non-targeting scrambled control  (Genechem)

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 carnosinase dipeptidase CNDP1 is a novel metabolic vulnerability in brain metastasis

doi: 10.1101/2025.03.18.644053

Figure Lengend Snippet: (A) qRT-PCR quantification of CNDP1 mRNA, relative to GAPDH and normalized to shNTC, in 10-230 BM (top) and 12-273 (bottom) cells expressing indicated shRNA. (B) Top. Proliferation curve performed by analyzing % confluency extracted from Incucyte image analysis normalized to day 0 of 10-230 BM cells expressing indicated tet-On shRNA (n=3, 96 h). Bottom. Proliferation curve performed by serial fixing and crystal violet staining of 12-273BM cells expressing indicated tet-On shRNA. Representative experiment shown of n=3 biological replicates. Statistics derived from one-way ANOVA testing between groups on the final time point. (C) Bar plots representing % cells distributed along cell cycle phases assessed by Edu differential staining (n=2) in 10-230 and 12-273 BM cells expressing indicated tet-On shRNA. Statistical analysis by one-way ANOVA with Dunnett multiple hypothesis testing correction. (D) Left. qRT-PCR quantification of CNDP1 mRNA, relative to hPPIA and then normalized to siNTC, in 10-230 BM. Right. Proliferation ratio of 10-230 BM cells transfected with indicated siRNAs after 72 h of culture normalized to 0h (n=3), indicated as % confluency extracted from Incucyte image analysis. Multiple biological replicates represented. Statistical analysis by one-way ANOVA. (E). Left. qRT-PCR quantification of CNDP1 mRNA, relative to hPPIA and then normalized to sINTC, in 12-273 BM. Right. Proliferation ratio of 12-273 BM cells transfected with indicated siRNAs after 96 h culture normalized to 0h (n=4), indicated as % confluency extracted from Incucyte image analysis technology. Multiple biological replicates represented. Statistical analysis by one-way ANOVA. See

Article Snippet: Tet-pLKO-puro was purchased from Addgene (RRID:Addgene_98398). shRNAs were cloned as previously described 115) into Tet-pLKO-puro using AgeI (NEB, Cat#R3552S) and EcoRI (Thermo, Cat#FD0275) restriction sites. pLKO tet-on scrambled non-targeting control (shNTC) was purchased from Addgene (RRID:Addgene_47541).

Techniques: Quantitative RT-PCR, Expressing, shRNA, Staining, Derivative Assay, Transfection

Journal: bioRxiv

Article Title: The carnosinase dipeptidase CNDP1 is a novel metabolic vulnerability in brain metastasis

doi: 10.1101/2025.03.18.644053

Figure Lengend Snippet: (A) Carnosine accumulation measured by ELISA represented as normalized values to Carnosine concentration in shNTC conditions (30 mins under culture) in 12-273 transduced with indicated tetON-shRNAs treated with doxycycline for 24h. Mean of two technical replicates shown. (B) Proliferation ratio of 10-230 BM cells cultured with 10 μm, 100 μm and 50 mM Carnosine concentrations during 0.5, 2, 6 and 24 h of culture normalized to not treated (NT) of their respective time points (n=3, technical replicates). Confluency obtained by treating the cells with Resazurin and measuring absorbance after 3 hours of treatment at 570 nm. Statistical analysis by one-way ANOVA. (C) Carnosine accumulation shown as normalized units to STC media in conditioned media and pellet after 30 minutes of treatment. (D) Proliferation ratio of SKMEL-239 cells transfected with indicated shRNAs after 96 h of culture normalized to 0h, indicated as % confluency extracted from Incucyte image analysis. (E) Proliferation ratio performed by serial fixing and crystal violet staining of Yumm3.2 BrafV600E/wt Cdkn2a-/- Pten-/- control or ectopically expressing Cndp1-HA after 96h as indicated. (F) Immunoblot showing Cndp1-HA expression in conditioned media and pellets of Yumm3.2 BrafV600E/wt Cdkn2a-/- Pten-/- control or ectopically expressing Cndp1-HA. See also Document S1 for details. (G) L-Histidine liberation assay as carnosinase activity read-out in pellets of Yumm3.2 BrafV600E/wt Cdkn2a-/- Pten-/- control or ectopically expressing Cndp1-HA. Three technical replicates shown. One representative technical replicate shown for all the above assays.

