Journal: Cell Cycle

Article Title: Identifying the hub gene in gastric cancer by bioinformatics analysis and in vitro experiments

doi: 10.1080/15384101.2020.1749789

Figure Lengend Snippet: Enrichment plots from GSEA. GSEA results showing that calcium signaling pathway, cell adhesion molecules, cytokine receptor interaction, dilated cardiomyopathy, ECM receptor interaction, focal adhesion, hedgehog signaling pathway, hematopoietic cell lineage, and hypertrophic cardiomyopathy were significantly enriched in SERPINH1 highly-expressed human GC tissues.

Article Snippet: An empty plasmid served as a negative control (scramble). siRNAs designed to specifically silence Serpinh1 was purchased from GenePharma (Shanghai, China).

Techniques:

Journal: Cell Cycle

Article Title: Identifying the hub gene in gastric cancer by bioinformatics analysis and in vitro experiments

doi: 10.1080/15384101.2020.1749789

Figure Lengend Snippet: Correlation of SERPINH1 expression with immune infiltration level in STAD (stomach adenocarcinoma). SERPINH1 expression is significantly negatively related to B cells and has a very weak correlation with macrophages and dendritic cell infiltration levels in STAD. But SERPINH1 expression has no significant correlations with tumor purity and infiltrating levels of CD8 + T cells, CD4 + T cells, and neutrophils in STAD.

Article Snippet: An empty plasmid served as a negative control (scramble). siRNAs designed to specifically silence Serpinh1 was purchased from GenePharma (Shanghai, China).

Techniques: Expressing

Journal: Cell Cycle

Article Title: Identifying the hub gene in gastric cancer by bioinformatics analysis and in vitro experiments

doi: 10.1080/15384101.2020.1749789

Figure Lengend Snippet: SERPINH1 expression is upregulated in GC. (a) and (b) Western blot analysis showed that the SERPINH1 expression level in matched GC tissues (t) and adjacent non-tumor tissues (n), and in normal gastric epithelial and GC cell lines. GAPDH was used as a loading control. (c) Representative immunohistochemistry staining results revealed the protein level expression of SERPINH1 in GC and normal tissues. *P < 0.05.

Article Snippet: An empty plasmid served as a negative control (scramble). siRNAs designed to specifically silence Serpinh1 was purchased from GenePharma (Shanghai, China).

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

Journal: Cell Cycle

Article Title: Identifying the hub gene in gastric cancer by bioinformatics analysis and in vitro experiments

doi: 10.1080/15384101.2020.1749789

Figure Lengend Snippet: SERPINH1 promotes cell proliferation, colony formation and migration in vitro. (a) The SERPINH1 protein level is increased after overexpression of SERPINH1 in BGC823 cells and decreased after knockdown of SERPINH1 in MKN45 cells. (b) Ectopic expression of SERPINH1 stimulates colony formation in BGC823 cells. Knockdown of SERPINH1 expression inhibits colony formation in MKN45 cells. (c) Ectopic expression of SERPINH1 promotes cell migration in BGC823 cells as demonstrated by transwell assays. Knockdown of SERPINH1 expression inhibits cell migration in MKN45 cells, Original magnification ×100. (d) Ectopic expression of SERPINH1 promotes cell proliferation in BGC823 cells whereas knockdown of SERPINH1 inhibits cell proliferation in MKN45 cells as determined by MTT assays. *P < 0.05.

Article Snippet: An empty plasmid served as a negative control (scramble). siRNAs designed to specifically silence Serpinh1 was purchased from GenePharma (Shanghai, China).

Techniques: Migration, In Vitro, Over Expression, Knockdown, Expressing

Journal: Cell Cycle

Article Title: Identifying the hub gene in gastric cancer by bioinformatics analysis and in vitro experiments

doi: 10.1080/15384101.2020.1749789

Figure Lengend Snippet: SERPINH1 influences the cell cycle progression and reduced the apoptotic rate by using flow cytometry. (a) Cell cycle detected by flow cytometry in BGC823 or MKN45 cells 48 h after transfection with pcDNA3.1 (+)-SERPINH1 or si-SERPINH1, respectively. Histogram represented the percentage of cells in G1, S and G2 cell-cycle phases. (b) Cell apoptosis was detected by Annexin-V/PI with flow cytometry in BGC823 or MKN45 cells 48 h after transfection with pcDNA3.1 (+)-SERPINH1 or si-SERPINH1, respectively. The apoptotic evaluation was calculated by the percentage of apoptotic cell number in total cell number. *P < 0.05.

