adeno associated virus serotype 9 aav9 vector  (Vector Biolabs)

Journal: Acta Neuropathologica Communications

Article Title: Tau modulation through AAV9 therapy augments Akt/Erk survival signalling in glaucoma mitigating the retinal degenerative phenotype

doi: 10.1186/s40478-024-01804-0

Figure Lengend Snippet: Modulation of Tau expression in C57BL/6 mice retinas A Scheme of AAV plasmid vector containing GFP, mTau, scrambled shRNA, and mTau-shRNAmir sequences. mTau (overexpression) and mTau-shRNAmir (knockdown, KD) sequences were cloned in AAV9 viral vector plasmid fused to the ampicillin-resistance gene. For mTau protein expression, a T2A self-cleaving peptide sequence was used. B Schematic depicting the experimental design and AAV administration experimental timeline. C Immunofluorescence images of retinal sections from control, AAV-GFP, AAV-mTau, AAV-Scramble and AAV-mTau KD groups showing GFP (green, FITC), tau (Green, Alexa Fluor 488), and ptau (Ser199/Ser202) (Red, Cy3). NeuN (red-Cy3) and nucleus (blue, DAPI) (representative images, Scale bar = 50 μm, arrows indicate the changes in the expressions; Antibody concentrations: GFP (1:1000), tau (1:800), ptau (Ser199/Ser202) (1:800), NeuN (1:1000)). D Quantification of GFP relative fluorescence intensity (RFI) percentage (**** P < 0.0001, n = 5). E Quantification of Tau RFI percentage (**** P < 0.0001, n = 5) F Quantification of pTau (Ser199/Ser202) RFI percentage (**** P < 0.0001, n = 5). Statistical significance was assessed by employing One-way ANOVA analysis with Tukey’s multiple comparison test

Article Snippet: For the over expression of the Tau, murine mutant Tau (P232S) cDNA (NCBI ref: BC014748) under the transcriptional control of the cytomegalovirus chicken β-actin (CAG2) hybrid promotor was inserted into the adeno-associated virus serotype 9 (AAV9) vector with the enhanced green fluorescence protein reporter (eGFP) and 2A linker in between GFP and the mTau sequence (AAV9-CAG2-eGFP-2A-mTau (P232S)-WPRE or AAV9- Tau) (Vector Biolabs, USA).

Techniques: Expressing, Plasmid Preparation, shRNA, Over Expression, Knockdown, Clone Assay, Sequencing, Immunofluorescence, Control, Fluorescence, Comparison

Journal: Acta Neuropathologica Communications

Article Title: Tau modulation through AAV9 therapy augments Akt/Erk survival signalling in glaucoma mitigating the retinal degenerative phenotype

doi: 10.1186/s40478-024-01804-0

Figure Lengend Snippet: Electrophysiological and structural alterations in C57BL/6 mice retinas subjected to AAV-tau overexpression and AAV-tau knockdown under normal IOP conditions. A Positive scotopic threshold responses (pSTRs) of the control, AAV-GFP, and AAV-mTau injected mice. B Quantification of amplitudes indicates a significant decrease in pSTR amplitudes in AAV9-Tau overexpressing mice (n = 10, P < 0.0001 t-test). C pSTRs of the control, AAV-scrambled, and AAV-tau shRNA expressing mice and D their quantification indicates a significant decrease of pSTR amplitudes in AAV9-KD mice retinas (n = 10, **** P < 0.0001, t-test). No significant difference in pSTR amplitudes was observed between the control and AAV9-GFP or AAV9-scramble-shRNA sequence-expressing eyes. E H and E staining of retinal sections shows GCL cell density loss in Tau overexpression and Tau KD retinas compared to the controls, arrows indicate the changes in cell densities (representative images, Scale bar = 50 μm). F Quantification of cell density in the GCL from the retinal H and E-stained images (**** P < 0.0001, One-way ANOVA analysis with Tukey’s multiple comparison test, n = 5 for each group)

Article Snippet: For the over expression of the Tau, murine mutant Tau (P232S) cDNA (NCBI ref: BC014748) under the transcriptional control of the cytomegalovirus chicken β-actin (CAG2) hybrid promotor was inserted into the adeno-associated virus serotype 9 (AAV9) vector with the enhanced green fluorescence protein reporter (eGFP) and 2A linker in between GFP and the mTau sequence (AAV9-CAG2-eGFP-2A-mTau (P232S)-WPRE or AAV9- Tau) (Vector Biolabs, USA).

