Journal: Non-coding RNA Research

Article Title: A novel enhancer RNA, Hmrhl, positively regulates its host gene, phkb, in chronic myelogenous leukemia

doi: 10.1016/j.ncrna.2019.08.001

Figure Lengend Snippet: Expression and coding potential analysis of Hmrhl. a. Quantitative real time PCR analysis of Hmrhl expression showed that it is expressed in all human tissues (Brain, Heart, Kidney, lung, liver, pancreas, spleen, thymus, small intestine, colon, skeletal muscle, testes, prostate, ovary, placenta, leukocyte, from left to right) examined. Lowest expression was found in skeletal muscle (SM) which was taken as control, the level of which was considered as 1 and all others were plotted in comparison to it. Highest expression was seen in spleen (spln) followed by pancreas (Pnc), testis (Tst) and other tissues. b. Northern blot detection of Hmrhl. Total RNA from HEK 293T and K562 cell lines were separated on agarose gel and subsequently hybridized with DIG labelled Hmrhl specific riboprobe to detect the transcript (i). In parallel, methylene blue staining was used to determine the size of HMRHL, using 28 S rRNA (5 kb) and 18s rRNA (1.9 kb) as reference (ii). Note that the size of Hmrhl is similar to that of 28s rRNA, revealing that Hmrhl is about 5 kb in size. c. Protein-coding potential as determined by Broad Institute's PhyloCSF data and visualized in UCSC Genome Browser, showing that Hmrhl has no coding potential. d. Circular phylogenetic tree built in iTOL (Interactive Tree of Life).

Article Snippet: Since Hmrhl locus exhibited enhancer properties in K562 Chronic Myelogenous Leukemia cells, we examined the expression profile of Hmrhl across various human cancers using a cancer specific cDNA panel (Origene, USA) by real time qPCR.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Comparison, Northern Blot, Agarose Gel Electrophoresis, Staining

Journal: Non-coding RNA Research

Article Title: A novel enhancer RNA, Hmrhl, positively regulates its host gene, phkb, in chronic myelogenous leukemia

doi: 10.1016/j.ncrna.2019.08.001

Figure Lengend Snippet: Hmrhl locus exhibits hallmarks of enhancer. a. ENCODE data visualized through Integrated Genome Viewer (IGV) for DNase hypersensitive sites, p300 binding, enhancer specific histone marks, H3K27Ac and H3K4Me1 and the promoter specific histone mark, H3K4Me3 at the 5′ end of Hmrhl, only in K562 but not in GM12878 cells. Note the two prominent peaks (red) for the enhancer mark H3K27Ac in K562. b-c. Chromatin immunoprecipitation with Ab8895 (anti-H3K4Me1 antibody) and Ab4729 (anti-H3K27Ac antibody) followed by qPCR in K562 cells. Note the enrichment of both the enhancer marks at the 5′ end of Hmrhl in the IP fraction as compared to input/PIS/gene desert region (GD), that serves as a negative control.

Article Snippet: Since Hmrhl locus exhibited enhancer properties in K562 Chronic Myelogenous Leukemia cells, we examined the expression profile of Hmrhl across various human cancers using a cancer specific cDNA panel (Origene, USA) by real time qPCR.

Techniques: Binding Assay, Chromatin Immunoprecipitation, Negative Control

Journal: Non-coding RNA Research

Article Title: A novel enhancer RNA, Hmrhl, positively regulates its host gene, phkb, in chronic myelogenous leukemia

