aav virus packaging  (Genechem)

Journal: International Journal of Biological Sciences

Article Title: SIRT5-related lysine demalonylation of GSTP1 contributes to cardiomyocyte pyroptosis suppression in diabetic cardiomyopathy

doi: 10.7150/ijbs.83306

Figure Lengend Snippet: Primers for the RT-qPCR

Article Snippet: Adeno-associated virus (AAV) packaged with short hairpin RNA (shRNA) targeting SIRT5 (AAV-shSIRT5), AAV-shGSTP1, AAV-SPI1, AAV-SIRT5, and control viruses (AAV-NC or AAV-shNC) with a mouse α-cardiac myosin heavy chain promoter were acquired from VectorBuilder (Guangzhou, Guangdong, China) with a viral titer of 2 × 10 11 GC/mL.

Techniques:

Journal: International Journal of Biological Sciences

Article Title: SIRT5-related lysine demalonylation of GSTP1 contributes to cardiomyocyte pyroptosis suppression in diabetic cardiomyopathy

doi: 10.7150/ijbs.83306

Figure Lengend Snippet: The significance of SIRT5 expression on DCM-related myocardial injury. (A) Strategy for SIRT5 KO mice. (B) Experimental procedure for mice. (C) Changes in FBG levels were assayed every 3 weeks. (D) Changes in body weight were assayed every 3 weeks. (E) M-mode echocardiogram of mice. (F) EF (%) and FS (%) of mice. (G) IVRT of mice. (H) Inflammatory infiltration in mouse myocardial tissues using HE staining. (I) Collagen deposition in myocardial tissues using Masson's staining. (J) The expression of SIRT1, SIRT3, SIRT4, SIRT5, and the Mal-Lys modification of the protein in mouse myocardial tissues using Western blot. All data are expressed as means ± SD (n = 10). *p < 0.05 vs. control group; #p < 0.05 vs. DCM (WT) group. Continuous data were compared by one-way/two-way ANOVA.

Article Snippet: Adeno-associated virus (AAV) packaged with short hairpin RNA (shRNA) targeting SIRT5 (AAV-shSIRT5), AAV-shGSTP1, AAV-SPI1, AAV-SIRT5, and control viruses (AAV-NC or AAV-shNC) with a mouse α-cardiac myosin heavy chain promoter were acquired from VectorBuilder (Guangzhou, Guangdong, China) with a viral titer of 2 × 10 11 GC/mL.

Techniques: Expressing, Staining, Modification, Western Blot, Control

Journal: International Journal of Biological Sciences

Article Title: SIRT5-related lysine demalonylation of GSTP1 contributes to cardiomyocyte pyroptosis suppression in diabetic cardiomyopathy

doi: 10.7150/ijbs.83306

Figure Lengend Snippet: SIRT5 deficiency deteriorates HG-induced cell damage. (A) Immunofluorescence staining of α-Sarcometric actin in primary cardiomyocytes extracted from WT and KO mice. (B) SIRT5 expression in WT and KO cardiomyocytes was examined using Western blot. (C) The viability of WT and KO cardiomyocytes was examined using CCK-8 assay. (D) The apoptosis of WT and KO cardiomyocytes was measured using flow cytometry. (E) Assessment of intracellular ROS levels by DCFH-DA staining. (F) WT and KO cardiomyocyte senescence was detected using β-galactosidase staining. (G) The release of pro-inflammatory factors IL-6 and TNF-α from WT and KO cardiomyocytes using ELISA. All data are expressed as means ± SD (n = 3). *p < 0.05 vs. WT:NG or KO:NG group; #p < 0.05 vs. WT:HG group. Continuous data were compared by two-way ANOVA.

Article Snippet: Adeno-associated virus (AAV) packaged with short hairpin RNA (shRNA) targeting SIRT5 (AAV-shSIRT5), AAV-shGSTP1, AAV-SPI1, AAV-SIRT5, and control viruses (AAV-NC or AAV-shNC) with a mouse α-cardiac myosin heavy chain promoter were acquired from VectorBuilder (Guangzhou, Guangdong, China) with a viral titer of 2 × 10 11 GC/mL.