Article Snippet: Tet-pLKO-puro was purchased from Addgene (RRID:Addgene_98398). shRNAs were cloned as previously described 115) into Tet-pLKO-puro using AgeI (NEB, Cat#R3552S) and EcoRI (Thermo, Cat#FD0275) restriction sites. pLKO tet-on scrambled non-targeting control (shNTC) was purchased from Addgene (RRID:Addgene_47541).

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay, Transduction, Cell Culture, Transfection, Staining, Control, Expressing, Western Blot, Activity Assay

Journal: bioRxiv

Article Title: The carnosinase dipeptidase CNDP1 is a novel metabolic vulnerability in brain metastasis

doi: 10.1101/2025.03.18.644053

Figure Lengend Snippet: (A) Workflow of RNA-sequencing and LC/MS-based nonpolar metabolomics integrative studio in 10-230BM cells transduced with indicated tetON-shRNA. (B) Differential gene expression/metabolite abundance between shNTC vs shCNDP1-1 and shCNDP1-2 by indicated adjusted p values. MetaboAnalyst executed hypergeometric testing by combining p-values for metabolites and transcripts according to their relative proportion in each pathway. (RNA-seq: DESeq2, Metabolomics: T-test, pathways enrichment FDR < 0.05). See Tables S5-7 (C) Heatmap of t-Aminoacyl synthetases expression across different conditions paired with a heatmap of expression of their corresponding amino acids. MS/MS-based proteomics and LC/MS-based metabolomics were conducted on lysates of 10-230 BM cells expressing the indicated shRNA treated with doxycycline for 72 hours in 10% dialyzed serum DMEM. Differentially expressed proteins were identified by unpaired, two-tailed t-test comparing 2 groups normalized abundance: shCNDP1-1 and shCNDP1-2 vs shNTC (Data represented by z-score as indicated). (D) Integrated Gene Set Enrichment analysis (GSEA) of 12-273 and 10-230 BM shNTC vs both shCNDP1 tetON-shRNA RNA sequencing represented by Normalized Enrichment Score using Reactome Gene Ontology Analysis (RNA-seq: DESeq2, adjust p value < 0.001 and FC > 1.5). See Table S6. (E) Differential expression testing by DESeq2 nominated ATF4 targets in both shCNDP1 vs shNTC conditions in 10-230 BM RNA sequencing. (Data represented by z-score as indicated). (F) Pathway analysis via the Seq-n-Slide pipeline tested for pathway enrichment in differentially expressed genes between shCNDP1 and shNTC 10-230 BM RNA sequencing (Adjust p value indicated). (G) Representative immunoblots of phosphoSerine51 eIF2a, total-eIF2a, ATF4 and housekeeping (HK) protein Vinculin in lysates of 10-230 BM transduced with indicated tetON-shRNA. See Document S1 for details. (H) Proliferation ratio of 12-273 BM cells transfected with indicated siRNAs for CNDP1 silencing after 96 h of culture normalized to 0h (n=4), either treated with vehicle (DMSO) or ISRIB (phospho-eIF2a inhibitor, 1 uM) performed by analyzing % confluency extracted from Incucyte image analysis. Statistical analysis by one-way ANOVA. Proliferation ratio for 12-230 BM siNTC/siCNDP1 also shown in E. See also

Article Snippet: Tet-pLKO-puro was purchased from Addgene (RRID:Addgene_98398). shRNAs were cloned as previously described 115) into Tet-pLKO-puro using AgeI (NEB, Cat#R3552S) and EcoRI (Thermo, Cat#FD0275) restriction sites. pLKO tet-on scrambled non-targeting control (shNTC) was purchased from Addgene (RRID:Addgene_47541).