doi: 10.1016/j.ncrna.2019.08.001

Figure Lengend Snippet: Hmrhl locus exhibits hallmarks of enhancer contd. a. Encode data shows the binding of various transcription and PolII at the 5′ end of Hmrhl. We have retained the H3K27Ac peaks in this figure also for a reference. b. Schematic for chromatin interaction analysis (ChiaPET data) for Hmrhl. The large purple-black peak representing histone marks on the extreme left denotes the promoter of phkb gene while the small purple peak at the far right represents the 5'end of Hmrhl. ChiaPET data shows the interaction of Hmrhl locus with phkb promoter, as represented by two black boxes (blue arrows) connected by a black line in b. The Hmrhl locus is expanded below in c , showing that this locus has enhancer properties only in K562 cell line (orange-yellow color), but not in other cell lines like GM12878, HepG2 or hESC. Genomic segments are colour coded by ENCODE as denoted in d , with red colour signifying active promoter ( phkb promoter at far left, black arrow in b ) while orange colour represents active enhancer at Hmrhl locus at far right (red arrow in b ).

Article Snippet: Since Hmrhl locus exhibited enhancer properties in K562 Chronic Myelogenous Leukemia cells, we examined the expression profile of Hmrhl across various human cancers using a cancer specific cDNA panel (Origene, USA) by real time qPCR.

Techniques: Binding Assay

Journal: Non-coding RNA Research

Article Title: A novel enhancer RNA, Hmrhl, positively regulates its host gene, phkb, in chronic myelogenous leukemia

doi: 10.1016/j.ncrna.2019.08.001

Figure Lengend Snippet: Hmrhl is differentially expressed in various cancers. a. Expression of Hmrhl in various normal and cancer samples as observed by qPCR. Note that Hmrhl is highly upregulated in several lymphoma samples (bracket) in comparison to normal range (arrow). In fact, of all cancers, the highest levels of Hmrhl are seen in some of the lymphoma samples. b-c. qPCR analysis of Hmrhl and PHKB expression showing that both are over expressed in K562 leukemia condition as compared to GM12878 normal lymphocytes.

Article Snippet: Since Hmrhl locus exhibited enhancer properties in K562 Chronic Myelogenous Leukemia cells, we examined the expression profile of Hmrhl across various human cancers using a cancer specific cDNA panel (Origene, USA) by real time qPCR.

Techniques: Expressing, Comparison

Journal: Non-coding RNA Research

Article Title: A novel enhancer RNA, Hmrhl, positively regulates its host gene, phkb, in chronic myelogenous leukemia

doi: 10.1016/j.ncrna.2019.08.001

Figure Lengend Snippet: Hmrhl functions as enhancer RNA for phkb gene. a. Lucifaerase assay showing the intense signal of reporter activity in K562 cells with insert 3 cloned in enhancer vector. Note the low level of luciferase signal obtained with insert 2 both with promoter and enhancer vectors. b. siRNA (Sigma) mediated down-regulation of Hmrhl causes down-regulation of PHKB in K562 cells treated with Hmrhl specific siRNA pool as compared to control cells without transfection and cells treated with scrambled siRNA as negative control. c-d. Smart pool siRNA (Dharmacon) were used against the Hmrhl region to downregulate Hmrhl and subsequently expression level of PHKB gene were checked by qPCR in both K562 and GM12878 cell lines. Scrambled siRNA was used as a negative control. Note the down regulation of PHKB only in K562.

Article Snippet: Since Hmrhl locus exhibited enhancer properties in K562 Chronic Myelogenous Leukemia cells, we examined the expression profile of Hmrhl across various human cancers using a cancer specific cDNA panel (Origene, USA) by real time qPCR.

Techniques: Activity Assay, Clone Assay, Plasmid Preparation, Luciferase, Control, Transfection, Negative Control, Expressing

