human chronic myelogenous leukemia cell line k562  (ATCC)

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: Aging Cell

Article Title: Overactivation of Cdc42 GTPase Impairs the Cytotoxic Function of NK Cells From Old Individuals Towards Senescent Fibroblasts

doi: 10.1111/acel.70398

Figure Lengend Snippet: Natural killer cells from old adults depict reduced conjugation with target cells. (A) Scheme illustrating the trafficking of secretory granules within NK cells towards the synapse between NK cells and the corresponding target cells. Synapse formation with the target cell is referred to as conjugation. (B) Flow cytometry analyses of conjugation of NK cells from young (average age 21 years) and old (average age 70 years) donors with senescent HDF following co‐cultures for 0, 60, and 90 min at an effector to target (E:T) cell ratio of 1:1. Senescent HDF were stained with CellTrace violet (450/50 filter), while NK cells from either young or old adults were stained with CellTrace CFSE (530/30 filter). The depicted data set is representative of 6 independent experiments. The dot plot was shown in bi‐exponential scale. (C) Graph shows quantification of the conjugation between either young or old NK cells with senescent HDF at the indicated time points after initiation of co‐cultures (0, 60, and 90 min). Data were presented as mean (conjugation of cells in percentage of total cells) ± SEM, N = 6. Two‐way ANOVA, followed by Bonferroni multiple comparison test was used to find the significance between the groups. (D) Flow cytometry analysis of HLA‐1 expression on non‐senescent (young) HDF, senescent HDF and the K562 leukemia cell line. The K562 cells served as conceptual control and IgG1 served as isotype control for each of the groups. (E) Quantification of HLA‐1 expression (MFI) in non‐senescent (young) HDF, senescent HDF and K562. Data were presented as percentage of mean (mean fluorescence intensity, MFI) ± SEM. N = 4. Two‐tailed t ‐test was used to find the significance between non‐senescent and senescent HDF. (F) Flow cytometry analysis of MICA/B expression on non‐senescent (young) HDF, senescent HDF and K562. IgG1 served as isotype control for each of the groups. (G) Quantification of MICA/B expression (MFI) in young and senescent HDF and K562. Data were presented as percentage of mean (MFI) ± SEM, N = 4. One‐way ANOVA, followed by Bonferroni multiple comparison test was used to find the significance. (H) Flow cytometry analysis of NKG2D/CD314 expression in NK cells from young and old donors. IgG1 served as isotype control for each of the groups. (I) Quantification of NKG2D/CD314 expression (MFI) on NK cells from either young or old adults. Data in the graphs were represented as percentage of mean (MFI) ± SEM, N = 4. Two‐tailed t ‐test was used to find the significance between NK cells from young and old donors.

Article Snippet: K562 cell line , ATCC , Cat. #CCL‐243.

Techniques: Conjugation Assay, Flow Cytometry, Staining, Comparison, Expressing, Control, Fluorescence, Two Tailed Test

Journal: Aging Cell

Article Title: Overactivation of Cdc42 GTPase Impairs the Cytotoxic Function of NK Cells From Old Individuals Towards Senescent Fibroblasts

