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: BioMed Research International

Article Title: Pegylated Gold Nanoparticles Induce Apoptosis in Human Chronic Myeloid Leukemia Cells

doi: 10.1155/2014/182353

Figure Lengend Snippet: (a) Cell viability rate and (b) cell viability count of K562 cells treated with the control medium, 5 and 10 mM gold nanoparticles (AuNPs), with their surface modified by polyethylene glycol (PEG-AuNPs) for 24–72 h. Results are means ± standard error (SE) based on triplicate experiments.

Article Snippet: Human chronic myeloid leukemia cell line K562 (ATCC, Manassas, VA, USA) was used in this study.

Techniques: Control, Modification

Journal: BioMed Research International

Article Title: Pegylated Gold Nanoparticles Induce Apoptosis in Human Chronic Myeloid Leukemia Cells

doi: 10.1155/2014/182353

Figure Lengend Snippet: K562 cells were stained with Liu's dye for morphological examinations. Cells were treated with medium only (upper panels), 10 mM AuNPs (middle panels), after 24–72 h of treatment. The arrow indicates an apoptotic cell (lower panels). Original magnification is 1,000x for all panels.

Article Snippet: Human chronic myeloid leukemia cell line K562 (ATCC, Manassas, VA, USA) was used in this study.

Techniques: Staining

Journal: BioMed Research International

Article Title: Pegylated Gold Nanoparticles Induce Apoptosis in Human Chronic Myeloid Leukemia Cells

doi: 10.1155/2014/182353

Figure Lengend Snippet: Morphological appearance of gold nanoparticles (AuNPs) with their surface modified by polyethylene glycol (PEG-AuNPs) imaged by transmission electronic microscopy. K562 cells were incubated with (a) medium only, 10 mM of PEG-AuNPs, for (b) 24 h, (c) 48 h, and (d) 72 h. Original magnification is 4,000x for all panels.

Article Snippet: Human chronic myeloid leukemia cell line K562 (ATCC, Manassas, VA, USA) was used in this study.

Techniques: Modification, Transmission Assay, Microscopy, Incubation

Journal: BioMed Research International

Article Title: Pegylated Gold Nanoparticles Induce Apoptosis in Human Chronic Myeloid Leukemia Cells

doi: 10.1155/2014/182353

Figure Lengend Snippet: Cell cycle distribution of K562 cells treated with medium only, PEG-AuNPs 10 mM, for (a) 24 h, (b) 48 h, and (c) 72 h (compared with the control medium: * P < 0.05 and ** P < 0.01). Cell cycle distribution was analyzed by flow cytometry and the percentages of cells in different cycle phases were automatically determined using ModFit cell cycle analysis software. Data are representative of three independent experiments.

Article Snippet: Human chronic myeloid leukemia cell line K562 (ATCC, Manassas, VA, USA) was used in this study.

Techniques: Control, Flow Cytometry, Cell Cycle Assay, Software

Journal: BioMed Research International

Article Title: Pegylated Gold Nanoparticles Induce Apoptosis in Human Chronic Myeloid Leukemia Cells

doi: 10.1155/2014/182353

Figure Lengend Snippet: K562 cells treated with 10 mM of gold nanoparticles (AuNPs) with their surface modified by polyethylene glycol (PEG-AuNPs) for 24 h. Fluorescence could be detected with emission wavelengths from 500–700 nm by (a) fluorescence microscopy: bright field images (left), fluorescent images (middle), and merged bright field and fluorescent images (right). Original magnification is 1,000x for all panels and (b) flow cytometry analysis.

Article Snippet: Human chronic myeloid leukemia cell line K562 (ATCC, Manassas, VA, USA) was used in this study.

Techniques: Modification, Fluorescence, Microscopy, Flow Cytometry

Journal: BioMed Research International

Article Title: Pegylated Gold Nanoparticles Induce Apoptosis in Human Chronic Myeloid Leukemia Cells

doi: 10.1155/2014/182353

Figure Lengend Snippet: Effect of gold nanoparticles (AuNPs) with their surface modified by polyethylene glycol (PEG-AuNPs) on mitochondrial transmembrane potential reduction in K562 cells. Bar chart representing the relative level of mitochondrial transmembrane potential between K562 cells treated with PEG-AuNP and control medium for 24–72 h (compared with the control medium: * P < 0.05 and ** P < 0.01).

Article Snippet: Human chronic myeloid leukemia cell line K562 (ATCC, Manassas, VA, USA) was used in this study.

Techniques: Modification, Control

Journal: Molecules

Article Title: Betulin, a Newly Characterized Compound in Acacia auriculiformis Bark, Is a Multi-Target Protein Kinase Inhibitor

doi: 10.3390/molecules26154599

Figure Lengend Snippet: In vitro effects of betulin on the viability of K562 chronic myelogenous leukemia (CML) cells and human peripheral blood lymphocytes (hPBLs). ( a ) The viability of K562S and K562R CML cell lines, which are respectively sensitive (S) and resistant (R) to treatment with doxorubicin, was studied with the MTS assay. Cell viability was measured after 48 h exposure to increasing doses of betulin. The IC 50 values were determined from the dose-response curves using GraphPad PRISM Software. Data are given as mean ± SD (n = 3) expressed in % of maximal viability (normal cells treated with the identical quantity of vehicle, DMSO, only). ( b ) The viability of K562S and K562R CML cell lines was measured after 48 h exposure to 0.1 or 1 µM of imatinib mesylate. Data are provided as mean ± SD (n = 3) expressed in % of maximal viability (relative to DMSO control, as in ( a )). ( c ) Cell viability 48 h after treatment with increasing concentrations of betulin (0.02– 100 µM), measured with MTS assay, showing the toxicity of betulin towards human PBL. Data are expressed as mean ± SD (N = 4, n = 3).

Article Snippet: K562 (ATCC ® , CCL-243, described here as K562S to indicate sensitivity to doxorubicin), a human chronic myelogenous leukemia cell line, was obtained from American Type Culture Collection (Manassas, VA, USA).

Techniques: In Vitro, MTS Assay, Software, Control