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: bioRxiv

Article Title: The Regulation of COX-2, ABCA1 and ABCG1 by the lncRNA PACERR links the inflammatory response and cholesterol homeostasis

doi: 10.1101/2025.06.02.657432

Figure Lengend Snippet: (A) PACERR expression in total RNA isolated from human carotid endarterectomy samples where advanced/central plaques are separated from peripheral plaque and sequencing was done on both. (B) scRNA-seq of carotid plaque samples. (C) THP-1 cells differentiated into macrophages using 10 nM PMA for 48–72 h then treated with ApoA1 (50µg/ml), HDL (50µg/ml), acLDL (37.5µg/ml), oxLDL (10µg/ml), or LPS (100ng/ml) for 24 hours followed by qPCR for gene expression analysis. (D) THP-1 macrophages were treated with 50µg/ml HDL, HDL2 or HDL3 for 24 hours and qPCR for gene expression was used, western blot to measure COX-2 protein levels and mass spectrometry was used to measure 6k-PGF1a/PGI2 and PGE2 in the cell media. (E) THP-1 macrophages were pre-treated with 5µM celecoxib for 1 hour before addition of 50µg/ml HDL or 100ng/ml LPS for 24 hours. (F) THP-1 macrophages were pre-treated with 5µM Bay-117082 for 1 hour before addition of 50µg/ml HDL or 100ng/ml LPS for 24 hours. (G) THP-1 macrophages stably expressing CREB shRNA were treated with 50 µg/ml or 100ng/ml LPS for 24 hours then qPCR was used to measure gene expression. Values are mean ±SEM of three independent experiments. *p<0.05, **p<0.01, ***p<0.005, ****p<0.001 versus untreated.

Article Snippet: Transient knockdown of CREB was done by designing shRNAs targeting a region to all CREB gene variants or a non-targeting control shRNA and cloned into the pLKO vector (Addgene, 8453) and transfected into HEK293T cells with packaging vectors psPAX2 (Addgene, 12260) and pMD2.G (Addgene, 12259).

Techniques: Expressing, Isolation, Sequencing, Gene Expression, Western Blot, Mass Spectrometry, Stable Transfection, shRNA