doi: 10.1111/acel.70398

Figure Lengend Snippet: CASIN treatment improves the cytotoxic ability of Natural killer cells from old humans and mice. (A) Graphical illustration of experimental plan, where young NK cells treated with vehicle and old NK cells treated with either vehicle or CASIN for 8 h and thereafter subjected to co‐culture with target senescent HDF exerting their differential killing ability. (B) Representative histograms depicting the killing ability of different experimental groups as measured by flow cytometry. Peak at the left side of histogram, showing the dead senescent HDF population with percentage of dead cells. (C) Quantification of the percentage of target senescent HDF death executed by young NK cells treated with vehicle, and old NK cells treated with either vehicle or CASIN. Data were represented as mean (Percentage of senescent fibroblast death) ± SEM. N = 4. (D) Representative histograms show the distribution of K562 killing by young NK cells treated with vehicle, and old NK cells treated with either vehicle or CASIN. Peak at the left side of histogram, showing the dead K562 population with percentage of dead cells. (E) Graph shows the percentage of target cell (K562) death mediated either by young NK cells treated with vehicle or by old NK cells treated with either vehicle or CASIN. Data were represented as mean (Percentage of K562 lysis) ± SEM. N = 4. One‐way ANOVA, followed by Bonferroni multiple comparison test was used to find the significance among the groups in C and E. (F) Illustration of the experimental design for treatment of young mice (average age 120 days) treated with vehicle and old mice (average age 650 days) treated with either vehicle or CASIN. Following treatment, NK cells were isolated from spleen and bone marrow and subjected to co‐cultures with murine dermal fibroblasts (MDF) derived from old mice (average age 650 days). (G) Flow cytometry with representative histograms depicting old/senescent MDF killing by NK cells isolated from bone marrow (left panel) and spleen (right panel) of vehicle and CASIN treated old mice. Peak at the left side of histogram, showing the dead old MDF population with percentage of dead cells. (H) Quantification of the percentage of old/senescent MDF killing by NK cells isolated from bone marrow and spleen of young and old mice treated with vehicle and old mice treated with CASIN. Data were represented as mean (percentage of old/senescent MDF lysis) ± SEM, N = 4, where each group contains pool of NK cells isolated from 4 different mice of same treatment group. Two‐way ANOVA, followed by Bonferroni multiple comparison test was used to find the significance among the groups. (I) Graphical summary. Unrestrained Cdc42 activity causes failure of old NK cells to kill senescent fibroblasts. Unrestrained Cdc42 activity disrupts the microtubular network and impaired mitochondrial ATP resulting in reduced conjugation, and impaired degranulation of lytic vesicles into the synaptic cleft with reduced cytotoxicity. CASIN can attenuate all these steps and in part attenuate the killing of senescent fibroblasts (senescent HDF).

Article Snippet: K562 cell line , ATCC , Cat. #CCL‐243.

Techniques: Co-Culture Assay, Flow Cytometry, Lysis, Comparison, Isolation, Derivative Assay, Activity Assay, Conjugation Assay

Journal: Aging Cell

Article Title: Overactivation of Cdc42 GTPase Impairs the Cytotoxic Function of NK Cells From Old Individuals Towards Senescent Fibroblasts

doi: 10.1111/acel.70398

Figure Lengend Snippet: Natural killer cells from old adults depict reduced conjugation with target cells. (A) Scheme illustrating the trafficking of secretory granules within NK cells towards the synapse between NK cells and the corresponding target cells. Synapse formation with the target cell is referred to as conjugation. (B) Flow cytometry analyses of conjugation of NK cells from young (average age 21 years) and old (average age 70 years) donors with senescent HDF following co‐cultures for 0, 60, and 90 min at an effector to target (E:T) cell ratio of 1:1. Senescent HDF were stained with CellTrace violet (450/50 filter), while NK cells from either young or old adults were stained with CellTrace CFSE (530/30 filter). The depicted data set is representative of 6 independent experiments. The dot plot was shown in bi‐exponential scale. (C) Graph shows quantification of the conjugation between either young or old NK cells with senescent HDF at the indicated time points after initiation of co‐cultures (0, 60, and 90 min). Data were presented as mean (conjugation of cells in percentage of total cells) ± SEM, N = 6. Two‐way ANOVA, followed by Bonferroni multiple comparison test was used to find the significance between the groups. (D) Flow cytometry analysis of HLA‐1 expression on non‐senescent (young) HDF, senescent HDF and the K562 leukemia cell line. The K562 cells served as conceptual control and IgG1 served as isotype control for each of the groups. (E) Quantification of HLA‐1 expression (MFI) in non‐senescent (young) HDF, senescent HDF and K562. Data were presented as percentage of mean (mean fluorescence intensity, MFI) ± SEM. N = 4. Two‐tailed t ‐test was used to find the significance between non‐senescent and senescent HDF. (F) Flow cytometry analysis of MICA/B expression on non‐senescent (young) HDF, senescent HDF and K562. IgG1 served as isotype control for each of the groups. (G) Quantification of MICA/B expression (MFI) in young and senescent HDF and K562. Data were presented as percentage of mean (MFI) ± SEM, N = 4. One‐way ANOVA, followed by Bonferroni multiple comparison test was used to find the significance. (H) Flow cytometry analysis of NKG2D/CD314 expression in NK cells from young and old donors. IgG1 served as isotype control for each of the groups. (I) Quantification of NKG2D/CD314 expression (MFI) on NK cells from either young or old adults. Data in the graphs were represented as percentage of mean (MFI) ± SEM, N = 4. Two‐tailed t ‐test was used to find the significance between NK cells from young and old donors.