Journal: Nature Communications

Article Title: EXPERT expands prime editing efficiency and range of large fragment edits

doi: 10.1038/s41467-025-56734-9

Figure Lengend Snippet: a Frequencies of intended edits and indels introduced by PE2max, PE3max, PE4max, PE5max and their corresponding EXPERTmax systems. Additional mismatches were introduced in the insertion-type edits. Bars represent the mean of n = 3 independent biological replicates. Data are presented as mean ± s.d. b Frequencies of intended edits and indels introduced by PE2max and EXPERTmax in K562 cells. Additional mismatches were introduced in the insertion-type edits. Bars represent the mean of n = 3 independent biological replicates. Data are presented as mean ± s.d. c Frequencies of intended edits and indels introduced by PE2max and EXPERTmax in Jurkat cells. Additional mismatches were introduced in the insertion-type edits. Bars represent the mean of n = 3 independent biological replicates. Data are presented as mean ± s.d. d Frequencies of intended edits and indels introduced by PE2max and EXPERTmax in Hela cells. Additional mismatches were introduced in the insertion-type edits. Bars represent the mean of n = 3 independent biological replicates. Data are presented as mean ± s.d. e Frequencies of intended edits, indels, and product purity introduced by PE2max and EXPERTmax in N2a cells. Bars represent the mean of n = 3 independent biological replicates. Data are presented as mean ± s.d. f Frequencies of intended edits, indels, and product purity introduced by PE2max and EXPERTmax in PFF cells. Additional mismatches were introduced in the insertion-type edits. Bars represent the mean of n = 3 independent biological replicates. Data are presented as mean ± s.d. g Schematic diagram of complex mutations in CFTR exon 4. The intended edits that carried four mutations were performed in HEK293T cells using PE2max, PE3max, EXPERTmax, and EXPERTmax + nicking sgRNA, respectively. h Frequencies of intended edits, indels, and product purity introduced by PE2max, PE3max, EXPERTmax, and EXPERTmax + nicking sgRNA, respectively. Bars represent the mean of n = 3 independent biological replicates. Data are presented as mean ± s.d. All sequencing data were collected from transfection-positive cells. Source data are provided as a file.

Article Snippet: HEK293T (ATCC, CRL-11268) and 293T-reporter cells were maintained in DMEM (Gibco) + 10% FBS (CELLiGENT) + 1% penicillin-streptomycin (Gibco); K562 (ATCC, CRL-3344) and Jurkat (ATCC, TIB-152) cells were maintained in RPMI Medium 1640 (Gibco) + 10% FBS (CELLiGENT) + 1% penicillin-streptomycin (Gibco); Hela (Procell, CL-0101), HFL1 (Procell, CL-0106) and N2a (Procell, CL-0168) cells were kindly provided by Procell Life Science & Technology Co., Ltd. Hela (Procell) and N2a (Procell) cells were maintained in MEM (Procell) + 10% FBS (CELLiGENT) + 1% penicillin-streptomycin (Gibco).

Techniques: Sequencing, Transfection

Journal: Journal of pharmacological sciences

Article Title: Aldo-keto reductase inhibitors increase the anticancer effects of tyrosine kinase inhibitors in chronic myelogenous leukemia.

doi: 10.1016/j.jphs.2021.05.001

Figure Lengend Snippet: Fig. 1. Continuous exposure to tyrosine kinase inhibitors (TKIs) leads to glucose accumulation in chronic myelogenous leukemia cells (A) K562 cells were treated with (0.1 or 0.3 mM) or without imatinib for 72 h and the amount of lactate released into the culture medium for the last 24 h was evaluated. Data represent means ± SD of three independent cell cultures. One-way ANOVA was followed by Bonferroni's test. **P < 0.01 (B) K562 cells were treated with a TKI (imatinib, nilotinib, or bosutinib) at the indicated concentrations for 8 days, and the intracellular glucose level was measured. Data represent means ± SD of three independent cell cultures. One-way ANOVA was followed by Bonferroni's test. **P < 0.01 (C) K562 cells were cultured in the presence of imatinib (0.1 or 0.3 mM) for 3, 6, and 9 days, and cell death was evaluated by flow cytometry after propidium iodide uptake. Data represent means ± SD of three independent cell cultures. One-way ANOVA was followed by Bonferroni's test. **P < 0.01, compared with the value at 0 days.

Article Snippet: K562 cells were stained with the Zombie NIRTM Fixable Viability Kit (BioLegend, San Diego, CA, USA) to distinguish dead cells from live cells.

