Journal: Bioinformation

Article Title: Genomic amplification of chromosome 7 in the Doxorubicin resistant K562 cell line

doi: 10.6026/97320630014587

Figure Lengend Snippet: K562/Dox cell line show resistance to different chemotherapeutics. K562 and K562/Dox were incubated with increasing concentrations (0.01-100 micro molar) of a) Doxorubicin, b) Daunorubicin,c) Idarubicin, and d) Etoposide for 48h. IC 50 was determined by nonlinear regression of data points. Data represents average of three independent experiments performed in triplicates.

Article Snippet: K562 cells (CLS GmBH, Germany) were cultured in RPMI media (Gibco, life technologies, USA) supplemented with 10% FBS at 37°C in a humidified incubator K562/Doxcell line was obtained by culturing K562 cells in gradually increasing dose of Dox (10nM- 200nM).

Techniques: Incubation

Journal: Bioinformation

Article Title: Genomic amplification of chromosome 7 in the Doxorubicin resistant K562 cell line

doi: 10.6026/97320630014587

Figure Lengend Snippet: Dox sensitivity in K562 and K562/Dox resistant cell line upon indicated treatment.

Article Snippet: K562 cells (CLS GmBH, Germany) were cultured in RPMI media (Gibco, life technologies, USA) supplemented with 10% FBS at 37°C in a humidified incubator K562/Doxcell line was obtained by culturing K562 cells in gradually increasing dose of Dox (10nM- 200nM).

Techniques:

Journal: Bioinformation

Article Title: Genomic amplification of chromosome 7 in the Doxorubicin resistant K562 cell line

doi: 10.6026/97320630014587

Figure Lengend Snippet: aCGH was performed for K562/Dox using K562 as the reference genome.Graphical view representing amplification in chromosomal 7 region showing gain of ABCB1 gene locus corresponding to 7q21.12 in K562/Dox as compared to K562 cells.

Article Snippet: K562 cells (CLS GmBH, Germany) were cultured in RPMI media (Gibco, life technologies, USA) supplemented with 10% FBS at 37°C in a humidified incubator K562/Doxcell line was obtained by culturing K562 cells in gradually increasing dose of Dox (10nM- 200nM).

Techniques: Amplification

Journal: Bioinformation

Article Title: Genomic amplification of chromosome 7 in the Doxorubicin resistant K562 cell line

doi: 10.6026/97320630014587

Figure Lengend Snippet: Relative ABCB1 gene expression in K562 and K562/Dox cell lines.

Article Snippet: K562 cells (CLS GmBH, Germany) were cultured in RPMI media (Gibco, life technologies, USA) supplemented with 10% FBS at 37°C in a humidified incubator K562/Doxcell line was obtained by culturing K562 cells in gradually increasing dose of Dox (10nM- 200nM).

Techniques: Expressing

Journal: Cancers

Article Title: Folic Acid-Appended Hydroxypropyl-β-Cyclodextrin Exhibits Potent Antitumor Activity in Chronic Myeloid Leukemia Cells via Autophagic Cell Death.

doi: 10.3390/cancers13215413

Figure Lengend Snippet: Figure 3. Antitumor effect of FA-HP-β-CyD. (a,b) Antitumor activity of FA-HP-β-CyD (0, 0.05, 0.1, 0.25, 0.5, and 1.0 mM) in K562 and BV173 cells. Cells were incubated with FA-HP-β-CyD for 72 h at 37 ◦C. (c) Antitumor activity of FA-HP-β-CyD and HP-β-CyD in A549 cells. Cells were incubated with 0, 2.5, 5, 7.5, and 10 mM of FA-HP-β-CyD or HP-β-CyD for 72 h at 37 ◦C. (d,e) Treatment with FA-HP-β-CyD in the presence or absence of FA. K562 and BV173 cells were incubated for 2 h at 37 ◦C with medium only (control), medium containing FA-HP-β-CyD (5 mM) and HP-β-CyD (5 mM) in the absence and presence of FA (2 mM). Each value represents the mean ± SEM of three experiments. * p < 0.05. The cell number ratio is the number of cells calculated with the number of cells in the control as 1.

Article Snippet: The human CML cell lines BV173 and K562 were purchased from DSMZ (Braunschweig, Germany) and the American Type Culture Collection (ATCC, Manassas, VA, USA), respectively.

Techniques: Activity Assay, Incubation, Control

Journal: Cancers

Article Title: Folic Acid-Appended Hydroxypropyl-β-Cyclodextrin Exhibits Potent Antitumor Activity in Chronic Myeloid Leukemia Cells via Autophagic Cell Death.

doi: 10.3390/cancers13215413

Figure Lengend Snippet: Figure 4. FA-HP-β-CyD induces apoptosis in K562 cells, BV173 cells, and Ba/F3BCR-ABL cells. (a) K562 cells, BV173 cells, and Ba/F3BCR-ABL cells were treated with 0, 0.5, 1.0, and 1.5 mM of FA-HP-β-CyD. After 72 h of culture, the Annexin V and PI-staining was done. Representative FACS plots are shown (n = 3). (b–d) Percentages of Annexin V-positive PI-negative cells exposed to FA-HP-β-CyD for 72 h are shown. Data represent the mean ± SD of three independent experiments. * p < 0.05.

