Journal: Haematologica

Article Title: A three-dimensional ex vivo tri-culture model mimics cell-cell interactions between acute myeloid leukemia and the vascular niche

doi: 10.3324/haematol.2016.157883

Figure Lengend Snippet: Three-dimensional mono-cultures for the culture of acute myeloid leukemia spheroids. Growth of (A) KG1a, (B) MOLM13, (C) MV4-11 and (D) OCI-AML3 cell lines was investigated in 3D. AML cells were cultured in starPEG-heparin hydrogels (upper left image) or in Matrigel™ (lower left image). Comparison of cell growth (upper right graph) and spheroid size (lower right graph) over 14 days was performed using three different cross-linking degrees of hydrogel (γ0.75, γ1, γ1.5). (E) Primary donor cells from two patients with AML were cultured in starPEG-heparin hydrogels as a mono-culture. Cell line experiments were performed at least twice with three technical replicates (n=2). Data are displayed as mean ± SD. pAML experiments were performed once in triplicate (n=1). The graph displays only the means. Cultures displayed heterogeneous distribution of cells, with each donor’s cell growing both as spheroids and single cells. Three photographs per technical replicate were utilized for ImageJ spheroid measurements as a minimum. *Indicates statistical significance: * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.

Article Snippet: KG1a, MOLM13, MV4-11 and OCI-AML3 cell lines were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ; Braunschweig, Germany) and used within 15 passages.

Techniques: Cell Culture, Comparison

Journal: Genome Biology

Article Title: RNA-binding protein RBM5 plays an essential role in acute myeloid leukemia by activating the oncogenic protein HOXA9

doi: 10.1186/s13059-023-03149-8

Figure Lengend Snippet: Disruption of RBM5 delays the growth of leukemia cells in vitro. a Competitive proliferation assay (CPA) was conducted in Cas9 stably expressed AML cells lines, including MOLM13, THP1, OCIAML2, U937, HEL, and TF1 after transduced with GFP reporter-based lentiviral sgRNAs (NT, RPA3, RBM5-sg1, RBM5-sg2) at about ~ 50% efficiency. The GFP% was quantified at days 3, 6, 9, 12, 15, and 18 by flow cytometry to evaluate the growth disadvantage. The guide RNA targeting the survival essential gene RPA3 was included as a positive control, and the guide RNA targeting the non-target (NT) gene was included as a negative control. Data shown are means ± SEM from three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, unpaired Student’s t -test. b The proliferation ability of AML cells was monitored by cell counting assay in MOLM13, THP1, and OCIAML2 with stably expressed Cas9 after transduced by two respective sgRNAs targeting RBM5 (RBM5-sg1, sg2) and one non-targeting sgRNA. The guide RNA targeting non-target gene (NT) was used as a negative control. Data shown are means ± SEM from three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, unpaired Student’s t -test. c Immunoblotting of RBM5 in RBM5 sgRNAs targeted cells. The bands were scanned and statistically analyzed. Data shown are means ± SEM from three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, unpaired Student’s t -test. The β-ACTIN was used as a reference. d Schematic diagram of the sgRNA-resistant cDNA mutagenesis. The 22 bp DNA sequence and corresponding amino acids close to the sgRNA PAM region (blue) in RBM5 wild-type (WT) and RBM5-sg1-resistant mutant (PAM-MUT) cDNA are shown with the non-sensed mutated nucleotides highlighted in different colors (red and orange). e Immunoblotting was conducted by infecting MOLM13 cells overexpressing ectopic Venus empty vector, RBM5-wild-type cDNA (WT), RBM5-sgRNA1-resistant mutant cDNA (PAM-MUT) with lentiviral-sgRNAs against non-target gene (NT), and RBM5 (RBM5-sg1), and the bands were scanned and statistically analyzed. Data shown are means ± SEM from three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, unpaired Student’s t -test. The β-ACTIN was used as a reference. f Competition-based proliferation assays comparing the impact of sgRNAs on MOLM13, THP1, and OCIAML2 cell fitness, in the context of co-transduction with Venus empty vector control, RBM5-wild-type cDNA (WT), RBM5-sgRNA1-resistant mutant cDNA (PAM-MUT) (all monitored by Venus reporter). Data shown are means ± SEM from three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, unpaired Student’s t -test

Article Snippet: MOLM13 was originally purchased from ACCEGEN.

