Journal: Blood Transfusion

Article Title: The tandem CD33-CLL1 CAR-T as an approach to treat acute myeloid leukemia

doi: 10.2450/BloodTransfus.786

Figure Lengend Snippet: ( A ) Expression of CD33 and CLL1 on the surface of human leukemia cell lines (HL-60, MV4–11, THP-1, U937, MOLM-13) was detected by flow cytometry. The gray line represents the isotype control. ( B ) Expression of CD33 and CLL1 in peripheral blood mononuclear cells (PBMCs, No.=22) and bone marrow (BM, No.=12) of Primary AML patients (down panel). A representative flow plot showed the expression of CD33 and CLL1 in tumor cells from two AML patients (up panel). ( C ) The structure of CD33-CAR-T (targeting CD33), CLL1-CAR-T (targeting CLL1), and CD33-CLL1- CAR-T (targeting both CD33 and CLL1). ( D ) The expression of CD45RO and CD62L on T cells before (un T)and after transduction (CAR-T) were detected using flow cytometry. CAR-T: chimeric antigen receptor T-cells; unT: untransfected T-cells.

Article Snippet: Human AML cell lines (Molm-13, THP-1, U937) were sourced from the Genentech Cell Line Repository and cultured in RPMI 1,640 medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM glutamine, and 1% penicillin-streptomycin at 37°C in a 5% CO 2 environment.

Techniques: Expressing, Flow Cytometry, Control, Transduction

Journal: Blood Transfusion

Article Title: The tandem CD33-CLL1 CAR-T as an approach to treat acute myeloid leukemia

doi: 10.2450/BloodTransfus.786

Figure Lengend Snippet: ( A ) Killing percentages of HL60, U937, THP-1, and MOLM-13 cells by unT, CD33-CLL1-CAR-T, CLL1-CAR-T, and CD33-CAR-T cells after 18 h co-culture in vitro . E: T ratios mean the ratios of the absolute number of CAR-T cells to target cells. ( B ) Cytotoxic effects of CD33-CAR-T, CLL1-CAR-T, and CD33-CLL1-CAR-T on 3 primary AML blasts (based on the expression of CD33 and CLL1) after 18 h co-culture in vitro (E: T=1:4). The data were represented as means ± SD and analyzed by One-way ANOVA and two-tailed unpaired Student’s t-test. *p<0.05; **p<0.01; ***p<0.001, ns, no significance.

Article Snippet: Human AML cell lines (Molm-13, THP-1, U937) were sourced from the Genentech Cell Line Repository and cultured in RPMI 1,640 medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM glutamine, and 1% penicillin-streptomycin at 37°C in a 5% CO 2 environment.

Techniques: Co-Culture Assay, In Vitro, Expressing, Two Tailed Test

Journal: Blood Advances

Article Title: Combined GLUT1 and OXPHOS inhibition eliminates acute myeloid leukemia cells by restraining their metabolic plasticity

doi: 10.1182/bloodadvances.2023009967

Figure Lengend Snippet: In vivo CRISPR-Cas9 screening identifies an essential role for Glut1 in MLL::AF9– driven AML. (A) Schematic representation of the experimental design for the pooled in vivo CRISPR screen in MLL::AF9 c-Kit + leukemia cells (n = 9 mice). (B) Bar plot of normalized median fold change of sgRNAs for the 20 genes with the strongest depletion scores in the screen. Fold change in sgRNA representation in leukemic cells harvested from the bone marrow was calculated as the number of reads after 12 days in vivo (final time point [T f ]) relative to input representation (initial time point [T 0 ]). (C) Waterfall plot showing the normalized fold change of individual sgRNAs for the top regulator Glut1 and 3 known regulators of MLL::AF9 AML ( Hoxa9, Cxcr4, and Cd47 ). A fold change of 10 was used to define depleted sgRNAs, denoted with a dotted line. Illustration in panel A created using BioRender. See also supplemental Figure 1 and supplemental Table 3 .