Techniques: Over Expression, Knockdown, Control, Injection, shRNA, Expressing, Sequencing, Staining, Comparison

Journal: Acta Neuropathologica Communications

Article Title: Tau modulation through AAV9 therapy augments Akt/Erk survival signalling in glaucoma mitigating the retinal degenerative phenotype

doi: 10.1186/s40478-024-01804-0

Figure Lengend Snippet: Tau overexpressing mice retinas shows exacerbated degenerative changes in the inner retina in high-IOP glaucoma condition. A Chronic elevation of IOP in C57BL/6 mice eyes was induced by intracameral microbead injections in control, AAV-GFP and AAV-mTau administered mice for 2 months. B Positive scotopic threshold response (pSTR) traces from control, glaucoma, glaucoma + AAV-GFP, and glaucoma + AAV-mTau overexpression mice retinas, as indicated and C their pSTR amplitudes quantification indicated a significant decline of the pSTR in the glaucoma AAV9-tau overexpression animals. (n = 10, *** P < 0.001 **** P < 0.0001) D Western blot analysis of Tau, pTau S199/202 and pTau S404 levels in control and glaucomatous retina. β-actin was used as a loading control E Fold change of Tau, pTau S199/202 and pTau. S404 showed a significant increase in retinas in high IOP conditions (*** P < 0.001, n = 3 each group, t-test). F H and E staining of retinal sections of control, glaucoma, glaucoma + AAV9-GFP, and glaucoma + AAV9-mTau mice (representative images, arrows indicate the changes in cell densities, Scale bar, 50 μm). G Quantification of the cell density in GCL indicated a significant decrease in the number of cells in glaucoma and AAV-tau overexpressed retinas compared to the controls (n = 5, **** P < 0.0001, One-way ANOVA analysis with Tukey’s multiple comparison test)

Article Snippet: For the over expression of the Tau, murine mutant Tau (P232S) cDNA (NCBI ref: BC014748) under the transcriptional control of the cytomegalovirus chicken β-actin (CAG2) hybrid promotor was inserted into the adeno-associated virus serotype 9 (AAV9) vector with the enhanced green fluorescence protein reporter (eGFP) and 2A linker in between GFP and the mTau sequence (AAV9-CAG2-eGFP-2A-mTau (P232S)-WPRE or AAV9- Tau) (Vector Biolabs, USA).

Techniques: Control, Over Expression, Western Blot, Staining, Comparison

Journal: Acta Neuropathologica Communications

Article Title: Tau modulation through AAV9 therapy augments Akt/Erk survival signalling in glaucoma mitigating the retinal degenerative phenotype

doi: 10.1186/s40478-024-01804-0

Figure Lengend Snippet: Silencing of Tau expression using AAV protects the inner retinal function and laminar structure in glaucomatous eyes. A Chronic elevation of IOP in C57BL/6 mice eyes was induced by intracameral microbead injections for 2 months. B Positive scotopic threshold response (pSTR) traces from control, glaucoma, glaucoma + scrambled and glaucoma + Tau KD mice retinas, as indicated. C Quantification of the pSTR amplitudes from the glaucoma AAV9-Tau KD mice eyes compared to glaucoma scrambled sequence expressing controls indicated significant protection against pSTR amplitudes loss in the glaucoma-AAV-Tau KD animals. (n = 10, *** P < 0.001, One-way ANOVA analysis with Tukey’s multiple comparison test). D H and E staining of retinal sections of the control, glaucoma, glaucoma + AAV9-scramble, and glaucoma + AAV9-tau KD mice eyes. (representative images, arrows indicate the changes in cell densities, Scale bar, 50 μm). E Quantification of the cell density in GCL indicated significant protection against cell loss in glaucoma AAV9-tau KD-subjected retinas compared to the microbead-injected control eyes. (n = 5, *** P < 0.001, **** P < 0.0001, One-way ANOVA analysis with Tukey’s multiple comparison test)

Article Snippet: For the over expression of the Tau, murine mutant Tau (P232S) cDNA (NCBI ref: BC014748) under the transcriptional control of the cytomegalovirus chicken β-actin (CAG2) hybrid promotor was inserted into the adeno-associated virus serotype 9 (AAV9) vector with the enhanced green fluorescence protein reporter (eGFP) and 2A linker in between GFP and the mTau sequence (AAV9-CAG2-eGFP-2A-mTau (P232S)-WPRE or AAV9- Tau) (Vector Biolabs, USA).

Techniques: Expressing, Control, Sequencing, Comparison, Staining, Injection

Journal: Acta Neuropathologica Communications

Article Title: Tau modulation through AAV9 therapy augments Akt/Erk survival signalling in glaucoma mitigating the retinal degenerative phenotype

doi: 10.1186/s40478-024-01804-0

Figure Lengend Snippet: Western blot (WB) analysis of retinal endoplasmic reticulum (ER) stress markers changes in glaucoma and Tau modulation conditions. A WBs of control and glaucoma C57BL/6 mice retinal lysates probed with GRP78(1:1000), CHOP(1:1000), P-PERK(1:1000), and β-actin (1:5000) antibodies and B-D their respective relative band intensities quantified as the fold change using β-actin as loading control (n = 3 **** P < 0.0001, t-test). E WB of retinal lysates of the control, glaucoma, AAV-GFP, AAV-mTau (overexpression), AAV-GFP + glaucoma, and AAV-mTau + glaucoma mice were probed with GRP-78, CHOP, P-PERK, and actin antibodies, and F-H their respective relative band intensities quantified as the fold change using β-actin as loading control (n = 3, ** P < 0.01, *** P < 0.001, **** P < 0.0001, One-way ANOVA analysis with Tukey’s multiple comparison test,). I WB of retinal lysates from control, glaucoma, AAV9-scramble, AAV-mTau KD, AAV9-scramble + glaucoma, AAV-mTau KD + glaucoma mice were probed with GRP 78, CHOP, P-PERK, and actin antibodies, and (J-L) their respective band intensities quantified as fold change using β-actin as a loading control (*** P < 0.001, **** P < 0.0001, One-way ANOVA analysis with Tukey’s multiple comparison test, n = 3 per group)

