non targeting shrna control  (Addgene inc)

Journal: bioRxiv

Article Title: Targeting HMGA1-driven leukemic transformation in myeloproliferative neoplasms with pacritinib

doi: 10.1101/2025.06.01.657170

Figure Lengend Snippet: (a) Efficient shRNA-mediated knockdown of HMGA1 (sh1, sh2 vs. sh-NC control) in HEL and UKE-1 cells. Left: Relative HMGA1 mRNA levels by qRT-PCR (mean ± SD, n = 3). Right: Western blot analysis of HMGA1 protein; ACTB served as loading control. (b) Lentiviral-mediated overexpression of HMGA1 (OE vs. CMV-NC control) in HEL and UKE-1 cells. Left: Relative HMGA1 mRNA levels by qRT-PCR (mean ± SD, n = 3). Right: Western blot analysis of HMGA1 protein; Tubulin served as loading control. (c) Lentiviral-mediated overexpression of Hmga1 (J/OE vs. J/NC control) in murine Ba/F3 ( Jak2 wild type, or Jak2 V617F ) and 32D-cl3 ( Jak2 wild type, or Jak2 V617F ) cells. Left: Relative Hmga1 mRNA levels by qRT-PCR (mean ± SD, n = 3). Right: Western blot analysis of Hmga1 protein; Tubulin served as loading control. Statistical analyses for (a-c) by two-sample t-test or one-way ANOVA, as appropriate. (d) HMGA1 overexpression exacerbates disease phenotype in a HEL xenograft model. Hematological parameters (WBC, white blood cell count; HGB, hemoglobin; HCT, hematocrit; PLT, platelet count) in NSG mice engrafted with HEL cells stably expressing control vector (CMV-NC, n = 6) or HMGA1 (OE, n = 6) at 35 days post-transplantation. Data are presented as mean ± SD. Two-sample t -test. (e) HMGA1 knockdown alters chromatin accessibility and HMGA1 binding at key cell cycle regulatory gene loci. Integrative Genomics Viewer (IGV) snapshots displaying ATAC-seq and HMGA1 CUT&Tag signals at representative E2F target genes ( E2F1 , CCNE1 , CCNE2 , CDK2 , RB1 ), G2M checkpoint genes ( CCNB1 , CCNB2 , CDC2 , WEE1 , CDC25C , PLK1 , AURKA , AURKB ), and common cell cycle regulators ( CCNA2 , CDKN1A / p21 , CDKN1B / p27 ) in HEL cells following control (NC) versus HMGA1 knockdown (KD). (f) Enhanced E2F target and G2M checkpoint gene signatures in sAML patient cells. UMAP projections of single-cell CITE-seq data (GSE185381) from control and sAML patients, with cells colored by enrichment scores for E2F target and G2M checkpoint gene sets. Corresponding density plots illustrate score distributions.

Article Snippet: A non-targeting shRNA control (pLKO.1-shSCR; Addgene plasmid #1864) was used.

Techniques: shRNA, Knockdown, Control, Quantitative RT-PCR, Western Blot, Over Expression, Cell Counting, Stable Transfection, Expressing, Plasmid Preparation, Transplantation Assay, Binding Assay

Journal: bioRxiv

Article Title: Targeting HMGA1-driven leukemic transformation in myeloproliferative neoplasms with pacritinib