Article Snippet: K562 cell, a chronic myelogenous leukemia cell line, was purchased from ATCC (ATCC, Cat. #CCL‐243).

Techniques: Conjugation Assay, Flow Cytometry, Staining, Comparison, Expressing, Control, Fluorescence, Two Tailed Test

Journal: Aging Cell

Article Title: Overactivation of Cdc42 GTPase Impairs the Cytotoxic Function of NK Cells From Old Individuals Towards Senescent Fibroblasts

doi: 10.1111/acel.70398

Figure Lengend Snippet: CASIN treatment improves the cytotoxic ability of Natural killer cells from old humans and mice. (A) Graphical illustration of experimental plan, where young NK cells treated with vehicle and old NK cells treated with either vehicle or CASIN for 8 h and thereafter subjected to co‐culture with target senescent HDF exerting their differential killing ability. (B) Representative histograms depicting the killing ability of different experimental groups as measured by flow cytometry. Peak at the left side of histogram, showing the dead senescent HDF population with percentage of dead cells. (C) Quantification of the percentage of target senescent HDF death executed by young NK cells treated with vehicle, and old NK cells treated with either vehicle or CASIN. Data were represented as mean (Percentage of senescent fibroblast death) ± SEM. N = 4. (D) Representative histograms show the distribution of K562 killing by young NK cells treated with vehicle, and old NK cells treated with either vehicle or CASIN. Peak at the left side of histogram, showing the dead K562 population with percentage of dead cells. (E) Graph shows the percentage of target cell (K562) death mediated either by young NK cells treated with vehicle or by old NK cells treated with either vehicle or CASIN. Data were represented as mean (Percentage of K562 lysis) ± SEM. N = 4. One‐way ANOVA, followed by Bonferroni multiple comparison test was used to find the significance among the groups in C and E. (F) Illustration of the experimental design for treatment of young mice (average age 120 days) treated with vehicle and old mice (average age 650 days) treated with either vehicle or CASIN. Following treatment, NK cells were isolated from spleen and bone marrow and subjected to co‐cultures with murine dermal fibroblasts (MDF) derived from old mice (average age 650 days). (G) Flow cytometry with representative histograms depicting old/senescent MDF killing by NK cells isolated from bone marrow (left panel) and spleen (right panel) of vehicle and CASIN treated old mice. Peak at the left side of histogram, showing the dead old MDF population with percentage of dead cells. (H) Quantification of the percentage of old/senescent MDF killing by NK cells isolated from bone marrow and spleen of young and old mice treated with vehicle and old mice treated with CASIN. Data were represented as mean (percentage of old/senescent MDF lysis) ± SEM, N = 4, where each group contains pool of NK cells isolated from 4 different mice of same treatment group. Two‐way ANOVA, followed by Bonferroni multiple comparison test was used to find the significance among the groups. (I) Graphical summary. Unrestrained Cdc42 activity causes failure of old NK cells to kill senescent fibroblasts. Unrestrained Cdc42 activity disrupts the microtubular network and impaired mitochondrial ATP resulting in reduced conjugation, and impaired degranulation of lytic vesicles into the synaptic cleft with reduced cytotoxicity. CASIN can attenuate all these steps and in part attenuate the killing of senescent fibroblasts (senescent HDF).

Article Snippet: K562 cell, a chronic myelogenous leukemia cell line, was purchased from ATCC (ATCC, Cat. #CCL‐243).

Techniques: Co-Culture Assay, Flow Cytometry, Lysis, Comparison, Isolation, Derivative Assay, Activity Assay, Conjugation Assay