Article Snippet: For the over expression of the Tau, murine mutant Tau (P232S) cDNA (NCBI ref: BC014748) under the transcriptional control of the cytomegalovirus chicken β-actin (CAG2) hybrid promotor was inserted into the adeno-associated virus serotype 9 (AAV9) vector with the enhanced green fluorescence protein reporter (eGFP) and 2A linker in between GFP and the mTau sequence (AAV9-CAG2-eGFP-2A-mTau (P232S)-WPRE or AAV9- Tau) (Vector Biolabs, USA).

Techniques: Western Blot, Control, Over Expression, Comparison

Journal: Theranostics

Article Title: Cardiomyocyte-derived OTUD7B promotes cardiac hypertrophy by deubiquitinating SERCA2a

doi: 10.7150/thno.129105

Figure Lengend Snippet: Cardiomyocyte-specific OTUD7B overexpression promotes cardiac hypertrophy by deubiquitinating SERCA2a at K628. (A) Schematic diagram of the TAC-induced mouse model. WT mice were injected with AAV9 cardiomyocyte-specific overexpressing SERCA2a WT or SERCA2a K628R , and OTUD7B OE or empty vector (EV) by the tail vein. After 2 weeks, mice were subjected to TAC. After 4 weeks, the cardiac function of mice was assessed using echocardiography. Mice were then euthanized, and samples were harvested. (B) Representative left ventricular M-mode echocardiographic images. (C-D) Values of ejection fraction (C) and fractional shortening (D). EF, ejection fraction; FS, fractional shortening; n = 6. (E) Representative whole heart images. (F) The ratio of heart weight to body weight (HW/BW). n = 6. (G) The ratio of heart weight to tibial length (HW/TL). n = 6. (H) Representative HE-stained images of cardiac tissue sections. (I-J) Representative images (I) and quantification (J) of wheat germ agglutinin (WGA)-stained cardiac tissue sections (n = 6). (K-L) Representative images (K) and quantification (L) from Masson's trichrome-stained cardiac tissue sections (n = 6). (M) Serum atrial natriuretic peptide (ANP) levels were detected using ELISA kits (n = 6). (N-P) mRNA levels of Myh7 (N), Nppa (O), and Nppb (P) in heart tissues (n = 6). Signal intensities were adjusted using Actb as the normalization control.

Article Snippet: Animals were maintained for 4 weeks after TAC or sham surgery. (3) OTUD7B (OTUD7B OE ) and SERCA2a (SERCA2a WT or SERCA2a K628R ) cardiomyocyte-specific overexpression was achieved using recombinant adeno-associated virus serotype 9 (AAV9) vectors driven by the cTNT promoter (Genechem, Shanghai, China).

Techniques: Over Expression, Injection, Plasmid Preparation, Staining, Enzyme-linked Immunosorbent Assay, Control

Journal: General Psychiatry

Article Title: MicroRNA-451a is a candidate biomarker and therapeutic target for major depressive disorder

doi: 10.1136/gpsych-2023-101291

Figure Lengend Snippet: Transgenic overexpression of miR-451a in the mPFC alleviated CRS-induced, depression-like behaviour and neuronal spinal loss in mice. (A) Diagram illustrating the experimental time course for AAV injection (Ad_OE-miR-451a for miR-451a overexpression and Ad_OE-scramble for negative control), CRS model establishment, the behavioural tests and the sacrifice of mice. (B) The representative image of the injected AAV in the mPFC: Cg1, cingulate cortex area 1; Cg2, cingulate cortex area 2; DP, dorsal peduncular cortex; FrA, frontal association cortex; IL, infralimbic cortex; M2, secondary motor cortex; MO, medial orbital cortex; PrL, prelimbic cortex; VO, ventral orbital cortex. (C) Typical fluorescent images of AAV_OE-miR-451a-GFP-infected cells (green) and neurons (red) in the mPFC. (D) The statistical graph shows the percentage of AAV_OE-miR-451a-GFP-infected neurons in total neurons (n=3 mice, 3 slices per mouse). (E) AAV _OE-miR-451a-GFP injection increased miR-451a level in the mPFC (n=4). (F) CRS-induced decrease in sucrose intake preference, which was reversed by prior injection of AAV virus expressing miR-451a (Ad_OE-miR-451a) into the mPFC (Ad_OE-scramble-CON n=17, Ad_OE-miR-451a-CON n=16, Ad_OE-scramble-CRS n=18, Ad_OE-miR-451a-CON n=16). (G) Similarly, overexpression of miR-451a in CRS mice normalised the immobile time in the forced swimming test (Ad_OE-scramble-CON n=17, Ad_OE-miR-451a-CON n=16, Ad_OE-scramble-CRS n=18, Ad_OE-miR-451a-CON n=16). (H) Representative images of dendritic segments and spines of neurons in the mPFC from controls and CRS-exposed mice injected with scrambled and miR-451a overexpression viruses. (I) Quantification revealed that miR-451a overexpression reversed decreases in spine densities of the mPFC neurons of the CRS mice (n=3 per group, 5 neurons per mouse). Data are mean±SEM and analysed by Student’s t-test (E) and two-way ANOVA with Tukey post-hoc test (F, G and I), respectively. AAV, adeno-associated virus serotype 9; ANOVA, analysis of variance; CON, control; CRS, chronic restraint stress; DAPI, diamidino-2-phenylindole; FST, forced swimming test; miR-451a, microRNA-451a; mPFC, medial prefrontal cortex; SEM, standard error of the mean.

