non targeting control shrna shctrl  (Addgene inc)

Journal: medRxiv

Article Title: Deficient QPRT drives trans-Golgi NAD + hyperinflation and pathological protein secretion in rheumatoid arthritis

doi: 10.1101/2024.10.27.24316032

Figure Lengend Snippet: Illustrative diagram of NAD + biosynthesis pathways: de novo , salvage and Priess handler pathway. b: A schematic representation of the NAD + biosensor structure and mechanism. The sensor comprises cpNLuc, NAD binding domain, and red fluorescent protein (RFP, mScarlet). When NAD + binds, it triggers a conformational change in the sensitive domain, bringing cpNLuc and RFP closer together to facilitate BRET. c: Localization of the biosensors (red) in the cytoplasm, ER and Golgi was identified using 488 phalloidin, ER tracker and Golgi trackers (green), respectively. d-f: Effect of shQPRT/shCtrl on cytoplasmic, ER, cis/medial-Golgi and trans-Golgi NAD + . h,i: Effect of TNFα (10ng/ml) on cis/medial-Golgi and trans-Golgi NAD + . j,k: Representative immunoblot (j) and quantification (k) of the cis/medial-Golgi marker GM130 and the trans-Golgi marker Golgin 97. l,m: Representative immunofluorescence image of Golgi marker GM130 in shQPRT/shCtrl transfected RA FLSs (l) and TNFα (10ng/ml) (m). Data are the mean ± s.e.m of independent replicates. P values were determined by two-tailed Student’s t-test ( d-i ) or two-way ANOVA ( k ): *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

Article Snippet: Non-targeting scrambled control (shCtrl) and QPRT-specific shRNAs in the pcDNA3.1+ vectors were packaged into lentiviral particles by cotransfecting HEK 293T cells with pMD2G (Addgene #12259) and psPAX2 (Addgene #394976) packaging vectors using polyethyleneimine (PEI, Yeasen, 40816ES03)transfection reagent.

Techniques: Binding Assay, Western Blot, Marker, Immunofluorescence, Transfection, Two Tailed Test

Journal: medRxiv

Article Title: Deficient QPRT drives trans-Golgi NAD + hyperinflation and pathological protein secretion in rheumatoid arthritis

doi: 10.1101/2024.10.27.24316032

Figure Lengend Snippet: Time series quantification on the effect of shQPRT/shCtrl on cis/medial-Golgi and trans-Golgi. c,d: Quantification of the effect of TNFα (10ng/ml) on cytoplasmic and ER NAD + . Data are the mean ± s.e.m of independent replicates. P values were determined by one-way ANOVA ( a,b ) or two-tailed Student’s t-test ( c,d ): *P < 0.05, **P < 0.01 and ****P < 0.0001.

Article Snippet: Non-targeting scrambled control (shCtrl) and QPRT-specific shRNAs in the pcDNA3.1+ vectors were packaged into lentiviral particles by cotransfecting HEK 293T cells with pMD2G (Addgene #12259) and psPAX2 (Addgene #394976) packaging vectors using polyethyleneimine (PEI, Yeasen, 40816ES03)transfection reagent.

Techniques: Two Tailed Test

Journal: medRxiv

Article Title: Deficient QPRT drives trans-Golgi NAD + hyperinflation and pathological protein secretion in rheumatoid arthritis

doi: 10.1101/2024.10.27.24316032

Figure Lengend Snippet: GSEA shows the enrichment of the epithelial to mesenchymal transition (EMT) pathway in shQPRT/shCtrl transfected RA FLSs. b: Normalized Enrichment Scores (NES) with −log 10 FDR for significant pathways identified in the GSEA analysis are presented. The FDR is zero for the EMT pathway, resulting in a −log 10 FDR of infinity. Detailed data can be found in Supplementary Table 2. c: Representative images of Transwell invasion assay. d: Quantification of relative invasive rate (relative to shCtrl group). e: Gene ontology (GO) analysis on positively regulated genes. BP, biological properties; CC, cellular components; MF, molecular function. GO bar plot was created using the SRplot web server . f: Correlation coefficient plot of RA-associated EMT-related genes in shQPRT/shCtrl transfected RA FLSs. The correlation coefficients of genes are depicted using a color scheme ranging from red (indicating negative correlation) to blue (indicating positive correlation), with white representing no correlation. FDR, false discovery rate; NES, normalized enrichment score. g,h: Representative immunoblotting images (f) and quantification (g) of EMT-associated secretome. i: Schematic representation of the coculture of endothelial cell line (EA.hy926) and macrophage cell line (THP1) with conditioned medium from shQPRT/shCtrl transfected RA FLSs. j: Capillary tube formation of EA.hy926 cultured in condition medium from shQPRT/shCtrl transfected RA FLSs. k,l: Quantification of the number of junctions and nodes analyzed by Angiogenesis Analyzer plugin on image J. m-q: mRNA expression levels of M1 macrophage markers TNFα, IL-1β, CXCL8, CXCL10 and ICAM in THP1 cells cultures in CM derived from shQPRT/shCtrl transfected RA FLSs. Data are the mean ± s.e.m of independent biological samples. P values were determined by two-tailed Student’s t-test ( d,k,l ), two-way ANOVA ( h ) or one-way ANOVA ( m-q ): *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