Article Snippet: An empty plasmid served as a negative control (scramble). siRNAs designed to specifically silence Serpinh1 was purchased from GenePharma (Shanghai, China).

Techniques: Flow Cytometry, Transfection

Journal: Human Gene Therapy

Article Title: AAV Delivery of Endothelin-1 shRNA Attenuates Cold-Induced Hypertension

doi: 10.1089/hum.2016.047

Figure Lengend Snippet: AAV delivery of ET1-shRNA prevented the development of cold-induced hypertension. (A) Systolic blood pressure. (B) Body weight. Data shown as means ± standard error of the mean (SEM); n = 8. AAV, adeno-associated virus; BP, blood pressure; ET1, endothelin-1; PBS, phosphate-buffered saline; shRNA, short-hairpin small interference RNA.

Article Snippet: Three ET1 and scrambled shRNA sequences were produced by Geno-Mechanix and high performance liquid chromatography purified.

Techniques: shRNA, Virus, Saline

Journal: Human Gene Therapy

Article Title: AAV Delivery of Endothelin-1 shRNA Attenuates Cold-Induced Hypertension

doi: 10.1089/hum.2016.047

Figure Lengend Snippet: AAV delivery ET1-shRNA attenuated the cold-induced increase in ET1 levels. Concentrations of ET1 in heart (A), aorta (B), mesenteric arteries (C), plasma (D), renal cortex (E), and renal medulla (F). These parameters were measured at 10 weeks after exposure to cold. Data shown as means ± SEM; n = 8. *p < 0.05, **p < 0.01, ***p < 0.001 vs. warm-PBS group. +p < 0.05 vs. the cold-SC-shRNA group.

Article Snippet: Three ET1 and scrambled shRNA sequences were produced by Geno-Mechanix and high performance liquid chromatography purified.

Techniques: shRNA, Clinical Proteomics

Journal: Human Gene Therapy

Article Title: AAV Delivery of Endothelin-1 shRNA Attenuates Cold-Induced Hypertension

doi: 10.1089/hum.2016.047

Figure Lengend Snippet: AAV delivery ET1-shRNA attenuated the cold-induced increase in superoxide production. (A) In situ superoxide levels in the heart (red, DHE staining). (B) Quantification of superoxide levels. These parameters were measured at 10 weeks after exposure to cold. Data shown as means ± SEM; n = 8. **p < 0.01 vs. warm-PBS group. ++p < 0.05 vs. the cold-SC-shRNA group.

Article Snippet: Three ET1 and scrambled shRNA sequences were produced by Geno-Mechanix and high performance liquid chromatography purified.

Techniques: shRNA, In Situ, Staining

Journal: Experimental & Molecular Medicine

Article Title: TWIK-1/TASK-3 heterodimeric channels contribute to the neurotensin-mediated excitation of hippocampal dentate gyrus granule cells

doi: 10.1038/s12276-018-0172-4

Figure Lengend Snippet: Electrical properties of DGGCs

Article Snippet: To produce the recombinant adenovirus vector, U6-loxP-CMV-mCherry-shRNA sequences from pSicoR-Scrambled (Sc) shRNA, pSicoR-TWIK-1 shRNA , and pSicoR-TASK-3 shRNA were cloned into pDONR TM 207 vectors (Invitrogen) and confirmed by DNA sequencing.

Techniques: shRNA, Membrane

Journal: Experimental & Molecular Medicine

Article Title: TWIK-1/TASK-3 heterodimeric channels contribute to the neurotensin-mediated excitation of hippocampal dentate gyrus granule cells