Article Snippet: For overexpression of miR-451a in vivo , viral constructs were designed using adeno-associated virus serotype 9 (AAV9) vectors (Genechem, China), encoding a pri-mmu-miR-451a-GFP fusion protein (Ad_OE-miR-451a) or green fluorescent protein (GFP) only as a negative control, under the control of the cytomegalovirus (CMV) promoter (Ad_OE-scramble).

Techniques: Transgenic Assay, Over Expression, Injection, Negative Control, Infection, Virus, Expressing

Journal: Biochemistry and Biophysics Reports

Article Title: Knockdown of ARHGAP24 reduces intimal hyperplasia through inhibiting the proliferation and phenotypic switching of smooth muscle cells possibly by inactivating both AKT and ERK1/2 signaling pathways

doi: 10.1016/j.bbrep.2023.101591

Figure Lengend Snippet: Primers for mRNA PCR (mouse).

Article Snippet: Recombinant adeno-associated virus serotype 9 (AAV9) vectors carrying Arhgap24 shRNA, Arhgap24 plasmid or negative control with a pCMV promoter (AAV9-siArhgap24, AAV9-Arhgap24 OE or AAV9-NC) were manufactured by GeneChem Co., Ltd (Shanghai, China).

Techniques: Sequencing

Journal: Biochemistry and Biophysics Reports

Article Title: Knockdown of ARHGAP24 reduces intimal hyperplasia through inhibiting the proliferation and phenotypic switching of smooth muscle cells possibly by inactivating both AKT and ERK1/2 signaling pathways

doi: 10.1016/j.bbrep.2023.101591

Figure Lengend Snippet: ARHGAP24 expression was significantly increased in dedifferentiated vascular smooth muscle cells in vivo and in vitro. A-D, qRT-PCR analysis of Acta2, Cnn1, Tagln and Arhgap24 expression at different time points in wire injured femoral arteries (n = 4 mice at different time points) compared to sham femoral arteries (n = 4 mice), normalized to Gapdh. Data were presented as mean ± SD. *P < 0.05 versus Uninjured group, ordinary one-way ANOVA followed by Holm-Sidak post-hoc test. E, HASMCs were incubated with the culture media containing 10 ng/ml PDGF-BB for the indicated time periods. The expressions of ACTA2, CNN1, TAGLN and ARHGAP24 in HASMCs were determined by Western blot. Data were presented as mean ± SD. n = 4, *P < 0.05 versus Ctrl group, one-way ANOVA followed by Holm-Sidak post-hoc test. F, HASMCs were incubated with the culture media containing 25 μM RepSox for the indicated time periods. The expressions of ACTA2, CNN1, TAGLN and ARHGAP24 in HASMCs were determined by Western blot. Data were presented as mean ± SD. n = 4, *P < 0.05 versus Ctrl group, one-way ANOVA followed by Holm-Sidak post-hoc test.

Article Snippet: Recombinant adeno-associated virus serotype 9 (AAV9) vectors carrying Arhgap24 shRNA, Arhgap24 plasmid or negative control with a pCMV promoter (AAV9-siArhgap24, AAV9-Arhgap24 OE or AAV9-NC) were manufactured by GeneChem Co., Ltd (Shanghai, China).

