Journal: Advanced Science

Article Title: Endothelial Retargeting of AAV9 In Vivo

doi: 10.1002/advs.202103867

Figure Lengend Snippet: Coating of rAAV2/9 with PAMAM dendrimers. A) Generation 2 polyamidoamine dendrimers modified stepwise by PEGylation with NHS‐PEG 2K ‐OPSS and conjugated to peptides via the N terminal cysteine residue. B) Representative transmission electron micrograph of AAV9 (left) or AAV9 coated with PEGylated G2 PAMAM (right). (Scale bars: 100 nm). C) Comparison of particle diameters of uncoated ( N = 153) and coated rAAV ( N = 69, p < 0.0001). D) Zeta potential of uncoated or G2 Cys coated AAV9 particles. E) Transduction efficacy of AAV9‐GFP on a nonendothelial (Human embryonic kidney = HEK293T cells) and an endothelial cell line (human umbilical vein endothelial cells = HUVECs) at MOI of 1 × 10 6 coated by incrementally increasing doses (pg/cell) of G2 Cys PAMAM (green: GFP, Scale bars: 100 µm).

Article Snippet: The following antibodies were used for immunofluorescence staining: EGFP (CAB4211, Thermo Fisher), murine CD31 (BM4086, Origene), porcine CD31 (LCI‐4, Bio‐Rad), CRISPR‐Cas9 (7A9‐3A3, Novus Biologicals), AAV9 intact capsid (ADK9, Origene).

Techniques: Modification, Transmission Assay, Transduction

Journal: Advanced Science

Article Title: Endothelial Retargeting of AAV9 In Vivo

doi: 10.1002/advs.202103867

Figure Lengend Snippet: Modulation of in vivo AAV9‐Cre transduction by G2 CNN coating in mTmG mice. Examples of hindlimb skeletal muscle transduction of mTmG mice expressing membrane dTomato (red) unless Cre recombinase activity enables expression of enhanced green fluorescence protein (EGFP, green). Cre was transduced by A) an unmodified rAAV2/9 vector with CMV promoter, B) unmodified AAV9 pCMV Cre, or C) G2 CNN coated AAV9 pEndo. Cre. Transduction with D) G2 CNN coated AAV9 with endoglin promoter was also carried out 30 min after preinjection with isotype control IgG k or E) anti‐ Stab2 antibody. Top: EGFP alone, bottom: merge of EGFP, dTomato and DAPI (blue). Scale bars represent 25 µm.

Article Snippet: The following antibodies were used for immunofluorescence staining: EGFP (CAB4211, Thermo Fisher), murine CD31 (BM4086, Origene), porcine CD31 (LCI‐4, Bio‐Rad), CRISPR‐Cas9 (7A9‐3A3, Novus Biologicals), AAV9 intact capsid (ADK9, Origene).

Techniques: In Vivo, Transduction, Expressing, Activity Assay, Fluorescence, Plasmid Preparation

Journal: Advanced Science

Article Title: Endothelial Retargeting of AAV9 In Vivo

doi: 10.1002/advs.202103867

Figure Lengend Snippet: Endothelial transduction of mTmG pigs with AAV9‐Cre. mT mG −1 reporter pigs were transduced with either unmodified AAV9.pCMV.Cre (left) or G2 CNN coated AAV9.pEndo.Cre (right) via local application into A) hindlimb muscle and B) left ventricle. Top: EGFP alone (green), bottom: merge of EGFP, dTomato (red), CD31 (white), and DAPI (blue). Scale bars represent A) 100 µm or B) 25 µm.

Article Snippet: The following antibodies were used for immunofluorescence staining: EGFP (CAB4211, Thermo Fisher), murine CD31 (BM4086, Origene), porcine CD31 (LCI‐4, Bio‐Rad), CRISPR‐Cas9 (7A9‐3A3, Novus Biologicals), AAV9 intact capsid (ADK9, Origene).