Techniques: Immunofluorescence, Staining, Expressing, Western Blot, CCK-8 Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Biological Sciences

Article Title: SIRT5-related lysine demalonylation of GSTP1 contributes to cardiomyocyte pyroptosis suppression in diabetic cardiomyopathy

doi: 10.7150/ijbs.83306

Figure Lengend Snippet: Deficiency of SIRT5 expression exacerbates HG-induced mitochondrial dysfunction and pyroptosis. (A) Effect of HG treatment on the contents of carbonyl, GSH, and SOD in WT and KO cardiomyocytes. (B) Effect of HG treatment on mtROS production in WT and KO cardiomyocytes. (C) The mitochondrial function of WT and KO cardiomyocytes was assessed by seahorse assay. (D) Effect of HG treatment on the pyroptosis of cardiomyocytes from WT and SIRT5 KO mice observed under a light microscope. (E) Effect of HG treatment on DNA damage in cardiomyocytes of WT and SIRT5 KO mice analyzed using TUNEL. (F) Effect of HG treatment on the expression of pyroptosis-related proteins in WT and KO cardiomyocytes using Western blot. (G) Effect of HG treatment on IL-1β and IL-18 release from WT and KO cardiomyocytes using ELISA. (H) Effect of HG and INF200 treatments on the pyroptosis of cardiomyocytes from SIRT5 KO mice observed under a light microscope. (I) Effect of HG and INF200 treatments on viability in cardiomyocytes of SIRT5 KO mice analyzed using TUNEL. (J) Effect of HG and INF200 treatments on DNA damage in cardiomyocytes of SIRT5 KO mice analyzed using TUNEL. (K) The identification of cardiac fibroblasts using Vimentin immunofluorescence staining. (L) Effect of the CM of WT and KO cardiomyocytes exposed to HG on ECM synthesis in cardiac fibroblasts. (M) Effect of the CM of WT and KO cardiomyocytes exposed to HG on the proliferative capacity of cardiac fibroblasts. All data are expressed as means ± SD (n = 3). *p < 0.05 vs. WT:NG or KO:NG group; #p < 0.05 vs. WT:HG group; &p < 0.05 vs. WT:HG:DMSO group; @p < 0.05 vs. KO:HG:DMSO group. Continuous data were compared by two-way ANOVA.

Article Snippet: Adeno-associated virus (AAV) packaged with short hairpin RNA (shRNA) targeting SIRT5 (AAV-shSIRT5), AAV-shGSTP1, AAV-SPI1, AAV-SIRT5, and control viruses (AAV-NC or AAV-shNC) with a mouse α-cardiac myosin heavy chain promoter were acquired from VectorBuilder (Guangzhou, Guangdong, China) with a viral titer of 2 × 10 11 GC/mL.

Techniques: Expressing, Light Microscopy, TUNEL Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining

Journal: International Journal of Biological Sciences

Article Title: SIRT5-related lysine demalonylation of GSTP1 contributes to cardiomyocyte pyroptosis suppression in diabetic cardiomyopathy

doi: 10.7150/ijbs.83306

Figure Lengend Snippet: SIRT5 stabilizes the GSTP1 protein via lysine demalonylation. (A) KEGG pathway enrichment analysis of proteins with significantly elevated Mal-Lys modifications in SIRT5 KO mice. (B) OTC, GSTP1, and HIBCH are expressed in mitochondria. (C) Immunohistochemical detection of GSTP1 expression in the myocardial tissues of mice. (D) GSTP1 expression in mitochondria of HG-treated WT and KO cardiomyocytes using Western blot. (E) Mal-Lys modification of GSTP1 protein in mitochondria of HG-treated WT and KO cardiomyocytes using Western blot. (F) Validation of Mal-Lys modification site of GSTP1 using Western blot. (G) Effect of oe-SIRT5 on SIRT5 and GSTP1 mRNA expression by RT-qPCR. (H) Effect of overexpression of SIRT5 on Mal-Lys modification of GSTP1 and its protein expression using Western blot. (I) Effect of overexpression of SIRT5 on the stability of GSTP1 protein. All data are expressed as means ± SD (n = 3 or 10). *p < 0.05 vs. Control, WT:NG or KO:NG group; #p < 0.05 vs. WT:HG group, &p < 0.05 vs. oe-NC group. Continuous data were compared by one-way or two-way ANOVA.