doi: 10.1038/s12276-018-0172-4

Figure Lengend Snippet: a The averaged current – voltage (I–V) relationship of the whole-cell currents from DGGCs infected with Ad-Sc or Ad-TASK-3 shRNAs or both Ad-TASK-3 shRNA and TWIK-1 shRNA as well as from naive DGGCs was measured in standard artificial cerebrospinal fluid in the presence of Cs + /TEA/4-AP (1 mM/5 mM/5 mM). Whole-cell currents were elicited by 1-s-duration ramp pulses descending from 50 mV to −150 mV from a holding potential of −70 mV. b A summary bar graph for a . The mean values of the current density in naive DGGCs ( n = 14 cells, N = 4 mice) or DGGCs expressing Sc shRNA ( n = 12 cells, N = 3 mice), TASK-3 shRNA ( n = 15 cells, N = 3 mice), or both TASK-3 and TWIK-1 shRNAs ( n = 15 cells, N = 3 mice) measured in the presence of Cs + /TEA/4-AP (1 mM/5 mM/5 mM) are shown. The current density values are depicted at +50 mV. c The TASK-3 shRNA- or both TASK-3 shRNA- and TWIK-1 shRNA-sensitive currents were determined by subtracting each of the shRNA averaged currents from the Sc shRNA averaged currents a . ** P < 0.01

Article Snippet: To produce the recombinant adenovirus vector, U6-loxP-CMV-mCherry-shRNA sequences from pSicoR-Scrambled (Sc) shRNA, pSicoR-TWIK-1 shRNA , and pSicoR-TASK-3 shRNA were cloned into pDONR TM 207 vectors (Invitrogen) and confirmed by DNA sequencing.

Techniques: Infection, shRNA, Expressing

Journal: Experimental & Molecular Medicine

Article Title: TWIK-1/TASK-3 heterodimeric channels contribute to the neurotensin-mediated excitation of hippocampal dentate gyrus granule cells

doi: 10.1038/s12276-018-0172-4

Figure Lengend Snippet: a Representative traces of the membrane potential to stepwise current injections recorded from naive DGGCs ( n = 27, N = 3) or DGGCs infected with Ad-Sc shRNA ( n = 21, N = 3), Ad-TWIK-1 shRNA ( n = 30, N = 3), Ad-TASK-3 shRNA ( n = 22, N = 3), or both Ad-TWIK-1 shRNA and Ad-TASK-3 shRNA ( n = 32, N = 3). The resting membrane potentials of the cells was maintained at −70 mV by constant current injections, and the depolarizing current was then injected stepwise in 5-pA increments. b The number of spikes indicated that the neuron infected with Ad-TWIK-1 shRNA and Ad-TASK-3 shRNA were more excitable compared to control mice. The recordings were performed in artificial cerebrospinal fluid containing 50 µM D-AP5, 10 µM CNQX, 10 µM bicuculline, 10 µM CGP 55845, 2 mM TEA, and 0.5 mM NiCl 2 , with a pipette solution containing 5 mM QX314. c Averaged values of rheobase currents in naive cells ( n = 27 cells, N = 3 mice) and cells expressing Ad-Sc shRNA ( n = 21 cells, N = 3 mice), Ad-TWIK-1 shRNA ( n = 30 cells, N = 3 mice), Ad-TASK-3 shRNA ( n = 12), or both Ad-TWIK-1 shRNA and Ad-TASK-3 shRNA ( n = 32 cells, N = 3 mice). All values are means ± SEM. * P < 0.05, *** P < 0.001

Article Snippet: To produce the recombinant adenovirus vector, U6-loxP-CMV-mCherry-shRNA sequences from pSicoR-Scrambled (Sc) shRNA, pSicoR-TWIK-1 shRNA , and pSicoR-TASK-3 shRNA were cloned into pDONR TM 207 vectors (Invitrogen) and confirmed by DNA sequencing.

Techniques: Membrane, Infection, shRNA, Injection, Control, Transferring, Expressing

Journal: Experimental & Molecular Medicine

Article Title: TWIK-1/TASK-3 heterodimeric channels contribute to the neurotensin-mediated excitation of hippocampal dentate gyrus granule cells

