Journal: Blood Advances

Article Title: UBE2O as a key regulator of drug-induced erythropoiesis in the context of myelodysplastic syndromes

doi: 10.1182/bloodadvances.2025017340

Figure Lengend Snippet: Erythropoietin-induced erythroid differentiation promotes UBE2O expression. (A) Percentages of benzidine-positive K562 cells (blue) treated with 10 IU/mL erythropoietin for 0 to 3 days, compared with nontreated control (NTC) cells. One-way analysis of variance (ANOVA) was used for statistical analysis (n = 3). (B) Representative images of benzidine staining of K562 cells treated with 10 IU/mL erythropoietin for 0 to 3 days. Red arrows indicate benzidine-positive cells (blue). The images are representative of 3 independent experiments. (C) Relative mRNA levels of UBE2O and GATA1 in K562 cells on days 0 to 3 after treatment with 10 IU/mL erythropoietin on day 0. One-way ANOVA was used for statistical analysis (n = 3). (D) Representative western blot analysis of UBE2O and GATA1 in K562 cells treated with 10 IU/mL erythropoietin for 0 to 3 days. Vinculin was used as a loading control. (E) Representative images of immunofluorescence staining for UBE2O in K562 cells treated with 10 IU/mL erythropoietin for 0 to 2 days, compared with NTC cells (n = 5). DAPI staining was used to highlight nuclei. (F) Relative number of UBE2O (green spots) in K562 cells treated with 10 IU/mL erythropoietin for 0 to 2 days (n = 5). Ordinary 1-way ANOVA was used for statistical analysis. (G) Correlation between UBE2O and GATA1 mRNA levels in K562 cells aftertreatment with 10 IU/mL erythropoietin on day 0, and the expression levels of the genes were compared on days 1 and 2. The black line indicates the linear regression fit. EPO, erythropoietin. ∗ P < .05; ∗∗ P < .01; and ∗∗∗∗ P < .0001.

Article Snippet: The human leukemic K562 cell line (catalog no. CCL-243; American Type Culture Collection, ATCC) was cultured in Iscove modified Dulbecco medium (IMDM) with L-glutamine (catalog no. AU-L0190-500; Aurogene) supplemented with 10% heat-inactivated fetal bovine serum (FBS; catalog no. EU-000-500; Immunological Sciences) and 1% penicillin/streptomycin (catalog no. AU-L022-100; Aurogene), and maintained in a humidified incubator with 5% CO 2 at 37°C.

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

Journal: Blood Advances

Article Title: UBE2O as a key regulator of drug-induced erythropoiesis in the context of myelodysplastic syndromes

doi: 10.1182/bloodadvances.2025017340

Figure Lengend Snippet: Luspatercept’s inhibition of the TGF-β pathway promotes UBE2O upregulation. (A) Percentages of benzidine-positive K562 cells (blue) treated with 1 μg/mL luspatercept for 0 to 3 days, compared with nontreated control (NTC) cells (n = 3). One-way ANOVA was used for statistical analysis (n = 3). (B) Representative images of benzidine staining of K562 cells treated with 1 μg/mL luspatercept for 0 to 3 days. The red arrows indicate benzidine-positive cells (blue). The images are representative of 3 independent experiments. (C) Relative mRNA levels of UBE2O and GATA1 in K562 cells on days 0 to 3 after treatment with 1 μg/mL luspatercept on day 0. One-way ANOVA was used for statistical analysis (n = 3). (D) Representative western blot analysis of UBE2O, GATA1, and p-Smad3 in K562 cells treated with 1 μg/mL luspatercept for 0 to 3 days. Vinculin and GAPDH were used as loading controls. (E) Representative images of immunofluorescence staining for UBE2O in K562 cells treated with 1 μg/mL luspatercept for 0 to 2 days, compared with NTC cells (n = 5). DAPI staining was used to highlight nuclei. (F) Relative number of UBE2O (green spots) in K562 cells treated with 1 μg/mL luspatercept for 0 to 2 days (n = 5). Ordinary 1-way ANOVA was used for statistical analysis. (G) Correlation between UBE2O and GATA1 mRNA levels in K562 cells aftertreatment with 1 μg/mL luspatercept on day 0, and the expression levels of the genes were compared on days 1 and 2. The black line indicates the linear regression fit. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LUSPA, luspatercept; p-Smad3, phosphorylated Smad3. ∗ P < .05; ∗∗ P < .01; and ∗∗∗∗ P < .0001.