Techniques: Cell Culture, Cytometry

Journal: Journal of pharmacological sciences

Article Title: Aldo-keto reductase inhibitors increase the anticancer effects of tyrosine kinase inhibitors in chronic myelogenous leukemia.

doi: 10.1016/j.jphs.2021.05.001

Figure Lengend Snippet: Fig. 2. Exposure to imatinib increases AKR1B expression in chronic myelogenous leukemia cells (A) K562 cells were cultured in the presence (0.1 mM) or absence of imatinib for 8 days, and AKR activity was evaluated. Data represent means ± SD of three independent cell cultures. Differences between groups were analyzed using Student's t-test. **P < 0.01 (B, C) Quantitative RT-PCR was performed to measure the mRNA levels of AKR1B1 and AKR1B10 in K562 cells treated with (0.1 mM (B) or 0.3 mM (C)) or without imatinib for 5 days. Data represent means ± SD of five inde- pendent cell cultures. Differences between groups were analyzed using Student's t- test. **P < 0.01.

Article Snippet: K562 cells were stained with the Zombie NIRTM Fixable Viability Kit (BioLegend, San Diego, CA, USA) to distinguish dead cells from live cells.

Techniques: Expressing, Cell Culture, Activity Assay, Quantitative RT-PCR

Journal: Journal of pharmacological sciences

Article Title: Aldo-keto reductase inhibitors increase the anticancer effects of tyrosine kinase inhibitors in chronic myelogenous leukemia.

doi: 10.1016/j.jphs.2021.05.001

Figure Lengend Snippet: Fig. 3. Higher glucose accumulation by imatinib induces AKR hyperactivation in chronic myelogenous leukemia cells. K562 cells were preincubated in low-glucose or normal medium for 72 h and then treated with (0.3 mM) or without imatinib for 5 days (A) Intracellular glucose levels were measured. Data represent means ± SD of three independent cell cultures. One-way ANOVA was followed by Bonferroni's test. **P < 0.01 (B) AKR activity was evaluated. Data represent means ± SD of three inde- pendent cell cultures. Differences between groups were analyzed using Student's t- test. N.S., not significant (C) Quantitative RT-PCR of AKR1B1 and AKR1B10 mRNA was performed. Data represent means ± SD of five independent cell cultures. One-way ANOVA was followed by Bonferroni's test. **P < 0.01 (D) Representative western blotting of nuclear and cytoplasmic protein extracts with anti-Nrf2, anti-HDAC2, and anti-GAPDH antibodies. IM, imatinib-treated lysates.

Article Snippet: K562 cells were stained with the Zombie NIRTM Fixable Viability Kit (BioLegend, San Diego, CA, USA) to distinguish dead cells from live cells.

Techniques: Activity Assay, Quantitative RT-PCR, Western Blot

Journal: Journal of pharmacological sciences

Article Title: Aldo-keto reductase inhibitors increase the anticancer effects of tyrosine kinase inhibitors in chronic myelogenous leukemia.

doi: 10.1016/j.jphs.2021.05.001

Figure Lengend Snippet: Fig. 4. Epalrestat (EPS) treatment along with imatinib increases intracellular oxidative stress in chronic myelogenous leukemia cells (A) K562 cells pretreated with or without EPS (80 mM) for 72 h were additionally treated with (0.3 mM) or without imatinib for 5 days. The intracellular GSH/GSSG ratio was evaluated. Data represent means ± SD of three independent cell cultures. One-way ANOVA was followed by Bonferroni's test. **P < 0.01 (B) Proliferation of the K562 cells was evaluated by CFSE dye dilution in 3-day cultures using flow cytometry. The CFSE concentration per cell is lower in highly proliferative cells and higher in slower-growing cells. Data are representative of three independent cell culture experiments.

Article Snippet: K562 cells were stained with the Zombie NIRTM Fixable Viability Kit (BioLegend, San Diego, CA, USA) to distinguish dead cells from live cells.