doi: 10.1038/s12276-018-0172-4

Figure Lengend Snippet: a Bath application of NT-mediated membrane depolarization of granule cells. A representative response of the membrane potential to stepwise current injections was recorded from naive DGGCs ( n = 15, N = 3) or DGGCs infected with Ad-Sc shRNA ( n = 16, N = 3), Ad-TWIK-1 shRNA ( n = 17, N = 3), Ad-TASK-3 shRNA ( n = 15, N = 3), or both Ad-TWIK-shRNA and Ad-TASK-3 shRNA ( n = 14, N = 3). The resting membrane potential of these cells was maintained at −70 mV by constant current injections, and depolarizing currents were then injected stepwise at 5-pA increments until the membrane potential reached the firing threshold. b , c Analyzed bar charts of spike numbers at 105 pA for a . b Resting membrane potential values of the whole-cell currents in naive dentate gyrus granule cells ( n = 15 cells, N = 3 mice) and cells expressing Sc shRNA ( n = 16 cells, N = 3 mice), TWIK-1 shRNA ( n = 17 cells, N = 3 mice), TASK-3 shRNA ( n = 15 cells, N = 3 mice), or TWIK-1/TASK-3 shRNAs ( n = 14 cells, N = 3 mice). c The extent of changes in the number of spikes following NT application in naive and Ad-Sc shRNA-infected DGGCs was larger than those in DGGCs transfected with Ad-TASK-1 shRNA alone, Ad-TWIK-1 shRNA alone, or both Ad-TWIK-1 shRNA and Ad-TASK-3 shRNAs. The data for Ad-TASK-3 shRNA alone, Ad-TWIK-1 shRNA alone, and both Ad-TWIK-1 shRNA and Ad-TASK-3 shRNAs showed no change in the numbers of spikes. The recordings were obtained in artificial cerebrospinal fluid containing 50 µM D-AP5, 10 µM CNQX, 10 µM bicuculline, 10 µM CGP55845, 2 mM TEA, and 0.5 mM NiCl 2 , with a pipette solution containing 5 mM QX314. All the data are presented as the means ± SEM. *** P < 0.001 was considered statistically significant

Article Snippet: To produce the recombinant adenovirus vector, U6-loxP-CMV-mCherry-shRNA sequences from pSicoR-Scrambled (Sc) shRNA, pSicoR-TWIK-1 shRNA , and pSicoR-TASK-3 shRNA were cloned into pDONR TM 207 vectors (Invitrogen) and confirmed by DNA sequencing.

Techniques: Membrane, Infection, shRNA, Injection, Expressing, Transfection, Transferring

Journal:

Article Title: Interleukin-1 regulates keratinocyte expression of T cell targeting chemokines through interleukin-1 receptor associated kinase-1 (IRAK1) dependent and independent pathways

doi: 10.1016/j.cellsig.2009.01.005

Figure Lengend Snippet: RNAi mediated down-regulation of IRAK1 expression. HaCaT cells were transfected with expression constructs encoding a scrambled negative control shRNA sequence or an IRAK1 specific shRNA. Geneticin selected cells were treated with medium only (Med) or 10 ng/ml IL-1β. After 24 hours cells were lysed in PBS with 2 M urea and 0.85% SDS and proteins examined by Western blotting. IRAK1 and GAPDH were detected using polyclonal anti-IRAK1 and anti-GAPDH rabbit antibodies, respectively.

Article Snippet: Control plasmid containing a scrambled shRNA sequence was also obtained from SuperArray.

Techniques: Expressing, Transfection, Construct, Negative Control, shRNA, Sequencing, Western Blot

Journal:

Article Title: Interleukin-1 regulates keratinocyte expression of T cell targeting chemokines through interleukin-1 receptor associated kinase-1 (IRAK1) dependent and independent pathways

doi: 10.1016/j.cellsig.2009.01.005

Figure Lengend Snippet: IRAK1 is required for CC chemokine secretion. (A and B), HaCaT cells were transfected with expression constructs encoding a scrambled negative control shRNA sequence (open bars) or IRAK1 specific shRNA (filled bars). (C and D), cells were transfected with either an empty (open bars) or IRAK1(1-217) (filled bars) expression construct. Geneticin selected cells were treated with medium only, 10 ng/ml IL-1β or 50 ng/ml TNF-α for 24 hours. Secretion of CCL5 (A and C) and CCL20 (B and D) was determined by ELISA. Expression of CCL5 and CCL20 in the control cells (open bars, scrambled shRNA (A and B) or empty vector (C and D)) was defined as 100 pct. Expression of CCL5 and CCL20 in the IRAK1 targeted cells (filled bars, IRAK1 specific shRNA (A and B) or IRAK1(1-217) expression vector (C and D)) is graphically represented relative to the control cells. * p < 0.05 compared to control cells transfected with scrambled shRNA plasmid (A and B) or empty vector (C and D) and receiving the same treatment.

Article Snippet: Control plasmid containing a scrambled shRNA sequence was also obtained from SuperArray.