Article Snippet: The human leukemic K562 cell line (catalog no. CCL-243; American Type Culture Collection, ATCC) was cultured in Iscove modified Dulbecco medium (IMDM) with L-glutamine (catalog no. AU-L0190-500; Aurogene) supplemented with 10% heat-inactivated fetal bovine serum (FBS; catalog no. EU-000-500; Immunological Sciences) and 1% penicillin/streptomycin (catalog no. AU-L022-100; Aurogene), and maintained in a humidified incubator with 5% CO 2 at 37°C.

Techniques: Inhibition, Control, Staining, Western Blot, Immunofluorescence, Expressing

Journal: Blood Advances

Article Title: UBE2O as a key regulator of drug-induced erythropoiesis in the context of myelodysplastic syndromes

doi: 10.1182/bloodadvances.2025017340

Figure Lengend Snippet: GATA1 regulates UBE2O expression during erythroid differentiation. (A) Graphic representation of the predicted GATA1 motif “CTAATCT” identified using the JASPAR 2020 database. (B) Table summarizing the statistical significance of the GATA1 motif, including its P value (7.38 × 10 −6 ) and q value (0.0146). (C) Genome browser snapshot showing the localization of the GATA1 motif (highlighted) at position chr17:76,453,056-76,453,066 relative to the transcriptional start site and adjacent regulatory elements. (D) Real-time qPCR of UBE2O, BCL-XL (positive control), and NFKBIA (negative control) DNA from K562 cells following ChIP performed 1 day after treatment with 10 IU/mL erythropoietin (n = 2). (E) Representative image of qualitative PCR of DNA from K562 cells following ChIP performed after 1 day of treatment with 10 IU/mL erythropoietin. (F) Real-time qPCR of UBE2O, BCL-XL (positive control), and NFKBIA (negative control) DNA from K562 cells following ChIP performed 1 day after treatment with 1 μg/mL luspatercept (LUSPA) (n = 2). (G) Representative image of qualitative PCR of DNA from K562 cells following ChIP performed after 1 day of treatment with 1 μg/mL luspatercept. CTR − , negative control; CTR + , positive control.

Article Snippet: The human leukemic K562 cell line (catalog no. CCL-243; American Type Culture Collection, ATCC) was cultured in Iscove modified Dulbecco medium (IMDM) with L-glutamine (catalog no. AU-L0190-500; Aurogene) supplemented with 10% heat-inactivated fetal bovine serum (FBS; catalog no. EU-000-500; Immunological Sciences) and 1% penicillin/streptomycin (catalog no. AU-L022-100; Aurogene), and maintained in a humidified incubator with 5% CO 2 at 37°C.

Techniques: Expressing, Positive Control, Negative Control

Journal: Frontiers in Oncology

Article Title: ZFAS1/STAT3 axis modulates imatinib resistance of chronic myeloid leukemia cells through glucose metabolism reprogramming

doi: 10.3389/fonc.2025.1603060

Figure Lengend Snippet: Elevated expression of lncRNA ZFAS1 and STAT3 in imatinib-resistant (IM-R) CML patient samples and IM-R K562 cell line. (A, B) Relative expressions of ZFAS1 and STAT3 in peripheral blood cells from imatinib-resistant (IM-R, n=30) and imatinib-sensitive (IM-S, n=30) CML patients. (C) Correlation analysis between ZFAS1 and STAT3 in CML patients samples. (D, E) Expression levels of lncRNA ZFAS1 and STAT3 in IM-R and IM-S K562 cells. Data are presented as mean ± SD. *** p <0.001 vs . IM-S.