doi: 10.1101/2025.06.01.657170

Figure Lengend Snippet: (a) Relative proliferation curves of human (HEL, UKE-1) and murine (Ba/F3, 32D-cl3 transduced with Jak2 wild-type or Jak2 V617F ) cell lines following HMGA1/Hmga1 overexpression (OE) or shRNA-meidated knockdown (sh1, sh2) compared to respective controls (CMV-NC or sh-NC)NC.) 32D-cl3 cells were cultured with IL-3. Data are mean ± SD. (n = 5 per group). Two-way ANOVA. (b) Flow cytometric analysis of CD11b expression on 32D-cl3 cells transduced with Jak2 wild-type (J WT ) or Jak2 V617F (J VF ), and co-transduced with control vector (NC) or HMGA1 overexpression (OE), following G-CSF (100 ng/mL) induced differentiation. (i) Representative histograms of CD11b-FITC fluorescence. (ii) Quantification of HMGA1-PE mean fluorescence intensity (MFI). (iii) Quantification of CD11b-FITC MFI (n = 5 per group). Data are mean ± SD. Two-sample t -test. (c) Quantification of human CD45 + CD117 + HEL cells in peripheral blood of NSG mice at day 35 post-transplant, comparing HMGA1-OE versus vector control (CMV-NC) groups (n=6 per group). Data are mean ± SD. Two-sample t -test. (d) Wright-Giemsa stained peripheral blood smears from NSG mice engrafted with HMGA1-OE or CMV-NC HEL cells at day 35. Quantification of HEL cells (% of total nucleated cells) is shown (n = 6 per group). Data are mean ± SD. Two-sample t -test. (e-f) Representative H&E and HMGA1 IHC staining (left panels of e and f, respectively) and quantification of HMGA1-positive cells (%) (right panels fo e and f, respectively) in (e) femur bone marrow and (f) spleen sections from NSG mice engrafted with HMGA1-OE or CMV-NC HEL cells. Scale bars: 50 µm. Data are mean ± SD. Two-sample t -test. (g) Representative images of spleens (left) and relative spleen weights (spleen weight/body weight %, right) from NSG mice at day 35 post-engraftment with HMGA1-OE or CMV-NC HEL cells (n = 6 per group). Data are mean ± SD. Two-sample t -test. (h) Kaplan-Meier survival curves for NSG mice injected with HMGA1-OE ro CMV-NC HEL cells (n = 6 per group). Median survival times are indicated. Log-rank (Mantel-Cox) test. (i) Heatmaps showing HMGA1 binding intensity (CUT&Tag, left) and chromatin accessibility (ATAC-seq, right) centered on transcription start site (TSS ± 3kb) for genes in HEL cells transduced with shNC or shHMGA1. Color scale indicates normalized read counts (Max/Min normalized). (j) Top de novo motifs identified by HOMER analysis within ATAC-seq peak regions that either lose accessibility (left) or gain accessibility (right) upon HMGA1 knockdown in HEL cells. P -value for motif enrichment are indicated. (k) Quantification of apoptosis by Annexin V-APC/7-AAD staining and flow cytometry in HEL and UKE-1 cells after transduction with shNC or HMGA1 shRNAs (sh1, sh2). Representative flow cytometry plots are shown. Data are mean ± SD. (n = 5 per group). One-way ANOVA with Tukey’s post-hoc test.

Article Snippet: A non-targeting shRNA control (pLKO.1-shSCR; Addgene plasmid #1864) was used.

Techniques: Transduction, Over Expression, shRNA, Knockdown, Cell Culture, Expressing, Control, Plasmid Preparation, Fluorescence, Staining, Immunohistochemistry, Injection, Binding Assay, Flow Cytometry

Journal: bioRxiv

Article Title: Targeting HMGA1-driven leukemic transformation in myeloproliferative neoplasms with pacritinib