Techniques: Transduction

Journal: Advanced Science

Article Title: Endothelial Retargeting of AAV9 In Vivo

doi: 10.1002/advs.202103867

Figure Lengend Snippet: Endothelial S1FG delivery via endothelial retargeted AAV9. A) AAV9 encoding S1FG, an artificial leukocyte adhesion molecule consisting of SDF‐1, the mucin domain of CX3CL1, and a GPI‐anchor, was injected to tail veins of wild type mice. B) Intravital microscopy of cremaster was carried out. C) Muscle venules (red outline) revealed enhanced adhesion of leukocytes (black dots) upon S1FG expression. D) Adhesion of leukocytes showed a significant increase with endothelial retargeting (G2 CNN coated AAV9.pEndo.S1FG) compared to untargeted delivery (unmodified AAV9.pCMV.S1FG) or control (unmodified AAV9.pCMV.lacZ); with cotreatment with CXCR4 antagonist AMD3100 abrogating this effect. Scale bars: 100 µm. Values represent the mean ± SEM. n = 4, 1‐way ANOVA with Dunnett post‐test.

Article Snippet: The following antibodies were used for immunofluorescence staining: EGFP (CAB4211, Thermo Fisher), murine CD31 (BM4086, Origene), porcine CD31 (LCI‐4, Bio‐Rad), CRISPR‐Cas9 (7A9‐3A3, Novus Biologicals), AAV9 intact capsid (ADK9, Origene).

Techniques: Injection, Intravital Microscopy, Expressing

Journal: Advanced Science

Article Title: Endothelial Retargeting of AAV9 In Vivo

doi: 10.1002/advs.202103867

Figure Lengend Snippet: Endothelial retargeted of Annexin A1 abrogates leukocyte recruitment in chronic atherosclerotic mouse model. A) Endothelial activation and subsequent macrophage recruitment is a crucial part of atherosclerotic plaque formation and progression. B) Unmodified or endothelial retargeted Annexin A1 encoding AAV9 were tail vein injected to ApoE −/‐ mice on high fat diet. Ly6C + , Ly6G + , and CD11b + leukocyte subpopulations were stained by intravenous injection of respective antibodies and visualized by intravital microscopy of carotid arteries. C) Adhesion of all three subtypes showed a significant decrease with endothelial retargeting (G2 CNN coated AAV9.pEndo.Anxa) in comparison to untargeted delivery (unmodified AAV9.pCMV.Anxa) or control (unmodified AAV9.pCMV.EGFP). Scale bars: 100 µm; n = 12, 1‐way ANOVA with Dunnett post‐test.

Article Snippet: The following antibodies were used for immunofluorescence staining: EGFP (CAB4211, Thermo Fisher), murine CD31 (BM4086, Origene), porcine CD31 (LCI‐4, Bio‐Rad), CRISPR‐Cas9 (7A9‐3A3, Novus Biologicals), AAV9 intact capsid (ADK9, Origene).

Techniques: Activation Assay, Injection, Staining, Intravital Microscopy

Journal: Advanced Science

Article Title: Endothelial Retargeting of AAV9 In Vivo

doi: 10.1002/advs.202103867

Figure Lengend Snippet: Blood pressure alteration by Cas9 mediated eNOS deletion with endothelial retargeted AAV9. A) Vasodilative effect of nitrous oxide synthesis by endothelial cells was targeted by deletion of exons 6 through 10 of the eNOS gene via CRISPR/Cas9, rendering overlaying smooth muscle cells incapable of relaxation. Conditional Cas9 knock‐in mice were transduced by unmodified AAV9.pEndo.Cre or G2 CNN coated AAV9.pEndo.Cre with sgRNA targeting eNOS. B–E) Noninvasive blood pressure measurements were taken at baseline, 4 and 8 weeks; along with cardiac catheterization at week 8. Systolic, diastolic, mean arterial, and developed pressure values were plotted ( n = 6, 1‐way ANOVA with Dunnett post‐test). F) Cas9 expression (green) was observed in CD31 + cells (red) in both heart (left) and muscle (right) (Scale bars: 25 µm). G) Edited alleles (indicated by arrows) in magnetically sorted CD31 + cells of skeletal muscle were evident upon agarose gel separation (NEB 1kb+ ladder).

Article Snippet: The following antibodies were used for immunofluorescence staining: EGFP (CAB4211, Thermo Fisher), murine CD31 (BM4086, Origene), porcine CD31 (LCI‐4, Bio‐Rad), CRISPR‐Cas9 (7A9‐3A3, Novus Biologicals), AAV9 intact capsid (ADK9, Origene).

Techniques: CRISPR, Knock-In, Expressing, Agarose Gel Electrophoresis

Journal: Aging (Albany NY)

Article Title: LncRNA SNHG7 promotes cardiac remodeling by upregulating ROCK1 via sponging miR-34-5p

doi: 10.18632/aging.103269

Figure Lengend Snippet: Silencing of SNHG7 inhibited cardiac remodeling after MI. ( A ) qRT-PCR analysis showing that AAV9-sh-SNHG7 reversed the up-regulation of SNHG7 in MI mice; GAPDH mRNA served as an internal control, and AAV9-sh-scramble served as an additional control. Data was presented as mean ± SEM; one-way ANOVA was used for the statistical analysis. n=6 mice per group. ( B , C ) 4 weeks after MI, echocardiographic findings showed that the silencing of SNHG7 improved ejection fraction (EF) and fraction shortening (FS). Data was presented as mean ± SEM; one-way ANOVA was used for the statistical analysis. n=12 mice per group. ( D ) mRNA expression of collagen 1α1 and collagen 3α1 were measured by qRT-PCR; GAPDH mRNA served as an internal control. Data was presented as mean ± SEM; one-way ANOVA was used for the statistical analysis. n=10 mice per group. ( E ) Western blotting analysis showing elevation of protein levels of collagen 1 and α-SMA in peri-infarcted tissue of left ventricle of mice after MI. Data was presented mean ± SEM; one-way ANOVA was used for the statistical analysis. n=5 mice per group. ** P <0.05.

Article Snippet: Animals were treated with adeno-associated virus 9 (AAV9) carrying short hairpin RNA for SNHG7 (AAV5-sh-SNHG7) and AAV9 carrying miR-34-5p precursor (AAV9-miR-34-5p) (Biowit Technology, Shenzhen, China) via intra-tracheal injection at a dose of 1x10^11 viral genome (vg) per mouse 7 days before MI.

Techniques: Quantitative RT-PCR, Expressing, Western Blot

Journal: Aging (Albany NY)

Article Title: LncRNA SNHG7 promotes cardiac remodeling by upregulating ROCK1 via sponging miR-34-5p

doi: 10.18632/aging.103269

Figure Lengend Snippet: Overexpression of miR-34-5p ameliorated cardiac fibrosis in the mice after MI. ( A ) Intravenous injection of AAV9-miR-34-5p via tail increased miR-34-5p expression in normal mice, as measured by qRT-PCR; GAPDH served as an internal control, and AAV9-scramble served as a negative control. Data was presented as mean ± SEM; two-tailed t test was used for the statistical analysis. n=5 independent cell cultures. ( B , C ) Four weeks after MI, echocardiographic imaging showed that the overexpression of miR-34-5p improved ejection fraction (EF) and fraction shortening (FS). Data was presented as mean ± SEM; one-way ANOVA was used for the statistical analysis. n=12 mice per group. ( D ) qRT-PCR analysis showing that AAV9-miR-34-5p injection reversed the up-regulation of collagen 1α1 and collagen 3α1 in MI mice; GAPDH mRNA served as an internal control, and AAV9-scramble served as an additional control. Data was presented as mean ± SEM; one-way ANOVA was used for the statistical analysis. n=6 mice per group. ( E ) Protein levels of collagen 1 and α-SMA were measured by western blot; GAPDH served as an internal control. Data was presented as mean ± SEM; one-way ANOVA was used for the statistical analysis. n=6 mice per group. ** P <0.05.

Article Snippet: Animals were treated with adeno-associated virus 9 (AAV9) carrying short hairpin RNA for SNHG7 (AAV5-sh-SNHG7) and AAV9 carrying miR-34-5p precursor (AAV9-miR-34-5p) (Biowit Technology, Shenzhen, China) via intra-tracheal injection at a dose of 1x10^11 viral genome (vg) per mouse 7 days before MI.

Techniques: Over Expression, Injection, Expressing, Quantitative RT-PCR, Negative Control, Two Tailed Test, Imaging, Western Blot

Journal: Cell Death & Disease

Article Title: FNDC5 prevents oxidative stress and neuronal apoptosis after traumatic brain injury through SIRT3-dependent regulation of mitochondrial quality control

doi: 10.1038/s41419-024-06748-w

Figure Lengend Snippet: a Schematic of MRI test and behavioral studies. b MRI scan of the brain in WT + TBI group and FNDC5-KO + TBI group; n = 6 for each group. c Quantification of lesion volume of MRI. d measurement of brain water content. * P < 0.05, ** P < 0.01 and *** P < 0.001 vs. WT + TBI group. e – g Several behavioral tests at different time points after TBI as measured by the grid-walking test ( e ), cylinder test ( f ) and adhesive removal test ( g ) ( n = 9 for each group). Significance was determined by Student’s t test ( c , d ) and two-way repeated ANOVA ( e – g ) with Bonferroni post hoc tests. * P < 0.05, ** P < 0.01 and *** P < 0.001 vs. WT+Sham group, # P < 0.05 and ## P < 0.01 vs. WT + TBI group. Values are presented as the mean ± SEM.