Techniques: Cytometry, Concentration Assay, Cell Culture

Journal: Journal of pharmacological sciences

Article Title: Aldo-keto reductase inhibitors increase the anticancer effects of tyrosine kinase inhibitors in chronic myelogenous leukemia.

doi: 10.1016/j.jphs.2021.05.001

Figure Lengend Snippet: Fig. 5. Epalrestat (EPS) enhances anticancer effects of tyrosine kinase inhibitors (TKIs) in chronic myelogenous leukemia cells (AeC) K562 cells pretreated with (80 mM) or without EPS for 72 h were then treated with vehicle (DMSO) only (A), imatinib (B), nilotinib, or bosutinib (C) for the indicated days. Cell death was then evaluated. Data represent means ± SD of three independent cell cultures. Two-way ANOVA was followed by Bonferroni's test. *P < 0.05, **P < 0.01 compared with corresponding value from cells not treated with EPS. #P < 0.05, ##P < 0.01.

Article Snippet: K562 cells were stained with the Zombie NIRTM Fixable Viability Kit (BioLegend, San Diego, CA, USA) to distinguish dead cells from live cells.

Techniques:

Journal: Journal of pharmacological sciences

Article Title: Aldo-keto reductase inhibitors increase the anticancer effects of tyrosine kinase inhibitors in chronic myelogenous leukemia.

doi: 10.1016/j.jphs.2021.05.001

Figure Lengend Snippet: Fig. 6. AKR1B10 inhibition enhances anticancer effects of imatinib in chronic myelogenous leukemia cells (A, B) K562 cells pretreated with HCCFA, a selective inhibitor of AKR1B10 (10 mM) for 72 h were then treated with vehicle (DMSO) only (A) or imatinib (B) for the indicated days. Cell death was then evaluated. Data represent means ± SD of three in- dependent cell cultures. Two-way ANOVA was followed by Bonferroni's test. *P < 0.05, **P < 0.01 compared with corresponding value from cells not treated with HCCFA. ##P < 0.01.

Article Snippet: K562 cells were stained with the Zombie NIRTM Fixable Viability Kit (BioLegend, San Diego, CA, USA) to distinguish dead cells from live cells.

Techniques: Inhibition

Journal: bioRxiv

Article Title: A Cellular Cytotoxicity Assay using Ready-to-Thaw Target Cells without Washing Steps

doi: 10.64898/2026.01.21.700863

Figure Lengend Snippet: Toxicity of different concentrations of the cryopreservation medium STEM-CELLBANKER ® EX (SCB) towards K562 cells, primary NK cells, and NK-92 cells; cell viability was assessed via flow cytometry after 24 h incubation. A: Relative viability of K562 cells incubated with 0–100% SCB at 37°C and room temperature (RT) (n = 3 independent experiments, each 1 technical replicate); statistical analysis: two-way ANOVA with Tukey post hoc test within RT and 37°C groups. B: Relative viability of primary NK cells with 0–100% SCB at RT, 37°C, and 37°C + 500 U/mL IL-2 (left, n = 1 experiment, 1 technical replicate; no statistics performed) and with 0–25% SCB at 37°C (right, n = 2 donors/experiments, each 3 technical replicates); statistical analysis: two-way ANOVA with Tukey post hoc test. C: Relative viability of NK-92 cells with 0–100% SCB (left, n = 1 experiment, 3 technical replicates) and 0– 25% SCB (right, n = 1 experiment, 3 technical replicates) at 37°C; statistical analysis: one-way ANOVA with Tukey post hoc test. Bars represent mean ± SEM (ns = not significant; *p < 0.05; **p < 0.01; ****p < 0.0001).

