shrna plasmid with pspax2  (Addgene inc)

Journal: Molecular Metabolism

Article Title: Primary cilia regulate GLP-1 signaling in pancreatic β cells

doi: 10.1016/j.molmet.2026.102357

Figure Lengend Snippet: Primary cilia are required for GLP-1-potentiated insulin secretion. ( A ) Dynamic insulin secretion from perifused mouse islets. Wild-type (WT, black) and β-cell-specific cilia knockout (βCKO, red) islets were sequentially exposed to 2 mM glucose (2G), 16 mM glucose (16G), 20 nM liraglutide (Lira), and 30 mM KCl as indicated. n = 6 replicates of 50 islets per genotype from 5 mice. ∗p < 0.05, two-way ANOVA with Sidak's multiple comparisons test. ( B ) Quantitative analysis of secretion traces: area under the curve (AUC) during initial glucose stimulation (minutes 10–30), liraglutide stimulation (minutes 30–50), KCl depolarization (minutes 60–70), and total AUC of the entire perifusion (minutes 0–70). βCKO islets secreted significantly less insulin in response to glucose and liraglutide but showed no defect in KCl-induced secretion. Data are mean ± SEM. ∗∗p < 0.01; ns, not significant, unpaired student's t-test. ( C-D ) IFT88 knockdown impairs GLP-1-augmented secretion in human islets. Static glucose-stimulated insulin secretion (GSIS) assays from islets of three male ( C ) and three female ( D ) donors. Islets transduced with control (Ctl, white) or IFT88 -targeting shRNA (IFT88 KD, red) were incubated at 1 mM glucose (1G), 11 mM glucose (11G), and 11G + 100 nM liraglutide (Lira). Donor ages are indicated. IFT88 KD significantly reduced liraglutide-potentiated insulin secretion. Data are mean ± SEM of triplicate samples per donor. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; ns, not significant; one-way ANOVA with Tukey's multiple comparisons test.

Article Snippet: IFT88 knockdown in human islets: Healthy non-diabetic human islets were transduced with adenoviral vectors encoding either GFP-tagged shRNA targeting human IFT88 (Ad-GFP-h-IFT88-shRNA) or scrambled control (Ad-GFP-U6-scrmb-shRNA, #1122N; Vector Biolabs).

Techniques: Knock-Out, Knockdown, Transduction, Control, shRNA, Incubation

Journal: Nucleic Acids Research

Article Title: m5C RNA methylation is dysregulated by oncogenic herpesviruses via c-Myc signaling to counteract host antiviral factors

doi: 10.1093/nar/gkag251

Figure Lengend Snippet: KSHV dysregulated NSUN2/1 expression via c-Myc. ( A and B ) Transcription factors (TFs) that regulate NSUN2/1 gene expression were predicted by analyzing public-domain ChIP-seq datasets. Venn diagram (A) showed the overlapped TFs across the indicated datasets. Six TF candidates including c-Myc (B) were listed. ( C ) Integrative genomics viewer (IGV) visualization of c-Myc occupancy near the NSUN2/1 promoter regions using the public-domain c-Myc ChIP-seq dataset of five lymphoma B cell lines ( GSE30726 ). ( D ) c-Myc binding motif near the promoter regions of NSUN2/1 was illustrated. ( E ) TREx.BCBL1.Rta cells were subjected to ChIP-PCR analysis for quantification of c-Myc association with the promoter regions of NSUN2/1 using an antibody recognizing c-Myc protein for its immunoprecipitation or a control IgG antibody, followed by qPCR analysis using three sets of primers (Set 1–3) targeting NSUN2 or NSUN1 promoter. ( F ) Public-domain RNA-seq data of KSHV-infected cell lines were collected and reanalyzed using the customized pipeline to identify the differentially expressed genes (adjust P -value < 0.05 as cutoff). The distinct gene expression level of c-Myc due to KSHV lytic reactivation was illustrated. ( G ) TREx.BCBL1.Rta, iSLK.BAC16, and iSLK.r219 cells were treated with Dox or mock, followed by protein immunoblotting analysis of c-Myc using its specific antibody. GAPDH was used as a loading control. ( H ) TREx.BCBL1.Rta cells were transduced with the shRNA targeting c-Myc or non-targeting control, followed by protein immunoblotting analysis of c-Myc, NSUN2, NSUN1, and KSHV K8.1. ( I and J ) TREx.BCBL1.Rta cells were treated with c-Myc inhibitors, EN4 (H) or 10074-G5 (I), at a series of concentrations or mock, followed by protein immunoblotting analysis of c-Myc, NSUN2, and NSUN1 using their specific antibodies. ( K ) TIME cells were transiently transfected with siRNAs (si1, si2) targeting c-Myc or siNT, followed by inoculation with KSHV.BAC16 viruses. These cells were harvested for nuclei staining with DAPI. GFP fluorescence signal indicating KSHV-infected cells was quantified (**** P <0.0001). ( L ) TIME cells were treated with the c-Myc inhibitor EN4 at the indicated concentrations, followed by transfection of NSUN1 or NSUN2 cDNAs. These cells were inoculated with KSHV.BAC16 viruses and lysed for protein immunoblotting assays of KSHV K8.1 protein.