Techniques: RNA Sequencing, Liquid Chromatography with Mass Spectroscopy, Transduction, shRNA, Gene Expression, Expressing, Tandem Mass Spectroscopy, Two Tailed Test, Quantitative Proteomics, Western Blot, Transfection

Journal: bioRxiv

Article Title: The carnosinase dipeptidase CNDP1 is a novel metabolic vulnerability in brain metastasis

doi: 10.1101/2025.03.18.644053

Figure Lengend Snippet: (A) 10-230 and 12-273 BM transduced with indicated tetON-shRNAs and treated with doxycycline during 72h, labeled with AHA (APC) for 4 hours and analyzed by flow cytometry. Cycloheximide (CHX, 50 ug/ml) was used as a positive control of translation shutdown. Statistics rendered by one-way ANOVA with Dunnet’s multiple hypothesis testing correction. (B) Left. Representative immunoblots of phospho-S6K1 p70 (Threonine 389), S6K1 p70, phosphoSer65 4E-BP, total 4EBP and housekeeping (HK) protein Hsp90 in lysates of 10-230 BM transduced with indicated tetON-shRNA. Right. Representative immunoblots of phospho-S6K1 p70 (Threonine 389), S6K1 p70, phosphoSer65 4E-BP, 4EBP and housekeeping (HK) protein Hsp90 in lysates of 10-230 BM cells transfected with siRNA NTC or CNDP1 as indicated. Band correspondent to p70 S6K1 is indicated. Results for HRI, phosphoSer51 and total eiF2α expression from same samples shown in and , respectively. Ratio of protein phosphorylation shown normalized to total protein and HK protein levels. See Document S1 for details. (C) GENI (gene set enrichment identifier124) was applied to TCGA melanoma samples (n = 472) using the Reactome 2022 pathway database. Five anticorrelating gene sets with FDR < 0.05 were represented. (D) Genes differentially expressed in shCNDP1 (combined) vs shNTC polysome heavy chain (log fc Translation vs log fc Transcription; p<0.05, lfc > 1) at translation, transcription and both transcription and translation levels in 10-230 BM cells. See Table 11. (E) Integrated Gene Enrichment analysis of Heavy Chain RNA sequencing of 10-230 BM shCNDP1 (combined) vs shNTC represented by Normalized Enrichment Score using Reactome Gene Ontology Analysis (RNA-seq: adjust p value < 0.05). See also

Article Snippet: Tet-pLKO-puro was purchased from Addgene (RRID:Addgene_98398). shRNAs were cloned as previously described 115) into Tet-pLKO-puro using AgeI (NEB, Cat#R3552S) and EcoRI (Thermo, Cat#FD0275) restriction sites. pLKO tet-on scrambled non-targeting control (shNTC) was purchased from Addgene (RRID:Addgene_47541).

Techniques: Transduction, Labeling, Flow Cytometry, Positive Control, Western Blot, shRNA, Transfection, Expressing, Phospho-proteomics, RNA Sequencing

Journal: bioRxiv

Article Title: The carnosinase dipeptidase CNDP1 is a novel metabolic vulnerability in brain metastasis

doi: 10.1101/2025.03.18.644053

Figure Lengend Snippet: (A) Integrated Gene Enrichment analysis of 12-273 BM heavy chain both shCNDP1 vs shNTC RNA sequencing represented by Normalized Enrichment Score using Reactome Gene Ontology Analysis (RNA-seq: adjust p value < 0.05). (B) Venn diagram including common transcripts changing as indicated at translation level (top) and transcription and translation level (bottom) in both 10-230 and 12-273 BM shCNDP1 (combined) vs shNTC polysome heavy chain (adjusted p value <0.05, n.i - non-identified). See Table S11. (C) Representative immunoblot of eIFs proteins and phophoSerine51 4E-BP1, 4E-BP1 and housekeeping (HK) protein Vinculin in lysates of 12-273 BM cells transfected with siRNA NTC or CNDP1 as indicated. See Document S1 for details.

Article Snippet: Tet-pLKO-puro was purchased from Addgene (RRID:Addgene_98398). shRNAs were cloned as previously described 115) into Tet-pLKO-puro using AgeI (NEB, Cat#R3552S) and EcoRI (Thermo, Cat#FD0275) restriction sites. pLKO tet-on scrambled non-targeting control (shNTC) was purchased from Addgene (RRID:Addgene_47541).