Techniques: Transfection, Expressing, Construct, Negative Control, shRNA, Sequencing, Enzyme-linked Immunosorbent Assay, Control, Plasmid Preparation

Journal:

Article Title: Interleukin-1 regulates keratinocyte expression of T cell targeting chemokines through interleukin-1 receptor associated kinase-1 (IRAK1) dependent and independent pathways

doi: 10.1016/j.cellsig.2009.01.005

Figure Lengend Snippet: IL-1β enhancement of IFN-γ induced CXC chemokine expression is IRAK1 independent. A, HaCaT cells were transfected with expression constructs encoding a scrambled negative control shRNA sequence (open bars) or IRAK1 specific shRNA (filled bars). B, cells were transfected with either an empty (open bars) or IRAK1(1-217) (filled bars) expression construct. Geneticin selected cells were treated with 20 ng/ml IFN-γ or 20 ng/ml IFN-γ plus 10 ng/ml IL-1β for 2 hours. Expression of CXC chemokine mRNAs was examined using real-time RT-PCR. Expression of the individual CXC chemokine mRNA in response to co-treatment with IFN-β+ IL-1β is graphically represented relative to expression induced by IFN-γ alone. * p < 0.05 compared to cells transfected with the same vector and treated with IFN-γ only. ** p < 0.01 compared to cells transfected with the same vector and treated with IFN-γ only. # p < 0.05 compared to control cells transfected with empty vector and receiving the same treatment.

Article Snippet: Control plasmid containing a scrambled shRNA sequence was also obtained from SuperArray.

Techniques: Expressing, Transfection, Construct, Negative Control, shRNA, Sequencing, Quantitative RT-PCR, Plasmid Preparation, Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Inhibition of Heat Shock Protein B8 Alleviates Retinal Dysfunction and Ganglion Cells Loss Via Autophagy Suppression in Mouse Axonal Damage

doi: 10.1167/iovs.63.6.28

Figure Lengend Snippet: RGCs loss and HspB8 expression changes after ONC. ( A ) Immunofluorescent staining of HspB8 ( red ) in normal retinal slices. ( B ) Western blot revealed that, compared with the control group (0.19 ± 0.02), HspB8/GAPDH increased in the ONC3 group (0.42 ± 0.02, P < 0.0001), and there was no significant difference in the ONC7 group (0.23 ± 0.02, P = 0.8998); n = 12. ( C ) Immunofluorescent staining of Brn3a. RGCs survival rates continued to decrease in the ONC3 group (76.62% ± 3.05%, P = 0.0022), ONC5 group (40.93% ± 1.32%, P = 0.0022), and ONC7 group (16.00% ± 1.12%, P = 0.0022) compared with the control group (100.00% ± 2.30%). The second areas on the retinal flat-mounts of each group were selected as example images ( n = 6). All data are presented as mean ± SEM. ONC3, ONC5, and ONC7 indicate 3, 5, and 7 days after ONC.

Article Snippet: An adeno-associated virus type 2 (AAV2) vector (GV478, U6-MCS-CAG-EGFP; Shanghai Genechem Co., Ltd., Shanghai, China) containing AAV2-scrambled HspB8 (shHspB8; U6-MCS-CAG-EGFP-HspB8-shRNA, targeting sequence 5′-CCGGAAGAGCTGATGGTAA) and AAV2-green fluorescent protein (GFP; empty GV478 vector without HspB8-shRNA, U6-MCS-CAG-EGFP) was constructed as an HspB8 silencing vector and negative control, respectively.

Techniques: Expressing, Staining, Western Blot

Journal: Investigative Ophthalmology & Visual Science

Article Title: Inhibition of Heat Shock Protein B8 Alleviates Retinal Dysfunction and Ganglion Cells Loss Via Autophagy Suppression in Mouse Axonal Damage

doi: 10.1167/iovs.63.6.28

Figure Lengend Snippet: ERG Characteristics in This Study

Article Snippet: An adeno-associated virus type 2 (AAV2) vector (GV478, U6-MCS-CAG-EGFP; Shanghai Genechem Co., Ltd., Shanghai, China) containing AAV2-scrambled HspB8 (shHspB8; U6-MCS-CAG-EGFP-HspB8-shRNA, targeting sequence 5′-CCGGAAGAGCTGATGGTAA) and AAV2-green fluorescent protein (GFP; empty GV478 vector without HspB8-shRNA, U6-MCS-CAG-EGFP) was constructed as an HspB8 silencing vector and negative control, respectively.