Article Snippet: K562 cells (ACC-10, DSMZ–German Collection of Microorganisms and Cell Cultures, Leibniz Institute, Braunschweig, Germany) were cultured in RPMI-1640 medium (Sigma-Aldrich, Schnelldorf, Germany, or PAN-Biotech, Aidenbach, Germany) supplemented with 10% fetal bovine serum (FBS; VWR, Darmstadt, Germany, or PAN-Biotech), 2 mM L-glutamine (Sigma-Aldrich), and 100 U/mL penicillin plus 100 μg/mL streptomycin (Sigma-Aldrich).

Techniques: Flow Cytometry, Incubation

Journal: bioRxiv

Article Title: A Cellular Cytotoxicity Assay using Ready-to-Thaw Target Cells without Washing Steps

doi: 10.64898/2026.01.21.700863

Figure Lengend Snippet: Effects of K562 cell cryopreservation in STEM-CELLBANKER ® EX (SCB) and post-thaw washing; all readouts normalized to pre-cryopreservation values. (A , B) K562 cells were cryopreserved at 1, 2, 3, 4, and 5 × 10^6 cells/mL in 1-mL cryotubes; after thawing, aliquots were either washed or not washed before flow-cytometric analysis. A: pooled relative viability (left), pooled viable recovery (middle), and pooled total recovery (right). B: relative viability shown separately for each freezing density (n = 1 experiment, 3 technical replicates); statistical analysis: two-way ANOVA with Tukey post hoc test. C: CellTrace™ Violet–labeled K562 cells were cryopreserved in 1-mL cryotubes and thawed at different time points; aliquots were washed or not washed prior to analysis (n = 1 experiment, 3 technical replicates); statistical analysis: two-way ANOVA with Tukey post hoc test. Bars represent mean ± SEM (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

Article Snippet: K562 cells (ACC-10, DSMZ–German Collection of Microorganisms and Cell Cultures, Leibniz Institute, Braunschweig, Germany) were cultured in RPMI-1640 medium (Sigma-Aldrich, Schnelldorf, Germany, or PAN-Biotech, Aidenbach, Germany) supplemented with 10% fetal bovine serum (FBS; VWR, Darmstadt, Germany, or PAN-Biotech), 2 mM L-glutamine (Sigma-Aldrich), and 100 U/mL penicillin plus 100 μg/mL streptomycin (Sigma-Aldrich).

Techniques: Labeling

Journal: bioRxiv

Article Title: A Cellular Cytotoxicity Assay using Ready-to-Thaw Target Cells without Washing Steps

doi: 10.64898/2026.01.21.700863

Figure Lengend Snippet: Cellular cytotoxicity assay using ready-to-thaw target cells without washing. CFSE-stained K562 targets were frozen in 40 µL aliquots of SCB with 20% FCS using isopropanol thermal buffering, thawed by direct addition of pre-warmed medium, and co-incubated with NK-92 cells for 4 h; EDTA and propidium iodide were then added for conjugate dissociation and dead-cell staining. A: Relative (left) and absolute (right) viability of target cells with-out NK-92 before cryopreservation, after thawing, and after the cytotoxicity assay (n = 1 experiment, 4–5 technical replicates: 5 before cryopreservation; 4 for both post-thaw conditions); statistical analysis: ordinary one-way ANOVA with Tukey post hoc test (both). B: Consistency of viable recovery (left) enables adjustment of pre-cryo-preservation target-cell density to achieve the desired post-thaw cell count (right) (n = 1 experiment, 4–5 technical replicates: 5 before cryopreservation; 4 after thawing); statistical analysis: unpaired t test (both). C: Specific lysis measured with NK-92 effectors by viability-based calculation (upper) and by volumetric absolute counts (lower) (n = 1 experiment, 4–5 technical replicates per E:T ratio: 4 for 0:1 and 9:1; 5 for 1:1 and 3:1). Bars represent mean ± SEM. (*p < 0.05; ****p < 0.0001).