Techniques: RNA Sequencing, Western Blot, Transfection

Journal: bioRxiv

Article Title: The carnosinase dipeptidase CNDP1 is a novel metabolic vulnerability in brain metastasis

doi: 10.1101/2025.03.18.644053

Figure Lengend Snippet: (A-E) Quantification of total mitochondria per cell (A) , mitochondria area (B) , circularity (C) , cristae number per mitochondria (D) and cristae area per mitochondria area (E) in 12-273 BM. Statistical analysis by one-way ANOVA. (F) Seahorse MitoStress analysis of OCR in 10-230 BM transduced with indicated tetON-shRNA and MDA-231 Brm2 treated as indicated. Statistical analysis by one-way ANOVA. Representative replicate shown (n=3, p<0.05 or indicated). (G) Normalized distributions of glutamine, glutamate, and TCA cycle metabolites, including α-ketoglutarate (αKG), succinate, fumarate, malate and citrate, in 10-230 BM cells treated with 10 10 μm, 100 μm and 50 mM Carnosine for 30 minutes are shown. (n = 3); data are shown as the mean ± SD. Statistical analysis by one-way ANOVA. P value indicated. Cells were cultured with [U- 13 C5] glutamine for 6 h before metabolite extraction and gas chromatography-mass spectrometry (GC-MS) analyses. (H) Carnosine accumulation measured by ELISA represented as normalized values to carnosine concentration in dialyzed media supplemented with indicated concentrations in 10-230 BM cells cultured with 10 μm, 100 μm and 50 mM Carnosine concentrations during 0.5, 2 h. Mean of two technical replicates shown. Results for 0.5 h also shown in Figure S2C. (I) 2logFold Change of HMOX protein abundance measured by proteomics (MS/MS) in 10-230 BM cells transduced with indicated tetON-shRNAs. Differentially expressed proteins identified by unpaired, two-tailed t-test comparing 2 groups: shCNDP1-1 and shCNDP1-2 versus shNTC. (J) 2logFoldChange of LCN2 transcript expression of both shCNDP1 tetON-shRNA vs shNTC in 10-230 BM and 12-230 BM cells. RNA sequencing data. p value < 0.05. (K) 2logFold Change of ATP7B and SLC46A3 transcript of Heavy Chain RNA sequencing of 10-230 BM shCNDP1 vs shNTC. p value < 0.05. (L) AUCell (Area Under the Curve) measurement of copper homeostasis gene signature (WikiPaths, WP3286) in CNDP1 high expression versus CNDP1 low expression in MBM cells from Biermann et al., 2022 data mining. Statistical analysis by one-way ANOVA, p<0.001. (M) Representative immunoblots of OGDH, PDH and housekeeping (HK) protein β-actin in lysates of 10-230 BM cells transfected with indicated sh-RNAs. See document S1 for details. (N) R Pearson correlation between melanoma cell lines classified by their MITF expression and resistance to Elesclomol treatment expressed as AUC of cell viability after treatment. Data mining from Depmap.

Article Snippet: Tet-pLKO-puro was purchased from Addgene (RRID:Addgene_98398). shRNAs were cloned as previously described 115) into Tet-pLKO-puro using AgeI (NEB, Cat#R3552S) and EcoRI (Thermo, Cat#FD0275) restriction sites. pLKO tet-on scrambled non-targeting control (shNTC) was purchased from Addgene (RRID:Addgene_47541).

Techniques: Transduction, shRNA, Cell Culture, Extraction, Gas Chromatography, Mass Spectrometry, Gas Chromatography-Mass Spectrometry, Enzyme-linked Immunosorbent Assay, Concentration Assay, Quantitative Proteomics, Tandem Mass Spectroscopy, Two Tailed Test, Expressing, RNA Sequencing, Western Blot, Transfection

Journal: Cancer Drug Resistance

Article Title: FGFR1 overexpression promotes resistance to PI3K inhibitor alpelisib in luminal breast cancer cells through receptor tyrosine kinase signaling-mediated activation of the estrogen receptor

doi: 10.20517/cdr.2024.181

Figure Lengend Snippet: FGFR1 knockdown restores alpelisib sensitivity in FGFR1-overexpressing MCF-7 cells. (A) Immunoblot analysis of FGFR1 protein levels in MCF-7/FGFR1 (MF) cells following transient transfection with FGFR1-targeting siRNA (MF/siFGFR1) or scramble control (MF/siC); (B) Dose-response curves of alpelisib treatment in MF cells with or without FGFR1 knockdown, demonstrating a significant reduction in IC 50 upon alpelisib treatment; (C) The statistical differences in IC 50 values between the two groups were analyzed using Welch’s t -test; (D and E) Clonogenic survival assays showing colony formation efficiency of MF cells treated with indicated concentrations of alpelisib (0.03-0.3 µM) following FGFR1 knockdown. Data represent triplicate measurements analyzed by two-way ANOVA. ** P < 0.01.