Techniques: Disruption, In Vitro, Proliferation Assay, Stable Transfection, Transduction, Flow Cytometry, Positive Control, Negative Control, Cell Counting, Western Blot, Mutagenesis, Sequencing, Plasmid Preparation, Control

Journal: Genome Biology

Article Title: RNA-binding protein RBM5 plays an essential role in acute myeloid leukemia by activating the oncogenic protein HOXA9

doi: 10.1186/s13059-023-03149-8

Figure Lengend Snippet: RBM5 knockdown impairs in vivo myeloid leukemia engraftment. a Western blot of RBM5 expression in MOLM13, OCIAML2, and THP1 cells after transduced with shNT (non-targeting control), sh#1 (shRBM5#1), and sh#2 (shRBM5#2) 6-day post-viral transduction. The β-ACTIN was used as a reference. b Cell counting assay was conducted on the sh#1, sh#2, and shNT targeted MOLM13, OCIAML2, and THP1 to monitor the ability of proliferation of AML cells. shNT was used as a negative control. Data shown are means ± SEM from three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, unpaired Student’s t -test. c Count of colonies formed by 300 MOLM13 and 500 OCIAML2 cells after shNT, sh#1, and sh#2 transduction. Data shown are means ± SEM from three independent experiments. ** P < 0.01, unpaired Student’s t -test. d Surface expression of CD11b and CD14 after lentiviral transduction of shRNA against control shNT and shRNAs targeting RBM5 (sh#1 and sh#2) in MOLM13 and OCIAML2 cells. MFI, mean fluorescence intensity. * P < 0.05, ** P < 0.01, unpaired Student’s t -test. e Schematic of in vivo transplantation of MOLM13-Luc-GFP cells infected with control shNT and shRNAs targeting RBM5 (sh#1 and sh#2). f Kaplan–Meier survival curves of recipient mice transplanted with MOLM13 cells transduced with shNT ( n = 7), sh#1 ( n = 5), and sh#2 ( n = 5). The P -value was determined by a log-rank Mantel-Cox test. ** P < 0.01. g The spleens of mice in the shNT ( n = 4) and shRBM5 ( n = 2, merged sh#1 and sh#2) groups were photographed and weighed 18-day post-transplantation. * P < 0.05, unpaired Student’s t -test. h Flow cytometry analysis of the percentage of human CD45 + and GFP + leukemia cells in bone marrow, spleen, and peripheral blood of recipient mice in the shNT ( n = 4) and shRBM5 ( n = 2) groups sacrificed after 18-day post-transplantation. Statistical analysis ( P -value) was performed using an unpaired Student’s t -test. * P < 0.05, *** P < 0.001. i Schematic diagram of the AML primary sample for CFC and cell differentiation assay. j Count of colonies formed by human primary AML cells after shNT, sh#1(shRBM5#1), and sh#2(shRBM5#2) transduction. ** P < 0.01, unpaired Student’s t -test. k Representative flow cytometry analysis of myeloid differentiation marker (CD14 and CD11b), in which AML primary samples were treated with lentiviral transduction of shRNA against control gene (shNT) and shRNAs targeting RBM5 (sh#1 and sh#2). MFI, mean fluorescence intensity. * P < 0.05, ** P < 0.01, unpaired Student’s t -test

Article Snippet: MOLM13 was originally purchased from ACCEGEN.

Techniques: Knockdown, In Vivo, Western Blot, Expressing, Transduction, Control, Cell Counting, Negative Control, shRNA, Fluorescence, Transplantation Assay, Infection, Flow Cytometry, Cell Differentiation, Marker

Journal: Genome Biology

Article Title: RNA-binding protein RBM5 plays an essential role in acute myeloid leukemia by activating the oncogenic protein HOXA9

doi: 10.1186/s13059-023-03149-8

Figure Lengend Snippet: Identification of RBM5 downstream target genes in AML. a The transcriptome analysis in three groups of MOLM13 cells, including the CTRL group (control cells with NT sgRNA treated), RBM5 KO group (RBM5 knockout cells with RBM5-sg1 treated), and RBM5 OE group (RBM5 overexpressed cells by lentiviral RBM5-P2A-venus vector). Each group was performed with three biological replicates. Integrated scatter plot analysis for all genes from the RNA-seq dataset by comparing RBM5 OE vs. CTRL and RBM5 KO vs. CTRL in MOLM13 cells (left panel) ( n = 13,178). The graph on the right panel shows the significant genes ( n = 154). False discovery rate [FDR] < 0.05. b Heat map showing filtered genes whose expression is significantly differentially expressed after RBM5 loss in three groups of MOLM13 cells, including the CTRL, RBM5 KO, and RBM5 OE. c Leading edge plot from gene enrichment analysis (GSEA) showing the enrichment of indicated pathway for genes in RBM5 OE compared to control. Enrichment scores, P -values, and FDR (false-discovery rate) are calculated as shown in the plot. d Leading edge plot from gene enrichment analysis (GSEA) showing the enrichment of indicated pathway for the decreased genes in RBM5 KO cells relative to control. Enrichment scores, P -values, and FDR (false-discovery rate) are calculated as shown in the plot. e Venn diagram showing the overlap between genes differentially expressed and genes undergoing altered splicing events after RBM5 knockout (from RNA-seq). f Sashimi plots the example gene PWWP3A and FLT3 in RBM5 KO and CTRL group in MOLM13 cells

Article Snippet: MOLM13 was originally purchased from ACCEGEN.

Techniques: Control, Knock-Out, Plasmid Preparation, RNA Sequencing, Expressing

Journal: Genome Biology

Article Title: RNA-binding protein RBM5 plays an essential role in acute myeloid leukemia by activating the oncogenic protein HOXA9

doi: 10.1186/s13059-023-03149-8

Figure Lengend Snippet: HOXA9 is a functional target gene of RBM5 in AML. a Scatter plot analysis comparing RBM5 mRNA levels and HOXA9 mRNA levels across acute myeloid leukemia cell lines (data was obtained from the Depmap). P -value is calculated by linear regression. b Immunoblotting of RBM5 and HOXA9 in various leukemia cell lines and normal bone marrow (NBM) samples (left panel). The bar chart displays the quantitative protein levels of RBM5 and HOXA9 in various cell lines depicted in the left panel (right panel). c Real-time-qPCR and immunoblotting analysis was conducted on the RBM5-sg1, RBM5-sg2, and sgNT targeted MOLM13 and OCIAML2 to monitor the expression of HOXA9 . Data shown are means ± SEM from three independent experiments. * P < 0.05, unpaired Student’s t -test. d Diagram of auxin-induced degron (AID) system to degrade endogenous RBM5. Endogenous RBM5 N-terminus AID knock-in KMT2A-r leukemia lines (SEM homozygous clones and MOLM13 bulk cells) were followed by constitutively expressing OsTIR1(F74G). With the presence of small molecule 5-Ph-IAA, the OsTIR1(F74G), as a substrate receptor in Skp1, Cullin, and F-box (SCF) complex, could target and mediate specific AID-tagged RBM5 proteins for ubiquitination and degradation. e The protein level of endogenous HA-miniAID-RBM5 of SEM homozygous clones can be acutely degraded with 5-Ph-IAA through degron-mediated proteasome degradation after 2-h post-treatment (left panel). HOXA9 mRNA level was significantly reduced after 2 h, 4 h, 6 h, and 24 h of treatment in SEM cells (right panel). f After 24 h of treatment with 5-Ph-IAA in HA-miniAID-RBM5 knock-in MOLM13 cells, the protein level of HA-miniAID-RBM5 decreased (left panel), and there was also a significant reduction in HOXA9 mRNA level (right panel). g The doxycycline-Tet-On system for ectopic overexpression of RBM5-HA in the MOLM13 cell line, after a 2-h treatment with doxycycline, a significant upregulation in HOXA9 mRNA levels was observed (right panel), accompanied by an increase in RBM5 expression (left panel). h Real-time-qPCR analysis was conducted in OCIAML2 cells transduced with RBM5 cDNA wild-type (WT) or six individual truncated mutants to validate the expression of HOXA9. Data shown are means ± SEM from three independent experiments. * P < 0.05, unpaired Student’s t -test. i ChIP-qPCR with anti-MYC tag antibody near H3K4me3-bound HOXA9 downstream region (Chr7:27,199,969–27,200,853) in OCIAML2-RBM5-MYC tag cells ( n = 3). The red arrows indicate the primer 1 and primer 2 located position, and the blue arrow indicates the negative control (NC) primer located position, where there is no H3K4me3 signal. Statistical analysis ( P -value) was performed using an unpaired Student’s t -test. All error bars represent mean ± SEM. j Rescued competitive proliferation assay was conducted by infecting OCIAML2-Cas9 and MOLM13-Cas9 cells overexpressing ectopic RBM5-wild-type cDNA (WT), RBM5-sgRNA1-resistant mutant cDNA (PAM-MUT), and HOXA9 cDNA (linked to Venus reporter) with lentiviral-sgRNAs against non-target (sgNT) and RBM5 (RBM5-sg1) at about 50% efficiency (all monitored by Venus reporter). Data shown are means ± SEM from three independent experiments. ** P < 0.01, unpaired Student’s t -test

