human chronic myeloid leukemia cml cell line k562  (ATCC)

Journal: Advanced Science

Article Title: Meisoindigo Acts as a Molecular Glue to Target PKMYT1 for Degradation in Chronic Myeloid Leukemia Therapy

doi: 10.1002/advs.202413676

Figure Lengend Snippet: Activity‐based proteomic identification of potential targets for meisoindigo. a) Chemical structure of meisoindigo (Mei). b) Synthesis of a Mei‐alkyne probe (MP). c) Cell viability of K562 cells treated with Mei or MP for 72 h (n = 3). d) Workflow for label‐free (LFQ) and tandem mass tag (TMT) quantitative proteomics to identify MP‐labeled proteins in K562 cells. e,f) Volcano plots of the first and second label‐free quantitative proteomics with MP (5 µM)/DMSO (negative control) (n = 3). g) Volcano plot of TMT quantitative proteomics with MP (5 µM)/DMSO (negative control) (n = 5). h) Venn diagram summarizing overlapping proteins from the quantitative proteomics data. i) Concentration‐dependent in situ fluorescence labeling of MP in K562 cells. The data are presented as the means ± SDs. Statistical significance was assessed via two‐tailed unpaired Student's t‐test. Figure (d) created with BioRender.com.

Article Snippet: The human chronic myeloid leukemia cell line K562 was purchased from Procell (Wuhan, China).

Techniques: Activity Assay, Quantitative Proteomics, Labeling, Negative Control, Concentration Assay, In Situ, Fluorescence, Two Tailed Test

Journal: Advanced Science

Article Title: Meisoindigo Acts as a Molecular Glue to Target PKMYT1 for Degradation in Chronic Myeloid Leukemia Therapy

doi: 10.1002/advs.202413676

Figure Lengend Snippet: Mei exerts antitumor effects by directly targeting PKMYT1. a) Western blot analysis of the PKMYT1 protein in protein affinity pull‐down assay in K562 cells, and the cells were treated with MP (5 µM) with or without Mei (25 µM). b) Concentration‐dependent fluorescence labeling of MP on recombinant PKMYT1 (75‐362). c) Mei treatment (10 µM) increased the thermal stability of PKMYT1 at the whole‐cell level, as measured by a temperature‐dependent cellular thermal shift assay (CETSA) (n = 3). d) Mei treatment increased the thermal stability of PKMYT1 in cell lysates, as measured by a concentration‐dependent CETSA at 47 °C (n = 3). e) MST assay of Mei binding to recombinant PKMYT1 (75‐362) (n = 3). f) Immunoblotting confirmed PKMYT1 knockdown in K562 cells via the CRISPR/Cas9 system (n = 3). g) Growth curves of wild‐type and PKMYT1‐knockdown K562 cells (n = 3). h) Viability of wild‐type and PKMYT1‐knockdown K562 cells after 48 h Mei treatment at various concentrations (n = 6). i) Schematic diagram of Mei administration in mice bearing wild‐type or PKMYT1‐knockdown K562 tumors. j) Images of tumors from mice (wild‐type or PKMYT1‐knockdown) treated with vehicle or Mei (150 mg kg −1 ) (n = 12). k) Dynamic changes in tumor volume (n = 12). l) Tumor weight (n = 12). The data are presented as the means ± SDs. Statistical significance was assessed via two‐tailed unpaired Student's t‐test. NS, not significant; ***P < 0.001 versus the wild‐type group. Figure (i) created with BioRender.com.

Article Snippet: The human chronic myeloid leukemia cell line K562 was purchased from Procell (Wuhan, China).

Techniques: Western Blot, Pull Down Assay, Concentration Assay, Fluorescence, Labeling, Recombinant, Thermal Shift Assay, Binding Assay, Knockdown, CRISPR, Two Tailed Test

Journal: Advanced Science

Article Title: Meisoindigo Acts as a Molecular Glue to Target PKMYT1 for Degradation in Chronic Myeloid Leukemia Therapy