Article Snippet: K562 cells (ACC-10, DSMZ–German Collection of Microorganisms and Cell Cultures, Leibniz Institute, Braunschweig, Germany) were cultured in RPMI-1640 medium (Sigma-Aldrich, Schnelldorf, Germany, or PAN-Biotech, Aidenbach, Germany) supplemented with 10% fetal bovine serum (FBS; VWR, Darmstadt, Germany, or PAN-Biotech), 2 mM L-glutamine (Sigma-Aldrich), and 100 U/mL penicillin plus 100 μg/mL streptomycin (Sigma-Aldrich).

Techniques: Cytotoxicity Assay, Staining, Incubation, Preserving, Cell Characterization, Lysis

Journal: Scientific Reports

Article Title: Neddylation status determines the therapeutic sensitivity of tyrosine kinase inhibitors in chronic myeloid leukemia

doi: 10.1038/s41598-025-04153-7

Figure Lengend Snippet: Neddylation inhibitor blunted the therapeutic function of BCR::ABL1-targeting TKIs. (A, B) K562 cells were treated with imatinib (0.5 µM), nilotinib (0.1 µM), ponatinib (2.5 nM) ( A ), or asciminib (50 nM) ( B ) in the presence or absence of MLN4924 (25 µM) for 24 h. Cell viability was evaluated by CCK8. ( C ) KU812 cells were treated with imatinib in the presence or absence of MLN4924 for 24 h. Cell viability was evaluated by CCK8. ( D ) K562 cells were treated with the indicated TKIs in the presence or absence of MLN4924 (25 µM) for 24 h, cell counting was performed following Hoechst staining. ( E ) K562 cells were treated with imatinib (0.5 µM) in the presence or absence of MLN4924 (25 µM) for 24 h. Cell apoptosis was evaluated by Annexin V/PI staining. ( F ) K562 cells were treated with imatinib (0.5 µM), nilotinib (0.1 µM), or ponatinib (2.5 nM) in the presence or absence of TAS4464 (1 µM) for 24 h. Cell viability was evaluated by CCK8. ( G ) K562 cells were treated with the indicated TKIs in the presence or absence of TAS4464 (1 µM) for 24 h, cell counting was performed following Hoechst staining. ( H ) UBA3 was silenced in K562 cells, followed by imatinib (0.5 µM) treatment for 24 h, cell viability was evaluated by CCK8. Unpaired, two-tailed Student’s t-test. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: K562 cells were treated with 0.5 μM imatinib in the presence or absence of 25 μM MLN4924 for 24 h. Cell apoptosis was evaluated using Annexin V-APC/PI Apoptosis Kit (Elabscience, Wuhan, China) following the manufacturer’s protocol.

Techniques: Cell Counting, Staining, Two Tailed Test

Journal: Scientific Reports

Article Title: Neddylation status determines the therapeutic sensitivity of tyrosine kinase inhibitors in chronic myeloid leukemia

doi: 10.1038/s41598-025-04153-7

Figure Lengend Snippet: Neddylation activator sensitizes the function of imatinib. ( A ) K562 cells were treated with imatinib (0.1 µM) in the presence or absence of VII-31 (80 nM) for 24 h. Cell viability was evaluated by CCK8. (B, C) K562 cells were treated with 10 µM cisplatin (CDDP) ( B ) or 0.5 µM doxorubicin ( C ) in the presence or absence of MLN4924 (25 µM) for 24 h. Cell viability was evaluated by CCK8. Data are presented as mean ± SEM. Unpaired, two-tailed Student’s t-test. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: K562 cells were treated with 0.5 μM imatinib in the presence or absence of 25 μM MLN4924 for 24 h. Cell apoptosis was evaluated using Annexin V-APC/PI Apoptosis Kit (Elabscience, Wuhan, China) following the manufacturer’s protocol.

