control scrambled shrna plasmid shctrl  (OriGene)

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: Oncogene

Article Title: AR-induced long non-coding RNA LINC01503 facilitates proliferation and metastasis via the SFPQ-FOSL1 axis in nasopharyngeal carcinoma

doi: 10.1038/s41388-020-01388-8

Figure Lengend Snippet: a Malignant, proliferation-related, and metastasis-related biological functions were enriched by gene set enrichment analysis (GSEA) in HK1 cells transfected with LINC01503 shRNA (sh1503) or shCtrl. NES normalized enrichment score. FDR < 0.25, p < 0.001. b Relative expression of LINC01503 upon specific shRNA knockdown in HK1 and SUNE1 cells. c LINC01503 knockdown inhibited the cell growth of HK1 and SUNE1 cells as tested by CCK-8 assays. d LINC01503 knockdown decreased cellular survival effects as evaluated by colony formation assays. e LINC01503 knockdown inhibited the cellular movement ability of HK1 and SUNE1 cells as assessed by wound healing assays. Scale bar, 100 μm. f , g LINC01503 knockdown inhibited the migration and invasion ability of HK1 and SUNE1 cells as determined by Transwell assays. Scale bar, 100 μm. Data are presented as the mean ± SD; p values were calculated with Student’s t test; * p < 0.05, ** p < 0.01.

Article Snippet: The shLINC01503 (sh1503) or its scramble control (shCtrl) plasmids were co-transfected into HEK293T cells with the lentivirus packaging plasmids pMD2G and psPAX2 (Addgene).

Techniques: Transfection, shRNA, Expressing, Knockdown, CCK-8 Assay, Migration

Journal: Oncogene

Article Title: AR-induced long non-coding RNA LINC01503 facilitates proliferation and metastasis via the SFPQ-FOSL1 axis in nasopharyngeal carcinoma

doi: 10.1038/s41388-020-01388-8

Figure Lengend Snippet: a Heatmap of clustering of differentially expressed genes in sh1503- and shCtrl-treated HK1 cells. b The expression of FOSL1 was increased in 20 NPC tissues compared with 16 normal tissues as determined by RT-qPCR. c LINC01503 expression was positively correlated with FOSL1 mRNA expression in 20 NPC tissues. d The RNA and protein levels of FOSL1 were decreased in sh1503-treated HK1 and SUNE1 cells as monitored by RT-qPCR and western blot. e The RNA and protein levels of FOSL1 were decreased in shSFPQ-treated HK1 and SUNE1 cells as monitored by RT-qPCR and WB. f Schematics of the 5′ region (-3000-0 nt) of the FOSL1. There are two SFPQ-binding sites in the promoter region of FOSL1, −1810 to −1797 and −1770 to −1758 nt. g Comparisons of SFPQ-binding sequences and the 5′ region of the FOSL1. The SFPQ-binding sequence was predicted by TRANSFAC analysis. h LINC01503 knockdown inhibited the enrichment of SFPQ on the FOSL1 promoter region in HK1 cells as indicated by chromatin immunoprecipitation (ChIP). i SFPQ overexpression increased the luciferase activity of the FOSL1 wild-type promoter construct, but not the mutant reporter gene construct, as determined by luciferase reporter assays in HEK293T cells. j LINC01503 and SFPQ knockdown decreased the chromatin accessibility at the promoter of the FOSL1 gene in HK1 cells as tested by DNase I digestion assays. Data are presented as mean ± SD; p values were calculated with Student’s t test; * p < 0.05, ** p < 0.01.

Article Snippet: The shLINC01503 (sh1503) or its scramble control (shCtrl) plasmids were co-transfected into HEK293T cells with the lentivirus packaging plasmids pMD2G and psPAX2 (Addgene).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Binding Assay, Sequencing, Knockdown, Chromatin Immunoprecipitation, Over Expression, Luciferase, Activity Assay, Construct, Mutagenesis

Journal: Oncogene

Article Title: AR-induced long non-coding RNA LINC01503 facilitates proliferation and metastasis via the SFPQ-FOSL1 axis in nasopharyngeal carcinoma

doi: 10.1038/s41388-020-01388-8

Figure Lengend Snippet: a Tumor growth curves of shCtrl and sh1503 HK1 cells in xenograft tumor growth model. b Representative image of xenograft tumors. c Tumor weight was smaller in the sh1503 group than in the shCtrl group. d Representative image of primary tumor in foot pads and metastatic inguinal lymph nodes in the inguinal lymph node metastasis model. e Representative image of the inguinal lymph node. f Inguinal lymph node volume was smaller in the sh1503 group. g H&E staining of foot pad primary tumors. h Pan-cytokeratin staining for inguinal lymph nodes as tested by IHC assay. i The metastatic ratio of inguinal lymph nodes was smaller in the sh1503 group. j Representative images of metastatic nodules on the lungs in the lung metastatic colonization model. k The number of metastatic nodule in lungs was lower in the sh1503 group. l H&E staining showed that the metastatic nodules were fewer and smaller in the sh1503 group. Data are presented as the mean ± SD; p values were calculated with Student’s t test; * p < 0.05, ** p < 0.01.

Article Snippet: The shLINC01503 (sh1503) or its scramble control (shCtrl) plasmids were co-transfected into HEK293T cells with the lentivirus packaging plasmids pMD2G and psPAX2 (Addgene).