Techniques: Expressing, In Vivo, In Vitro, Quantitative RT-PCR, Incubation, Western Blot

Journal: Biochemistry and Biophysics Reports

Article Title: Knockdown of ARHGAP24 reduces intimal hyperplasia through inhibiting the proliferation and phenotypic switching of smooth muscle cells possibly by inactivating both AKT and ERK1/2 signaling pathways

doi: 10.1016/j.bbrep.2023.101591

Figure Lengend Snippet: Silencing ARHGAP24 inhibited HASMCs dedifferentiation and proliferation in the absence or present PDGF-BB. A, HASMCs were transfected with negative control (NC) or siARHGAP24 at 100 nM in the absence of PDGF-BB. The expressions of ACTA2, CNN1, TAGLN and ARHGAP24 in HASMCs were determined by Western blot. Data were presented as mean ± SD. n = 4, *P < 0.05 versus NC group, non-parametric paired t -test. B, Cell proliferation was detected using EdU detection kits (Thermo, USA) to analyze the incorporation of EdU during DNA synthesis. Scale bar = 100 μm. Data were presented as mean ± SD. n = 4, *P < 0.05 versus NC group, non-parametric paired t -test. C, HASMCs were transfected with NC or siARHGAP24 at 100 nM in the present of PDGF-BB. The expressions of ACTA2, CNN1 and TAGLN in HASMCs were determined by Western blot. Data were presented as mean ± SD. n = 4, *P < 0.05 versus NC group; # P < 0.05 versus NC + PDGF-BB group, one-way ANOVA followed by Holm-Sidak post-hoc test. D, Cell proliferation was detected using EdU detection kits (Thermo, USA) to analyze the incorporation of EdU during DNA synthesis. Scale bar = 100 μm. Data were presented as mean ± SD. n = 4, *P < 0.05 versus NC group; # P < 0.05 versus NC + PDGF-BB group, one-way ANOVA followed by Holm-Sidak post-hoc test.

Article Snippet: Recombinant adeno-associated virus serotype 9 (AAV9) vectors carrying Arhgap24 shRNA, Arhgap24 plasmid or negative control with a pCMV promoter (AAV9-siArhgap24, AAV9-Arhgap24 OE or AAV9-NC) were manufactured by GeneChem Co., Ltd (Shanghai, China).

Techniques: Transfection, Negative Control, Western Blot, DNA Synthesis

Journal: Biochemistry and Biophysics Reports

Article Title: Knockdown of ARHGAP24 reduces intimal hyperplasia through inhibiting the proliferation and phenotypic switching of smooth muscle cells possibly by inactivating both AKT and ERK1/2 signaling pathways

doi: 10.1016/j.bbrep.2023.101591

Figure Lengend Snippet: AAV9-mediated silencing of Arhgap24 alleviated intimal hyperplasia in a mouse model. A, Representative immunofluorescence of Arhgap24 in injured femoral arteries from wild type mice (WT) treated with AAV9-NC or AAV9-siArhgap24 after 4 weeks post-femoral wire injury (n = 4). Note: Green (Arhgap24), Blue (Dapi that reflects total cells). Scale bar: 20 μm. B, Quantification of the Arhgap24 expression in VSMCs in the injured femoral arteries (n = 4). Data were presented as mean ± SD. * P < 0.05 versus AAV9-NC, non-parametric paired t -test. C, Representative immunofluorescence of Ki67 and Acta2 in injured femoral arteries from WT mice treated with AAV9-NC or AAV9-siArhgap24 after 4 weeks post-femoral wire injury (n = 4). Note: Green (Ki67), Blue (Dapi that reflects total cells), Red (Acta2 in VSMCs). Scale bar: 20 μm. D, Quantification of the Ki67 and Acta2 expression in VSMCs in the injured femoral arteries (n = 4). Data were presented as mean ± SD. * P < 0.05 versus AAV9-NC, non-parametric paired t -test. E, Hematoxylin and eosin staining of serial cross sections from femoral arteries from WT treated with AAV9-NC or AAV9-siArhgap24 after 4 weeks post-femoral wire injury (n = 4). Scale bar: 50 μm. F, Morphometric measurements of the ratio of intimal and media area (I/M ratio) in the injured femoral arterial sections (n = 4). Data are presented as mean ± SD of I/M ratio. * P < 0.05 versus AAV9-NC, non-parametric paired t -test. L, Lumen; A, Adventitia. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Recombinant adeno-associated virus serotype 9 (AAV9) vectors carrying Arhgap24 shRNA, Arhgap24 plasmid or negative control with a pCMV promoter (AAV9-siArhgap24, AAV9-Arhgap24 OE or AAV9-NC) were manufactured by GeneChem Co., Ltd (Shanghai, China).

Techniques: Immunofluorescence, Expressing, Staining

Journal: Biochemistry and Biophysics Reports

Article Title: Knockdown of ARHGAP24 reduces intimal hyperplasia through inhibiting the proliferation and phenotypic switching of smooth muscle cells possibly by inactivating both AKT and ERK1/2 signaling pathways

