Journal: bioRxiv

Article Title: Dual-acting gene therapy targeting HIF1A and HIF2A by RNA interference mitigates retinal degeneration in two mouse models of AMD

doi: 10.1101/2024.11.29.626080

Figure Lengend Snippet: (A) Graphical representation of tissue changes leading to hypoxia and degenerative processes during aging and AMD. (B) Gene expression analysis of 661W cells stably expressing shCtrl, shHif1 or shHif2 after normoxic or hypoxic (24 h, 0.2% O 2 ) exposure. Values were normalized to beta Actin ( Actb ) and expressed relatively to normoxic 661W-shCtrl (set to 1). Shown are individual values (n=3) and means ± SD. Significance was tested using a one-way ANOVA with Tukey’s multiple comparison test. *: statistically significant; p-values of all comparisons are given in Table S1. (C) Western blot analysis for HIF1A, HIF2A and ACTB in 661W cells stably expressing shCtrl, shHif1 or shHif2 after normoxic and hypoxic exposure. (D) Schematic representation of the DNA constructs packaged into AAV vectors. AAV-shHif: the construct consists of the GRK1 promotor driving photoreceptor-specific expression of shHif1a embedded in the miR-E scaffold in the 3’UTR of GFP, followed by the VMD2 promotor controlling RPE-specific expression of shHif2a embedded in the miR-E scaffold in the 3’UTR of mCherry. AAV-shCtrl: a scrambled non-targeting control shRNA sequence replaced shHif1a and shHif2a. AAV-shHif1: the GRK1 promotor drives photoreceptor-specific expression of shHif1a embedded in the miR-E scaffold in the 3’UTR of GFP. AAV-shHif2: the VMD2 promotor controls RPE-specific expression of shHif2a embedded in the miR-E scaffold in the 3’UTR of mCherry. Sequences of shRNAs are provided in Fig. S1 and supplemental data SD1. CC: choriocapillaris, BM: Bruch’s membrane, RPE: retinal pigment epithelium, PS: photoreceptor segments, ONL: outer nuclear layer.

Article Snippet: shRNAmiR DNA fragments containing shHif1a , shHif2a or shCtrl sequences were designed using the miR-E backbone following the recommendation by Fellmann ( ) (Fig. S1A-C) and produced by Bio-Synthesis (Bio-Synthesis Inc. TX, USA).

Techniques: Expressing, Stable Transfection, Comparison, Western Blot, Construct, Control, shRNA, Sequencing, Membrane

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: Advanced Science

Article Title: Single‐Cell Chromatin Accessibility Analysis Reveals Subgroup‐Specific TF‐NTR Regulatory Circuits in Medulloblastoma

doi: 10.1002/advs.202309554

Figure Lengend Snippet: Inhibiting NTRs in MB cells affects the in vitro sphere‐forming ability and in vivo tumorigenic capacity. A,B) Extreme limiting dilution assays (ELDAs) were conducted to assess the ability of cells to form colonies, demonstrating a decrease in neurosphere frequency upon NTR inhibition in Group 3 (HTB‐187) or Group 3/4 (HTB‐185) cells. HTB‐185 and HTB‐187 cells were seeded in 24‐well ultra‐low attachment plates at various concentrations, including 100, 50, 25, 10, and 5 cells per well. Each sphere‐forming well was counted and the dilution ratio was plotted based on the number of diluted cells and the number of sphere‐forming wells. **** P < 0.0001. C) Display of representative spheres derived from Group 3 or Group 3/4 cells expressing either control shRNA (shCtrl), shHTR2C, or shGABRG2. Images of colonies were captured after 1 week of incubation. D,E) Relative HTR2C or GABRG2 expression was measured by qRT‐PCR in shCtrl, shHTR2C, and shGABRG2 Group3/4 cells. Data are shown as the mean ± SEM. ** P < 0.01. F) left, HE staining of the maximum cross‐sectional area of G3/4 intracranial orthotopic xenograft model among each group. G) Statistical results of the percentage of medulloblastoma tumor size divide the whole section. Data are expressed as dots. ** P < 0.01. H) Immunofluorescence staining of GFAP, IBA1, NeuN revealed less GFAP and increased IBA1 signal in shHTR2C and shGABRG2 groups. Shown are representative images from at least three mice with similar results. I,J) Quantification of GFAP and IBA1 of each group described in H. * P < 0.05, ** P < 0.01, **** P < 0.0001. (Scale bars: 50 µm in C, 2 mm in F, 100 µm in H).

Article Snippet: Subsequently, 1 × 10 6 shHTR2C, shGABRG2, and shCtrl Group 3/4 cells in 2 μl PBS were carefully injected into the cerebellum of 6–8week‐old female nude mice (BABL/c, Gempharmatech Co., Ltd, China) at the coordinates 2 mm lateral to midline and 1 mm posterior to lambda.

