Journal: Frontiers in Cell and Developmental Biology

Article Title: CRISPR/Cas9-mediated CH2 deletion of Fel d1 triggers transcriptomic reprogramming and disease-associated pathways in feline cells

doi: 10.3389/fcell.2025.1716808

Figure Lengend Snippet: Generation of CH2-Knockout Monoclonal Cells. (A) Monoclonal selection of cells after puromycin screening. Scale bar: 100 µm. (B) Sanger sequencing showing a three-base insertion mutation at the target site in the CH2-KO clone. (C) qRT-PCR results for CH2 mRNA expression in control and KO cells. (D) Western blot analysis of Fel d1 protein expression in control and KO cells. (E) Quantification of Fel d1 protein expression levels. Data were obtained from three independent experiments. Data are presented as mean ± standard deviation, and P values were calculated by two-tailed unpaired t-test. Significant differences are indicated by **** (P < 0.0001).

Article Snippet: Total mRNA was isolated from control and CH2-KO cells by using mRNA isolation kit (QIAGEN, Germany, 80,284), and gene expression was assessed using a Thermo Fisher Scientific 6/7/12K Flex RT-PCR system, with the PCR procedures detailed in .

Techniques: Knock-Out, Selection, Sequencing, Mutagenesis, Quantitative RT-PCR, Expressing, Control, Western Blot, Standard Deviation, Two Tailed Test

Journal: Frontiers in Cell and Developmental Biology

Article Title: CRISPR/Cas9-mediated CH2 deletion of Fel d1 triggers transcriptomic reprogramming and disease-associated pathways in feline cells

doi: 10.3389/fcell.2025.1716808

Figure Lengend Snippet: Transcriptomic convergence of RA and HCM pathways in CH2-KO cells. (A) Genome-wide expression dynamics and PCA of RNA-seq data from rheumatoid arthritis (left) and hypertrophic cardiomyopathy (right) models. The upper plots show expression trend curves, middle heatmaps display sample clustering, and lower plots present PCA distribution, indicating transcriptional remodeling and sample separation between disease and control groups. (B) Venn diagram illustrating the overlap of differentially expressed genes in models of rheumatoid arthritis (green circles) and hypertrophic cardiomyopathy (blue circles). Numbers indicate unique and shared genes. (C) Relative mRNA expression of TGFB2 and TGFB3 . (D) Relative mRNA expression of ITGA2 , ITGB3 and MYBPC3 . (E) Relative mRNA expression of MMP1 , MMP3 and TLR4 . Data were obtained from three independent experiments. Data are presented as mean ± standard deviation, and P values were calculated by two-tailed unpaired t-test. Significant differences are indicated by * (P < 0.05), ** (P < 0.001) and *** (P < 0.001).

Article Snippet: Total mRNA was isolated from control and CH2-KO cells by using mRNA isolation kit (QIAGEN, Germany, 80,284), and gene expression was assessed using a Thermo Fisher Scientific 6/7/12K Flex RT-PCR system, with the PCR procedures detailed in .

Techniques: Genome Wide, Expressing, RNA Sequencing, Control, Standard Deviation, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Circular RNA hsa_circ_0043280 inhibits cervical cancer tumor growth and metastasis via miR-203a-3p/PAQR3 axis

doi: 10.1038/s41419-021-04193-7

Figure Lengend Snippet: A RIP assay showing the association between AGO2 and hsa_circ_0043280. Top, IP efficiency of the AGO2-antibody in Western blots. Bottom, relative enrichment representing RNA levels associated with AGO2 relative to the input control. An IgG antibody served as a control. B , C Schematic model and Sanger sequencing for wild-type or mutant transcripts of hsa_circ_0043280 or PAQR3 3′ UTR luciferase reporters (left). Luciferase reporter activity of hsa_circ_0043280 and the PAQR3 3′ UTR in HEK-293T cells co-transfected with miR-203a-3p mimics or mimics NC (right). D RAP assays showed that miR-203a-3p could be pulled down by a biotinylated probe designed for the hsa_circ_0043280 back-splicing site in MS751 and HeLa cells. E Hsa_circ_0043280 and PAQR3 mRNA were pulled down and enriched with the 3′-end biotinylated miR-203a-3p by miRNA capture assays. F RNA FISH indicated the colocalization of hsa_circ_0043280 and miR-203a-3p. Scale bar, 20 μm. G PAQR3 expression in MS751 and HeLa cells transfected with miR-203a-3p mimics alone or co-transfected with hsa_circ_0043280. Each experiment was performed at least three times independently. ** P < 0.01; *** P < 0.001.

Article Snippet: The biotin-coupled mRNA–miRNA complex was then pulled down by incubating the cell lysates with streptavidin magnetic beads (Life Technologies) for 1 h at room temperature followed by RNA extraction.