Article Snippet: Human leukemic cell line K562 cells were procured from ATCC in Manassas, VA, USA, and maintained in RMPI 1640 medium supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin.

Techniques: Expressing

Journal: Frontiers in Oncology

Article Title: ZFAS1/STAT3 axis modulates imatinib resistance of chronic myeloid leukemia cells through glucose metabolism reprogramming

doi: 10.3389/fonc.2025.1603060

Figure Lengend Snippet: Effects of ZFAS1 knockdown on imatinib-resistant K562 cells. (A) PCR analysis showing the efficiency of ZFAS1 knockdown in IM-R K562 cells. (B) Drug sensitivity assay (CCK-8) showing relative cell viability of si-NC and si-ZFAS1 IM-R K562 cells treated with varying concentrations of IM. (C) IC 50 values of imatinib in si-ZFAS1 cells and si-NC cells. (D) Colony formation assay showing the number of colonies formed by si-NC and si-ZFAS1 IM-R K562 cells treated with or without 2 µM IM. (E) Flow cytometry analysis showing the apoptosis rate of si-NC and si-ZFAS1 IM-R K562 cells treated with 2 µM IM. Data are presented as mean ± SD. NTC: non-treated control; ** p <0.01, *** p <0.001 vs . si-NC; ### p <0.001 vs . IM+si-NC.

Article Snippet: Human leukemic cell line K562 cells were procured from ATCC in Manassas, VA, USA, and maintained in RMPI 1640 medium supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin.

Techniques: Knockdown, Sensitive Assay, CCK-8 Assay, Colony Assay, Flow Cytometry, Control

Journal: Frontiers in Oncology

Article Title: ZFAS1/STAT3 axis modulates imatinib resistance of chronic myeloid leukemia cells through glucose metabolism reprogramming

doi: 10.3389/fonc.2025.1603060

Figure Lengend Snippet: Effects of ZFAS1 knockdown on glucose metabolism in IM-R K562 cells. (A) Relative glucose uptake levels in si-NC and si-ZFAS1 IM-R K562 cells. (B) Relative lactate production in si-NC and si-ZFAS1 IM-R K562 cells. (C) Relative ATP levels in si-NC and si-ZFAS1 IM-R K562 cells. (D) Extracellular acidification rate (ECAR) in si-NC and si-ZFAS1 IM-R K562 cells. (E) Oxygen consumption rate (OCR) in si-NC and si-ZFAS1 IM-R K562 cells. (F) ECAR vs . OCR plot illustrating the metabolic shift in si-ZFAS1 IM-R K562 cells. (G) Comprehensive metabolic profile showing the impact of ZFAS1 knockdown on IM-R K562 cell metabolism. Data are presented as mean ± SD. ** p <0.01, *** p <0.001 vs . si-NC.

Article Snippet: Human leukemic cell line K562 cells were procured from ATCC in Manassas, VA, USA, and maintained in RMPI 1640 medium supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin.

Techniques: Knockdown

Journal: Frontiers in Oncology

Article Title: ZFAS1/STAT3 axis modulates imatinib resistance of chronic myeloid leukemia cells through glucose metabolism reprogramming

doi: 10.3389/fonc.2025.1603060

Figure Lengend Snippet: STAT3 overexpression reverses the effects of ZFAS1 knockdown on imatinib sensitivity and glucose metabolism reprogramming in IM-resistant K562 cells, including si-NC+NC, si-ZFAS1+NC, and si-ZFAS1+STAT3 IM-R K562 cells. (A) Drug sensitivity assay by CCK-8 showing relative cell viability of cells treated with varying concentrations of IM. (B) IC50 values were determined via CCK-8 assay after 48 hours of imatinib treatment. (C) Relative glucose uptake levels. (D) Relative lactate production. (E) Relative ATP levels. (F) ECAR over time. (G) Glycolysis and glycolytic capacity. (H) OCR over time. (I) Summary of basal respiration and maximal respiration. ** p <0.01, *** p <0.001 vs . si-NC+NC; # p <0.05, ## p <0.01, ### p <0.001 vs . si-ZFAS1+NC.