Article Snippet: MOLM13 was originally purchased from ACCEGEN.

Techniques: Functional Assay, Western Blot, Expressing, Knock-In, Clone Assay, Ubiquitin Proteomics, Over Expression, Transduction, ChIP-qPCR, Negative Control, Proliferation Assay, Mutagenesis

Journal: Genome Biology

Article Title: RNA-binding protein RBM5 plays an essential role in acute myeloid leukemia by activating the oncogenic protein HOXA9

doi: 10.1186/s13059-023-03149-8

Figure Lengend Snippet: HOXA9 partially restores the gene transcriptional defect in RBM5 null leukemia cells. a Integrated scatter plot analysis for all genes ( P < 0.05) from the RNA-seq dataset by comparing HOXA9 vs. RBM5 KO and RBM5 KO vs. CTRL in MOLM13 cells (left panel) ( n = 17,079). The graph at the right shows the differentially expressed genes (DEG) in RBM5 KO vs. CTRL ( n = 174). False discovery rate [FDR] < 0.05. Pearson r and P -value are calculated by correlation. b Heat map of RNA-seq analysis shows the decreased genes after RBM5 loss respectively in CTRL, RBM5 KO, and RBM5 KO with HOXA9 overexpression groups. c Enrichment of target genes involved in MLL signature genes, NUP98-HOXA9 and FLT3 targets for the increased genes in RBM5 KO cells with HOXA9 overexpression relative to control, as shown by GSEA. d . Real-time-qPCR analysis was performed on samples of CTRL, RBM5 KO (R5 KO), and RBM5 KO MOLM13 cells with HOXA9 cDNA transduction (R5 KO + HOXA9) to validate the expression of RBM5 , HOXA9 , FLT3 , PIM1 , and DDIT4 genes. Data shown are means ± SEM from three independent experiments. *** P < 0.001, **** P < 0.0001, unpaired Student’s t -test. e Western blot of HOXA9 and FLT3 were performed on samples of CTRL, R5 KO, and RBM5 KO MOLM13 cells with HOXA9 cDNA transduction (R5 KO + HOXA9). The β-ACTIN was used as a reference. f Immunoblotting was conducted by infecting OCIAML2 cells overexpressing ectopic Venus empty vector and FLT3 cDNA. The β-ACTIN was used as a reference. g Rescued competitive proliferation assay was conducted by infecting OCIAML2-Cas9 cells overexpressing ectopic RBM5-wild-type cDNA (WT), RBM5-sgRNA1-resistant mutant cDNA (PAM-MUT), and FLT3 cDNA (linked to Venus reporter) with lentiviral-sgRNAs against non-target (sgNT) and RBM5 (RBM5-sg1) at about 50% efficiency (all monitored by Venus reporter). Data shown are means ± SEM from three independent experiments. ** P < 0.01, unpaired Student’s t -test. h Scatter plot analysis comparing RBM5 mRNA levels and FLT3 mRNA levels across leukemia cell lines (data was obtained from the DepMap). P -value is calculated by linear regression

