k562 whole cell lysate standard  (Promega)

Journal: Journal of Proteome Research

Article Title: Comprehensive Protein Inference Analysis with PyProteinInference Elucidates Biological Understanding of Tandem Mass Spectrometry Data

doi: 10.1021/acs.jproteome.4c00734

Figure Lengend Snippet: Upset plot showing the overlap of identified proteins at a 1% protein false discovery rate (FDR) from the K562 whole cell lysate data set. The X axis indicates the individual method or overlapping methods examined, while the Y axis indicates the intersection size between methods on the X axis. Peptide-centric protein groups are expanded out to individual proteins to facilitate comparisons to other methods that only output protein group leads; individual methods show total counts for comparison purposes (not only counts unique to the method).

Article Snippet: For algorithm validation of pyProteinInference, 0.5 μg of K562 whole cell lysate (Promega, Madison WI; Catalog #V6951 was loaded into a 25 cm × 75 μm ID, 1.6 μm C18 IonOpticks Aurora Series column (IonOpticks, AUR2-25075C18A) on a Thermo UltiMate 3000 high-performance liquid chromatography (HPLC) system (Thermo Fisher Scientific) at a flow rate of 400 nl min –1 .

Techniques: 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: 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

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Imaging of Cell-Cell Communication in a Vertical Orientation Reveals High-Resolution Structure of Immunological Synapse and Novel PD-1 Dynamics

doi: 10.4049/jimmunol.1403143

Figure Lengend Snippet: Optical geometry and device design. (A) The optical geometry (left) for imaging the IS by conventional methods and corresponding simulated point-spread-function (PSF, right) of the excitation beam. (B) The optical geometry (left) for imaging the IS by the VCP system and corresponding simulated PSF (right) of the excitation beam. (C) Overall design of the microfluidic platform and flow pathways during cell loading. VCP ver.3 is shown. (D) Wide field fluorescent microscopic image merged with bright field image of the microfluidic device after cell loading. Red and green channels correspond to K562 and KHYG-1 cells, respectively. Scale bar indicates 100 µm (left) and 20 µm (right), respectively. (E) Percentage of trap-captured cells in each step during the cell loading procedure. The graph shows average and standard deviation (SD) of cell capture efficiency over four different area of the device. The results are from three independent experiments.

Article Snippet: Plasmids and transduction of CD16-KHYG-1 and K562 cell lines To generate the PD-1-GFP construct, full length of PD-1-GFP fusion protein sequence (OriGene, MD) was amplified by the primers 5’-AATCCGGAATTCGCCGCCGCGATCGCCATGC-3’ (Forward) and 5’-AATCGCGGATCCTTAAACTCTTTCTTCACC-3’ (Reverse).

Techniques: Imaging, Standard Deviation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Imaging of Cell-Cell Communication in a Vertical Orientation Reveals High-Resolution Structure of Immunological Synapse and Novel PD-1 Dynamics

doi: 10.4049/jimmunol.1403143

Figure Lengend Snippet: Time-lapse images of ‘Dispersed→stay-Centralized’ (D→sC) PD-1/PD-L1 clusters (9.1%, 2 out of 22 observations) by 3D confocal fluorescence microscope. (A) Schematic model (left) and time series taken from Supplemental Movie 3 (right). Fluorescent images of selected time points from live cell imaging by using the VCP ver.2 system for IS formation between PD-L1-mCherry+ K562 (bottom, red) and PD-1-GFP+ CD16-KHYG-1 (top, green) cells. PD-1-GFP, PD-L1-mCherry, bright field, merged, and colocalization of PD-1 and PD-L1 are presented. Scale bar, 10 µm. (B) Track path of the clusters in the colocalized fluorescence image. Black dashed line indicates central cluster region. (C) Fluorescent image merged with bright field image after disconnecting negative pressure.

Article Snippet: Plasmids and transduction of CD16-KHYG-1 and K562 cell lines To generate the PD-1-GFP construct, full length of PD-1-GFP fusion protein sequence (OriGene, MD) was amplified by the primers 5’-AATCCGGAATTCGCCGCCGCGATCGCCATGC-3’ (Forward) and 5’-AATCGCGGATCCTTAAACTCTTTCTTCACC-3’ (Reverse).