doi: 10.1101/2025.06.01.657170

Figure Lengend Snippet: (a) Prognostic significance of HMGA1 expression in the OHSU BeatAML MPN-sAML cohort (n = 31). Genes are ranked by their hazard ratio (HR) for overall survival (OS). Points are colored based on FDR significance: grey ( FDR > 0.05), blue ( FDR < 0.05 & HR < 1, good prognosis), red ( FDR < 0.05 & HR ≥ 1, poor prognosis) (b) Gene Set Enrichment Analysis (GSEA) showing Hallmark pathways enriched among genes associated with poor prognosis (ranked by HR) in the OHSU BeatAML MPN-sAML cohort. Heatmap displays NES for selected pathways. Asterisks (*) indicate FDR < 0.05. (c) Kaplan-Meier OS curves for MPN-sAML patients from the OHSU BeatAML cohort (n = 31, top panel) and an independent in-house cohort (n = 21, bottom pnel), stratified by high versus low HMGA1 expression (HMGA1 expression levels for BeatAML in-house cohort using median cut-off). Log-rank (Mantel-Cox) test. (d) Representative immunohistochemical (IHC) staining for HMGA1 in bone marrow biopsies from in-house MPN-sAML cohort patients, illustrating HMGA1 expression changes with therapy and clinical outcome. (i) HMGA1-low patient achieving complete remission (CR) post-ruxolitinib. (ii) HMGA1-low patient achieving CR post-decitabine + venetoclax. (iii) HMGA1-high patient with progressive disease (PD) despite 5-azacytidine + venetoclax, showing increased HMGA1 at relapse. (iv) HMGA1-high patient achieving durable remission with decreased HMGA1 staining post-allogeneic hematopoietic stem cell transplantation (allo-HCT). Scale bars: 80µm (overview), 20µm (insets). (e) Comparison of HMGA1 expression levels between MPN-sAML patients achieving complete remission (CR, includes CRh, CRi) and those not achieving CR (Non-CR). Top panel: HMGA1 transcript levels (Log2 normalized counts) in the OHSU BeatAML cohort (n=31). Bottom panel: Percentage of HMGA1-positive cells (IHC score) in the in-house cohort (n=21). Data are mean ± SD. Two-sample t -test. (f) Heatmap illustrating Hallmark GSEA of differentially expressed genes in HEL cells treated with DMSO (vehicle), ruxolitinib (Rux), fedratinib (Fed), pacritinib (Pac), or momelotinib (Mmb) for 4 hours or 48 hours (GSE229712) and in HEL cells with acquired resistance to ruxolitinib (Rux-Persistent, GSE190517) compared to DMSO control. Color intensity represents NES. * indicate FDR < 0.05. (g) Dose-response curves depicting cell viability of parental (NC) control versus ruxolitinib-persistent (Rux-P) HEL (left) and UKE-1 (right) cells treated with indicated concentrations of ruxolitinib for 72 hours. IC 50 values (mean± SD) are shown. Two-way ANOVA comparing IC 50 values. (h) HMGA1 mRNA expression (RNA-seq, normalized counts) in HEL cells: non-targeting control (NC), ruxolitinib-persistent (Rux-P), and fedratinib-persistent (Fed-P). (I) Immunoblot analysis of HMGA1 protein levels in parental (NC) and and ruxolitinib-persistent (Rux-P) HEL and UKE-1 cells. Tublin serves as a loading control. (J) Dose-response curve showing cell viability of HEL cells transduced with control vector (NC), HMGA1 overexpression (OE), or HMGA1 shRNA (Sh1) constructs, treated with indicated concentrations of ruxolitinib (Rux), fedratinib (Fed), pacritinib (Pac), and momelotinib (Mmb) for 72 hours. IC50 values (mean ± SD) are shown. Two-way ANOVA comparing IC50 values between OE/Sh1 and respective NC. (k) Schematic representation of the in vivo pacritinib treatment study in NSG mice engrafted with luciferase-expressing HEL cells (transduced with CMV-NC vector or HMGA1-OE construct). Following leukemia engraftment (Day 0-21), mice received pacritinib (100 mg/kg, BID) or vehicle orally for 14 days (Day 21-35). Endpoint analyses included bioluminescence imaging, spleen weight, flow cytometry, Wright-Giemsa staining, H&E, and IHC, alongside survival monitoring. (l) Representative bioluminescence image (left) and quatification of total tumor bioluminescence (total flux, right) at day 35 in NSG mice engrafted with CMV-NC or HMGA1-OE HEL cells and treated with vehicle or pacritinib (n=6 mice/group). Data are shown in mean ± SD. One-way ANOVA with Tukey’s post-hoc test.

Article Snippet: A non-targeting shRNA control (pLKO.1-shSCR; Addgene plasmid #1864) was used.

Techniques: Expressing, Immunohistochemical staining, Immunohistochemistry, Staining, Transplantation Assay, Comparison, Control, RNA Sequencing, Western Blot, Transduction, Plasmid Preparation, Over Expression, shRNA, Construct, In Vivo, Luciferase, Imaging, Flow Cytometry

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: iScience

Article Title: Myoglobin modulates the Hippo pathway to promote cardiomyocyte differentiation.

doi: 10.1016/j.isci.2024.109146

Figure Lengend Snippet: Figure 1. Deletion of endogenous myoglobin inhibits cardiomyocyte differentiation (A) Representative western blot and quantification of Mb in NRVMs treated with control (Ctrl) or Mb targeted siRNA (Mb). N = 3. (B) Representative immunofluorescence images of NRVMs stained for cardiac troponin T (cTNT; red) to visualize sarcomeric structures and DAPI for nuclei (blue). Relative quantification of troponin fiber lengths in Mb cells relative to control cells. Scale bar: 10 mm. N = 3. (C) Relative mRNA expression of cardiac structural differentiation markers-mlc-2v (myosin light chain), myh6 (myosin heavy chain), tnnt2 (cardiac troponinT)- in NRVM compared to control undifferentiated cells after Mb knockdown. N = 3. (D) Relative mRNA expression levels of cardiac structural differentiation markers in control and Mb H9C2 cells. N = 4–5. Data are mean G SEM; *p < 0.05, **p < 0.01.

Article Snippet: Transient knockdown in mouse HL-1 cells was performed with siRNA particles targeted to mouse myoglobin (sc-35994, Santa Cruz) or non-targeting scramble siRNA (sc-37007, Santa Cruz) following the same protocol.