Article Snippet: MOLM13 was originally purchased from ACCEGEN.

Techniques: RNA Sequencing, Over Expression, Control, Transduction, Expressing, Western Blot, Plasmid Preparation, Proliferation Assay, Mutagenesis

Journal: Cancer research

Article Title: FGF2 from marrow microenvironment promotes resistance to FLT3 inhibitors in acute myeloid leukemia

doi: 10.1158/0008-5472.CAN-15-3569

Figure Lengend Snippet: A) MOLM14 cells were added to a 384-well plate containing cytokines, chemokines, and growth factors with 10 nM AC220 (quizartinib). Viability was measured using MTS reagent, averaged over all concentrations (1, 10, and 100 ng/ml), and the results sorted according to highest average viability. Average viability >2 standard deviations above the mean are highlighted in red and by an *. B) MOLM14 cells were cultured in lower concentrations of recombinant proteins (1, and 0.1 ng/ml) in 96-well plates plus 10 nM AC220. Viability was assessed after 48 hours with MTS reagent and data plotted as percent of the respective untreated control. All wells were plated in triplicate with standard deviation indicated by error bars. C) MOLM14 cells were cultured continuously in 10 nM AC220 with FL, FGF2, or media alone as indicated (N=4 for each). Fresh media, AC220 and cytokine were replaced every 2–3 days over the indicated time period. Viable cell numbers were analyzed every 2–3 days with Guava ViaCount.

Article Snippet: MOLM14 cells were generously provided by Dr. Yoshinobu Matsuo (Fujisaki Cell Center, Hayashibara Biochemical Labs, Okayama, Japan) ( 19 ).

Techniques: Cell Culture, Recombinant, Control, Standard Deviation

Journal: Cancer research

Article Title: FGF2 from marrow microenvironment promotes resistance to FLT3 inhibitors in acute myeloid leukemia

doi: 10.1158/0008-5472.CAN-15-3569

Figure Lengend Snippet: A) MOLM14 cells were cultured in media alone or media supplemented with 10 ng/ml FGF2, treated with 10 nM AC220, and exposed to 25, 50, and 100 nM of the FGFR inhibitor PD173074. Viability was measured by MTS assay after 48 hours and normalized to their respective untreated control (media alone, FGF2, 25 nM PD173074, etc.) B) MOLM14 cells were electroporated with siRNAs targeting FGFR1, FGFR2, FGFR3, FGFR4 and a non-specific (NS) control. After 48 hours the cells were pelleted and resuspended in media +/− FGF2 and +/− 10 nM AC220; and viability assessed by MTS after 48 hours. * indicates p<0.01 by t test. C) Western blot was used to evaluate FGFR1 protein expression after siRNA (see also supplemental Figure S1). D) BaF3 cells expressing FLT3-ITD (20) were retrovirally transfected with either empty pMX vector (pMX neg) or vector containing FGFR1, selected with puromycin, and analyzed by Western blot to show FGFR1 expression. The FLT3-ITD Baf3 cells with E) empty pMX vector or F) pMX with FGFR1 were then treated with a gradient of AC220 in either media alone or supplemented with 10 ng/ml FGF2. Viability was assessed after 48 hours with MTS reagent and data plotted as percent of the respective untreated control. All cell line experiments were performed in triplicate and error bars indicate standard deviation. Thawed frozen viable (see methods) and fresh primary FLT3-ITD AML samples were plated with media alone (no ligand) or with FGF2 at 1, 10 and 100 ng/ml concentration. The cells were then exposed to a gradient of AC220 and viability was measured after 72 hours by MTS. G) An example of ex vivo FGF2-mediated protection from AC220 with a primary FLT3-ITD AML sample. H) The area under the curve was calculated for 23 patient samples in the absence or presence of FGF2 ligand (see 2G) and graphed with mean and standard error of the mean shown. One-tailed t-tests were performed with p values indicated by *<0.05, **<0.005, and ***=0.0007.

