, 4870s) purchased from CST (MA, USA); and anti-OPA1(1:1000, 612606) purchased from BD Biosciences (NY, USA) at 4 °C overnight.

Techniques: Western Blot, Staining, Microscopy, Fluorescence

Journal: Cell Death Discovery

Article Title: CTRP3 alleviates mitochondrial dysfunction and oxidative stress injury in pathological cardiac hypertrophy by activating UPRmt via the SIRT1/ATF5 axis

doi: 10.1038/s41420-024-01813-x

Figure Lengend Snippet: A Representative western blotting of SIRT1 protein level in the heart tissues of SIRT1-KO mice. GAPDH serves as a loading control. B Top panel, representative echocardiographic images 4 weeks after TAC surgery. Bottom two panels, LVEF and LVFS determined by analyzing echocardiographic images ( n = 10). C Left panel, representative images of heart sections stained with HE and Masson’s trichrome. Right panel, quantification of left ventricular cross-sectional area and collagen volume ( n = 5, 10–15 random fields were analyzed per sample). D Representative western blotting and quantification of β-MHC, ANP, collagen-1, and α-SAM protein levels in heart tissues. GAPDH served as a loading control ( n = 4). E Representative western blotting and quantification of ATF5, ClpP, Lonp1, HSP10, and HSP60 protein levels in heart tissues. GAPDH served as a loading control ( n = 4). F Top panel, representative electron microscope images of a cardiomyocyte mitochondrion of the heart. Bottom two panels, quantification of mitochondria density, cristae/mitochondrial area in heart tissues ( n = 5, 10–15 random fields were analyzed per sample). G Quantification of ATP levels in heart tissues ( n = 5). H Left panel, representative fluorescence images of cardiac sections stained with DHE (red) to indicate ROS levels; nuclei were stained with DAPI (blue). Right panel, relative quantification of ROS fluorescence intensity in heart tissues ( n = 6, 10–15 random fields were analyzed per sample). I Quantification of MDA levels in heart tissues ( n = 10). J Quantification of SOD activities in heart tissues ( n = 10). Data were analyzed by one-way ANOVA and presented as mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; ns not significant, LVEF left ventricular ejection fraction, LVFS left ventricular fraction shortening, MDA malondialdehyde, TAC transverse aortic constriction.

Article Snippet: Membranes were then incubated with the following diluted primary antibodies: anti-SIRT1 (1:1000, 21535-1-AP), anti-β-MHC (1:1000, 22280-1-AP), anti-ANP (1:1000, 27426-1-AP), anti-collagen-3 (1:1000, 22734-1-AP), anti-Cyt-b (1:1000, 55090-1-AP), anti-TOMM20 (1:1000, 11802-1-AP), anti-ClpP (1:1000, 15698-1-AP), and anti-Lonp1 (1:1000, 15440-1-AP) purchased from Proteintech (Wuhan, China); anti-α-SMA (1:5000, ab7817), anti-MTCO1 (1:1000, ab14705), and anti-ATF5 (1:2000, ab184923) purchased from ABCAM (Cambridge, UK); anti-GAPDH (1:5000, AT0002) purchased from Engibody (WI, USA); anti-HSP10 (1:1000, A5580) purchased from ABclonal (Wuhan, China); anti-HSP60 (1:1000, 4870s) purchased from CST (MA, USA); and anti-OPA1(1:1000, 612606) purchased from BD Biosciences (NY, USA) at 4 °C overnight.

Techniques: Western Blot, Staining, Microscopy, Fluorescence

Journal: Cell Death Discovery

Article Title: CTRP3 alleviates mitochondrial dysfunction and oxidative stress injury in pathological cardiac hypertrophy by activating UPRmt via the SIRT1/ATF5 axis

doi: 10.1038/s41420-024-01813-x

Figure Lengend Snippet: A – D Representative western blotting analysis and quantification of SIRT1, ATF5, ClpP, Lonp1, HSP10, and HSP60 protein levels in NRCMs. GAPDH served as the loading control ( n = 4). Data were analyzed by one-way ANOVA and presented as mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; ns not significant, NRCM neonatal rat cardiomyocyte.

Article Snippet: Membranes were then incubated with the following diluted primary antibodies: anti-SIRT1 (1:1000, 21535-1-AP), anti-β-MHC (1:1000, 22280-1-AP), anti-ANP (1:1000, 27426-1-AP), anti-collagen-3 (1:1000, 22734-1-AP), anti-Cyt-b (1:1000, 55090-1-AP), anti-TOMM20 (1:1000, 11802-1-AP), anti-ClpP (1:1000, 15698-1-AP), and anti-Lonp1 (1:1000, 15440-1-AP) purchased from Proteintech (Wuhan, China); anti-α-SMA (1:5000, ab7817), anti-MTCO1 (1:1000, ab14705), and anti-ATF5 (1:2000, ab184923) purchased from ABCAM (Cambridge, UK); anti-GAPDH (1:5000, AT0002) purchased from Engibody (WI, USA); anti-HSP10 (1:1000, A5580) purchased from ABclonal (Wuhan, China); anti-HSP60 (1:1000, 4870s) purchased from CST (MA, USA); and anti-OPA1(1:1000, 612606) purchased from BD Biosciences (NY, USA) at 4 °C overnight.

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Circulating Heat Shock Protein 60 Levels Are Elevated in HIV Patients and Are Reduced by Anti-Retroviral Therapy

doi: 10.1371/journal.pone.0045291

Figure Lengend Snippet: (A) Plasma Hsp10 levels in 20 HIV patients before and after cART, and Hsp10 levels in samples from 23 HIV-negative individuals, as measured by an in-house Hsp10 capture ELISA. Statistical comparisons between groups by Kolmogorov-Smirnov test. (HIV negative samples were obtained from both plasma and commercially available serum samples; Hsp10 levels were not significantly different in plasma and serum – ). (B) Hsp10 levels (the same data as shown in A) with data from each patient treated as a matched pair and represented by a single line. Statistics by Wilcoxon matched-pair signed-rank test.

Article Snippet: The assay used anti-Hsp10 goat polyclonal antibody (R&D Systems, Minneapolis, MN, USA) as both the immobilised capture and detection antibodies, and was optimised and validated by spiking plasma and serum with undetectable Hsp10 levels with recombinant Hsp10 .

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay