Review

murine shrna plasmid (Santa Cruz Biotechnology)

Name: murine shrna plasmid
Brand: Santa Cruz Biotechnology
Rating: 93 (1 reviews)

Santa Cruz Biotechnology is a verified supplier

About

News

Press Release

Team

Advisors

Partners

Contact

Bioz Stars

Bioz vStars

Buy from Supplier

Structured Review

Santa Cruz Biotechnology murine shrna plasmid
Murine Shrna Plasmid, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/murine shrna plasmid/product/Santa Cruz Biotechnology
Average 93 stars, based on 1 article reviews

murine shrna plasmid - by Bioz Stars, 2026-02

93/100 stars

Images

Image Search Results

ATF4 expression is upregulated in ccRCC and is associated with poor outcomes. a The mRNA levels of ATF4 were analyzed in the ccRCC tissues in the TCGA dataset compared with the normal controls. b Kaplan–Meier analysis of OS for all ccRCC patients with low or high ATF4 expression levels in the TCGA dataset. c Western blot experiment was performed to evaluate the protein level of ATF4 in five paired ccRCC tissue samples (“T” means tumor tissue, and “N” means paired normal tissue). d Representative images of ATF4 IHC staining with high or low expression level in ccRCC tumor tissues by TMAs. (e) IHC representative images and analysis of ATF4 expression in tumor tissues (n = 199) and normal tissues (n = 143) by TMAs. f Kaplan–Meier analysis of ccRCC over survival based on ATF4 expression level in ccRCC TMAs. The p-value was calculated using the log-rank test. g Comparison of ATF4 IHC scores among groups with different tumor TNM stages and tumor grades. The p-values were calculated using the Mann–Whitney U test. h Association of ATF4 expression with clinical and pathological characteristics of 199 ccRCC patients in TMAs. The data are presented in terms of means ± SEM. The p-values were estimated using the Mann–Whitney U test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

Journal: Discover Oncology

Article Title: ATF4/NUPR1 axis promotes cancer cell survival and mediates immunosuppression in clear cell renal cell carcinoma

doi: 10.1007/s12672-024-01485-0

Figure Lengend Snippet: ATF4 expression is upregulated in ccRCC and is associated with poor outcomes. a The mRNA levels of ATF4 were analyzed in the ccRCC tissues in the TCGA dataset compared with the normal controls. b Kaplan–Meier analysis of OS for all ccRCC patients with low or high ATF4 expression levels in the TCGA dataset. c Western blot experiment was performed to evaluate the protein level of ATF4 in five paired ccRCC tissue samples (“T” means tumor tissue, and “N” means paired normal tissue). d Representative images of ATF4 IHC staining with high or low expression level in ccRCC tumor tissues by TMAs. (e) IHC representative images and analysis of ATF4 expression in tumor tissues (n = 199) and normal tissues (n = 143) by TMAs. f Kaplan–Meier analysis of ccRCC over survival based on ATF4 expression level in ccRCC TMAs. The p-value was calculated using the log-rank test. g Comparison of ATF4 IHC scores among groups with different tumor TNM stages and tumor grades. The p-values were calculated using the Mann–Whitney U test. h Association of ATF4 expression with clinical and pathological characteristics of 199 ccRCC patients in TMAs. The data are presented in terms of means ± SEM. The p-values were estimated using the Mann–Whitney U test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

Article Snippet: All cell lines were cultured in the recommended medium supplemented with 10% fetal bovine serum (FBS; Evergreen Co. Ltd., China) and 1% penicillin–streptomycin at 37 °C in 5% CO 2 under mycoplasma-free conditions. pLKO-shATF4 plasmids with short hairpin RNAs (shRNAs) targeting human and murine ATF4 were designed by Invitrogen.

Techniques: Expressing, Western Blot, Immunohistochemistry, Comparison, MANN-WHITNEY

Association of ATF4 expression with clinical and pathological characteristics of 199 ccRCC patients in TMAs

Journal: Discover Oncology

Article Title: ATF4/NUPR1 axis promotes cancer cell survival and mediates immunosuppression in clear cell renal cell carcinoma

doi: 10.1007/s12672-024-01485-0

Figure Lengend Snippet: Association of ATF4 expression with clinical and pathological characteristics of 199 ccRCC patients in TMAs

Techniques: Expressing

ATF4 knockdown attenuates ccRCC cell proliferation, migration, and invasion and induces apoptosis. a Relative ATF4 protein levels in HEK293T, HKC cells, and a panel of ccRCC cell lines. b ATF4 expression levels in 786-O and SN12 cells transduced with shATF4 or NC for four days were detected via Western blot. c The indicated cells are transduced with shATF4 for four days, followed by bright-field imaging (left) and quantification (right). Optical density (OD) values at 590 nm were used to compare cell growth ability between different groups. d Transwell assay indicates impaired abilities of migration and invasion of 786-O and SN12 cells treated with shATF4. Representative graphs (left). Bar graph showing the statistical results (right). e Effect of ATF4 knockdown on the apoptosis rate in 786-O and SN12 cells. f Western blot analysis of mTOR, EMT, and apoptosis-related gene expression in 786-O cells with NC and shATF4. g BALB/c nude mice underwent orthotopic implantation of SN12 cells, stably expressing control shRNA or shATF4, and then tumor masses were resected on day 21. Tumor weights were expressed in terms of mean ± SEM, n = 6 for each group. h H&E and IHC analyses of tissues harvested from the mice tumor masses stained for ki67, and cleaved caspase-3. All functional assays above were independently repeated thrice in triplicate. The data are presented herein as mean ± SEM. p-values were calculated using the two-tailed Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

Journal: Discover Oncology

Article Title: ATF4/NUPR1 axis promotes cancer cell survival and mediates immunosuppression in clear cell renal cell carcinoma

doi: 10.1007/s12672-024-01485-0

Figure Lengend Snippet: ATF4 knockdown attenuates ccRCC cell proliferation, migration, and invasion and induces apoptosis. a Relative ATF4 protein levels in HEK293T, HKC cells, and a panel of ccRCC cell lines. b ATF4 expression levels in 786-O and SN12 cells transduced with shATF4 or NC for four days were detected via Western blot. c The indicated cells are transduced with shATF4 for four days, followed by bright-field imaging (left) and quantification (right). Optical density (OD) values at 590 nm were used to compare cell growth ability between different groups. d Transwell assay indicates impaired abilities of migration and invasion of 786-O and SN12 cells treated with shATF4. Representative graphs (left). Bar graph showing the statistical results (right). e Effect of ATF4 knockdown on the apoptosis rate in 786-O and SN12 cells. f Western blot analysis of mTOR, EMT, and apoptosis-related gene expression in 786-O cells with NC and shATF4. g BALB/c nude mice underwent orthotopic implantation of SN12 cells, stably expressing control shRNA or shATF4, and then tumor masses were resected on day 21. Tumor weights were expressed in terms of mean ± SEM, n = 6 for each group. h H&E and IHC analyses of tissues harvested from the mice tumor masses stained for ki67, and cleaved caspase-3. All functional assays above were independently repeated thrice in triplicate. The data are presented herein as mean ± SEM. p-values were calculated using the two-tailed Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

Techniques: Knockdown, Migration, Expressing, Transduction, Western Blot, Imaging, Transwell Assay, Stable Transfection, Control, shRNA, Staining, Functional Assay, Two Tailed Test

ATF4 deficiency downregulates NUPR1 expression in ccRCC. a Heatmap analysis of DEG function clustering in the ccRCC from NC and shATF4 groups. b Kyoto KEGG pathway enrichment and GO enrichment analysis among DEGs in 786-O cells. c Venn diagram illustrating overlap among DEGs and target gene lists. DEGs 1 (NC vs. shATF4-1), DEGs 2 (NC vs. shATF4-2), target genes (the predicted gene list transcription-regulated by ATF4). d Volcano plot depicting the gene expression differences between NC and shATF4 groups. The horizontal axis represents the log-ratio (gene expression fold change in different samples), while the vertical axis represents the probability for each gene of being differentially expressed. The 75 genes in ( c ) are shown. e NUPR1 was verified via qRT-PCR experiment in ATF4 knockdown 786-O and SN12 cells. Data are presented in terms of mean ± SEM, n = 3. The p-values were calculated using the two-tailed Student’s t-test (*p < 0.05, ** p < 0.01, ***p < 0.001, ****p < 0.0001). (f) NUPR1 expression levels were detected by Western blot in ATF4 knockdown 786-O and SN12 cells. g Expression correlation of ATF4 and NUPR1 based on the IHC score in ccRCC tissue by TMAs (n = 199), representative IHC staining images in two ccRCC patients (left), the statistical result of correlation analysis (right)

Journal: Discover Oncology

Article Title: ATF4/NUPR1 axis promotes cancer cell survival and mediates immunosuppression in clear cell renal cell carcinoma

doi: 10.1007/s12672-024-01485-0

Figure Lengend Snippet: ATF4 deficiency downregulates NUPR1 expression in ccRCC. a Heatmap analysis of DEG function clustering in the ccRCC from NC and shATF4 groups. b Kyoto KEGG pathway enrichment and GO enrichment analysis among DEGs in 786-O cells. c Venn diagram illustrating overlap among DEGs and target gene lists. DEGs 1 (NC vs. shATF4-1), DEGs 2 (NC vs. shATF4-2), target genes (the predicted gene list transcription-regulated by ATF4). d Volcano plot depicting the gene expression differences between NC and shATF4 groups. The horizontal axis represents the log-ratio (gene expression fold change in different samples), while the vertical axis represents the probability for each gene of being differentially expressed. The 75 genes in ( c ) are shown. e NUPR1 was verified via qRT-PCR experiment in ATF4 knockdown 786-O and SN12 cells. Data are presented in terms of mean ± SEM, n = 3. The p-values were calculated using the two-tailed Student’s t-test (*p < 0.05, ** p < 0.01, ***p < 0.001, ****p < 0.0001). (f) NUPR1 expression levels were detected by Western blot in ATF4 knockdown 786-O and SN12 cells. g Expression correlation of ATF4 and NUPR1 based on the IHC score in ccRCC tissue by TMAs (n = 199), representative IHC staining images in two ccRCC patients (left), the statistical result of correlation analysis (right)

Techniques: Expressing, Quantitative RT-PCR, Knockdown, Two Tailed Test, Western Blot, Immunohistochemistry

Knockdown of ATF4 induces ferroptosis and inhibits cell proliferation by targeting NUPR1. a Western blot indicates ATF4 protein expression in 786-O cells following treatment with erastin (0–5 μM) for 24 h. b ATF4 knockdown inhibited erastin-induced (2.5 μM, 24 h) NUPR1 protein expression. c Scanning electron microscopy of 786-O cells transfected with NC or shATF4. Mitochondrion (arrow). d , e Representative immunofluorescent microscope images and flow cytometry analyses of lipid peroxidation levels reported by BODIPY-C11 in 786-O cells transfected with NC or shATF4 in the absence or presence of ferrostatin-1. f Representative immunofluorescent microscope images of FerroOrange (left) and statistical result (right) for relative Fe. 2+ level of 786-O cells transfected with NC or shATF4. g 786-O cells were transduced with shATF4 for four days in the absence or presence of ferrostatin-1, followed by bright-field imaging (left) and quantification (right). OD values at 450 nm were used to compare cell growth ability between different groups. The data are presented as mean ± SEM, n = 3. The p-values were estimated using the two-tailed Student’s t-test (*p < .05, ** p < .01, ***p < .001, ****p < .0001)

Journal: Discover Oncology

Article Title: ATF4/NUPR1 axis promotes cancer cell survival and mediates immunosuppression in clear cell renal cell carcinoma

doi: 10.1007/s12672-024-01485-0

Figure Lengend Snippet: Knockdown of ATF4 induces ferroptosis and inhibits cell proliferation by targeting NUPR1. a Western blot indicates ATF4 protein expression in 786-O cells following treatment with erastin (0–5 μM) for 24 h. b ATF4 knockdown inhibited erastin-induced (2.5 μM, 24 h) NUPR1 protein expression. c Scanning electron microscopy of 786-O cells transfected with NC or shATF4. Mitochondrion (arrow). d , e Representative immunofluorescent microscope images and flow cytometry analyses of lipid peroxidation levels reported by BODIPY-C11 in 786-O cells transfected with NC or shATF4 in the absence or presence of ferrostatin-1. f Representative immunofluorescent microscope images of FerroOrange (left) and statistical result (right) for relative Fe. 2+ level of 786-O cells transfected with NC or shATF4. g 786-O cells were transduced with shATF4 for four days in the absence or presence of ferrostatin-1, followed by bright-field imaging (left) and quantification (right). OD values at 450 nm were used to compare cell growth ability between different groups. The data are presented as mean ± SEM, n = 3. The p-values were estimated using the two-tailed Student’s t-test (*p < .05, ** p < .01, ***p < .001, ****p < .0001)

Techniques: Knockdown, Western Blot, Expressing, Electron Microscopy, Transfection, Microscopy, Flow Cytometry, Transduction, Imaging, Two Tailed Test

ATF4 knockdown remodels TME and promotes antitumor immunity. a BALB/c mice underwent orthotopic implantation of RenCa cells, stably expressing control shRNA or shATF4 respectively, and then tumor masses were resected on day 14. N = 4 for each group. b Representative H&E staining images and quantitative analysis for the amount of lungs with metastatic nodules (black arrow indicates metastatic nodules). c IHC analysis of tissues harvested from the mice tumor masses stained for NUPR1, CD8, CD4, and granzyme B. d Representative images and statistical results of CD8 and CD4 IHC staining with ATF4 high or low expression level in ccRCC tumor tissues by TMAs (n = 199). e Tumor growth of RenCa cells with ZZW115 and/or anti-PD1 therapy treated in BALB/c mice. n = 5 for each group. f IHC analysis of tumor tissue harvested from the mice treated with DMSO or ZZW115 stained for CD8 and CD4. The data are represented in terms of mean ± SEM, and p-values were calculated using the Mann–Whitney U test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

Journal: Discover Oncology

Article Title: ATF4/NUPR1 axis promotes cancer cell survival and mediates immunosuppression in clear cell renal cell carcinoma

doi: 10.1007/s12672-024-01485-0

Figure Lengend Snippet: ATF4 knockdown remodels TME and promotes antitumor immunity. a BALB/c mice underwent orthotopic implantation of RenCa cells, stably expressing control shRNA or shATF4 respectively, and then tumor masses were resected on day 14. N = 4 for each group. b Representative H&E staining images and quantitative analysis for the amount of lungs with metastatic nodules (black arrow indicates metastatic nodules). c IHC analysis of tissues harvested from the mice tumor masses stained for NUPR1, CD8, CD4, and granzyme B. d Representative images and statistical results of CD8 and CD4 IHC staining with ATF4 high or low expression level in ccRCC tumor tissues by TMAs (n = 199). e Tumor growth of RenCa cells with ZZW115 and/or anti-PD1 therapy treated in BALB/c mice. n = 5 for each group. f IHC analysis of tumor tissue harvested from the mice treated with DMSO or ZZW115 stained for CD8 and CD4. The data are represented in terms of mean ± SEM, and p-values were calculated using the Mann–Whitney U test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

Techniques: Knockdown, Stable Transfection, Expressing, Control, shRNA, Staining, Immunohistochemistry, MANN-WHITNEY

DB1055 and DB818 affect cell survival in human HOXA9‐positive acute myeloid leukemia (AML) cells and induce cell death. (A, B) Cell viability measurement upon treatment of DB818 or DB1055 on 14 human AML cell lines relative to their level of HOXA9 gene expression. Mean IC 50 values (CellTiter 96® AQueous One Solution Cell Proliferation Assay kit) are plotted over HOXA9 expression (absolute quantitative reverse‐transcription polymerase chain reaction quantification relative to TBP, normalized to 10 ng of total purified messenger RNA). Linear regression curves are plotted. Corresponding R (coefficient of determination), R 2 (coefficient of correlation), and p ‐values are embedded. T, THP1; E, Eol‐1; U, U937 cell lines. (C) Quantification of relative cell proliferation using CellTrace™ loaded THP‐1 cells For HOXA9 invalidation, cells were transduced (~98% GFP‐positive transduced cells) with lentivirus expressing shHOXA9‐1F2, ‐1F3, or shCTR and washed prior to being loaded for 5 min with CellTrace™‐Violet and washed prior to cell culture. For drug treatments, cells were loaded with CellTrace™‐CFSE (compatible with drug intrinsic fluorescence) for 5 min and washed prior to the addition of 10 µM of DB1055 or DB818 or none. Each treatment was performed in triplicate. Cells were analyzed at the indicated days by flow cytometry. Graphs are expressed as the % of proliferation, normalized to the corresponding control (untreated or shCTR). (D) Effect of DB818 or DB1055 treatment or HOXA9 invalidation on colony‐forming assays in THP‐1 model. THP‐1 cells were transduced with short hairpin RNA (shRNA) expressing lentivirus or treated with increasing concentrations of DB818, DB1055, or AraC treatment (µM) prior to being plated at a density of 1000 cells/well for 14 days ( n = 6). Results are expressed as the percentage of colonies eac wells relative to the corresponding controls (shCTR or untreated “0.” (E) Cell death induction analysis was evidenced using propidium iodide (PI) staining. THP‐1 cells were transduced with shRNA expressing lentiviruses or treated with either DB818, DB1055 (µM), or PMA as a positive control (2.5 ng/mL) for 7 days prior to PI staining of dead cells and flow cytometry analysis ( n = 6). (F) Apoptotic cell death induction was analyzed by Annexin V and PI double staining. Similarly treated cells were grown for 7 days prior to AnnexinV/PI labeling and flow cytometry analyses. Additive columns represent the percentage of annexinV‐positive cells being also either PI‐positive staining in gray or PI‐negative in white ( n = 6). Student t ‐test: **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05; or as specified on graphs.

Journal: HemaSphere

Article Title: Induction of AML cell differentiation using HOXA9/DNA binding inhibitors as a potential therapeutic option for HOXA9‐dependent AML

doi: 10.1002/hem3.77

Figure Lengend Snippet: DB1055 and DB818 affect cell survival in human HOXA9‐positive acute myeloid leukemia (AML) cells and induce cell death. (A, B) Cell viability measurement upon treatment of DB818 or DB1055 on 14 human AML cell lines relative to their level of HOXA9 gene expression. Mean IC 50 values (CellTiter 96® AQueous One Solution Cell Proliferation Assay kit) are plotted over HOXA9 expression (absolute quantitative reverse‐transcription polymerase chain reaction quantification relative to TBP, normalized to 10 ng of total purified messenger RNA). Linear regression curves are plotted. Corresponding R (coefficient of determination), R 2 (coefficient of correlation), and p ‐values are embedded. T, THP1; E, Eol‐1; U, U937 cell lines. (C) Quantification of relative cell proliferation using CellTrace™ loaded THP‐1 cells For HOXA9 invalidation, cells were transduced (~98% GFP‐positive transduced cells) with lentivirus expressing shHOXA9‐1F2, ‐1F3, or shCTR and washed prior to being loaded for 5 min with CellTrace™‐Violet and washed prior to cell culture. For drug treatments, cells were loaded with CellTrace™‐CFSE (compatible with drug intrinsic fluorescence) for 5 min and washed prior to the addition of 10 µM of DB1055 or DB818 or none. Each treatment was performed in triplicate. Cells were analyzed at the indicated days by flow cytometry. Graphs are expressed as the % of proliferation, normalized to the corresponding control (untreated or shCTR). (D) Effect of DB818 or DB1055 treatment or HOXA9 invalidation on colony‐forming assays in THP‐1 model. THP‐1 cells were transduced with short hairpin RNA (shRNA) expressing lentivirus or treated with increasing concentrations of DB818, DB1055, or AraC treatment (µM) prior to being plated at a density of 1000 cells/well for 14 days ( n = 6). Results are expressed as the percentage of colonies eac wells relative to the corresponding controls (shCTR or untreated “0.” (E) Cell death induction analysis was evidenced using propidium iodide (PI) staining. THP‐1 cells were transduced with shRNA expressing lentiviruses or treated with either DB818, DB1055 (µM), or PMA as a positive control (2.5 ng/mL) for 7 days prior to PI staining of dead cells and flow cytometry analysis ( n = 6). (F) Apoptotic cell death induction was analyzed by Annexin V and PI double staining. Similarly treated cells were grown for 7 days prior to AnnexinV/PI labeling and flow cytometry analyses. Additive columns represent the percentage of annexinV‐positive cells being also either PI‐positive staining in gray or PI‐negative in white ( n = 6). Student t ‐test: **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05; or as specified on graphs.

Article Snippet: The short hairpin RNA (shRNA) directed against HOXA9 shHOXA9‐1F2 or −1F3 were subcloned in the shLuc‐pRRL lentiviral vector used as control (Prof. Bob Weinberg, #19125; Addgene).

Techniques: Gene Expression, Proliferation Assay, Expressing, Reverse Transcription, Polymerase Chain Reaction, Purification, Cell Culture, Fluorescence, Flow Cytometry, Control, Transduction, shRNA, Staining, Positive Control, Double Staining, Labeling

Journal: HemaSphere

Article Title: Induction of AML cell differentiation using HOXA9/DNA binding inhibitors as a potential therapeutic option for HOXA9‐dependent AML

doi: 10.1002/hem3.77

Figure Lengend Snippet: Identification of commonly deregulated genes and processes using transcriptomic analysis in THP‐1 cell model. Gene expression profile was obtained in quadruplicate for THP‐1 cells invalidated for HOXA9 (shHOXA9‐1F3) versus control (shCTR) for 48 h or THP‐1 cells treated or not using DB818 or DB1055 for 24 h. (A) Venn diagrams for the intersection of genes being statistically significant ( p < 0.05) and differently expressed in each condition (relative to the corresponding control) with (top Venn diagram, UP&DOWN , fold change (FC) > 1.5 or <−1.5), statistically significant upregulated (middle Venn diagram, UP , FC > 1.5), and statistically significant downregulated (bottom Venn diagram, DOWN , FC < −1.5). (B) Volcano plots for all deregulated genes in shHOXA9‐1F3 transduced cells (top plot) or from DB1055 (middle plot) or DB818 (bottom plot) gene expression effect within the shHOXA9‐1F3 deregulated gene list. (C) Top 20 of the deregulated cellular networks identified by ingenuity pathway analysis (IPA) with significant | z ‐score| of at least 1.65 and ordered on the top ‐log p ‐values highlights the implication of differentiation, cell death leukocyte activation, and migration/movement processes. (D) Gene Set Enrichment Analysis (GSEA) assays highlight commonly deregulated processes and correlate with HOXA9 and MLL‐associated curated gene set terms. NES values are plotted for each GSEA from shHOXA9‐1F3, DB1055, or DB818 deregulated gene sets (GSEA version 6.2). Corresponding GSEA plots with NES, p ‐values, and Venn diagrams are presented in Supporting Information S1: Figures and together with other related GSEA‐CGP results.

Techniques: Gene Expression, Control, Activation Assay, Migration

Comparison of DB1055 and DB818 deregulated genes with ChIP‐seq and DNase‐seq data. (A) “HESS TARGETS OF HOXA9 AND MEIS1 DN” and “TAKEDA TARGETS OF NUP98‐HOXA9 FUSION 8D DN” curated gene sets terms (C2, CGP: chemical and genetic perturbations) highlighted from GSEA for both shHOXA9 and DB818 or DB1055 cell treatment. The normalized enrichment scores (NES), nominal p ‐values, and FDR are shown in each caption. Corresponding Venn diagrams are presented on the right of the panel and a heatmap for implicated genes is given in Supporting Information S1: Figure . (B) Heatmap for the expression change of the 138 genes commonly significantly deregulated in the three conditions (HOXA9 invalidation and treatment with DB818 or DB1055) and position of different genes of interest and identification of the presence of a peak from HOXA9 ChIP in MV4‐11 cells. <xref ref-type=

³² The intensity of the blue color depends on the distance to TSS as defined in the figure. In white: genes in which the distance to TSS was over 100 kb. Circular diagram represents the percentage of the 138 commonly dysregulated genes (UP or DOWN) that present (plain color) or not (dashed color) a ChIP‐peak identified for HOXA9 binding in the MV4‐11 cell line. (C) Venn diagrams for comparison of the lists of deregulated genes from transcriptomic analyses and genes identified at the proximity of differential peaks from global DNase‐seq analyses as depicted in the Supporting Information Material and Methods section. (D) IGV visualization of some peaks attributed to some of the genes differentially expressed in panel b (arrows). Potential HOXA9 binding sites (based on aTGATT(T)A/TAA(A)TCAt sequence as a HOX/PBX consensus site) are highlighted for TREM1, KCNA3, and MTSS1 and the sequences indicated, whereas no such potential site is present at the increased peak for CDKN1A. " width="100%" height="100%">

Journal: HemaSphere

Article Title: Induction of AML cell differentiation using HOXA9/DNA binding inhibitors as a potential therapeutic option for HOXA9‐dependent AML

doi: 10.1002/hem3.77

Figure Lengend Snippet: Comparison of DB1055 and DB818 deregulated genes with ChIP‐seq and DNase‐seq data. (A) “HESS TARGETS OF HOXA9 AND MEIS1 DN” and “TAKEDA TARGETS OF NUP98‐HOXA9 FUSION 8D DN” curated gene sets terms (C2, CGP: chemical and genetic perturbations) highlighted from GSEA for both shHOXA9 and DB818 or DB1055 cell treatment. The normalized enrichment scores (NES), nominal p ‐values, and FDR are shown in each caption. Corresponding Venn diagrams are presented on the right of the panel and a heatmap for implicated genes is given in Supporting Information S1: Figure . (B) Heatmap for the expression change of the 138 genes commonly significantly deregulated in the three conditions (HOXA9 invalidation and treatment with DB818 or DB1055) and position of different genes of interest and identification of the presence of a peak from HOXA9 ChIP in MV4‐11 cells. ³² The intensity of the blue color depends on the distance to TSS as defined in the figure. In white: genes in which the distance to TSS was over 100 kb. Circular diagram represents the percentage of the 138 commonly dysregulated genes (UP or DOWN) that present (plain color) or not (dashed color) a ChIP‐peak identified for HOXA9 binding in the MV4‐11 cell line. (C) Venn diagrams for comparison of the lists of deregulated genes from transcriptomic analyses and genes identified at the proximity of differential peaks from global DNase‐seq analyses as depicted in the Supporting Information Material and Methods section. (D) IGV visualization of some peaks attributed to some of the genes differentially expressed in panel b (arrows). Potential HOXA9 binding sites (based on aTGATT(T)A/TAA(A)TCAt sequence as a HOX/PBX consensus site) are highlighted for TREM1, KCNA3, and MTSS1 and the sequences indicated, whereas no such potential site is present at the increased peak for CDKN1A.

Techniques: Comparison, ChIP-sequencing, Expressing, Binding Assay, Sequencing

Global gene deregulation analysis identified common activation of differentiation process upon shHOXA9 as well as DB818 or DB1055 treatment of THP‐1. (A) Gene Set Enrichment Analysis assays for GO term “regulation of leukocyte differentiation” for both HOXA9 invalidation and cell treatment with DB818/1055. The normalized enrichment scores (NES), nominal p ‐values, and FDR are shown in each caption and the corresponding Venn diagram is presented on the right panel. (B) Heatmap for the expression of genes associated with the “cell differentiation” item from Ingenuity Pathway Analysis (localized by a red arrow in Figure ) ranked on the expression from shHOXA9‐1F3 gene list. #, genes of interest further validated using quantitative reverse transcription polymerase chain reaction (qRT‐PCR). (C) Validation of gene expression deregulation by qRT‐PCR for a series of deregulated genes identified in (C) (four to eight independent experiments). Corresponding significant statistics (Student t ‐test) are calculated relatively to the corresponding control (CTR for DB818 or DB1055 treatments and shCTR for shHOXA9‐1F3) and indicated on the graphs: **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05; or as specified on graphs. (D) Enzyme‐linked immunosorbent assay‐derived DNA binding inhibition assay. Double‐stranded oligonucleotides containing the HOXA9 putative binding site at EMP1 and TLR4 gene promoters identified from MV4‐11 ChIP‐peaks <xref ref-type=

³⁴ were evaluated with human HOXA9 protein expressed from reticulocyte lysate in the presence of increasing concentrations of DB818 or DB1055 ( n = 4–6) as described. ²¹ The unselective DB828 compound was used as a control. The concentrations of DB818 and DB1055 that inhibit 50% of the HOXA9/DNA complex are indicated in the graph. " width="100%" height="100%">