Techniques: Staining

Journal: Scientific Reports

Article Title: Ten-Eleven Translocation-2 (Tet2) Is Involved in Myogenic Differentiation of Skeletal Myoblast Cells in Vitro

doi: 10.1038/srep43539

Figure Lengend Snippet: ( A ) Knockdown of Tet2 by using shRNA. Upper : C2C12 cells were transfected with Tet2-specific shRNA plasmid (shTet2) or a control shRNA plasmid (shCtrl) and then selected with puromycin. The mRNA levels of Tet family genes or myoblast differentiation-associated genes in puromycin-selected cells were detected by qRT-PCR. Lower : the cells were induced to differentiation and the mRNA levels of Tets or myoblast differentiation-associated genes were detected 4 d after differentiation induction. Myog, myogenin; MyoM, myomaker. ( B ) Western blot confirmation of Tet2 protein decline in shRNA- or siRNA-mediated Tet2 knockdown cells. β-actin was used as an internal control. The expected Tet2 band (NW: 224 kDa) is shown. Full-length blots are presented in . ( C ) Evaluation of myotube formation of Tet2 knockdown C2C12 cells. Myotubes were visualized by immunostaining for MyHC 6 d after differentiation induction (left). Nuclei were stained with DAPI. Scale bar, 100 μm. The fusion index was calculated as the ratio of the number of nuclei in MyHC-positive cells to the total number of nuclei present in the observation field. The quantitative analysis revealed a significant decrease in the fusion index of shTet2 C2C12 as compared with shCtrl cells (right). Data are presented as means ± SEM (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: To achieve persistent knockdown of Tet2, C2C12 cells was transfected with a Tet2 shRNA plasmid (shTet2) or a control scrambled shRNA plasmid (shCtrl) (Origene Technologies).

Techniques: shRNA, Transfection, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Immunostaining, Staining

Journal: Scientific Reports

Article Title: Ten-Eleven Translocation-2 (Tet2) Is Involved in Myogenic Differentiation of Skeletal Myoblast Cells in Vitro

doi: 10.1038/srep43539

Figure Lengend Snippet: C2C12 cells transfected with Tet2 shRNA (shTet2) or control shRNA (shCtrl) were cultured in growth medium. Methylation status of promoters of myogenin ( A ), Myf6 ( B ) or myomaker ( C ) was analyzed by using bisulfite sequencing. Numbers with a minus sign indicate the position of CpG relative to the transcription starting site. Each row represents an individual clone sequenced; black and white circles represent methylated and unmethylated CpGs, respectively. Percentage of methylation indicates the proportion of methylated CpG sites relative to the whole CpG sites examined.

Article Snippet: To achieve persistent knockdown of Tet2, C2C12 cells was transfected with a Tet2 shRNA plasmid (shTet2) or a control scrambled shRNA plasmid (shCtrl) (Origene Technologies).

Techniques: Transfection, shRNA, Cell Culture, Methylation, Methylation Sequencing

Journal: Scientific Reports

Article Title: Ten-Eleven Translocation-2 (Tet2) Is Involved in Myogenic Differentiation of Skeletal Myoblast Cells in Vitro

doi: 10.1038/srep43539

Figure Lengend Snippet: C2C12 cells transfected with Tet2 shRNA (shTet2) or control shRNA (shCtrl) were cultured in growth medium with or without vitamin C (VC; 500 μM). ( A ) Immunostaining for 5hmC in shTet2 vs shCtrl C2C12 cells with or without VC treatment. Nuclei were stained with DAPI. Scale bar, 50 μm. ( B ) Quantification of fluorescence intensities of 5hmC. The quantitative analysis revealed that Tet2 knockdown decreased the ability of VC to enhance 5hmC formation. ( C ) qRT-PCR analysis for the expression of myogenin, Myf6 and myomaker in shTet2 vs shCtrl C2C12 cells with or without VC treatment. Gapdh was used as an internal control. Data are presented as means ± SEM (n = 3). Asterisks above columns represent significant difference among groups (p < 0.05).

Article Snippet: To achieve persistent knockdown of Tet2, C2C12 cells was transfected with a Tet2 shRNA plasmid (shTet2) or a control scrambled shRNA plasmid (shCtrl) (Origene Technologies).

Techniques: Transfection, shRNA, Cell Culture, Immunostaining, Staining, Fluorescence, Quantitative RT-PCR, Expressing

Journal: Scientific Reports

Article Title: Ten-Eleven Translocation-2 (Tet2) Is Involved in Myogenic Differentiation of Skeletal Myoblast Cells in Vitro

doi: 10.1038/srep43539

Figure Lengend Snippet: C2C12 cells transfected with Tet2 shRNA (shTet2) or control shRNA (shCtrl) were subdivided into two experimental groups: non-vitamin C treatment group (indicated as VC−), in which VC was absent in both growth medium and differentiation medium, and VC-treatment group (indicated as VC+), in which the cells were first cultured for 48 h in growth medium containing 500 μM VC, and then were shifted to differentiated medium containing 500 μM VC. ( A ) Evaluation of myoblast differentiation in different treatment groups. Cells after 6 d of differentiation induction were immunostained with anti-MyHC antibodies to mark the myotubes. Nuclei were stained with DAPI. Scale bar, 100 μm. ( B ) Quantification analysis for differentiation efficiency in different treatment groups. The fusion index was calculated as the ratio of the number of nuclei in MyHC-positive cells to the total number of nuclei present in the observation field. ( C ) qRT-PCR analysis for the expression of myogenin, Myf6 and myomaker in C2C12 cells after 4 d of differentiation induction in different treatment groups. Gapdh was used as an internal control. Data are presented as means ± SEM (n = 3). Asterisks above columns represent significant difference among groups (p < 0.05).

Article Snippet: To achieve persistent knockdown of Tet2, C2C12 cells was transfected with a Tet2 shRNA plasmid (shTet2) or a control scrambled shRNA plasmid (shCtrl) (Origene Technologies).

Techniques: Transfection, shRNA, Cell Culture, Staining, Quantitative RT-PCR, Expressing