Article Snippet: Non-targeting scrambled control (shCtrl) and QPRT-specific shRNAs in the pcDNA3.1+ vectors were packaged into lentiviral particles by cotransfecting HEK 293T cells with pMD2G (Addgene #12259) and psPAX2 (Addgene #394976) packaging vectors using polyethyleneimine (PEI, Yeasen, 40816ES03)transfection reagent.

Techniques: Transfection, Transwell Invasion Assay, Western Blot, Cell Culture, Expressing, Derivative Assay, Two Tailed Test

Journal: medRxiv

Article Title: Deficient QPRT drives trans-Golgi NAD + hyperinflation and pathological protein secretion in rheumatoid arthritis

doi: 10.1101/2024.10.27.24316032

Figure Lengend Snippet: mRNA expression levels of QPRT, ZEB1, SNAIL, TWIST, PDPN and CDH2 in shQPRT/shCtrl transfected RA FLSs. b: Gene ontology (GO) analysis on downregulated genes. BP, biological properties; CC, cellular components; MF, molecular function. c: mRNA expression levels of EMT-related secretome d-g: mRNA expression levels of M2 macrophage markers IL-10, TGFβ, CD206 and CLEC1A in THP1 cell cultures in CM derived from shQPRT/shCtrl transfected RA FLSs. h-j: TNF (h), VEGF (i) and CTGF (j) levels in the supernatant were measured by ELISA. k: Representative immunoblots of CXCL8, IL6 and VEGF in the supernatant of shQPRT/shCtrl transfected RA FLSs. Data are mean ± s.e.m.; P values were determined by two-way ANOVA ( a,c ), one-way ANOVA ( d-g ) or two-tailed Student’s t-test ( h-j ): *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

Article Snippet: Non-targeting scrambled control (shCtrl) and QPRT-specific shRNAs in the pcDNA3.1+ vectors were packaged into lentiviral particles by cotransfecting HEK 293T cells with pMD2G (Addgene #12259) and psPAX2 (Addgene #394976) packaging vectors using polyethyleneimine (PEI, Yeasen, 40816ES03)transfection reagent.

Techniques: Expressing, Transfection, Derivative Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Two Tailed Test

Journal: medRxiv

Article Title: Deficient QPRT drives trans-Golgi NAD + hyperinflation and pathological protein secretion in rheumatoid arthritis

doi: 10.1101/2024.10.27.24316032

Figure Lengend Snippet: Volcano plot showing the log2fold change (FC) against -log10Pvalue comparing from shQPRT/shCtrl transfected RA FLSs (n=3 per group, P < 0.05, |logFC|>1.0). b: Heatmap plot of differentially expressed proteins. The complete cluster algorithm and Euclidean distance metric were used. c,d: Gene ontology (GO) analysis on upregulated (c) and downregulated (d) proteins. BP, biological properties; CC, cellular components; MF, molecular function. e,f: Representative immunoblot (e) and quantification (f) of PARP12 and Poly/Mono-ADP ribose levels. Representative and collective data from three biological and two technical replicates. g,h: Representative immunoblot (g) and quantification (h) of the cis-Golgi marker GM130 and the trans-Golgi marker Golgin 97 in shPARP12/shCtrl transfected RA FLSs . i,j: Representative immunoblotting images (i) and quantification (i) of EMT-related secretome in shPARP12/shCtrl transfected RA FLSs. Data are the means ± s.e.m. Statistical comparisons were made using two-way ANOVA: * P < 0.05, ** P < 0.01, *** P < 0.001. The volcano plot ( a ), heatmap ( b ) and GO bar plot( c,d ) were created using the SRplot web server .