Article Snippet: Adeno-associated virus (AAV) packaged with short hairpin RNA (shRNA) targeting SIRT5 (AAV-shSIRT5), AAV-shGSTP1, AAV-SPI1, AAV-SIRT5, and control viruses (AAV-NC or AAV-shNC) with a mouse α-cardiac myosin heavy chain promoter were acquired from VectorBuilder (Guangzhou, Guangdong, China) with a viral titer of 2 × 10 11 GC/mL.

Techniques: Immunohistochemical staining, Expressing, Western Blot, Modification, Biomarker Discovery, Quantitative RT-PCR, Over Expression, Control

Journal: International Journal of Biological Sciences

Article Title: SIRT5-related lysine demalonylation of GSTP1 contributes to cardiomyocyte pyroptosis suppression in diabetic cardiomyopathy

doi: 10.7150/ijbs.83306

Figure Lengend Snippet: SIRT5-controlled GSTP1 mediates HG-induced cardiomyocyte injury. (A) The effect of sh-GSTP1 on GSTP1 protein expression using Western blot. WT cardiomyocytes were transfected with oe-SIRT5 alone (with oe-NC as control) or in combination with sh-GSTP1 (with oe-SIRT5 + sh-NC as control), followed by HG. (B) The viability of HG-treated cardiomyocytes was examined using a CCK-8 assay. (C) The oxidative stress levels and inflammatory factor release in HG-treated cardiomyocytes using ELISA. (D) HG-treated cardiomyocyte senescence was detected using β-galactosidase staining. (E) The mtROS production in HG-treated cardiomyocytes using MitoSox Red staining. (F) The mitochondrial function of HG-treated cardiomyocytes was assessed by seahorse assay. (G) The pyroptosis of cardiomyocytes from WT and SIRT5 KO mice was observed under a light microscope. (H) DNA damage in cardiomyocytes treated with HG was analyzed using TUNEL. (I) The expression of pyroptosis-related proteins in HG-treated cardiomyocytes using Western blot. (J) Effect of the CM of cardiomyocytes after transfection and exposure to HG on the proliferative capacity of cardiac fibroblasts. (K) Effect of cardiomyocytes after transfection and exposure to HG on ECM synthesis in cardiac fibroblasts. All data are expressed as means ± SD (n = 3). *p < 0.05 vs. oe-SIRT5 + sh-NC group; #p < 0.05 vs. oe-NC group. Continuous data were compared by one-way or two-way ANOVA.

Article Snippet: Adeno-associated virus (AAV) packaged with short hairpin RNA (shRNA) targeting SIRT5 (AAV-shSIRT5), AAV-shGSTP1, AAV-SPI1, AAV-SIRT5, and control viruses (AAV-NC or AAV-shNC) with a mouse α-cardiac myosin heavy chain promoter were acquired from VectorBuilder (Guangzhou, Guangdong, China) with a viral titer of 2 × 10 11 GC/mL.

Techniques: Expressing, Western Blot, Transfection, Control, CCK-8 Assay, Enzyme-linked Immunosorbent Assay, Staining, Light Microscopy, TUNEL Assay

Journal: International Journal of Biological Sciences

Article Title: SIRT5-related lysine demalonylation of GSTP1 contributes to cardiomyocyte pyroptosis suppression in diabetic cardiomyopathy

doi: 10.7150/ijbs.83306

Figure Lengend Snippet: SIRT5-mediated GSTP1 overexpression alleviates myocardial injury in DCM mice. (A) Schematic diagram of the DCM mouse model and the AAV treatment schedule. (B) Changes in FBG levels were assayed every 3 weeks. (C) Changes in body weight were assayed every 3 weeks. (D) M-mode echocardiogram of mice. (E) EF (%) and FS (%) of mice. (F) IVRT of mice. (G) The protein expression of SIRT5, GSTP1, Cleaved-caspase1, NLRP3, GSDMD-N, and Collagen I and III in mouse myocardial tissues using Western blot. (H) Inflammatory infiltration in mouse myocardial tissues using HE staining. (I) Collagen deposition in myocardial tissues using Masson's staining. All data are expressed as means ± SD (n = 10). *p < 0.05 vs. AAV-NC group; #p < 0.05 vs. AAV-SIRT5 + AAV-shNC group. Continuous data were compared by one-way/two-way ANOVA.

Article Snippet: Adeno-associated virus (AAV) packaged with short hairpin RNA (shRNA) targeting SIRT5 (AAV-shSIRT5), AAV-shGSTP1, AAV-SPI1, AAV-SIRT5, and control viruses (AAV-NC or AAV-shNC) with a mouse α-cardiac myosin heavy chain promoter were acquired from VectorBuilder (Guangzhou, Guangdong, China) with a viral titer of 2 × 10 11 GC/mL.

Techniques: Over Expression, Expressing, Western Blot, Staining

Journal: International Journal of Biological Sciences

Article Title: SIRT5-related lysine demalonylation of GSTP1 contributes to cardiomyocyte pyroptosis suppression in diabetic cardiomyopathy

doi: 10.7150/ijbs.83306

Figure Lengend Snippet: SPI1 promotes the transcriptional expression of SIRT5. (A) SPI1 targets the SIRT5 promoter region in cardiac tissue samples. (B) Detection of SPI1 expression in the myocardial tissues of DCM mice by immunohistochemical assay. (C) The effect of HG treatment on SPI1 protein expression in WT cardiomyocytes using Western blot. (D) The enrichment ability of SPI1 on SIRT5 promoter using ChIP-qPCR. (E) The effect of overexpression of SPI1 on SPI1 and SIRT5 mRNA expression in WT cardiomyocytes using RT-qPCR. (F) The effect of overexpression of SPI1 on SPI1 and SIRT5 protein expression in WT cardiomyocytes using Western blot. (G) The effect of overexpression of SPI1 on the transcriptional activity of SIRT5 promoter using a dual-luciferase reporter assay. All data are expressed as means ± SD (n = 10 or 3). *p < 0.05 vs. Control, NG, IgG group; #p < 0.05 vs. oe-NC group. Continuous data were compared by unpaired t-test or two-way ANOVA.

Article Snippet: Adeno-associated virus (AAV) packaged with short hairpin RNA (shRNA) targeting SIRT5 (AAV-shSIRT5), AAV-shGSTP1, AAV-SPI1, AAV-SIRT5, and control viruses (AAV-NC or AAV-shNC) with a mouse α-cardiac myosin heavy chain promoter were acquired from VectorBuilder (Guangzhou, Guangdong, China) with a viral titer of 2 × 10 11 GC/mL.

Techniques: Expressing, Immunohistochemical staining, Western Blot, ChIP-qPCR, Over Expression, Quantitative RT-PCR, Activity Assay, Luciferase, Reporter Assay, Control

Journal: International Journal of Biological Sciences

Article Title: SIRT5-related lysine demalonylation of GSTP1 contributes to cardiomyocyte pyroptosis suppression in diabetic cardiomyopathy

doi: 10.7150/ijbs.83306

Figure Lengend Snippet: SPI1-mediated transcriptional activation of SIRT5 inhibits HG-induced cardiomyocyte injury. (A) Interference efficiency of sh-SIRT5 in cardiomyocytes by RT-qPCR. WT cardiomyocytes were transfected with oe-SPI1 alone (with oe-NC as control) or in combination with sh-SIRT5 (with oe-SPI1 + sh-NC as control), followed by HG. (B) The expression of SIRT5, GSTP1, and pyroptosis-related proteins in HG-treated cardiomyocytes using Western blot. (C) The pyroptosis of HG-treated cardiomyocytes was observed under a light microscope. (D) DNA damage in HG-treated cardiomyocytes was analyzed using TUNEL. (E) The viability of HG-treated cardiomyocytes was examined using CCK-8 assays. (F) The oxidative stress levels and inflammatory factor release in HG-treated cardiomyocytes using ELISA. (G) HG-treated cardiomyocyte senescence was detected using β-galactosidase staining. (H) The mtROS production in HG-treated cardiomyocytes using MitoSox Red staining. (I) The mitochondrial function of HG-treated cardiomyocytes was assessed by seahorse assay. (J) Effect of the CM of cardiomyocytes after transfection and exposure to HG on the proliferative capacity of cardiac fibroblasts. (K) Effect of cardiomyocytes after transfection and exposure to HG on ECM synthesis in cardiac fibroblasts. All data are expressed as means ± SD (n = 3). *p < 0.05 vs. oe-SPI1 + sh-NC group; #p < 0.05 vs. oe-NC group. Continuous data were compared by one-way or two-way ANOVA.

Article Snippet: Adeno-associated virus (AAV) packaged with short hairpin RNA (shRNA) targeting SIRT5 (AAV-shSIRT5), AAV-shGSTP1, AAV-SPI1, AAV-SIRT5, and control viruses (AAV-NC or AAV-shNC) with a mouse α-cardiac myosin heavy chain promoter were acquired from VectorBuilder (Guangzhou, Guangdong, China) with a viral titer of 2 × 10 11 GC/mL.

Techniques: Activation Assay, Quantitative RT-PCR, Transfection, Control, Expressing, Western Blot, Light Microscopy, TUNEL Assay, CCK-8 Assay, Enzyme-linked Immunosorbent Assay, Staining

Journal: International Journal of Biological Sciences

Article Title: SIRT5-related lysine demalonylation of GSTP1 contributes to cardiomyocyte pyroptosis suppression in diabetic cardiomyopathy

doi: 10.7150/ijbs.83306

Figure Lengend Snippet: Protective effect of SPI1 on myocardial injury in DCM mice is elicited through the activation of SIRT5. DCM mice were further administrated with AAV-SPI1 (AAV-NC as control) or in combination with AAV-shSIRT5 (AAV-SPI1 + AAV-shNC as control). (A) Changes in FBG levels were assayed every 3 weeks. (B) Changes in body weight were assayed every 3 weeks. (C) M-mode echocardiogram of mice. (D) IVRT of mice. (E) EF (%) and FS (%) of mice. (F) Inflammatory infiltration in mouse myocardial tissues using HE staining. (G) Collagen deposition in myocardial tissues using Masson's staining. (H) The protein expression of SPI1, SIRT5, GSTP1, Cleaved-caspase1, NLRP3, GSDMD-N, and Collagen I and III in mouse myocardial tissues using Western blot. All data are expressed as means ± SD (n = 10). *p < 0.05 vs. AAV-NC group; #p < 0.05 vs. AAV-SPI1 + AAV-shNC group. Continuous data were compared by one-way/two-way ANOVA.

Article Snippet: Adeno-associated virus (AAV) packaged with short hairpin RNA (shRNA) targeting SIRT5 (AAV-shSIRT5), AAV-shGSTP1, AAV-SPI1, AAV-SIRT5, and control viruses (AAV-NC or AAV-shNC) with a mouse α-cardiac myosin heavy chain promoter were acquired from VectorBuilder (Guangzhou, Guangdong, China) with a viral titer of 2 × 10 11 GC/mL.

Techniques: Activation Assay, Control, Staining, Expressing, Western Blot

Journal: International Journal of Biological Sciences

Article Title: SIRT5-related lysine demalonylation of GSTP1 contributes to cardiomyocyte pyroptosis suppression in diabetic cardiomyopathy

doi: 10.7150/ijbs.83306

Figure Lengend Snippet: Schematic diagram of the mechanism. SPI1 inhibits oxidative stress-induced NLRP3 activation and subsequent cell pyroptosis and attenuates pyroptosis-induced cardiac fibroblast activation and ECM synthesis by promoting the transcription of SIRT5 and mediating the lysine demalonylation of GSTP1.

Article Snippet: Adeno-associated virus (AAV) packaged with short hairpin RNA (shRNA) targeting SIRT5 (AAV-shSIRT5), AAV-shGSTP1, AAV-SPI1, AAV-SIRT5, and control viruses (AAV-NC or AAV-shNC) with a mouse α-cardiac myosin heavy chain promoter were acquired from VectorBuilder (Guangzhou, Guangdong, China) with a viral titer of 2 × 10 11 GC/mL.

Techniques: Activation Assay