Techniques:

Journal: Investigative Ophthalmology & Visual Science

Article Title: Inhibition of Heat Shock Protein B8 Alleviates Retinal Dysfunction and Ganglion Cells Loss Via Autophagy Suppression in Mouse Axonal Damage

doi: 10.1167/iovs.63.6.28

Figure Lengend Snippet: Silencing of HspB8 protected RGCs and retinal function. ( A ) The AAV2-shHspB8 group had lower HspB8 protein levels (0.11 ± 0.02) compared with the control group (0.19 ± 0.02) ( P = 0.0436), as did the AAV2-shHspB8 + ONC5 group (0.19 ± 0.02) compared with the AAV2-GFP + ONC5 group (0.29 ± 0.03) ( P = 0.0089). Consistent with previous results, the ONC5 group had increased HspB8 levels (0.27 ± 0.03) compared with the control group ( P = 0.0148). No significant differences were found for the AAV2-GFP group (0.21 ± 0.02; P = 0.4383) compared with the control group or for the AAV2-GFP + ONC5 group compared with the ONC5 group ( P = 0.6461); n = 9. ( B ) Representative RGCs immunofluorescent images stained by Brn3a: ONC5 group, AAV2-shHspB8 + ONC5 group, and AAV2-GFP + ONC5 group. ( C ) The RGCs survival rate of the AAV2-shHspB8 + ONC5 group was higher (52.32% ± 2.38%) than that for the AAV2-GFP + ONC5 group (42.14% ± 1.95%) ( P = 0.0040), whereas the AAV2-GFP + ONC5 group showed no significant change compared with the ONC5 group (40.93% ± 1.32%) ( P > 0.9999; n = 6). ( D ) There were no significant differences among the visual acuities of the ONC5 group, AAV2-shHspB8 + ONC5 group, and AAV2-GFP + ONC5 group ( P = 0.3672; n = 10). ( E ) Typical ERG waves indicated that the AAV2-shHspB8 + ONC5 group had enhanced b-wave amplitudes compared with the other two groups. ( F ) The b-wave amplitudes at 3.0 and 10.0 cd·s/m² ( n = 10). ( G ) The a- and b-wave latencies demonstrated no significant changes among the three groups. The AAV2-shHspB8 + ONC5 group had higher b-wave amplitudes than those in the AAV2-GFP + ONC5 group at 3.0 and 10.0 cd·s/m² ( P = 0.0011 and P = 0.0007, respectively), whereas no difference was observed between the AAV2-GFP + ONC5 group and the ONC5 group ( P > 0.9999 and P = 0.9968, respectively). Although similar changes were found in the a-wave amplitudes, none of these differences was statistically significant. Data are shown in the ( n = 10). ( H ) Amplitudes of OPs at 3.0 cd·s/m 2 ( n = 10). Data are shown in the . All data are presented as mean ± SEM.

Article Snippet: An adeno-associated virus type 2 (AAV2) vector (GV478, U6-MCS-CAG-EGFP; Shanghai Genechem Co., Ltd., Shanghai, China) containing AAV2-scrambled HspB8 (shHspB8; U6-MCS-CAG-EGFP-HspB8-shRNA, targeting sequence 5′-CCGGAAGAGCTGATGGTAA) and AAV2-green fluorescent protein (GFP; empty GV478 vector without HspB8-shRNA, U6-MCS-CAG-EGFP) was constructed as an HspB8 silencing vector and negative control, respectively.

Techniques: Staining

Journal: Investigative Ophthalmology & Visual Science

Article Title: Inhibition of Heat Shock Protein B8 Alleviates Retinal Dysfunction and Ganglion Cells Loss Via Autophagy Suppression in Mouse Axonal Damage