Techniques: Fluorescence, Microscopy, Live Cell Imaging

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Imaging of Cell-Cell Communication in a Vertical Orientation Reveals High-Resolution Structure of Immunological Synapse and Novel PD-1 Dynamics

doi: 10.4049/jimmunol.1403143

Figure Lengend Snippet: Comparison of IS structure by conventional (Conv.) and VCP systems imaged by confocal fluorescence microscopy. CD16-KHYG-1 (NK) cell conjugated with K562 (target) cell on cover glass coated with poly-L-lysine (A) and vertical stack of NK-target cell pair by VCP ver.2 (B) are fixed, permeabilized, and stained for F-actin (red), perforin (green), and α-tubulin (cyan). Scale bars indicate 5 µm. The three-dimensional (3D) fluorescent images centered at the IS are z-projected. Magnified areas (8×8 µm, white boxes) from the fluorescent microscope image obtained by using coverslips coated with poly-L-lysine (C) and by using the VCP ver.2 system (D). Perforin (green) and F-actin (red) are visualized. Scale bars indicate 2 µm. (E) Fluorescence intensity profile of F-actin and perforin was measured across the white line. Fluorescence intensity of the perforin and F-actin is shown in green and red line, respectively. (F) Full width at half maximum (FWHM) and standard error of fluorescence intensity for individual perforin granule along the vertical line (blue line in C and D). (G) Pearson’s correlation coefficient (r) was calculated for perforin and F-actin from 3D colocalization analysis. Each dot represents each pair (n = 46 for conventional, n = 45 for VCP imaging). (H) Pearson’s correlation coefficient (r) was calculated from 2D colocalization analysis. The same set of data as (G) was used. (I) Costes P-value (indicating the reliability of Pearson’s correlation coefficient analysis) obtained during 2D colocalization analysis. VCP ver.3 was used for (G-I).

Article Snippet: Plasmids and transduction of CD16-KHYG-1 and K562 cell lines To generate the PD-1-GFP construct, full length of PD-1-GFP fusion protein sequence (OriGene, MD) was amplified by the primers 5’-AATCCGGAATTCGCCGCCGCGATCGCCATGC-3’ (Forward) and 5’-AATCGCGGATCCTTAAACTCTTTCTTCACC-3’ (Reverse).

Techniques: Fluorescence, Microscopy, Staining, Imaging

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Imaging of Cell-Cell Communication in a Vertical Orientation Reveals High-Resolution Structure of Immunological Synapse and Novel PD-1 Dynamics

doi: 10.4049/jimmunol.1403143

Figure Lengend Snippet: Kinetics of cytotoxicity mediated by NK cells imaged under wide-field fluorescence microscope. (A) Live cell time-lapse images from the VCP ver.3 system used to establish kinetics of CD16-KHYG-1 (red)-mediated cytotoxicity against K562 (green) target cell. Scale bars indicate 100 µm (left) and 10 µm (right), respectively. (B) Mean fluorescent intensity (MFI) of K562 cells paired with CD16-KHYG-1 cells (gray), K562 cells alone (green), and CD16-KHYG-1 cells alone (red) during the 6-hour acquisition. (C–F) Classification of killing kinetics of NK cells when paired with K562 target cells. (C) ‘Slow decay’ of normalized MFI from K562 cells conjugated with NK cells for 6 hrs. (D) ‘Single-drop’ of normalized MFI from K562 cells conjugated with NK cells (left) and quantification of ‘single-drop’ occurrence in K562 cells after conjugation with NK cells (right). (E) ‘Fast decay’ of normalized MFI from K562 cells conjugated with NK cells for 6 hrs. (F) ‘Multiple-drop’ of normalized MFI from K562 cells conjugated with NK cells (left) and quantification of ‘multiple-drop’ occurrence in K562 cells after conjugation with NK cells (right). Black lines indicate average of the each population.

Article Snippet: Plasmids and transduction of CD16-KHYG-1 and K562 cell lines To generate the PD-1-GFP construct, full length of PD-1-GFP fusion protein sequence (OriGene, MD) was amplified by the primers 5’-AATCCGGAATTCGCCGCCGCGATCGCCATGC-3’ (Forward) and 5’-AATCGCGGATCCTTAAACTCTTTCTTCACC-3’ (Reverse).

Techniques: Fluorescence, Microscopy, Conjugation Assay