Article Snippet: The human CML cell lines BV173 and K562 were purchased from DSMZ (Braunschweig, Germany) and the American Type Culture Collection (ATCC, Manassas, VA, USA), respectively.

Techniques: Staining

Journal: Cancers

Article Title: Folic Acid-Appended Hydroxypropyl-β-Cyclodextrin Exhibits Potent Antitumor Activity in Chronic Myeloid Leukemia Cells via Autophagic Cell Death.

doi: 10.3390/cancers13215413

Figure Lengend Snippet: Figure 5. Intracellular distribution of FA-HP-β-CyD. (a) Cellular association of TRITC-FA-HP-β-Cy in K562 cells (left) and BV173 cells (right). The fluorescence intensity derived from TRITC was determined by flow cytometry at 1 h after incubation at 37 ◦C. Control: medium only. (b,c) Intra- cellular distribution of TRITC-FA-HP-β-CyD. CML cells were treated with medium only (control), TRITC-FA-HP-β-CyD (1 mM), TRITC-FA-HP-β-CyD (1 mM) and FA (2 mM), or TRITC-HP-β-CyD (10 mM) for 1 h. The experiments were performed three times independently and representative images are shown.

Article Snippet: The human CML cell lines BV173 and K562 were purchased from DSMZ (Braunschweig, Germany) and the American Type Culture Collection (ATCC, Manassas, VA, USA), respectively.

Techniques: Derivative Assay, Cytometry, Incubation, Control

Journal: Cancers

Article Title: Folic Acid-Appended Hydroxypropyl-β-Cyclodextrin Exhibits Potent Antitumor Activity in Chronic Myeloid Leukemia Cells via Autophagic Cell Death.

doi: 10.3390/cancers13215413

Figure Lengend Snippet: Figure 6. Induction of autophagy by treatment with FA-M-β-CyD. (a) Detection of autophagy in CML cells. K562 cells treated with FA-HP-β-CyD and HP-β-CyD for 2 h were exposed to Cyto-ID for 30 min. For observation, fluorescence microscopy was used. (b) Effect of HP-β-CyDs on LC3B expression in K562 cells. Cells were treated with medium only (control), FA-HP-β-CyD, HP-β-CyD, imatinib (IM), and rapamycin (Rap) for 2 h. LC3B protein levels were detected by western blotting. (c) The graph shows the fluorescence intensity of the bands. * p < 0.05 compared with the control. (d,e) Effects of chloroquine, bafilomycin A1, and LY294002 on the antitumor activity of FA-HP-β-CyD (d) and HP-β-CyD (e) in BV173 cells. Cells were incubated for 24 h. * p < 0.05 compared with FA-HP-β-CyD.

Article Snippet: The human CML cell lines BV173 and K562 were purchased from DSMZ (Braunschweig, Germany) and the American Type Culture Collection (ATCC, Manassas, VA, USA), respectively.

Techniques: Microscopy, Expressing, Control, Western Blot, Activity Assay, Incubation

Journal: Cancers

Article Title: Folic Acid-Appended Hydroxypropyl-β-Cyclodextrin Exhibits Potent Antitumor Activity in Chronic Myeloid Leukemia Cells via Autophagic Cell Death.

doi: 10.3390/cancers13215413

Figure Lengend Snippet: Figure 7. Detection of mitophagy, measurement of intracellular ATP levels, and ROS generation assay in CML cells. (a,c) Detection of mitophagy in K562 and BV173 cells. The cells were treated with Mtphagy dye, a mitophagy detection reagent, and then incubated with medium only (control), 1 mM of FA-HP-β-CyD, and 10 mM of HP-β-CyD, followed by Lyso dye, a lysosome detection reagent. Representative images are shown (n = 3). (b,d) Fluorescence intensities of Mtphagy dye and Lyso dye in the control, in HP-β-CyD-treated, and in FA-HP-β-CyD-treated cells are shown in bar graphs. * p < 0.05 compared with the control. (e) Measurement of intracellular ATP levels. K562 and BV173 cells were incubated with medium only (control), 1 mM of FA-HP-β-CyD, and 10 mM of HP-β-CyD for 2 h. Then, the cells were treated with ATP detection reagent. Bar graphs represent the mean ± SEM (n = 3 per group). * Significant difference with p < 0.05 compared with the control and FA-HP-β-CyD. (f) ROS generation assay. K562 and BV173 cells were incubated with medium only (control), 1 mM of FA-HP-β-CyD, and 10 mM of HP-β-CyD, and detected by flow cytometry using ROS detection reagents. Blue: control; orange: HP-β-CyD; and red: FA-HP-β-CyD.

Article Snippet: The human CML cell lines BV173 and K562 were purchased from DSMZ (Braunschweig, Germany) and the American Type Culture Collection (ATCC, Manassas, VA, USA), respectively.

Techniques: Incubation, Control, Fluorescence, Cytometry

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, 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

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, Transfection, Negative Control, Expressing