Article Snippet: In contrast to murine AML cells in which Glut1 levels are predominant ( E), human AML cell lines (DepMap, Broad Institute) and samples from patients with AML from The Cancer Genome Atlas (TCGA) database had comparably high levels of GLUT3 ( C-D).

Techniques: In Vivo, CRISPR

Journal: Blood Advances

Article Title: Combined GLUT1 and OXPHOS inhibition eliminates acute myeloid leukemia cells by restraining their metabolic plasticity

doi: 10.1182/bloodadvances.2023009967

Figure Lengend Snippet: GLUT1 is required for AML cell growth and survival. (A) Flow cytometric analysis of GLUT1 expression in MLL::AF9 LSC-enriched (c-Kit + ) cells and their normal bone marrow c-Kit + counterparts. In panels B to F, MLL::AF9 cells were transduced with Glut1 sgRNAs (Glut1 sgRNA1 and sgRNA2) or a nontargeting control cloned into GFP-expressing lentiviral vectors. (B) Genetic editing in the Glut1 locus was quantified by deep sequencing within sorted GFP + cells, 3 days after transduction. (C) Representative histogram of GLUT1 expression measured by flow cytometry within GFP + leukemia cells, 4 days after transduction. (D) Quantification of GFP + MLL::AF9 leukemia cells in the bone marrow of mice 12 days after transplantation with c-Kit + leukemia cells transduced with Glut1 sgRNAs or nontargeting control. The percentage of GFP + cells at day 12 was normalized to the input percentage of GFP + cells before transplantation, 2 days after transduction (T 0 ). (E) Ex vivo competition proliferation assay as measured by the percentage of GFP + leukemia cells on day 2, 5, 8, and 10 after transduction, normalized to the input percentage at day 2 (T 0 ). (F) Kaplan-Meier survival analysis of mice that received transplantation with sorted GFP + leukemia cells 2 days after transduction (n = 5 mice per group; log-rank test). Data are represented as mean ± standard deviation (SD) with an n = 3, unless otherwise stated. Significance was measured by 1-way analysis of variance (ANOVA) with the following thresholds: ∗∗∗ P < .001 and ∗∗∗∗ P < .0001. Refer to supplemental Figure 2 and supplemental Table 2 .

Article Snippet: In contrast to murine AML cells in which Glut1 levels are predominant ( E), human AML cell lines (DepMap, Broad Institute) and samples from patients with AML from The Cancer Genome Atlas (TCGA) database had comparably high levels of GLUT3 ( C-D).

Techniques: Expressing, Transduction, Control, Clone Assay, Sequencing, Flow Cytometry, Transplantation Assay, Ex Vivo, Proliferation Assay, Standard Deviation

Journal: Blood Advances

Article Title: Combined GLUT1 and OXPHOS inhibition eliminates acute myeloid leukemia cells by restraining their metabolic plasticity

doi: 10.1182/bloodadvances.2023009967

Figure Lengend Snippet: Autophagy is induced as a metabolic adaptation in AML cells after Glut1 disruption. (A-D) Mechanistic investigation of Glut1 disruption was performed in LSC-enriched (c-Kit + ) bone marrow–derived MLL::AF9 cells, 4 days after transduction. (A) Representative histograms showing flow cytometric quantification of autophagic vesicle load (autophagosomes and autolysosomes) using the cell-permeant aliphatic Autophagy Probe Red. Mean fluorescent intensity (MFI) expression is depicted, unstained control is shown in gray. (B) Bar chart showing MFI of LC3B autophagy marker in Glut1 -disrupted AML cells compared with cells transduced with nontargeting control. (C) Representative western blot and (D) its corresponding quantification of LC3B-I and LC3B-II. Data were normalized to expression in nontargeting control and to actin as a loading control (n = 2). (E) mRNA expression of Atg7 in sorted GFP + (sgRNA-expressing) leukemia cells, 3 days after transduction, represented as FPKM values (n = 4). (F) Flow cytometric quantification of viable MLL::AF9 cells transduced with Glut1 sgRNAs or nontargeting control and then treated with dimethyl sulfoxide (DMSO) (vehicle) or 1 μM chloroquine (CQ) for 72 hours. Data were normalized to corresponding DMSO-treated controls, and statistics measured by unpaired 2-tailed Student t test. Data are shown as mean ± SD (n = 3) and statistical testing was performed by 1-way ANOVA, unless otherwise stated. ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001; and ∗∗∗∗ P < .0001.