Article Snippet: The FGFR1-targeting siRNA oligonucleotide (sequence: 5’-GCAAGATTGGCCCAGACAA-3’) and a non-targeting scrambled control siRNA were synthesized by RiboBio Co., Ltd. (Guangzhou, China).

Techniques: Knockdown, Western Blot, Transfection, Control

Journal: Cancer Drug Resistance

Article Title: FGFR1 overexpression promotes resistance to PI3K inhibitor alpelisib in luminal breast cancer cells through receptor tyrosine kinase signaling-mediated activation of the estrogen receptor

doi: 10.20517/cdr.2024.181

Figure Lengend Snippet: FGFR1 knockdown reverses alpelisib resistance in T47D/FGFR1 cells. (A) Immunoblot analysis of FGFR1 protein levels in T47D/FGFR1 (TF) cells following transfection with FGFR1-targeting siRNA (TF/siFGFR1) or scramble control (TF/siC); (B) CCK-8 measurement of the dose-response curves of TF/siC and TF/siFGFR1 cells followed by IC 50 calculation; (C) Comparison of the IC 50 values between the two groups analyzed with Welch’s t -test; (D and E) Clonogenic assays of TF/siC and TF/siFGFR1 cells. Twenty-four h after siRNA transfection, the cells were reseeded and treated with alpelisib at indicated concentrations. Quantified colony formation efficiencies were analyzed with a two-way ANOVA test. ** P < 0.01.

Article Snippet: The FGFR1-targeting siRNA oligonucleotide (sequence: 5’-GCAAGATTGGCCCAGACAA-3’) and a non-targeting scrambled control siRNA were synthesized by RiboBio Co., Ltd. (Guangzhou, China).

Techniques: Knockdown, Western Blot, Transfection, Control, CCK-8 Assay, Comparison

Journal: Oncogene

Article Title: A TBX2-driven signaling switch from androgen receptor to glucocorticoid receptor confers therapeutic resistance in prostate cancer

doi: 10.1038/s41388-024-03252-5

Figure Lengend Snippet: A RNA-seq analysis in PC3 TBX2DN compared with PC3 Neo control showing a significant increase in the expression of AR (60-fold) and AR target genes; B q RT-PCR analysis showing increased AR mRNA expression in PC3 TBX2DN and C4-2B TBX2DN when compared with the respective PC3 Neo and C4-2B Neo controls, and conversely a decreased AR in LNCaP TBX2OE cells when compared with LNCaP Neo control; C Western blot analysis showing increased AR protein expression in PC3 TBX2DN and C4-2B TBX2DN cells when compared with the respective PC3 Neo and C4-2B Neo controls, and conversely decreased AR protein in LNCaP TBX2OE cells when compared with LNCaP Neo control; D qRT-PCR analysis showing increased mRNA expression of some of the known AR target genes in PC3 TBX2DN and C4-2B TBX2DN cells when compared with the respective PC3 Neo and C4-2B Neo controls, and conversely decreased expression of the AR target genes in LNCaP TBX2OE when compared with LNCaP Neo control; E Western blot analysis showing decreased TBX2 and increased AR protein expression in PC3 shTBX2 cells when compared with the PC3 NTSCR control cells; F Western blot analysis showing increased AR protein expression in DU145 TBX2DN cells compared with the DU145 Neo control cells. Data represent the average of triplicates ±S.D; Student’s unpaired 2-tailed t -tests were performed to compare the two groups ** p ≤ 0.01; *** p ≤ 0.001; and **** p ≤ 0.0001.

Article Snippet: The custom constructs for sh-TBX2 and its non-targeting scrambled RNA duplex siRNA control (NTSCR) were procured from Sigma Aldrich (RNAi single clones, Millipore sigma, Burlington, MA).

Techniques: RNA Sequencing, Control, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Quantitative RT-PCR

Journal: Oncogene

Article Title: A TBX2-driven signaling switch from androgen receptor to glucocorticoid receptor confers therapeutic resistance in prostate cancer

doi: 10.1038/s41388-024-03252-5

Figure Lengend Snippet: A q RT-PCR analysis showing decreased GR mRNA levels in LNCaP TBX2OE cells that were transfected with si-GR compared with si-controls, with or without enzalutamide treatment; B Cell viability assay showing increased enzalutamide sensitivity in LNCaP TBX2OE cells that were transfected with si-GR compared with si-controls. One-way ANOVA was performed where (n = 3), *** p < 0.001;****, p < 0.0001; C Schematic representing that GR knockdown in LNCaP TBX2OE cells leads to reversal to enzalutamide sensitivity; D , E Prostatospheres (PSs) formed from C4-2B NTSCR cells showing no significant changes in the number or size of PSs formed upon enzalutamide treatment either in the presence or absence of dexamethasone. One-way ANOVA was performed where (n = 3), *** p < 0.001;****, p < 0.0001; F , G Prostatospheres (PSs) formed from C4-2B shTBX2 cells showing a significant decrease in the number of PSs formed upon enzalutamide treatment, and a significant increase in the size and number of PSs formed upon dexamethasone treatment which failed to respond to continued enzalutamide treatment. One-way ANOVA was performed where (n = 3), *** p < 0.001;****, p < 0.0001; H Schematic representing that activation of GR in C4-2B shTBX2 cells leads to reversal to enzalutamide resistance.

Article Snippet: The custom constructs for sh-TBX2 and its non-targeting scrambled RNA duplex siRNA control (NTSCR) were procured from Sigma Aldrich (RNAi single clones, Millipore sigma, Burlington, MA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Transfection, Viability Assay, Knockdown, Activation Assay

Journal: Oncogene

Article Title: A TBX2-driven signaling switch from androgen receptor to glucocorticoid receptor confers therapeutic resistance in prostate cancer

doi: 10.1038/s41388-024-03252-5

Figure Lengend Snippet: A Western blot analyses of the CoIP in SP2509-treated (1 µM) C4-2B cells showing disruption of TBX2-GR and TBX2-LSD1 protein-protein interactions. Samples were immunoprecipitated with an anti-TBX2 antibody using species matched control IgG and immunoblotted for anti-GR and anti-LSD1 antibodies; B Western blot analyses of SP2509-treated (1 µM) C4-2B cells showing reduced expression of GR, TBX2, and LSD1; C Western blot analyses of the CoIP in SP2509-treated (2 µM) LNCaP EnzaR cells showing disruption of TBX2-GR and TBX2-LSD1 protein-protein interactions. Samples were immunoprecipitated with an anti-TBX2 antibody using species matched control IgG and immunoblotted for anti-GR and anti-LSD1 antibodies; D Western blot analyses of SP2509-treated (2 µM) LNCaP EnzaR cells showing reduced expression of GR, TBX2, and LSD1; E Western blot analyses of SP2509- (2 µM) or DMSO-treated LNCaP EnzaR shTBX2 cells or the control LNCaP EnzaR NTSCR cells showing a greater reduction of TBX2 and GR levels in LNCaP EnzaR shTBX2 compared with LNCaP EnzaR NTSCR control cells; F Cell proliferation assay in LNCaP EnzaR shTBX2 cells and LNCaP EnzaR NTSCR control cells at 72 and 96 h in the presence of enzalutamide (20 µM) and SP2509 (1 µM and 2 µM) showing significantly reduced proliferation in LNCaP EnzaR shTBX2 cells compared with LNCaP EnzaR NTSCR control cells.

Article Snippet: The custom constructs for sh-TBX2 and its non-targeting scrambled RNA duplex siRNA control (NTSCR) were procured from Sigma Aldrich (RNAi single clones, Millipore sigma, Burlington, MA).

Techniques: Western Blot, Disruption, Protein-Protein interactions, Immunoprecipitation, Control, Expressing, Proliferation Assay