Journal: HemaSphere

Article Title: Induction of AML cell differentiation using HOXA9/DNA binding inhibitors as a potential therapeutic option for HOXA9‐dependent AML

doi: 10.1002/hem3.77

Figure Lengend Snippet: Global gene deregulation analysis identified common activation of differentiation process upon shHOXA9 as well as DB818 or DB1055 treatment of THP‐1. (A) Gene Set Enrichment Analysis assays for GO term “regulation of leukocyte differentiation” for both HOXA9 invalidation and cell treatment with DB818/1055. The normalized enrichment scores (NES), nominal p ‐values, and FDR are shown in each caption and the corresponding Venn diagram is presented on the right panel. (B) Heatmap for the expression of genes associated with the “cell differentiation” item from Ingenuity Pathway Analysis (localized by a red arrow in Figure ) ranked on the expression from shHOXA9‐1F3 gene list. #, genes of interest further validated using quantitative reverse transcription polymerase chain reaction (qRT‐PCR). (C) Validation of gene expression deregulation by qRT‐PCR for a series of deregulated genes identified in (C) (four to eight independent experiments). Corresponding significant statistics (Student t ‐test) are calculated relatively to the corresponding control (CTR for DB818 or DB1055 treatments and shCTR for shHOXA9‐1F3) and indicated on the graphs: **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05; or as specified on graphs. (D) Enzyme‐linked immunosorbent assay‐derived DNA binding inhibition assay. Double‐stranded oligonucleotides containing the HOXA9 putative binding site at EMP1 and TLR4 gene promoters identified from MV4‐11 ChIP‐peaks ³⁴ were evaluated with human HOXA9 protein expressed from reticulocyte lysate in the presence of increasing concentrations of DB818 or DB1055 ( n = 4–6) as described. ²¹ The unselective DB828 compound was used as a control. The concentrations of DB818 and DB1055 that inhibit 50% of the HOXA9/DNA complex are indicated in the graph.

Techniques: Activation Assay, Expressing, Cell Differentiation, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Biomarker Discovery, Gene Expression, Control, Enzyme-linked Immunosorbent Assay, Derivative Assay, Binding Assay, Inhibition

In vivo validation of THP1 as an HOXA9‐dependant mice cell model. (A) Representative images for bioluminescence kinetic analyses after i.p. inoculation in NSG mice ( n = 6 per group) of THP‐1‐Luc cells (1 × 10 6 ) that were previously transduced by short hairpin RNA (shRNA)‐expressing lentiviruses. (B) Quantification of the time course for bioluminescent THP‐1‐Luc responses to HOXA9 invalidation. Quantification is expressed in photons/second over time (in days). (C) Spleen weight of mice from euthanized altogether on Day 41. Results are presented as mean ± SEM. Statistics, Student t ‐test: **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05; ns, nonspecific or p ‐values as indicated in graphs. (D) Quantification by flow cytometry of GFP status of THP‐1 (hCD45‐positive) cells at 48 h after lentiviral infection or in spleen isolated from mice at end‐point sacrifice. (E) Kaplan–Meier plot for mice survival upon HOXA9 invalidation. THP‐1‐Luc (WT) cells (2 × 10 6 cells) were transduced or not with shRNAs (WT THP‐1, black line, n = 5; shCTR, blue line, n = 6; shHOXA9‐1F2, green line, n = 6; and shHOXA9‐1F3, red line, n = 6) 24 h before engraftment. Statistics were performed using log‐rank analysis of the Kaplan–Meier survival curve.

Journal: HemaSphere

Article Title: Induction of AML cell differentiation using HOXA9/DNA binding inhibitors as a potential therapeutic option for HOXA9‐dependent AML

doi: 10.1002/hem3.77

Figure Lengend Snippet: In vivo validation of THP1 as an HOXA9‐dependant mice cell model. (A) Representative images for bioluminescence kinetic analyses after i.p. inoculation in NSG mice ( n = 6 per group) of THP‐1‐Luc cells (1 × 10 6 ) that were previously transduced by short hairpin RNA (shRNA)‐expressing lentiviruses. (B) Quantification of the time course for bioluminescent THP‐1‐Luc responses to HOXA9 invalidation. Quantification is expressed in photons/second over time (in days). (C) Spleen weight of mice from euthanized altogether on Day 41. Results are presented as mean ± SEM. Statistics, Student t ‐test: **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05; ns, nonspecific or p ‐values as indicated in graphs. (D) Quantification by flow cytometry of GFP status of THP‐1 (hCD45‐positive) cells at 48 h after lentiviral infection or in spleen isolated from mice at end‐point sacrifice. (E) Kaplan–Meier plot for mice survival upon HOXA9 invalidation. THP‐1‐Luc (WT) cells (2 × 10 6 cells) were transduced or not with shRNAs (WT THP‐1, black line, n = 5; shCTR, blue line, n = 6; shHOXA9‐1F2, green line, n = 6; and shHOXA9‐1F3, red line, n = 6) 24 h before engraftment. Statistics were performed using log‐rank analysis of the Kaplan–Meier survival curve.