doi: 10.1167/iovs.63.6.28

Figure Lengend Snippet: Intravitreal injection of AAV2 had no impact on RGCs survival and visual function. ( A ) GFP immunofluorescent staining of retinal section in the AAV2-shHspB8 group. ( B ) Representative pictures of retinal flat-mounts stained by Brn3a in the control group, AAV2-shHspB8 group, and AAV2-GFP group. ( C ) RGCs survival rates showed no evident decrease in the AAV2-shHspB8 group (101.20% ± 2.18%) or AAV2-GFP group (100.90% ± 1.63%) compared with the control group (100.00% ± 2.30%) ( P = 0.8925 and P = 0.9367, respectively; n = 6). ( D ) Visual acuity appeared to be unaffected by intravitreal injection of AAV2-shHspB8 (0.41 ± 0.03 cyc/deg) compared with the control group (0.41 ± 0.02 cyc/deg) ( P > 0.9999; n = 10). ( E ) No differences in latencies were statistically significant (10.0 cd·s/m 2 ), nor were differences in amplitudes (3.0 and 10.0 cd·s/m², respectively) of a-waves ( P > 0.9999, P = 0.9989, and P = 0.9341, respectively) and b-waves ( P > 0.9999, P = 0.5400, and P = 0.7851, respectively) in the control group and the AAV2-shHspB8 group ( n = 10). Data are presented in the . ( F ) Neither the latency nor the amplitude of PhNRs showed significant differences between the AAV2-shHspB8 group (114.20 ± 6.51 ms, 40.64 ± 2.89 µV) and the control group (116.40 ± 5.16 ms, 36.09 ± 4.01 µV) ( P = 0.9416 and P = 0.5336, respectively; n = 7). ( G ) Example ff-ERG waves of the control group and AAV2-shHspB8 group. All data are presented as mean ± SEM.

Article Snippet: An adeno-associated virus type 2 (AAV2) vector (GV478, U6-MCS-CAG-EGFP; Shanghai Genechem Co., Ltd., Shanghai, China) containing AAV2-scrambled HspB8 (shHspB8; U6-MCS-CAG-EGFP-HspB8-shRNA, targeting sequence 5′-CCGGAAGAGCTGATGGTAA) and AAV2-green fluorescent protein (GFP; empty GV478 vector without HspB8-shRNA, U6-MCS-CAG-EGFP) was constructed as an HspB8 silencing vector and negative control, respectively.

Techniques: Injection, Staining

Journal: Investigative Ophthalmology & Visual Science

Article Title: Inhibition of Heat Shock Protein B8 Alleviates Retinal Dysfunction and Ganglion Cells Loss Via Autophagy Suppression in Mouse Axonal Damage

doi: 10.1167/iovs.63.6.28

Figure Lengend Snippet: The ONC enhanced autophagy level was suppressed by AAV2-shHspB8. ( A ) For the LC3II/LC3I ratios for the AAV2-shHspB8 group (0.29 ± 0.04) compared with the control group (0.37 ± 0.05), P = 0.2219; for the ONC5 group (0.52 ± 0.05) compared with the control group, P = 0.0444; for the AAV2-shHspB8 + ONC5 group (0.32 ± 0.05) compared with the AAV2-GFP + ONC5 group (0.53 ± 0.05), P = 0.0045; and for the AAV2-GFP + ONC5 group compared with the ONC5 group, P = 0.8111. For the p62 levels for the AAV2-shHspB8 group (0.98 ± 0.08) compared with the control group (0.61 ± 0.06), P = 0.0013; for the ONC5 group (0.90 ± 0.06) compared with the control group, P = 0.0170; for the AAV2-shHspB8 + ONC5 group (1.07 ± 0.07) compared with the AAV2-GFP + ONC5 (0.79 ± 0.05) group, P = 0.0260; and for the AAV2-GFP + ONC5 group compared with the ONC5 group, P = 0.7142 ( n = 9). All data are presented as mean ± SEM. ( B ) Immunofluorescent retinal sections of LC3 ( red ) and p62 ( red ); protein levels illustrated by fluorescence intensity in these five groups showed synchronized trends for the western blot results ( n = 3).

Article Snippet: An adeno-associated virus type 2 (AAV2) vector (GV478, U6-MCS-CAG-EGFP; Shanghai Genechem Co., Ltd., Shanghai, China) containing AAV2-scrambled HspB8 (shHspB8; U6-MCS-CAG-EGFP-HspB8-shRNA, targeting sequence 5′-CCGGAAGAGCTGATGGTAA) and AAV2-green fluorescent protein (GFP; empty GV478 vector without HspB8-shRNA, U6-MCS-CAG-EGFP) was constructed as an HspB8 silencing vector and negative control, respectively.