Article Snippet: Non-targeting scrambled control (shCtrl) and QPRT-specific shRNAs in the pcDNA3.1+ vectors were packaged into lentiviral particles by cotransfecting HEK 293T cells with pMD2G (Addgene #12259) and psPAX2 (Addgene #394976) packaging vectors using polyethyleneimine (PEI, Yeasen, 40816ES03)transfection reagent.

Techniques: Transfection, Western Blot, Marker

Journal: medRxiv

Article Title: Deficient QPRT drives trans-Golgi NAD + hyperinflation and pathological protein secretion in rheumatoid arthritis

doi: 10.1101/2024.10.27.24316032

Figure Lengend Snippet: Immunoblotting analysis was performed on lysate from shCtrl, shQPRT (a), or shPARP12 (b) transfected RA FLSs. GRASP55 phosphorylation was assessed using phos-tag gels, and the asterisk indicates p-GRASP55. mTORc1 activity was assessed by the phosphorylation of ribosomal protein S6 (S6) and eukaryotic initiation factor 4E-binding protein 1(4E-BP1). c-f: Quantification of the proteins. g: Co-immunoprecipitation and immunoblotting experiments confirm PARP12 interaction with GRASP55. h: ADP ribosylation of GRASP55 in shQPRT or shPARP12 transfected RA FLSs. i: ADP ribosylation of GRASP55 in PARP12 overexpressing RA FLSs in the absence or presence of 100 μM NAD+ for 24 h (n = 3). j: Co-IP analysis of GRASP55 interaction with the autophagosome marker LC3B, and multivesicular body (MVB) marker CHMP2A in shCtrl, shQPRT and shPARP12 transfected RA FLSs. Data are the means ± s.e.m. Statistical significance was assessed by two-way ANOVA ( c,d ) or two-tailed Student’s t-test ( e,f ): ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Non-targeting scrambled control (shCtrl) and QPRT-specific shRNAs in the pcDNA3.1+ vectors were packaged into lentiviral particles by cotransfecting HEK 293T cells with pMD2G (Addgene #12259) and psPAX2 (Addgene #394976) packaging vectors using polyethyleneimine (PEI, Yeasen, 40816ES03)transfection reagent.

Techniques: Western Blot, Transfection, Activity Assay, Binding Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Marker, Two Tailed Test

Journal: medRxiv

Article Title: Deficient QPRT drives trans-Golgi NAD + hyperinflation and pathological protein secretion in rheumatoid arthritis

doi: 10.1101/2024.10.27.24316032

Figure Lengend Snippet: Immunoblotting analysis was performed on lysate from shCtrl or shQPRT transfected RA FLSs. GRASP65 phosphorylation was assessed using phos-tag gels, and the asterisk indicates p-GRASP65. b: Representative immunofluorescence image of GRASP55 expression in shQPRT/shCtrl transfected RA FLSs. c-f: Immunoblotting analysis of autophagy markers LC3B and P62 in shQPRT (c,d) or shPARP12 (e,f) transfected RA FLSs compared to control. Data are the means ± s.e.m. Statistical significance was assessed by two-way ANOVA ( d,f ): *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Non-targeting scrambled control (shCtrl) and QPRT-specific shRNAs in the pcDNA3.1+ vectors were packaged into lentiviral particles by cotransfecting HEK 293T cells with pMD2G (Addgene #12259) and psPAX2 (Addgene #394976) packaging vectors using polyethyleneimine (PEI, Yeasen, 40816ES03)transfection reagent.

Techniques: Western Blot, Transfection, Immunofluorescence, Expressing, Control

Journal: bioRxiv

Article Title: Maternal sterol 27-hydroxylase is crucial for securing fetal development

doi: 10.1101/2023.11.08.566330

Figure Lengend Snippet: (A) Western blot analysis to validate the knockdown efficiency of shRNA against Fau (shFau). Non-targeting control shRNA (shCtrl) was used as a control. (B) Protein synthesis rate analysis using OP-Puro staining. A representative flow cytometry analysis of OP-Puro intensity in the LLC expressing shCtrl or shFau is shown. (C) Polysome profiling of LLC expressing shFau driven by the Tet-On inducible system. (D) A schema of the in vitro culture of fetal lung expressing shFau. (E) Representative photos of the fetal lungs cultured in the air-liquid interface system. The scale bar represents 1 mm. (F) The number of branching points in the fetal lungs transduced with lentiviruses. (G) Representative Western blot analysis of the fetal lungs transduced with lentiviruses. (H) The relative signal of Fau and Sftpc. The intensity of the band was normalized using Actb, and the fold changes in the shFau samples compared to the DMSO control is shown. * p < 0.05, ** p < 0.01.