doi: 10.1016/j.bbrep.2023.101591

Figure Lengend Snippet: AAV9-mediated overexpression of Arhgap24 exacerbates intimal hyperplasia in a mouse model. A, Representative immunofluorescence of Arhgap24 in injured femoral arteries from wild type mice (WT) treated with AAV9-NC or AAV9-Arhgap24 OE after 4 weeks post-femoral wire injury (n = 4). Note: Green (Arhgap24), Blue (Dapi that reflects total cells). Scale bar: 20 μm. B, Quantification of the Arhgap24 expression in VSMCs in the injured femoral arteries (n = 4). Data were presented as mean ± SD. * P < 0.05 versus AAV9-NC, non-parametric paired t -test. C, Representative immunofluorescence of Ki67 and Acta2 in injured femoral arteries from WT mice treated with AAV9-NC or AAV9-Arhgap24 OE after 4 weeks post-femoral wire injury (n = 4). Note: Green (Ki67), Blue (Dapi that reflects total cells), Red (Acta2 in VSMCs). Scale bar: 20 μm. D, Quantification of the Ki67 and Acta2 expression in VSMCs in the injured femoral arteries (n = 4). Data were presented as mean ± SD. * P < 0.05 versus AAV9-NC, non-parametric paired t -test. E, Hematoxylin and eosin staining of serial cross sections from femoral arteries from WT treated with AAV9-NC or AAV9-Arhgap24 OE after 4 weeks post-femoral wire injury (n = 4). Scale bar: 50 μm. F, Morphometric measurements of the ratio of intimal and media area (I/M ratio) in the injured femoral arterial sections (n = 4). Data are presented as mean ± SD of I/M ratio. * P < 0.05 versus AAV9-NC, non-parametric paired t -test. L, Lumen; A, Adventitia. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Recombinant adeno-associated virus serotype 9 (AAV9) vectors carrying Arhgap24 shRNA, Arhgap24 plasmid or negative control with a pCMV promoter (AAV9-siArhgap24, AAV9-Arhgap24 OE or AAV9-NC) were manufactured by GeneChem Co., Ltd (Shanghai, China).

Techniques: Over Expression, Immunofluorescence, Expressing, Staining

Journal: Biochemistry and Biophysics Reports

Article Title: Knockdown of ARHGAP24 reduces intimal hyperplasia through inhibiting the proliferation and phenotypic switching of smooth muscle cells possibly by inactivating both AKT and ERK1/2 signaling pathways

doi: 10.1016/j.bbrep.2023.101591

Figure Lengend Snippet: Down-regulation of ARHGAP24 might affect both AKT and ERK1/2 to inhibit SMC dedifferentiation and proliferation. HASMCs were transfected with NC or siARHGAP24 at 100 nM in the present of PDGF-BB. The expressions of p -JNK, JNK, p-p38, p38, p -AKT, AKT, p -ERK1/2 and ERK1/2 in HASMCs were determined by Western blot. Data were presented as mean ± SD. n = 4, *P < 0.05 versus NC group; # P < 0.05 versus NC + PDGF-BB group, one-way ANOVA followed by Holm-Sidak post-hoc test.

Article Snippet: Recombinant adeno-associated virus serotype 9 (AAV9) vectors carrying Arhgap24 shRNA, Arhgap24 plasmid or negative control with a pCMV promoter (AAV9-siArhgap24, AAV9-Arhgap24 OE or AAV9-NC) were manufactured by GeneChem Co., Ltd (Shanghai, China).

Techniques: Transfection, Western Blot

Journal: Autophagy

Article Title: Autophagic degradation of CCN2 (cellular communication network factor 2) causes cardiotoxicity of sunitinib

doi: 10.1080/15548627.2021.1965712

Figure Lengend Snippet: Autophagy contributes to the cardiotoxicity of sunitinib. (A-C) Inhibition of autophagy rescues the apoptosis of cardiomyocyte. CCC-HEH-2 cells were transfected with nontargeting siRNA (NC) or siRNA targeting ATG7 or ATG5, and then treated with or without sunitinib for 24 h. (A-B) The expression of ATG7 or ATG5, LC3-I/II and c-CASP3 was detected by western blot, using TUBA as a loading control. Western blot was repeated three times and densitometric analysis was carried out. (C) Cell morphology was obtained by microscope. Scale bars: 100 µm. (D) C57BL/6 mice with heterozygous cardiac-specific Atg7 knockout (Atg7−/+) were generated by crossing Atg7-floxed C57BL/6 mice (Atg7flox/flox, Atg7f/f) with Myh6-MerCreMer transgenic C57BL/6 mice and then treated with 25 mg/kg tamoxifen for 3 days. 2 weeks after induction, Atg7f/+ and Atg7−/+ mice were treated with saline or sunitinib (40 mg/kg) for 42 days (n = 5 per group) by means of intragastric administration. Heart tissues were harvested after the mice were sacrificed, and total tissue lysates were prepared. (E) The deletion efficiency was determined by detecting the levels of ATG7 in total heart tissue lysates using GAPDH as a loading control. (F) The protein expression levels of ATG7 and LC3-I/II were analyzed in lysates from total heart tissues from 3 mice of each group. Densitometric analysis was carried out (G) Heart weight to body weight ratio. (H) Serum was analyzed for CK-MB and TNNT2 levels. (I-J) Cardiac function was examined by echocardiography, and the percentage of Ejection Fraction (EF) and Fractional Shortening (FS) was shown. n = 5. (K) Heart sections were stained with H&E. Scale bars: 100 µm. The p value was calculated by one-way ANOVA and the Sidak test. *: p < 0.05, **: p< 0.01, ***: p < 0.001, #: p < 0.05, ##: p < 0.01 and ###: p < 0.001. Abbreviations: SUNI, sunitinib; HW, heart weight; BW, body weight; CK-MB, creatine kinase myocardial band; TNNT2, troponin T2, cardiac.