Techniques: In Vivo, Biomarker Discovery, shRNA, Expressing, Flow Cytometry, Infection, Isolation

Evaluation of the effect of the selected inhibitors on cell survival of blasts from patients and patient‐derived xenografts (PDX) mouse models. (A) A series of 42 blast cells were obtained from acute myeloid leukemia (AML) patients. All samples are individually numbered and cytogenetically and molecularly defined according to patients' information referred to in Supporting Information S1: Table . All of the AML blast samples were quantified by quantitative reverse‐transcription polymerase chain reaction for HOXA9 expression relative to TBP expression. Values presented from 12.45 to −12.4 correspond to the individual ΔC t values for HOXA9 expression relative to that of the housekeeping gene TBP used as a normalization control. Moreover, means Δ C t from 2.9 to −11.4 are calculated in the right column for each defined cytogenetically/molecularly different AML subtype defined on the left (the number of samples for each alteration is indicated as a value). This validates that our samples with the defined alterations are in agreement with known HOXA9‐positive (such as NPM1 or MLL) or ‐negative (C/EBPa, inv(16), t(8;21) subtypes of AML. (B) Graph for correlation analysis between HOXA9 expression relative to TBP and DB818 response analyzed by diphenyltetrazolium bromide (MTT). The coefficient of determination and p ‐values are embedded in the graph. (C) Graph comparing the mean ± SEM of individual Δ C t values (dots) for 50% more DB818‐sensitive or ‐resistant AML samples determined using MTT assays after 96 h incubation with increasing concentrations of DB818 as defined in the Materials and Methods section. (D) Graph presenting the IC 50 of DB818 treatment (MTT assays at 96 h treatment) of AML blasts from NPM1 and MLL (HOXA9‐positive subtypes) relative to cEBPα‐mutated AMLs (HOXA9‐negative subtype). (E, F) DB1055 treatment of PDX from two AML patient blasts cells (E) PDX‐1: AML‐M1, NPM1‐mutated, Flt‐3‐ITD, IDH2‐mutated, WT1+; (F) PDX‐2: AML‐M4, NPM1‐mutated, Flt‐3‐TKD, IDH1‐mutated, as indicated in Supporting Information S1: Table ). Top: treatment schedules after intratibial (IT) injection of 5 × 10 6 blast cells amplified in a first recipient mice series. Medium: measurement of spleens (middle pictures) and comparison of spleen weight (bottom graphs). Bottom: quantification of human CD45‐positive (identifying human patient AML cells) per spleen or femur (bone marrow cells). Statistics, Student t ‐test: *** p < 0.001; ** p < 0.01; * p < 0.05; ns, nonspecific.

Journal: HemaSphere

Article Title: Induction of AML cell differentiation using HOXA9/DNA binding inhibitors as a potential therapeutic option for HOXA9‐dependent AML

doi: 10.1002/hem3.77

Figure Lengend Snippet: Evaluation of the effect of the selected inhibitors on cell survival of blasts from patients and patient‐derived xenografts (PDX) mouse models. (A) A series of 42 blast cells were obtained from acute myeloid leukemia (AML) patients. All samples are individually numbered and cytogenetically and molecularly defined according to patients' information referred to in Supporting Information S1: Table . All of the AML blast samples were quantified by quantitative reverse‐transcription polymerase chain reaction for HOXA9 expression relative to TBP expression. Values presented from 12.45 to −12.4 correspond to the individual ΔC t values for HOXA9 expression relative to that of the housekeeping gene TBP used as a normalization control. Moreover, means Δ C t from 2.9 to −11.4 are calculated in the right column for each defined cytogenetically/molecularly different AML subtype defined on the left (the number of samples for each alteration is indicated as a value). This validates that our samples with the defined alterations are in agreement with known HOXA9‐positive (such as NPM1 or MLL) or ‐negative (C/EBPa, inv(16), t(8;21) subtypes of AML. (B) Graph for correlation analysis between HOXA9 expression relative to TBP and DB818 response analyzed by diphenyltetrazolium bromide (MTT). The coefficient of determination and p ‐values are embedded in the graph. (C) Graph comparing the mean ± SEM of individual Δ C t values (dots) for 50% more DB818‐sensitive or ‐resistant AML samples determined using MTT assays after 96 h incubation with increasing concentrations of DB818 as defined in the Materials and Methods section. (D) Graph presenting the IC 50 of DB818 treatment (MTT assays at 96 h treatment) of AML blasts from NPM1 and MLL (HOXA9‐positive subtypes) relative to cEBPα‐mutated AMLs (HOXA9‐negative subtype). (E, F) DB1055 treatment of PDX from two AML patient blasts cells (E) PDX‐1: AML‐M1, NPM1‐mutated, Flt‐3‐ITD, IDH2‐mutated, WT1+; (F) PDX‐2: AML‐M4, NPM1‐mutated, Flt‐3‐TKD, IDH1‐mutated, as indicated in Supporting Information S1: Table ). Top: treatment schedules after intratibial (IT) injection of 5 × 10 6 blast cells amplified in a first recipient mice series. Medium: measurement of spleens (middle pictures) and comparison of spleen weight (bottom graphs). Bottom: quantification of human CD45‐positive (identifying human patient AML cells) per spleen or femur (bone marrow cells). Statistics, Student t ‐test: *** p < 0.001; ** p < 0.01; * p < 0.05; ns, nonspecific.

Techniques: Derivative Assay, Reverse Transcription, Polymerase Chain Reaction, Expressing, Control, Incubation, Injection, Amplification, Comparison

Review

Structured Review

Images

Similar Products

Image Search Results