Article Snippet: MOLM14 cells were generously provided by Dr. Yoshinobu Matsuo (Fujisaki Cell Center, Hayashibara Biochemical Labs, Okayama, Japan) ( 19 ).

Techniques: Cell Culture, MTS Assay, Control, Western Blot, Expressing, Transfection, Plasmid Preparation, Standard Deviation, Concentration Assay, Ex Vivo, One-tailed Test

Journal: Cancer research

Article Title: FGF2 from marrow microenvironment promotes resistance to FLT3 inhibitors in acute myeloid leukemia

doi: 10.1158/0008-5472.CAN-15-3569

Figure Lengend Snippet: A) Naïve MOLM14 cells in media alone were exposed to a matrix of an AC220 gradient (0, 1.4, 4, 12, 37, 111, 333, 1000 nM) overlayed with the FGFR inhibitor PD173074 (0, 1.4, 4, 12, 37, 111, 333, 1000 nM) and compared to B) FGF2- and C) FL-dependent resistant MOLM14 cells (Figure 1C) in the same conditions. Viability was measured after 48 hours and average viability graphed as surface plots with gray scale denoting 20% increments. Graphs at right indicate viability with AC220 alone, PD173074 alone, and equimolar combination to highlight synergy. Calculation of the combination index is included in Supplemental Table 1 and synergy (CI<0.1) indicated by an *. All experiments were done in triplicate with average viability scaled to untreated condition (100%). Error bars represent standard deviation.

Article Snippet: MOLM14 cells were generously provided by Dr. Yoshinobu Matsuo (Fujisaki Cell Center, Hayashibara Biochemical Labs, Okayama, Japan) ( 19 ).

Techniques: Standard Deviation

Journal: Cancer research

Article Title: FGF2 from marrow microenvironment promotes resistance to FLT3 inhibitors in acute myeloid leukemia

doi: 10.1158/0008-5472.CAN-15-3569

Figure Lengend Snippet: A) Naïve MOLM14 cells were treated for 24 hours −/+ 10 nM AC220 (first two lanes) and comparedto FGF2- and FL-dependent resistant cells (grown continuously in FGF2 or FL, lanes 3–10). The FGF2- and FL-dependent resistant cells were harvested 24 hours after addition of fresh media, recombinant protein and AC220. The cells were then lysed as described with Western blot analysis as per Materials and Methods. The FGF2-dependent resistant culture that developed a Y842C mutation is indicated.

Article Snippet: MOLM14 cells were generously provided by Dr. Yoshinobu Matsuo (Fujisaki Cell Center, Hayashibara Biochemical Labs, Okayama, Japan) ( 19 ).

Techniques: Recombinant, Western Blot, Mutagenesis

Journal: Cancer research

Article Title: FGF2 from marrow microenvironment promotes resistance to FLT3 inhibitors in acute myeloid leukemia