Article Snippet: The AAV9 virus expressing FNDC5 (AAV-FNDC5) or AAV9-CON323 (AAV-Ctrl) from Genchem (Shanghai, China) was used for stereotaxic injection.

Techniques: Adhesive

Journal: Cell Death & Disease

Article Title: FNDC5 prevents oxidative stress and neuronal apoptosis after traumatic brain injury through SIRT3-dependent regulation of mitochondrial quality control

doi: 10.1038/s41419-024-06748-w

Figure Lengend Snippet: a , b Representative DHE staining images and the quantitative results ( n = 6 for each group). Scale bar: 200 μm. c The effects of FNDC5 on MDA levels. d The effects of FNDC5 on mitochondrial MnSOD activity. e , f Representative images and statistical analysis of TUNEL staining of the perilesional cortex after TBI. Scale bar: 200 μm. g , h Representative Western blots and statistical analysis of the levels of Bax and Bcl-2 ( n = 6 for each group). Significance was determined by one-way ANOVA ( b – d , f , h ) with Bonferroni post hoc tests. * P < 0.05 and ** P < 0.01 vs. sham group, # P < 0.05 and ## P < 0.01 vs. TBI + NC group. Values are presented as the mean ± SEM.

Article Snippet: The AAV9 virus expressing FNDC5 (AAV-FNDC5) or AAV9-CON323 (AAV-Ctrl) from Genchem (Shanghai, China) was used for stereotaxic injection.

Techniques: Staining, Activity Assay, TUNEL Assay, Western Blot

Journal: Cell Death & Disease

Article Title: FNDC5 prevents oxidative stress and neuronal apoptosis after traumatic brain injury through SIRT3-dependent regulation of mitochondrial quality control

doi: 10.1038/s41419-024-06748-w

Figure Lengend Snippet: a , b Western blot and statistical analysis of FNDC5 expression. c Representative MitoTracker fluorescence images illustrating mitochondrial morphology. Scale bar: 10 μm. d , e Mitochondrial morphological characteristics were quantified by ImageJ software. f ATP content of HT22 cells. g NAD + /NADH of HT22 cells. h , i Representative fluorescence intensity of JC-1 staining illustrating the MMP. Scale bar: 20 μm. j , k Representative MitoSOX fluorescence images of mitochondria-derived ROS. Scale bar: 50 μm ( n = 6 for each group). l , m Western blot and statistical analysis of SIRT3 expression. n Relative mRNA level of SIRT3. o Total acetylation level of mitochondrial proteins. p Relative acetylation level of mitochondrial proteins. Significance was determined by Student’s t test ( b , m , n , p ) and one-way ( d – g , i , k ) ANOVA with Bonferroni post hoc tests. * P < 0.05 and ** P < 0.01 vs. Control group, # P < 0.05 and ## P < 0.01 vs. FNDC5-KO + vel group. Values are presented as the mean ± SEM.

Article Snippet: The AAV9 virus expressing FNDC5 (AAV-FNDC5) or AAV9-CON323 (AAV-Ctrl) from Genchem (Shanghai, China) was used for stereotaxic injection.

Techniques: Western Blot, Expressing, Fluorescence, Software, Staining, Derivative Assay, Control

Journal: Cell Death & Disease

Article Title: FNDC5 prevents oxidative stress and neuronal apoptosis after traumatic brain injury through SIRT3-dependent regulation of mitochondrial quality control

doi: 10.1038/s41419-024-06748-w

Figure Lengend Snippet: a Representative DHE staining images in SIRT3 CKO mice and SIRT3 f/f mice. Scale bar: 200 μm. b The quantitative results of DHE staining images. c The effects of FNDC5 on MDA levels in SIRT3 CKO mice and in SIRT3 f/f mice. d The effects of FNDC5 on mitochondrial MnSOD activity in SIRT3 CKO mice and in SIRT3 f/f mice. e Representative images of TUNEL staining of the perilesional cortex 24 h in SIRT3 CKO mice and SIRT3 f/f mice. Scale bar: 200 μm. f , g Representative Western blot and statistical analysis of the levels of Bax and Bcl-2 in SIRT3 CKO mice and in SIRT3 f/f mice. ( n = 6 for each group). Significance was determined by two-way repeated ANOVA ( b , c , d , g ) with Bonferroni post hoc tests. * P < 0.05 and ** P < 0.01 vs. sham group, # P < 0.05 and ## P < 0.01 vs. TBI + NC group. Values are presented as the mean ± SEM.