Article Snippet: In contrast to murine AML cells in which Glut1 levels are predominant ( E), human AML cell lines (DepMap, Broad Institute) and samples from patients with AML from The Cancer Genome Atlas (TCGA) database had comparably high levels of GLUT3 ( C-D).

Techniques: Disruption, Derivative Assay, Transduction, Expressing, Control, Marker, Western Blot

Journal: Blood Advances

Article Title: Combined GLUT1 and OXPHOS inhibition eliminates acute myeloid leukemia cells by restraining their metabolic plasticity

doi: 10.1182/bloodadvances.2023009967

Figure Lengend Snippet: GLUT1 and OXPHOS inhibition has synergistic antileukemic efficacy in human AML cells. Assessment of synergistic effects between pharmacological inhibition of GLUT1 (BAY-876) and OXPHOS (IACS-010759) in (A) THP-1 and (B) Mono Mac 6. Quantification of viable cell counts after 72-hour treatment with increasing doses of BAY-876 alone (orange) or together with 20 nM IACS-010759 (purple) assessed by flow cytometry. Data were normalized to corresponding DMSO-treated controls. (C) Viability of primary AML samples treated ex vivo with indicated concentrations of BAY-876 alone (orange) or together with 100 nM IACS-010759 (purple), normalized to corresponding DMSO-treated controls. (D) Stratification of AML patients into “responders” and “nonresponders.” (E) Viability of CD34 + normal bone marrow cells relative to primary AML cells. (F) Comparison of viability in RUNX1 -mutated vs wild-type adult AML after dual treatment with BAY-876 and 100 nM IACS-010759. (D-F) Data were normalized to viability corresponding to 100 nM IACS-010759 treatment alone and are shown as mean ± SD (n = 3-4). Synergistic effects have been marked with the letter “S.” Statistical testing was performed by unpaired 2-tailed Student t test; ∗ P < .05. Refer to supplemental Figures 6-9 and supplemental Table 4 .

Article Snippet: In contrast to murine AML cells in which Glut1 levels are predominant ( E), human AML cell lines (DepMap, Broad Institute) and samples from patients with AML from The Cancer Genome Atlas (TCGA) database had comparably high levels of GLUT3 ( C-D).

Techniques: Inhibition, Flow Cytometry, Ex Vivo, Comparison

Journal: BMB Reports

Article Title: Diesel exhaust particles disrupt blood–retina barrier integrity via TLR2 and TLR4 activation

doi: 10.5483/BMBRep.2025-0013

Figure Lengend Snippet: TNF-α and IL-1β mRNA expression in U937 human macrophages at 6, 24, and 48 h after DEP exposure followed by washing. (A) Schematic representation of the experimental procedure. (B, C) TNF-α and IL-1β mRNA expression. TNF-α (B) and IL-1β (C) mRNA levels were significantly elevated at each time point following DEP exposure compared with those in the control. *P < 0.05 vs. control.

Article Snippet: U937 human macrophages (Korean Cell Line Bank, Seoul, Korea) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco, MA, USA) for 3 days at 37°C in a 5% CO 2 incubator.

