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: Oncology Letters

Article Title: Inhibition of autophagy enhances apoptosis induced by bortezomib in AML cells

doi: 10.3892/ol.2020.12370

Figure Lengend Snippet: Downregulation of Beclin-1 enhances apoptosis and reduces cell viability induced by bortezomib in NB4 cells. Cells were infected with lentiviruses expressing shRNAs (non-targeting control or Beclin-1). Puromycin-resistant cells were pooled after each infection. (A) Cells transfected with the control shRNA and shBeclin-1 were treated with or without bortezomib (20 nM) for 24 h, and the expression levels of cleaved caspase-3, cleaved PARP, Bcl-2, p62 and Beclin-1, and LC3-I to LC3-II conversion were determined by western blotting. (B) Ratio of Bcl-2 and β-actin expression levels. Data are presented as the mean ± standard deviation of three independent repeats. *P<0.05 and **P<0.01. (C) Ratio of LC3-II and β-actin expression levels. Data are presented as the mean ± standard deviation of three independent repeats. **P<0.01. (D) Cells transfected with the control shRNA and shBeclin-1 were treated with bortezomib (20 nM) for 0 and 24 h, and cell viability was assessed using a water-soluble tetrazolium salts-8 assay. Data are presented as the mean ± standard deviation of three independent repeats. ***P<0.001. shRNA/sh, short hairpin RNA; CTRL, control; PARP, poly(ADP-ribose) polymerase.

Article Snippet: Lentiviral particles containing short hairpin RNA (shRNA/sh) Beclin-1 (cat. no. 131209BZ; 5′-CCGACTTGTTCCTTACGGAAA-3′) and shRNA negative control (shCTRL; cat. no. 131127CZ; 5′-TTCTCCGAACGTGTCACGTTTC-3′) expression vectors were obtained from Shanghai GenePharma Co., Ltd., and were then cloned into pGLV3/H1/GFP-Puro vector (Shanghai GenePharma Co., Ltd.).

Techniques: Infection, Expressing, Transfection, shRNA, Western Blot, Standard Deviation

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