Article Snippet: The AAV9 virus expressing FNDC5 (AAV-FNDC5) or AAV9-CON323 (AAV-Ctrl) from Genchem (Shanghai, China) was used for stereotaxic injection.

Techniques: Staining, Activity Assay, TUNEL Assay, Western Blot

Journal: Cell Death & Disease

Article Title: FNDC5 prevents oxidative stress and neuronal apoptosis after traumatic brain injury through SIRT3-dependent regulation of mitochondrial quality control

doi: 10.1038/s41419-024-06748-w

Figure Lengend Snippet: a , b Western blot and statistical analysis of the levels of NRF2 expression in normal HT22 cell line and FNDC5 KO HT22 cell line. c Relative mRNA level of SIRT3 after treating with an NRF2 inhibitor (ML385). d , e Western blot and statistical analysis of the levels of SIRT3 expression. f FNDC5 deficiency does not affect the mRNA of NRF2. g The protein level of NRF2 was degraded faster over time in FNDC5 knockout cells after incubating with the mRNA synthesis inhibitor CHX. h statistical analysis of the levels of NRF2. i The protein level of NRF2 expression after incubating with the proteasome inhibitor MG132. j Statistical analysis of the levels of NRF2. k The protein level of NRF2 expression after incubating with the lysosomal inhibitor NH4Cl. l statistical analysis of the levels of NRF2. m Ubiquitination level of NRF2. Significance was determined by Student’s t test ( b , f ) and one-way ANOVA ( c , e ) or two-way repeated ANOVA ( h , j , l ) with Bonferroni post hoc tests. * P < 0.05 and ** P < 0.01 vs. Control group, # P < 0.05 and ## P < 0.01 vs. TBI + vector group, & P < 0.05 and && P < 0.01 vs. TBI + FNDC5 + veh group. Values are presented as the mean ± SEM.

Article Snippet: The AAV9 virus expressing FNDC5 (AAV-FNDC5) or AAV9-CON323 (AAV-Ctrl) from Genchem (Shanghai, China) was used for stereotaxic injection.

Techniques: Western Blot, Expressing, Knock-Out, Ubiquitin Proteomics, Control, Plasmid Preparation

Journal: Brazilian Journal of Medical and Biological Research

Article Title: High expression of P4HA3 in obesity: a potential therapeutic target for type 2 diabetes

doi: 10.1590/1414-431X2022e11741

Figure Lengend Snippet: P4HA3 was significantly upregulated in obese and type 2 diabetes mellitus (T2DM) patients. A , Venn diagram of the differentially expressed (DE) genes in 2 pairwise comparisons: lean obese, obese (non-diabetic) vs diabetic. B , Top ranked DE genes in both groups (|logFC| ≥1 and P<0.05), with their log 2 fold-changes. C , P4HA3 expression in adipose tissue from 30 individuals with normal body mass index (BMI), 30 obese patients, and 30 obese patients with T2DM. Pearson correlation between P4HA3 expression level in adipose tissue and BMI in 30 obese patients ( D ) and between P4HA3 expression level in adipose tissue and Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) of 30 obese with T2DM patients ( E ). **P<0.01 (ANOVA).

Article Snippet: AAV9 virus containing shRNA targeting P4HA3 was produced by SunBio (China), with scramble non-targeting shRNA used as control.

Techniques: Expressing

Journal: Brazilian Journal of Medical and Biological Research

Article Title: High expression of P4HA3 in obesity: a potential therapeutic target for type 2 diabetes

doi: 10.1590/1414-431X2022e11741

Figure Lengend Snippet: P4HA3 knockdown inhibited adipocyte differentiation in 3T3-L1 cells. A , Expression of P4HA3 following siRNA treatment was detected by qRT-PCR. B and C , C/EBP-α and PPAR-γ expression during the course of adipocytes differentiation was quantified using RT-qPCR and western blot assay after P4HA3 knockdown. D , Representative Oil Red O staining of differentiated adipocytes with or without P4HA3 knockdown (scale bar, 20 μm). E and F , Triglycerides (TG) and glucose uptake determination in differentiated adipocytes with or without P4HA3 knockdown. NC: negative control. Data are reported as means±SD. **P<0.01; ***P<0.001 (ANOVA and t -test)

Article Snippet: AAV9 virus containing shRNA targeting P4HA3 was produced by SunBio (China), with scramble non-targeting shRNA used as control.