Techniques: Western Blot, Control, Staining, Expressing, Knockdown

Journal: iScience

Article Title: Myoglobin modulates the Hippo pathway to promote cardiomyocyte differentiation.

doi: 10.1016/j.isci.2024.109146

Figure Lengend Snippet: Figure 5. Deletion of Mb inhibits YAP phosphorylation in neonatal mouse hearts (A) Western blot and relative quantification of pYAP to T-YAP ratios in embryonic (E14.5), fetal (postnatal day 3) and adult (10-week-old) mice. N = 3 mice per age. (B) Representative western blot and relative quantification of pYAP to T-YAP ratios in postnatal day 7 mouse hearts after injection of AAV9 virus encoding a scramble control or Mb targeting shRNA into postnatal day 1 mice. N = 5–7 mice per group. (C) Representative tissue sections and quantification of phospho histone 3 (H3P) staining from mice injected with control or Mb targeted shRNA expressing AAV9. N = 6 mice per group. Arrow indicates H3P+ nuclei and green represents the GFP cardiomyocytes infected with AAV9. Scale bar: 50 mm. Data are mean G SEM. *p < 0.05, **p < 0.01.

Article Snippet: Transient knockdown in mouse HL-1 cells was performed with siRNA particles targeted to mouse myoglobin (sc-35994, Santa Cruz) or non-targeting scramble siRNA (sc-37007, Santa Cruz) following the same protocol.

Techniques: Phospho-proteomics, Western Blot, Injection, Virus, Control, shRNA, Staining, Expressing, Infection

Journal: Molecular Biology of the Cell

Article Title: The microvillar protocadherin CDHR5 associates with EBP50 to promote brush border assembly

doi: 10.1091/mbc.e23-02-0065

Figure Lengend Snippet: Figure 8. Loss of EBP50 results in severe BB assembly defects in CACO-2BBE cells. (A) Confocal images of 12-day polarized CACO-2BBE cells stably expressing either a scramble shRNA construct or shRNAs targeting EBP50 (KD 37 and 64), stained for EBP50 (green) and F-

Article Snippet: Knockdown studies utilized a non-targeting scramble control shRNA (Addgene; plasmid #46896), CDHR5 KD shRNA (TRC clone TRCN000054168; KD68), and EBP50 KD shRNA clones (TRCN0000043733; KD33), (TRCN0000043734; KD34), (TRCN0000043735; KD35), (TRCN0000043737; KD37), (TRCN0000440444; KD44) and (TRCN0000437164; KD64) sequences that were expressed from the pLKO.1 plasmid.

Techniques: Stable Transfection, Expressing, shRNA, Construct, Staining

Journal: Molecular Biology of the Cell

Article Title: The microvillar protocadherin CDHR5 associates with EBP50 to promote brush border assembly

doi: 10.1091/mbc.e23-02-0065

Figure Lengend Snippet: Figure 9. Loss of CDHR5 results in BB assembly defects in CACO-2BBE cells. (A) Confocal images of 12-day polarized CACO-2BBE cells stably expressing either a scramble shRNA construct or an shRNA targeting CDHR5, stained for CDHR5, EBP50, Ezrin, and Phospho-ERM (P-ERM) (green) and F-actin (red). Boxed regions denote the area in zoomed image panels. Scale bars, 10 μm. Loss of CDHR5 expression disrupts proper apical assembly, but does not block apical targeting of Ezrin, activated Ezrin (detected by P-ERM antibody) and EBP50. (B) Scatterplot quantification of apical/total signal ratios of Ezrin, activated Ezrin (P-ERM) and EBP50 in scramble and CDHR5 KD CACO-2BBE cells. Each data point represents the ratio of the entire apical signal found in the x-z section compared to the total signal of the x-z section. Measurements of apical to total signal: EBP50 signal, scramble n=78, KD n=65; Ezrin signal, scramble n=74, KD n=56; P-ERM signal, scramble n=62, KD n=69. ns = not significant, *p < 0.01, two-tailed t test. (C) Cartoon proposing the new interactome of the IMAC, in which CDHR5 is cross-linked to the actin cytoskeleton via EBP50-Ezrin.

Article Snippet: Knockdown studies utilized a non-targeting scramble control shRNA (Addgene; plasmid #46896), CDHR5 KD shRNA (TRC clone TRCN000054168; KD68), and EBP50 KD shRNA clones (TRCN0000043733; KD33), (TRCN0000043734; KD34), (TRCN0000043735; KD35), (TRCN0000043737; KD37), (TRCN0000440444; KD44) and (TRCN0000437164; KD64) sequences that were expressed from the pLKO.1 plasmid.

Techniques: Stable Transfection, Expressing, shRNA, Construct, Staining, Blocking Assay, Two Tailed Test