Article Snippet: Mission lentiviral shRNA plasmids targeting Fau (shFau: TRCN0000104130, TRCN0000104131, TRCN0000104132, and TRCN0000104133), non-targeting shRNA control (shCtrl: SHC016) were purchased from Sigma-Aldrich.

Techniques: Western Blot, shRNA, Staining, Flow Cytometry, Expressing, In Vitro, Cell Culture, Transduction

Journal: Nucleic Acids Research

Article Title: The function of RAD52 N-terminal domain is essential for viability of BRCA-deficient cells

doi: 10.1093/nar/gkaa1145

Figure Lengend Snippet: Effect of RAD52 WT or RAD52 1–209 expression on viability of BRCA1- and BRCA2-deficient cells. BRCA1 −/− MDA-MB-436 ( A ), HCC1937 ( B ) and BRCA2 −/− CAPAN1 ( C ) cells expressing RAD52 WT or RAD52 1–209 or empty vector were treated with shRAD52 targeting 3′UTR. After 14 days, the number of colonies were counted. Clonal survival is expressed in percentage after normalizing to cells expressing the empty vector and control shRNA (shCtrl). Clonogenic survival graph are shown on the left and representative crystal violet stained colonies are shown on the right for each panel. Error bars indicate S.D. ( n = 4) and statistical analysis was performed using one-way ANOVA with Tukeys multiple comparison test; ns P > 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and **** P ≤ 0.0001.

Article Snippet: pLKO vector expressing shRNA targeting 3′UTR of RAD52 (shRAD52) (Sigma, SHCLND-NM_134424.2–1462s21c1) or Non-Target shRNA control (shCtrl) (Sigma, SHC202) were used.

Techniques: Expressing, Plasmid Preparation, shRNA, Staining

Journal: Nucleic Acids Research

Article Title: The function of RAD52 N-terminal domain is essential for viability of BRCA-deficient cells

doi: 10.1093/nar/gkaa1145

Figure Lengend Snippet: RAD52 1–209 promotes homology dependent repair (HDR) of DSBs by gene conversion in BRCA1-deficient MDA-MB-436 cells. ( A ) The scheme of the HDR assay. ( B ) Efficiency of I-Sce induced HDR in MDA-MB-436 BRCA1 +/+ and MDA-MB-436 BRCA1 −/− treated with shRAD52 or a control shRNA (shCtrl). The relative change of HDR efficiency for each group was calculated by normalizing each to the HDR efficiency obtained for BRCA1 +/+ cells. ( C ) Efficiency of I-Sce induced HDR in BRCA1 −/− cells expressing RAD52 WT or RAD52 1–209 or empty vector treated with shRAD52 or control shRNA(shCtrl). ( D ) Efficiency of I-Sce induced HDR in BRCA1 −/− cells expressing RAD52 WT or RAD52 YI65–66AA or RAD52 K102A/K133A or empty vector treated with shRAD52.The relative change of HDR efficiency for each group in panels (C) and (D) was calculated by normalizing to the HDR efficiency obtained for cells expressing empty vector and shCtrl. ( E ) Efficiency of I-Sce induced HDR in BRCA1 −/− cells expressing RAD52 1–209 with shRAD52 and treated with D-I03 (50 μM) or DMSO (1%). Data normalized to the HDR efficiency obtained for cells treated with DMSO. Error bars represent S.D ( n = 3), statistical analysis was performed using One-way ANOVA with Tukeys multiple comparison test in (B), (C) and (D) and Two-way ANOVA with Bonferroni's multiple comparisons test in (E); ** P ≤ 0.01, **** P ≤ 0.0001 and ns P > 0.05.

Article Snippet: pLKO vector expressing shRNA targeting 3′UTR of RAD52 (shRAD52) (Sigma, SHCLND-NM_134424.2–1462s21c1) or Non-Target shRNA control (shCtrl) (Sigma, SHC202) were used.

Techniques: shRNA, Expressing, Plasmid Preparation