Article Snippet: Cardiomyocyte-specific overexpression of Ccn2 Adeno-associated virus serotype 9 (AAV9) vectors carrying the Tnnt2 promoter and Ccn2 cDNA (AAV9- Tnnt2-Ccn2 ) were constructed and packaged by Vigene Biosciences (Shandong, China), and the titers of the AAV vectors (viral genomes/mL) were determined by quantitative real-time PCR (qPCR), as described previously [ 91 ].

Techniques: Inhibition, Transfection, Expressing, Western Blot, Control, Microscopy, Knock-Out, Generated, Transgenic Assay, Saline, Staining

Journal: Autophagy

Article Title: Autophagic degradation of CCN2 (cellular communication network factor 2) causes cardiotoxicity of sunitinib

doi: 10.1080/15548627.2021.1965712

Figure Lengend Snippet: Maladaptive autophagy promotes the cardiotoxicity of sunitinib via degradation of CCN2. (A) CCC-HEH-2 cells were treated with 5 µM sunitinib for different periods of time, as indicated (between 0 and 48 h). (B) CCC-HEH-2 cells were treated with 24 h of sunitinib for different concentrations, as indicated (between 0 and 12.5 µM). (A-B) The expression of CCN2 was measured by western blot. Western blot was repeated for three times independently and densitometric analysis was carried out. (C) Total heart tissue lysates from saline and sunitinib treated mice were analyzed by western blot using an anti-CCN2 antibody. TUBA was used as a loading control. Heart tissues from 3 mice of each group were used and densitometric analysis was carried out. (D) Immunocytochemistry for CCN2 and nuclear staining by DAPI in CCC-HEH-2 cells. CCC-HEH-2 cells were treated with or without 5 µM sunitinib for 24 h. The expression of CCN2 was measured by immunofluorescence assay. Scale bar: 50 µm (upper) and 20 µm (lower). (E-F) CCC-HEH-2 cells were transfected with CCN2 shRNA (#1, #2) and vector (Vsh) lentivirus and then collected 72 h later. (E) Representative western blot showing the effect of CCN2 knockdown on the apoptosis of CCC-HEH-2 cells. (F) Cells were harvested and stained with ANXA5 and PI, apoptosis rates were analyzed by flow cytometry and representative images are shown. The experiments were performed four times independently, and data are expressed as the mean ± SD. (G-L) AAV9-Tnnt2-Ccn2-null or AAV9-Tnnt2-NC virus was injected into C57BL/6 mice through the tail vein to specifically knockdown the expression of Ccn2 in the heart (n = 9), and the hearts were collected 4 weeks later. (G) Immunofluorescence assays were performed to detect the knockdown efficiency of Ccn2 in heart tissue achieved by AAV. Scale bars: 50 µm. (H) Heart weight to body weight ratio was calculated. (I-J) Cardiac function was examined by echocardiography. (J) Quantification of EF, FS and the rates of heartbeats. (K) Heart tissues from the mice were collected, and were stained with H&E for histopathological analysis. Scale bars: 50 µm. (L) Sirius Red staining was performed to detect fibrosis in heart tissue. Scale bars: 100 µm. The p value was calculated by Student’s t-test (unpaired, two-tailed, 2 groups) or by one-way ANOVA and Dunnett’s multiple comparisons test (multiple groups). *: p < 0.05, **: p < 0.01 and ***: p < 0.001. Abbreviations: SUNI, sunitinib; HW, heart weight; BW, body weight.

Article Snippet: Cardiomyocyte-specific overexpression of Ccn2 Adeno-associated virus serotype 9 (AAV9) vectors carrying the Tnnt2 promoter and Ccn2 cDNA (AAV9- Tnnt2-Ccn2 ) were constructed and packaged by Vigene Biosciences (Shandong, China), and the titers of the AAV vectors (viral genomes/mL) were determined by quantitative real-time PCR (qPCR), as described previously [ 91 ].

