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