Article Snippet: Human leukemic cell line K562 cells were procured from ATCC in Manassas, VA, USA, and maintained in RMPI 1640 medium supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin.

Techniques: Over Expression, Knockdown, Sensitive Assay, CCK-8 Assay

Journal: Frontiers in Oncology

Article Title: ZFAS1/STAT3 axis modulates imatinib resistance of chronic myeloid leukemia cells through glucose metabolism reprogramming

doi: 10.3389/fonc.2025.1603060

Figure Lengend Snippet: ZFAS1 promotes HIF1α upregulation through STAT3 in IM-resistant K562 cells. (A) Relative expression of STAT3 in IM-R K562 cells transfected with STAT3 plasmid compared to negative control (NC). (B) Western blot analysis showing the protein expression levels of HIF1α, LDHA, and PDK1 in si-NC, si-ZFAS1, and si-ZFAS1 + STAT3 transfected IM-R K562 cells. Data are presented as mean ± SD. *** p <0.001 vs . NC.

Article Snippet: Human leukemic cell line K562 cells were procured from ATCC in Manassas, VA, USA, and maintained in RMPI 1640 medium supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin.

Techniques: Expressing, Transfection, Plasmid Preparation, Negative Control, Western Blot

Journal: Frontiers in Oncology

Article Title: ZFAS1/STAT3 axis modulates imatinib resistance of chronic myeloid leukemia cells through glucose metabolism reprogramming

doi: 10.3389/fonc.2025.1603060

Figure Lengend Snippet: Effect of 2-DG on STAT3 -mediated imatinib resistance in IM-R K562 cells. (A) CCK-8 assay shows relative cell viability of IM-R K562 cells with negative control (NC), STAT3 overexpression ( STAT3 ), and STAT3 treated with 2-DG ( STAT3 + 2-DG) under varying concentrations of imatinib. (B) IC50 values were determined via CCK-8 assay after 48 hours of imatinib treatment. ** p <0.01 vs . NC; # p <0.05 vs . STAT3.

Article Snippet: Human leukemic cell line K562 cells were procured from ATCC in Manassas, VA, USA, and maintained in RMPI 1640 medium supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin.

Techniques: CCK-8 Assay, Negative Control, Over Expression

Journal: Experimental & Molecular Medicine

Article Title: VGF and the VGF-derived peptide AQEE30 stimulate osteoblastic bone formation through the C3a receptor

doi: 10.1038/s12276-025-01419-y

Figure Lengend Snippet: a , Secretion fractionation protocol. Premature K562 cells (pre-MKs) were treated with PMA and then differentiated into mature MK-like cells for 3 days. CM of pre-MKs and MKs was collected, concentrated and then divided into 96 fractions by size-exclusion chromatography. b , The viability of MC3T3-E1 cells after treatment with each pair of pre-MK-CM and MK-CM was measured using a Cell Counting Kit-8 ( n = 2). c , Secreted proteins commonly identified in fractions 51 and 52 of the MK-CM. d , Real-time PCR analysis of VGF , HMGB1 and HDGF in pre-MKs and MKs ( n = 3). e , K562 cells were transfected with scrambled siRNA or VGF siRNA, and the knockdown efficiency was measured by real-time PCR ( n = 3). f , K562 cells were transfected with scrambled siRNA and VGF siRNA and then differentiated into mature MKs. MC3T3-E1 cells were treated with their CM, and cell viability was measured ( n = 4). g , Murine fetal liver cells isolated from Vgf −/− mice and their WT littermates were differentiated into mature MKs by thrombopoietin treatment. The viability of MC3T3-E1 cells after treatment with CM from mature MKs was measured ( n = 5). h , CM was collected as described in g . Bone nodule formation in MC3T3-E1 cells was tested after treatment with CM from mature MKs. Mineralized samples were stained with Alizarin red S (right) and measured at 570 nm (left, n = 4). The data represent the mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001 versus the control groups.