Techniques: Expressing, Western Blot, Saline, Control, Immunocytochemistry, Staining, Immunofluorescence, Transfection, shRNA, Plasmid Preparation, Knockdown, Flow Cytometry, Virus, Injection, Two Tailed Test

Journal: Autophagy

Article Title: Autophagic degradation of CCN2 (cellular communication network factor 2) causes cardiotoxicity of sunitinib

doi: 10.1080/15548627.2021.1965712

Figure Lengend Snippet: Overexpression of Ccn2 rescues the cardiotoxicity caused by sunitinib. (A) Schematic representation of AAV9-Tnnt2-Ccn2 used in this study. (B) AAV9-Tnnt2-Ccn2 or AAV9-Tnnt2-VEC virus was injected into C57BL/6 mice through the tail vein to specifically reduce the expression of CCN2 in the heart, which was followed by saline or sunitinib (40 mg/kg) treatment for 42 days by means of intragastric administration (n = 5). (C) Representative western blots showing CCN2 expression in heart tissue lysates. GAPDH was used as a loading control. Total heart tissues from 3 mice of each group were used and densitometric analysis was carried out. (D) Heart weight to body weight ratio. (E-G) Cardiac function was examined by echocardiography. (H) Serum was analyzed for CK-MB and TNNT2 levels. (I) Heart sections were stained with H&E. Scale bars: 100 µm. The p value was calculated by one-way ANOVA (Dunnett’s multiple comparisons test). **: p < 0.01, ***: p < 0.001, ##: p < 0.01 and ###: p < 0.001. Abbreviations: AAV9, adeno-associated virus 9; SUNI, sunitinib; VEC, vector; HW, heart weight; BW, body weight; CK-MB, creatine kinase myocardial band; TNNT2, troponin T2, cardiac.

Article Snippet: Cardiomyocyte-specific overexpression of Ccn2 Adeno-associated virus serotype 9 (AAV9) vectors carrying the Tnnt2 promoter and Ccn2 cDNA (AAV9- Tnnt2-Ccn2 ) were constructed and packaged by Vigene Biosciences (Shandong, China), and the titers of the AAV vectors (viral genomes/mL) were determined by quantitative real-time PCR (qPCR), as described previously [ 91 ].

Techniques: Over Expression, Virus, Injection, Expressing, Saline, Western Blot, Control, Staining, Plasmid Preparation

Journal: Autophagy

Article Title: Autophagic degradation of CCN2 (cellular communication network factor 2) causes cardiotoxicity of sunitinib

doi: 10.1080/15548627.2021.1965712

Figure Lengend Snippet: Inhibition of HMGB1 by GA confers functional protection against sunitinib-induced cardiotoxicity. (A) CCC-HEH-2 cells were treated with various concentrations of GA, as indicated (between 0 and 100 µM) after 24 h of exposure. Representative western blots showing HMGB1 expression upon GA treatment using TUBA as a loading control. (B) CCC-HEH-2 cells were exposed to GA (50 µM) with or without sunitinib (5 µM) for 24 h. LC3-I/II and CCN2 protein levels were measured by western blot analysis using TUBA as a loading control. Western blot was repeated at least three times and densitometric analysis was carried out. (C) Autophagic flux assays showed the effect of HMGB1 inhibition by GA on sunitinib-induced autophagy in MCMs cells. Scale bars: 10 µm. Right panel: quantification of the percentage of mCherry-positive, GFP-positive puncta per nucleus. n = 100 cells pooled from three independent experiments. (D) CCC-HEH-2 cells were exposed to GA (50 µM) with or without sunitinib (5 µM) for 24 h. c-PARP and c-CASP3 protein levels were measured by western blot analysis using TUBA as a loading control. Western blot was repeated at least three times and densitometric analysis was carried out. (E) Cells were treated with or without sunitinib, in the absence or presence of GA for 24 h, and cell morphology was obtained by microscopy. Scale bars: 100 µm. (F) CCC-HEH-2 cells were exposed to GA (50 µM) with or without sunitinib (5 µM) for 72 h. SRB assay showed the effect of GA on sunitinib-induced inhibition in the survival fraction of CCC-HEH-2 cells. Three independent experiments were performed. (G-J) C57BL/6 mice were treated with saline, sunitinib (40 mg/kg), GA (10 mg/kg) or GA plus sunitinib for 42 days by means of intragastric administration (n = 5 per group). (G) Heart weight to body weight ratio. (H) Serum was analyzed for CK-MB and TNNT2 levels. (I) Cardiac function was examined by echocardiography. (J) Heart tissues from the mice were collected, and stained with H&E for histopathological analysis. Scale bars: 100 µm. The p value was calculated by one-way ANOVA (Dunnett’s multiple comparisons test or Sidak test). **: p < 0.01, ***: p < 0.001, #: p < 0.05, ##: p < 0.01 and ###: p < 0.001. Abbreviations: SUNI, sunitinib; HW, heart weight; BW, body weight; CK-MB, creatine kinase myocardial band; TNNT2, troponin T2, cardiac; GA, glycyrrhizic acid; MCMs, adult mouse cardiomyocyte.

Article Snippet: Cardiomyocyte-specific overexpression of Ccn2 Adeno-associated virus serotype 9 (AAV9) vectors carrying the Tnnt2 promoter and Ccn2 cDNA (AAV9- Tnnt2-Ccn2 ) were constructed and packaged by Vigene Biosciences (Shandong, China), and the titers of the AAV vectors (viral genomes/mL) were determined by quantitative real-time PCR (qPCR), as described previously [ 91 ].

Techniques: Inhibition, Functional Assay, Western Blot, Expressing, Control, Microscopy, Sulforhodamine B Assay, Saline, Staining