Techniques: Fluorescence, Western Blot

Journal: Investigative Ophthalmology & Visual Science

Article Title: Inhibition of Heat Shock Protein B8 Alleviates Retinal Dysfunction and Ganglion Cells Loss Via Autophagy Suppression in Mouse Axonal Damage

doi: 10.1167/iovs.63.6.28

Figure Lengend Snippet: AAV2-shHspB8 inhibited autophagy and protected subcellular structures. N, nuclei of RGCs; white arrowheads , autolysosomes in the soma of RGCs; black # , normal mitochondria in the soma of RGCs; red # , normal mitochondria in nerve fibers; regions with blue border , unmyelinated nerve fibers and neurotubules inside; blue rectangle , normal Golgi apparatus and endoplasmic reticulum in the soma of RGCs; region with red border , apoptosis RGCs and its pyknotic nucleus; red circles , vesicular expansions of the endoplasmic reticulum; black arrowhead , autophagosome in nerve fiber. *Swollen or vacuolar mitochondria.

Article Snippet: An adeno-associated virus type 2 (AAV2) vector (GV478, U6-MCS-CAG-EGFP; Shanghai Genechem Co., Ltd., Shanghai, China) containing AAV2-scrambled HspB8 (shHspB8; U6-MCS-CAG-EGFP-HspB8-shRNA, targeting sequence 5′-CCGGAAGAGCTGATGGTAA) and AAV2-green fluorescent protein (GFP; empty GV478 vector without HspB8-shRNA, U6-MCS-CAG-EGFP) was constructed as an HspB8 silencing vector and negative control, respectively.

Techniques:

Journal: Investigative Ophthalmology & Visual Science

Article Title: Inhibition of Heat Shock Protein B8 Alleviates Retinal Dysfunction and Ganglion Cells Loss Via Autophagy Suppression in Mouse Axonal Damage

doi: 10.1167/iovs.63.6.28

Figure Lengend Snippet: AAV2-shHspB8 played a neuroprotective role by inhibiting autophagy. ( A ) Representative images of RGCs immunofluorescent staining (Brn3a). ( B ) For the ONC5-3MA group (51.53% ± 2.39%) compared with the ONC5-DMSO group (39.82% ± 2.92%), P = 0.0024; for the ONC5-Rapa group (30.71% ± 1.62%) compared with the ONC5-DMSO group, P = 0.0238; for the ONC5 group (40.93% ± 1.32%) compared with the ONC5-DMSO group, P = 0.9981; for the ONC5 group compared with the AAV2-shHspB8 + ONC5 group (52.32% ± 2.38%), P = 0.0032; for the AAV2-shHspB8 + ONC5 + Rapa group (37.95% ± 1.62%) compared with the AAV2-shHspB8 + ONC5 group, P = 0.0002 ( n = 6). ( C ) Visual acuity in the OMR test ( n = 10). ( D ) The a- and b-wave amplitudes under different flash light intensions ( n = 10). ( E ) Example ERG waves at 3.0 cd·s/m 2 . ( F ) The a-wave amplitudes at 3.0 and 10.0 cd·s/m 2 and b-wave amplitudes at 3.0 and 10.0 cd·s/m 2 : for the ONC5-3MA group compared with the ONC5-DMSO group, P = 0.0004, P = 0.4801, P = 0.0021, and P = 0.0008, respectively; for the ONC5-Rapa group compared with the ONC5-DMSO group, P = 0.0012, P = 0.0198, P = 0.0034, and P = 0.0004, respectively; for the ONC5 group compared with the ONC5-DMSO group, P = 0.0733, P = 0.3971, P = 0.5879, and P = 0.9839, respectively; for the ONC5 group compared with the AAV2-shHspB8 + ONC5 group, P = 0.1531, P = 0.2154, P < 0.0001, and P = 0.0002, respectively; and for the AAV2-shHspB8 + ONC5 + Rapa group compared with the AAV2-shHspB8 + ONC5 group, P = 0.3198, P = 0.2154, P < 0.0001, and P < 0.0001, respectively ( n = 10). Data are shown in the . ( G ) Amplitudes of OPs at 3.0 cd·s/m 2 ( n = 10). Data are shown in the .

Article Snippet: An adeno-associated virus type 2 (AAV2) vector (GV478, U6-MCS-CAG-EGFP; Shanghai Genechem Co., Ltd., Shanghai, China) containing AAV2-scrambled HspB8 (shHspB8; U6-MCS-CAG-EGFP-HspB8-shRNA, targeting sequence 5′-CCGGAAGAGCTGATGGTAA) and AAV2-green fluorescent protein (GFP; empty GV478 vector without HspB8-shRNA, U6-MCS-CAG-EGFP) was constructed as an HspB8 silencing vector and negative control, respectively.

Techniques: Staining