Article Snippet: Murine osteoblastic MC3T3-E1 and human leukemic K562 cells were purchased from the American Type Culture Collection and cultured in α-MEM and RPMI 1640 medium, both supplemented with 10% fetal bovine serum (FBS; Gibco), respectively.

Techniques: Fractionation, Size-exclusion Chromatography, Cell Counting, Real-time Polymerase Chain Reaction, Transfection, Knockdown, Isolation, Staining, Control

Journal: Nature Communications

Article Title: Exploitation of the fibrinolytic system by B-cell acute lymphoblastic leukemia and its therapeutic targeting

doi: 10.1038/s41467-024-54361-4

Figure Lengend Snippet: A Immunoblot for the indicated proteins in lysates of empty vector + , BCR-ABL1 + and MLL-AF9 + BA/F3 cells treated with vehicle (circles) or IGF1 (squares) and quantification (right) of the band intensity for Rictor and pAKT S473 normalized to GAPDH ( P = 0.049, P = 0.004, two-way ANOVA, Sidak test, n = 6 biological replicates, mean ± SD). B Immunoblot for the indicated proteins in lysates of sorted GFP (BCR-ABL1 or MLL-AF9) + BM cells from individual WT recipient mice with BCR-ABL1 + B-ALL (circles) or MLL-AF9 + AML (squares) and quantification of the band intensity for Rictor and pAKT S473 normalized to β-actin ( P = 0.0001, P = 0.016, two-way ANOVA, Sidak test, n = 4 biological replicates, mean ± SD). C Representative immunoblot for the indicated proteins in lysates of K562, NALM6 or THP1 cells treated with vehicle (circles) or for 5 (squares) or 10 (triangles) minutes human recombinant IGF1 (12 ng/ml) and its quantification (right) of the band intensities for Rictor and pAKT S473 normalized to β-actin ( P = 0.018, P = 0.011, two-way ANOVA, Dunnett test, n = 4 biological replicates, mean ± SD). The samples are derived from the same experiment, but one gel was used for mTOR, Rictor, pAktS473, Akt and β -actin. Another gel was used for Raptor, pS6K1 and S6K1. D Percentage of IGF1-receptor (IGF1R) + cells of all GFP (BCR-ABL1) + B-ALL (circles) versus all GFP (MLL-AF9) + AML cells (squares) in the murine system ( P < 0.0001, two-tailed t test, B-ALL n = 4, AML n = 7 biological replicates, mean ± SD). E Percentage of IGF1-receptor (IGF1R) + of all K562 (circles) versus SUPB15 (squares) cells ( P < 0.0001, two-tailed t test, n = 4 biological replicates, mean ± SD). F Representative immunoblot for the indicated proteins in lysates of K562 and SUPB15 cells treated with vehicle (circles) or human recombinant IGF1 (squares) and quantification (right) of the band intensities for Rictor and pAKT S473 normalized to GAPDH ( P = 0.026, P = 0.0868, two-way ANOVA, Sidak test, n = 6 biological replicates, mean ± SD). Source data are provided as a Source Data file.

Article Snippet: The human leukemic cell lines K562 (BCR-ABL1 + ; myeloid, model of CML) (ACC10), NALM6 (lymphoid, model of BCR-ABL1-negative B-ALL) (ACC128), THP1 (MLL-AF9 + ; myeloid, model of AML) (ACC16), SUPB15 (lymphoid, model of BCR-ABL1-positive B-ALL) (ACC389) and Kasumi (AML1-ETO + ; myeloid, model of AML) (ACC220) were purchased from the DSMZ and cultured in RPMI, 10% FBS, 1% penicillin/streptomycin and 1% L-glutamine.

Techniques: Western Blot, Plasmid Preparation, Recombinant, Derivative Assay, Two Tailed Test