Techniques: In Vitro, In Vivo, Inhibition, Derivative Assay, Expressing, Control, shRNA, Incubation, Quantitative RT-PCR, Staining, Immunofluorescence

Journal: Cellular & Molecular Biology Letters

Article Title: Extrachromosomal circular DNA promotes prostate cancer progression through the FAM84B/CDKN1B/MYC/WWP1 axis

doi: 10.1186/s11658-024-00616-3

Figure Lengend Snippet: FAM84B overexpression promotes the growth and metastasis of PCa cells. PCa cells were infected with shCtrl, shLig 3 1#, shLig 3 1# + oeCtrl, shLig 3 1# + oeFAM84B. A Detection of FAM84B expression by RT–qPCR. B , C The PCa cell proliferation and DNA synthesis activity were assessed using CCK8 and EdU staining. D , E The migratory and invasion of PCa cells were assessed using transwell assays. F The growth rate of xenograft tumors formed by subcutaneously inoculated PC-3 cells in mice ( n = 5). G Lig3, FAM84B, and Ki67 protein expression in xenograft tumors ( n = 5) was examined using IHC. H Observation of tumor cell metastasis formed by intracardiac injection of PC-3 cells by bioluminescence imaging ( n = 5). Experiments were repeated three times with multiple wells. The bars indicate SD. * p < 0.05 (one-way/two-way ANOVA). Scale bar = 50 μm

Article Snippet: Overexpression lentivirus (oeFAM84B, oeWWP1, oeCDKN1B) based on mammalian gene expression lentiviral vector (pLV[Exp]-EGFP:T2A:Puro-EF1A), shLig3 1, 2, 3#, shWWP1 1, 2, 3# based on mammalian short hairpin (sh)RNA interference lentiviral vector (pLV[shRNA]-EGFP:T2A:Puro-U6), and their respective controls (oeCtrl and shCtrl) were purchased from VectorBuilder (Guangzhou, Guangdong, China).

Techniques: Over Expression, Infection, Expressing, Quantitative RT-PCR, DNA Synthesis, Activity Assay, Staining, Injection, Imaging

Journal: Cellular & Molecular Biology Letters

Article Title: Extrachromosomal circular DNA promotes prostate cancer progression through the FAM84B/CDKN1B/MYC/WWP1 axis

doi: 10.1186/s11658-024-00616-3

Figure Lengend Snippet: MYC promotes WWP1 expression to drive PCa tumor progression. PCa cells were treated with DMSO, MYCi361, oeCtrl + MYCi361, and oeWWP1 + MYCi361 ( A ) WWP1 mRNA expression in PCa cells was examined using RT–qPCR. B , C The PCa cell proliferation and DNA synthesis activity were assessed using CCK8 and EdU staining. D The growth rate of xenograft tumors formed by subcutaneously inoculated PC-3 cells in mice treated with MYCi361 or DMSO ( n = 5). E MYC, WWP1, and Ki67 protein expression in xenograft tumors ( n = 5) was examined using IHC. F , G The migratory and invasive capacity of PCa cells were examined using transwell assays. H Observation of tumor cell metastasis formed by intracardiac injection of PC-3 cells in mice treated with MYCi361 or DMSO by bioluminescence imaging ( n = 5). Experiments were repeated three times with multiple wells. The bars indicate SD. * p < 0.05 (one-way/two-way ANOVA). Scale bar = 50 μm

Article Snippet: Overexpression lentivirus (oeFAM84B, oeWWP1, oeCDKN1B) based on mammalian gene expression lentiviral vector (pLV[Exp]-EGFP:T2A:Puro-EF1A), shLig3 1, 2, 3#, shWWP1 1, 2, 3# based on mammalian short hairpin (sh)RNA interference lentiviral vector (pLV[shRNA]-EGFP:T2A:Puro-U6), and their respective controls (oeCtrl and shCtrl) were purchased from VectorBuilder (Guangzhou, Guangdong, China).

Techniques: Expressing, Quantitative RT-PCR, DNA Synthesis, Activity Assay, Staining, Injection, Imaging

Journal: Cellular & Molecular Biology Letters

Article Title: Extrachromosomal circular DNA promotes prostate cancer progression through the FAM84B/CDKN1B/MYC/WWP1 axis

doi: 10.1186/s11658-024-00616-3

Figure Lengend Snippet: CDKN1B is an effector of FAM84B in PCa. PCa cells were treated with oeCtrl, oeFAM84B, oeFAM84B + oeCtrl, and oeFAM84B + oeCDKN1B. A FAM84B and CDKN1B protein expression in PCa cells was examined using western blot assays. B , C The PCa cell proliferation and DNA synthesis activity were assessed using CCK8 and EdU staining. D , E The migratory and invasive capacity of PCa cells were examined using transwell assays. F The growth rate of xenograft tumors formed by subcutaneously inoculated PC-3 cells in mice ( n = 5). G Observation of tumor cell metastasis formed by intracardiac injection of PC-3 cells in mice by bioluminescence imaging ( n = 5). Experiments were repeated three times with multiple wells. The bars indicate SD. * p < 0.05 (one-way/two-way ANOVA). Scale bar = 50 μm

Article Snippet: Overexpression lentivirus (oeFAM84B, oeWWP1, oeCDKN1B) based on mammalian gene expression lentiviral vector (pLV[Exp]-EGFP:T2A:Puro-EF1A), shLig3 1, 2, 3#, shWWP1 1, 2, 3# based on mammalian short hairpin (sh)RNA interference lentiviral vector (pLV[shRNA]-EGFP:T2A:Puro-U6), and their respective controls (oeCtrl and shCtrl) were purchased from VectorBuilder (Guangzhou, Guangdong, China).

Techniques: Expressing, Western Blot, DNA Synthesis, Activity Assay, Staining, Injection, Imaging