Journal: Frontiers in Pharmacology

Article Title: Pharmacologic Targeting of BET Proteins Attenuates Hyperuricemic Nephropathy in Rats

doi: 10.3389/fphar.2021.636154

Figure Lengend Snippet: I-BET151 reduces Brd4 levels in the kidney of HN rats. (A) A rat model of HN was established and treated with I-BET151 as indicated in “Material and Methods.” After 3 weeks, kidneys were taken for immunoblot analysis for Brd2, Brd3, Brd4 or GAPDH. Expression levels of Brd2 (B) , Brd3 (C) , Brd4 (D) were quantified by densitometry analysis and then normalized with GAPDH. (E) Photomicrographs (original magnification, ×400) illustrate immunohistochemical Brd4 staining of kidney tissues. (F) Brd4 staining graphic presentation of quantitative data. Data are represented as the mean ± SEM. * p < 0.05; ** p < 0.01.

Article Snippet: Brd2 antibody was purchased from Boster Biological Technology (Wuhan, China), OAT1 and OAT3 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), I-BET151 was purchased from Target Mol (MA, United States).

Techniques: Western Blot, Expressing, Immunohistochemical staining, Staining

Journal: Journal of Visualized Experiments : JoVE

Article Title: The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

doi: 10.3791/2331

Figure Lengend Snippet:

Article Snippet: Order the fosmid clone of Gene of interest (G.O.I.) from GeneService (Cambridge, UK) using Wormbase as a guide.

Techniques: Bacteria

Journal: Cancers

Article Title: The NOTCH-RIPK4-IRF6-ELOVL4 Axis Suppresses Squamous Cell Carcinoma

doi: 10.3390/cancers15030737

Figure Lengend Snippet: Transcriptome profiling of Ripk4 KO mouse keratinocytes. ( A ) Schematics showing the experimental workflow of RNA sequencing in Ripk4 knock-out keratinocytes and in vivo CRISPR screen of top 63 downregulated genes. LSL-Pik3ca H1047R ; LSL-Cas9-GFP embryonic (E9.5) mice were transduced with sgNTC (non-targeting control) or sgRipk4. At postnatal day P4, the transduced skin was harvested and processed for RNA sequencing. Differential gene expression analysis revealed 63 significantly downregulated genes in Ripk4 KO compared to sgNTC control mice. These 63 genes were selected to construct a targeted CRISPR library and screened in LSL-Pik3ca H1047R ; LSL-Cas9-GFP mice to identify the Ripk4 downstream genes that themselves act as tumor suppressor genes (image created with biorender.com). ( B ) Heat map showing the differential expression of genes in the sgNTC (control) versus sgRipk4 keratinocytes. ( C ) Gene set enrichment analysis (GSEA) revealed downregulation of the synthesis of very-long-chain fatty acids pathway in Pik3ca H1047R ; Ripk4 KO skin. ( D ) METASCAPE pathway analysis revealed several upregulated and downregulated pathways including epidermis development.

Article Snippet: Plasmid pDS1 (Addgene #158032) expressing U6-sgRNA stuffer-tracr cassette and hPGK-driven Cre recombinase (Cre) (19) was used in all the experiments involving CRISPR methodology. sgRNAs targeting genes of interest (63 downregulated gene library) and non-targeting sgRNAs (2), were ordered as a pooled oligo chip (CustomArray Inc., Redmond, WA, USA) and cloned into pDS1 using BsmBI restriction sites.

Techniques: RNA Sequencing Assay, Knock-Out, In Vivo, CRISPR, Transduction, Expressing, Construct

Journal: Cancers

Article Title: The NOTCH-RIPK4-IRF6-ELOVL4 Axis Suppresses Squamous Cell Carcinoma

doi: 10.3390/cancers15030737

Figure Lengend Snippet: Elovl4 is a tumor suppressor downstream of Ripk4. ( A ) Representative image of tumors in an LSL- Pik3ca H1047R ; LSL-Cas9-GFP mouse transduced with LV carrying Cre recombinase and sgRNAs targeting the genes downregulated in Ripk4-deficient keratinocytes. ( B ) Tumor-free survival for LSL-Pik3ca H1047R ; LSL-Cas9-GFP mouse transduced with downregulated genes library ( n ≥ 8 per group; p < 0.0001, log-rank test). ( C ) Pie chart showing the genes that have their corresponding sgRNAs enriched in the tumors collected from mice transduced with the CRISPR gene library targeting downregulated Ripk4 target genes. ( D ) Real-time PCR results showing relative expression of indicated genes in mouse keratinocytes carrying CRISPR/Cas9-mediated ablation of Ripk4 when compared to non-targeting control. Data are shown as means ± SEM ( n = 3). * Denotes p -value < 0.05. (E) Tumor-free survival for tumor-prone Pik3ca H1047R ;Cas9 mice transduced with an independent sgRNA against Elovl4. sgNTC served as a control ( n ≥ 5 per group; p < 0.0001, log-rank test). (F) Representative images of tumors in the head and neck region of Pik3ca H1047R ; Cas9 mice transduced with sgElovl4 LV-Cre.

Article Snippet: Plasmid pDS1 (Addgene #158032) expressing U6-sgRNA stuffer-tracr cassette and hPGK-driven Cre recombinase (Cre) (19) was used in all the experiments involving CRISPR methodology. sgRNAs targeting genes of interest (63 downregulated gene library) and non-targeting sgRNAs (2), were ordered as a pooled oligo chip (CustomArray Inc., Redmond, WA, USA) and cloned into pDS1 using BsmBI restriction sites.

Techniques: Transduction, CRISPR, Real-time Polymerase Chain Reaction, Expressing