Article Snippet: Short hairpin RNA (shRNA) targeting c-Myc was purchased from Addgene (Cat#15662).

Techniques: Expressing, Gene Expression, ChIP-sequencing, Binding Assay, Immunoprecipitation, Control, RNA Sequencing, Infection, Western Blot, Transduction, shRNA, Transfection, Staining, Fluorescence

Journal: Nucleic Acids Research

Article Title: m5C RNA methylation is dysregulated by oncogenic herpesviruses via c-Myc signaling to counteract host antiviral factors

doi: 10.1093/nar/gkag251

Figure Lengend Snippet: EBV also reduced expression of NSUN2/1 that restrict its lytic infection. ( A ) BJAB cells were inoculated with EBV.BX viruses. Cell lysates were collected at 48 h post infection and were followed by protein immunoblotting analysis using specific antibodies recognizing NSUN2 and NSUN1. ( B ) Akata and Akata BX cells were treated with a human IgG antibody to induce EBV lytic reactivation, followed by protein immunoblotting analysis using specific antibodies recognizing NSUN2, NSUN1, and c-Myc. GAPDH was used as a loading control. ( C ) Public-domain RNA-seq data of EBV-infected cell lines were collected and reanalyzed using the customized pipeline to identify the differentially expressed genes (adjust P -value < 0.05 as cutoff). The distinct gene expression level of NSUN2/1 due to EBV lytic reactivation was illustrated. ( D and E ) Akata cells were transiently transfected with siRNAs targeting NSUN2/1 (D) or TRIM25 (E), or siNT. The RNAs were extracted and subjected to RT-qPCR analysis of EBV viral genes (BZLF1, BMRF1), which are normalized to GAPDH. ( F–H ) AGS.BX1 cells were transfected with siRNA targeting NSUN1 (F), NSUN2 (G), or TRIM25 (H). EBV viral gene expression was measured by RT-qPCR. Results of at least three biological replicates were presented as mean ± SD (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, Student’s t -test). ( I ) A model that human gamma-herpesviruses (KSHV, EBV) downregulate NSUN2/1 and decrease m5C modification of TRIM25 mRNA to favor their lytic replication. c-Myc is downregulated due to KSHV/EBV lytic replication, which reduces NSUN2/1 expression. Coordinately, NSUN2/1-mediated m5C modification of TRIM25 mRNA is inhibited, which impairs its stability. As TRIM25 is a key E3 ubiquitin ligase in RIG-I signal transduction, its inhibition disrupts RIG-I mediated antiviral sensing and thus favors KSHV/EBV lytic replication.

Article Snippet: Short hairpin RNA (shRNA) targeting c-Myc was purchased from Addgene (Cat#15662).

Techniques: Expressing, Infection, Western Blot, Control, RNA Sequencing, Gene Expression, Transfection, Quantitative RT-PCR, Modification, Ubiquitin Proteomics, Transduction, Inhibition