Techniques: Expressing, Control

Journal: BMB Reports

Article Title: Diesel exhaust particles disrupt blood–retina barrier integrity via TLR2 and TLR4 activation

doi: 10.5483/BMBRep.2025-0013

Figure Lengend Snippet: Claudin-5 and ZO-1 expression and permeability in human retinal endothelial cells (HRECs) treated with conditioned media from U937 macrophages exposed to DEP. (A) Schematic representation of the experimental procedure. (B) Immunocytochemical staining. Claudin-5 and ZO-1 expression in HRECs was markedly reduced after treatment with conditioned media from DEP-exposed U937 macrophages. (C, D) Western blot analysis. Claudin-5 (C) and ZO-1 (D) protein levels in HRECs were significantly decreased following treatment with DEP-exposed U937-conditioned media compared with the control. (E) TEER permeability assay. Permeability in HRECs was significantly increased after treatment with DEP-exposed U937-conditioned media compared with the control. Scale bar = 30 μm, *P < 0.05 vs. control.

Article Snippet: U937 human macrophages (Korean Cell Line Bank, Seoul, Korea) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco, MA, USA) for 3 days at 37°C in a 5% CO 2 incubator.

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

Journal: BMB Reports

Article Title: Diesel exhaust particles disrupt blood–retina barrier integrity via TLR2 and TLR4 activation

doi: 10.5483/BMBRep.2025-0013

Figure Lengend Snippet: Expression levels of TLR2/TLR4 and TNF-α/IL-1β mRNA in U937 macrophages exposed to DEPs with TLR2 (C29) or TLR4 (TAK242) inhibitors. (A) Schematic representation of the experimental procedure. (B, C) TLR2 and TLR4 expression levels. Compared with those in the control, TLR2 (B) and TLR4 (C) levels in U937 macrophages were significantly elevated following DEP exposure. TLR2 and TLR4 expression levels were significantly restored following treatment with C29 and TAK242 before DEP exposure, respectively. (D, E) TNF-α and IL-1β mRNA expression. TNF-α (D) and IL-1β (E) mRNA levels were significantly lower in U937 macrophages treated with C29 or TAK242 before DEP exposure than in cells that received DEP exposure alone without TLR inhibitors. *P < 0.05 vs. control, † P < 0.05 vs. DEP.

Article Snippet: U937 human macrophages (Korean Cell Line Bank, Seoul, Korea) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco, MA, USA) for 3 days at 37°C in a 5% CO 2 incubator.

Techniques: Expressing, Control

Journal: BMB Reports

Article Title: Diesel exhaust particles disrupt blood–retina barrier integrity via TLR2 and TLR4 activation

doi: 10.5483/BMBRep.2025-0013

Figure Lengend Snippet: Claudin-5 and ZO-1 expression and permeability in HRECs treated with conditioned media from U937 macrophages treated with C29 or TAK242 before exposure to DEPs. (A) Schematic representation of the experimental procedure. (B) Immunocytochemical staining. Claudin-5 and ZO-1 expression was markedly preserved in HRECs cultured in conditioned media from U937 macrophages treated with C29 or TAK242 before DEP exposure, than in HRECs that received DEP exposure alone without pretreatment with TLR inhibitors. (C, D) Western blot analysis. Claudin-5 (C) and ZO-1 (D) protein levels in HRECs were significantly restored following treatment with conditioned media from U937 macrophages treated with C29 or TAK242 before DEP exposure. (E) TEER permeability assay. Permeability was significantly lower in HRECs treated with conditioned media from U937 macrophages treated with C29 or TAK242 before DEP exposure, than in HRECs that received DEP exposure alone without pretreatment with TLR inhibitors. Scale bar = 30 μm, *P < 0.05 vs. control, † P < 0.05 vs. DEP.

Article Snippet: U937 human macrophages (Korean Cell Line Bank, Seoul, Korea) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco, MA, USA) for 3 days at 37°C in a 5% CO 2 incubator.

Techniques: Expressing, Permeability, Staining, Cell Culture, Western Blot, Control