Techniques: Knockdown, Expressing, Quantitative RT-PCR, Western Blot, Staining, Negative Control

Journal: Brazilian Journal of Medical and Biological Research

Article Title: High expression of P4HA3 in obesity: a potential therapeutic target for type 2 diabetes

doi: 10.1590/1414-431X2022e11741

Figure Lengend Snippet: P4HA3 knockdown counteracted high fat diet (HFD)-induced obesity and improved insulin resistance in db/db mice. A , C57BL6 db/db mice were fed with HFD, and then injected with AAV-shRNA-P4HA3 or AAV-scrambled shRNA. The body weight was monitored during the experiment. B and C , Fasting blood glucose (FBG) and insulin levels were determined after 6-h fasting at 20 weeks. D and E , Free fatty acid (FFA) and triglycerides (TG) levels in the blood were determined after 6-h fasting at 20 weeks. F , OGTT (oral glucose tolerance test) was performed at 20 weeks. G , Glucose AUC (area under the curve) was determined by OGTT. H , Insulin tolerance tests (ITT) was performed at 20 weeks. I , Insulin AUC was determined by ITTs. NC: negative control. Data are reported as means±SD. ***P<0.001 ( t -test).

Article Snippet: AAV9 virus containing shRNA targeting P4HA3 was produced by SunBio (China), with scramble non-targeting shRNA used as control.

Techniques: Knockdown, Injection, shRNA, Negative Control

Journal: Brazilian Journal of Medical and Biological Research

Article Title: High expression of P4HA3 in obesity: a potential therapeutic target for type 2 diabetes

doi: 10.1590/1414-431X2022e11741

Figure Lengend Snippet: P4HA3 silencing modulated adipocyte differentiation and insulin sensitivity gene expressions in adipose tissue. A , Epididymal and inguinal fat pad weight (g) of db/db mice were measured. B , The cross-sectional area of adipocytes in adipose tissue was analyzed by HE staining (scale bar, 200 μm). C and D , C/EBP-α, PPAR-γ, and inflammatory factors (MCP1, interleukin (IL)-6, and IL-1) expression in epididymal and inguinal adipose tissues of db/db mice with or without P4HA3 knockdown was quantified using RT-qPCR. Mice were sacrificed after 6-h fasting for tissue collection. E, The phosphorylation levels of FoxO1, IRS, PI3K, and AKT in epididymal and inguinal adipose tissues of db/db mice were analyzed by western blotting. HFD: high fat diet; NC: negative control. Data are reported as means±SD. **P<0.01; ***P<0.001 ( t -test).

Article Snippet: AAV9 virus containing shRNA targeting P4HA3 was produced by SunBio (China), with scramble non-targeting shRNA used as control.

Techniques: Staining, Expressing, Knockdown, Quantitative RT-PCR, Phospho-proteomics, Western Blot, Negative Control

Journal: Brazilian Journal of Medical and Biological Research

Article Title: High expression of P4HA3 in obesity: a potential therapeutic target for type 2 diabetes

doi: 10.1590/1414-431X2022e11741

Figure Lengend Snippet: P4HA3 improves hepatic glucose homeostasis and steatosis in db/db mice. A , The expression levels of gluconeogenesis genes ( G6PC and PCK1 ) and glycogenolytic gene ( PYGL ) in liver tissues of db/db mice were determined by RT-qPCR. Mice were sacrificed after 6-h fasting for tissue collection. B , The phosphorylation levels of FoxO1, IRS, PI3K, and AKT in liver tissues of db/db mice with or without P4HA3 knockdown were analyzed using western blotting. C , Hepatic steatosis and liver triglycerides (TG) levels in db/db mice with or without P4HA3 knockdown were examined by HE staining (scale bar, 20 μm) and TG kit. HFD: high fat diet; NC: negative control. Data are reported as means±SD. **P<0.01, ***P<0.001 ( t -test).

Article Snippet: AAV9 virus containing shRNA targeting P4HA3 was produced by SunBio (China), with scramble non-targeting shRNA used as control.

Techniques: Expressing, Quantitative RT-PCR, Phospho-proteomics, Knockdown, Western Blot, Staining, Negative Control