Techniques: Two Tailed Test

Journal: Scientific Reports

Article Title: Neddylation status determines the therapeutic sensitivity of tyrosine kinase inhibitors in chronic myeloid leukemia

doi: 10.1038/s41598-025-04153-7

Figure Lengend Snippet: RNA sequencing analysis of the transcriptomal features in CML cells. (A, B) K562 cells were treated with imatinib (0.5 µM) in the presence or absence of MLN4924 (25 µM) for 12 h, RNA sequencing was performed. PCA analysis ( A ) and sample correlation analysis ( B ) were performed. ( C ) GO analysis of the DEGs between imatinib group and imatinib + MLN4924 group. (D-H) Construction of WGCNA network of all RNA sequencing samples: ( D ) Dendrogram of gene topological matrix branch. ( E ) Dendrogram and correlation heatmap of 10 eigengenes in each module. ( F ) Heatmap of correlations between gene modules and each group of RNA-seq samples. The Pearson correlation coefficient (R) between different gene modules and clinical traits were shown in the heatmap. ( G ) Scatter plot of gene significance for selected groups with module membership (MM) in the most significantly positively correlated module. The brown module showed significant positive correlations with both MLN and IMA + MLN groups. ( H ) GO enrichment analysis of genes in blue, red, turquoise, and brown modules.

Article Snippet: K562 cells were treated with 0.5 μM imatinib in the presence or absence of 25 μM MLN4924 for 24 h. Cell apoptosis was evaluated using Annexin V-APC/PI Apoptosis Kit (Elabscience, Wuhan, China) following the manufacturer’s protocol.

Techniques: RNA Sequencing

Journal: Scientific Reports

Article Title: Neddylation status determines the therapeutic sensitivity of tyrosine kinase inhibitors in chronic myeloid leukemia

doi: 10.1038/s41598-025-04153-7

Figure Lengend Snippet: ABL1 is a novel neddylation substrate. ( A ) The binding of NEDD8 to ABL1 domain was evaluated by co-IP in K562 cells. ( B ) ABL1 structure predicted by an AI large-molecule structure predicting tool ESM-fold. The light-green sites represent K1080, K87, and K756. ( C ) ABL1 structure predicted by AlphaFold3. The orange sites represent K1080, K87, and K756. ( D ) ABL1 structure before and after deneddylation as predicted by ESM-fold. The blue color represents structure after deneddylation of ABL1, the purple color indicates ABL1 bound to a NEDD8 molecule. The pink color represents the NEDD8 molecule. The red site is K1080, the yellow site is K87, the green site is K756. ( E ) ABL1 structure before and after deneddylation as predicted by AlphaFold3. The yellow color indicates ABL1 structure after deneddylation, the pink color represents ABL1 neddylated by one NEDD8 molecule. The purple color represents NEDD8.

Article Snippet: K562 cells were treated with 0.5 μM imatinib in the presence or absence of 25 μM MLN4924 for 24 h. Cell apoptosis was evaluated using Annexin V-APC/PI Apoptosis Kit (Elabscience, Wuhan, China) following the manufacturer’s protocol.

Techniques: Binding Assay, Co-Immunoprecipitation Assay

Journal: Scientific Reports

Article Title: Neddylation status determines the therapeutic sensitivity of tyrosine kinase inhibitors in chronic myeloid leukemia

doi: 10.1038/s41598-025-04153-7

Figure Lengend Snippet: A low neddylation status inhibits the effectiveness of imatinib therapy in murine CML model. (A, B) K562 cells were subcutaneously inoculated into nude mice ( n = 6/group), followed by the administration of imatinib (100 mg/kg), MLN4924 (50 mg/kg), or imatinib plus MLN4924, tumor growth was monitored ( A ), tumor weight was evaluated at the experimental endpoint ( B ). Data are presented as mean ± SEM. Unpaired, two-tailed Student’s t-test. * p < 0.05.

Article Snippet: K562 cells were treated with 0.5 μM imatinib in the presence or absence of 25 μM MLN4924 for 24 h. Cell apoptosis was evaluated using Annexin V-APC/PI Apoptosis Kit (Elabscience, Wuhan, China) following the manufacturer’s protocol.

Techniques: Two Tailed Test