doi: 10.1158/0008-5472.CAN-15-3569

Figure Lengend Snippet: The time to development of exponential growth (indicated by gray) was plotted over time for the extended cultures in Figure 1C. The timepoints at which Sanger or next-generation sequencing (at right, average read depth ~1500) was performed is indicated by arrows. If a mutation could not be detected it is indicated with “−”. A) MOLM14 cells cultured in media and AC220 alone (no ligand) developed exponential growth later than FGF2- and FL-dependent cultures (panels B and C) and developed early resistance mutations: D839V and D835H. B) FGF2-dependent and C) FL-dependent resistant cultures (cultured continuously in 10 ng/ml FGF2 or FL plus 10 nM AC220) were split at 4 months and 1×107 cells placed into fresh media without recombinant protein with continued 10 nM AC220 treatment. Fresh media and 10 nM AC220 were replaced every 2–3 days over the indicated time period and viable cell number was analyzed using Guava ViaCount. D) Naïve MOLM14 cells and resistant MOLM14 cells with FLT3 D839V mutation (R-1), FLT3 D842C mutation (FGF2 R-1), and non-mutated resistant cells (FL R1 after FL subtraction) were exposed to a gradient of FLT3 inhibitors: AC220 (quizartinib), crenolanib, sorafenib, and ponatinib. Viability was measured after 48 hours by MTS assay with average viability scaled relative to untreated cells (100%). The experiment was done in triplicate and error bars represent standard deviation.

Article Snippet: MOLM14 cells were generously provided by Dr. Yoshinobu Matsuo (Fujisaki Cell Center, Hayashibara Biochemical Labs, Okayama, Japan) ( 19 ).

Techniques: Next-Generation Sequencing, Mutagenesis, Cell Culture, Recombinant, MTS Assay, Standard Deviation

Journal: Cancer research

Article Title: FGF2 from marrow microenvironment promotes resistance to FLT3 inhibitors in acute myeloid leukemia

doi: 10.1158/0008-5472.CAN-15-3569

Figure Lengend Snippet: A) The human stromal cell lines HS-5 and HS-27 were analyzed for FGF2 expression by Western blot. B) MOLM14 cells were cultured in media alone, 10 ng/ml FGF2, or in 1 μm transwells over HS-5 or HS-27 stromal cell lines. The cells were treated with 10 nM AC220 and/or 250 nM PD173074 as indicated and MOLM14 cells were analyzed for viable cell number after 4 days. Viable cells were plotted as percentage of untreated control. C) The corresponding stromal cells from the transwell co-culture experiments were also evaluated by MTS assay for viability. Results are shown relative to the untreated condition. All experiments were done in triplicate, error bars represent standard deviation, and * indicates p<0.05.

Article Snippet: MOLM14 cells were generously provided by Dr. Yoshinobu Matsuo (Fujisaki Cell Center, Hayashibara Biochemical Labs, Okayama, Japan) ( 19 ).

Techniques: Expressing, Western Blot, Cell Culture, Control, Co-Culture Assay, MTS Assay, Standard Deviation

Journal: Journal of Translational Medicine

Article Title: Naturally selected CD7 CAR-T therapy without genetic editing demonstrates significant antitumour efficacy against relapsed and refractory acute myeloid leukaemia (R/R-AML)

doi: 10.1186/s12967-022-03797-7

Figure Lengend Snippet: CD7 CAR-T cells exhibit powerful antitumour functions in vitro A CD7 expression levels in CCRF-CEM, KG-1a, MOLM-13, K562, and K562-CD7 cells. B , D – H Specific lysis of CCRF-CEM, KG-1a, MOLM-13, K562-CD7, and K562 cells by CD7 CAR-T cells and NTR at various E:T ratios (n = 3). C , I Cytotoxic effects of CD7 CAR or NTR on primary CD7 + R/R-AML blasts at E:T ratios of 1:1, 2:1, and 4:1 (n = 3). J Cytokine secretion (IFN-γ, TNF-α, and IL-2) of CD7 CAR or NTR on KG-1a cells at E:T (1:1) (n = 3). ns, not significant; * P < 0.01; ** P < 0.01; *** P < 0.001; **** P < 0.0001

Article Snippet: Fig. 4 CD7 CAR-T cells exhibit powerful antitumour functions in vitro A CD7 expression levels in CCRF-CEM, KG-1a, MOLM-13, K562, and K562-CD7 cells.

Techniques: In Vitro, Expressing, Lysis