Techniques: Western Blot, Sequencing, Mutagenesis, Luciferase, Activity Assay, Transfection, Expressing

Journal: Viruses

Article Title: Downregulation of miRNA-26a by HIV-1 Enhances CD59 Expression and Packaging, Impacting Virus Susceptibility to Antibody-Dependent Complement-Mediated Lysis

doi: 10.3390/v16071076

Figure Lengend Snippet: RNA-seq mRNA and miRNA profiles in infected CD4 + T cells compared to uninfected cells. ( A ) Volcano plot representing the differential expression (FC = fold change) of mRNAs in infected (GFP-positive, GFP-pos) CD4 + T cells as compared to uninfected cells. The mRNAs with a log2FC of >0.585 and a DeSeq2 base mean > 100 are in red, while those with a log2FC < −0.585 are in blue. ( B ) Graphs representing the upregulated (left panel) or downregulated (right panel) biological processes in infected (GFP-pos) as compared to uninfected cells as determined using g:Profiler ( https://biit.cs.ut.ee/gprofiler/gost (accessed on 26 January 2024)). ( C ) Volcano plot representing the differential expression of miRNAs in infected (GFP-pos) CD4 + T cells as compared to uninfected cells. Dots of upregulated miRNAs with a log2FC of >0.585 and a DeSeq2 base mean > 100 are in red, while those downregulated with a log2FC < −0.585 are in blue. ( D ) The heatmap depicts the 37 miRNAs specifically dysregulated in the GFP-pos population detected in this study. The scale bar is in log2FC. MiRNAs with forms (1) and (2) are identical miRNAs produced by two distinct loci. NDE stands for non-differentially expressed. See also .

Article Snippet: The same samples were used for miRNA-seq and mRNA-seq using the Illumina TruSeq mRNA or Small RNA system (Illumina Technologies, San Diego, CA, USA) at the IRCM Molecular Biology and Functional Genomics Core Facility.

Techniques: RNA Sequencing, Infection, Quantitative Proteomics, Produced

Journal: Viruses

Article Title: Downregulation of miRNA-26a by HIV-1 Enhances CD59 Expression and Packaging, Impacting Virus Susceptibility to Antibody-Dependent Complement-Mediated Lysis

doi: 10.3390/v16071076

Figure Lengend Snippet: Validation of the differential expression of predicted CD59-targeting miRNAs (miRNAs-21, miRNA-26a and miRNA-29a) and CD59 upon HIV-1 infection. Expression levels of miRNAs-21, -26a and -29a ( A ) and CD59 mRNA ( B ) in uninfected and productively infected (GFP-pos) CD4 + T cells (n = 6) were measured via real-time qPCR. Shown are the mean fold-changes compared to uninfected cells (in gray, which is set at 1). Error bars represent the standard error of the mean (SEM). Statistical significance was determined using the nonparametric Mann–Whitney’s test, values: ** p < 0.01. ( C ) CD59 cell-surface expression was evaluated via flow cytometry, and the mean fluorescent intensity (MFI) was compared between uninfected and infected CD4 + T cells (GFP-positive) obtained from healthy blood donors. The left panel is representative data from cells from one donor, the middle panel is the MFI of cells from three donors and the right panel is normalized data (as compared to uninfected cells). Error bars represent the SEM (n = 3). Mann–Whitney’s test, values: * p = 0.05.

Article Snippet: The same samples were used for miRNA-seq and mRNA-seq using the Illumina TruSeq mRNA or Small RNA system (Illumina Technologies, San Diego, CA, USA) at the IRCM Molecular Biology and Functional Genomics Core Facility.

Techniques: Biomarker Discovery, Quantitative Proteomics, Infection, Expressing, Flow Cytometry

Journal: Viruses

Article Title: Downregulation of miRNA-26a by HIV-1 Enhances CD59 Expression and Packaging, Impacting Virus Susceptibility to Antibody-Dependent Complement-Mediated Lysis

doi: 10.3390/v16071076

Figure Lengend Snippet: CD59 mRNA is a target of miRNA-26a. ( A ) CD59 mRNA levels were measured in miRNAs-21, -26a and -29a mimics or control-nucleofected primary CD4 + T cells via real-time qPCR. Shown is the mean fold-change compared to control transfected cells (in gray, which is set at 1) (n = 4). Error bars represent the SEM. Statistical significance was determined using the nonparametric Kruskal Wallis test, values: ** p < 0.01. ( B ) CD59 surface expression was evaluated in miRNAs-21 and -26a mimics or control-nucleofected primary CD4 + T cells via flow cytometry. Shown is the cell surface expression of CD59 from CD4 + T cells of four blood donors (normalized to CTRL, which was set to 100%) with a representative result on the left (MFI are shown). Error bars represent the SEM. Statistical significance was determined using the nonparametric Kruskal Wallis test, values: * p < 0.05. ( C ) The MIR-Report system was used to validate whether the CD59 3′ UTR was a target of miRNA-26a. MiRNA blue nucleotides represent those predicted to interact with the CD59 3′ UTR. WT or mutated (substituted nucleotides in green) fragments of the 3′ UTR encompassing the predicted sequences recognized by miRNA-26a were fused to the F-Luciferase (F-Luc) gene. ( D ) HEK-293T cells were transfected with miRNA-26 mimics, the reporter plasmids with the indicated fragment of the WT (in yellow) or mutated (in black) 3′ UTR and a Renilla-Luciferase (R-Luc) normalizing control. Following cell lysis, relative light units (RLUs) were measured, and F-Luc was normalized to the negative control (in grey, which is set to 1). Error bars represent the SEM. Statistical significance was determined using the nonparametric Kruskal Wallis test, values: * p < 0.05.

Article Snippet: The same samples were used for miRNA-seq and mRNA-seq using the Illumina TruSeq mRNA or Small RNA system (Illumina Technologies, San Diego, CA, USA) at the IRCM Molecular Biology and Functional Genomics Core Facility.

Techniques: Control, Transfection, Expressing, Flow Cytometry, Luciferase, Lysis, Negative Control