doi: 10.1002/advs.202413676

Figure Lengend Snippet: Mei promotes PKMYT1 degradation via K48‐linked ubiquitination. a) Western blot analysis of PKMYT1 protein levels in K562 cells treated with different concentrations of Mei for 12 h (n = 5). b) Western blot analysis of PKMYT1 protein levels in K562 cells treated with Mei (10 µM) at different time points (n = 5). c) Real‐time qPCR assessment of PKMYT1 mRNA levels in K562 cells treated with different concentrations of Mei for 4 h (n = 3). d) Time‐dependent qPCR analysis of PKMYT1 mRNA levels in K562 cells following treatment with Mei (10 µM) (n = 3). e) Western blot analysis of PKMYT1 degradation in K562 cells treated with cycloheximide (CHX) with or without Mei (10 µM) at different time points (n = 3). f) A proteasome inhibitor rescued the reduction of PKMYT1 in K562 cells, and the cells were treated with Mei alone or in combination with MG‐132 (10 µM) for 6 h. g) Effect of the lysosome inhibitor bafilomycin A1 (Baf‐A1, 200 nM) on Mei‐mediated PKMYT1 degradation in K562 cells. h) Co‐IP assay demonstrating Mei‐induced K48‐linked ubiquitination of PKMYT1. The data are presented as the means ± SDs. Statistical significance was assessed via two‐tailed unpaired Student's t‐test, *P < 0.05, **P < 0.01, ***P < 0.001 versus the control group.

Article Snippet: The human chronic myeloid leukemia cell line K562 was purchased from Procell (Wuhan, China).

Techniques: Ubiquitin Proteomics, Western Blot, Co-Immunoprecipitation Assay, Two Tailed Test, Control

Journal: Advanced Science

Article Title: Meisoindigo Acts as a Molecular Glue to Target PKMYT1 for Degradation in Chronic Myeloid Leukemia Therapy

doi: 10.1002/advs.202413676

Figure Lengend Snippet: PKMYT1 knockdown inhibits K562 cell growth. a) The mRNA expression of PKMYT1 in peripheral blood from the GSE100026 database in healthy individuals, patients with chronic myeloid leukemia (CML) in the chronic phase, and patients with CML in the blast crisis. b) Key genes linked to PKMYT1 in the STRING database. c) The effect of PKMYT1 knockdown on G2/M cell cycle transition in K562 cells was assessed by flow cytometry (n = 3). d) The effects of PKMYT1 knockdown on cell cycle proteins in K562 cells were examined by immunoblotting. e) Detection of the effect of PKMYT1 knockdown on K562 cell proliferation via a soft agar colony formation assay (n = 3). f) Detection of the effect of PKMYT1 knockdown on K562 cell proliferation via EdU staining (n = 5). g) Effect of PKMYT1 knockdown on ROS levels in K562 cells (n = 5). h) Effect of PKMYT1 knockdown on the mitochondrial membrane potential in K562 cells (n = 5). i) Oxygen consumption rate (OCR) levels in wild‐type or PKMYT1‐knockdown cells were assessed using a Seahorse XF24 analyzer (n = 6). j‐l), Basal respiration, ATP production, and Maximum respiration were assessed (n = 6). For 6c,e‐h,j‐l, the data are presented as the means ± SDs. Statistical significance was assessed via two‐tailed unpaired Student's t‐test, *P < 0.05, **P < 0.01, ***P < 0.001 versus the control group.

Article Snippet: The human chronic myeloid leukemia cell line K562 was purchased from Procell (Wuhan, China).

Techniques: Knockdown, Expressing, Flow Cytometry, Western Blot, Soft Agar Assay, Staining, Membrane, Two Tailed Test, Control

Journal: Advanced Science

Article Title: Meisoindigo Acts as a Molecular Glue to Target PKMYT1 for Degradation in Chronic Myeloid Leukemia Therapy

doi: 10.1002/advs.202413676

Figure Lengend Snippet: PKMYT1 knockdown inhibits leukemia cell proliferation and delays leukemia progression in vivo. a) Flowchart of the chronic myeloid leukemia orthotopic xenograft model. b) Survival of mice inoculated with wild‐type K562 and PKMYT1‐knockdown K562 cells (n = 13). c) The body weights of the mice inoculated with wild‐type K562 or PKMYT1‐knockdown K562 cells were measured every 3–4 days (n = 8). d) Spleen image and spleen indices of PKMYT1‐knockdown and wild‐type group mice (n = 8). e) Image and weights of metastatic tumors from PKMYT1‐knockdown and wild‐type group mice (n = 8). f) Blood smear results for PKMYT1‐knockdown and wild‐type group mice (n = 8). g‐h) Human CD45 + and CD34 + cell content in the peripheral blood of PKMYT1‐knockdown and wild‐type group mice (n = 7). For 7e, narrow spacing between metastatic tumors indicates that these tumors originate from the same mouse, whereas wide spacing suggests that the tumors originate from different mice. For 7c, the data are presented as the means ± SEMs; for 7d,e,g,h, the data are presented as the means ± SDs. Statistical significance was assessed via two‐tailed unpaired Student's t‐test, *P < 0.05, **P < 0.01, ***P < 0.001 versus the wild‐type group. Figure a created with figdraw.com.

Article Snippet: The human chronic myeloid leukemia cell line K562 was purchased from Procell (Wuhan, China).

Techniques: Knockdown, In Vivo, Two Tailed Test