Journal: EBioMedicine

Article Title: A Minimally-invasive Blood-derived Biomarker of Oligodendrocyte Cell-loss in Multiple Sclerosis

doi: 10.1016/j.ebiom.2016.06.031

Figure Lengend Snippet: Murine brain and spinal cord show differential methylation in the MOG gene, due to their O4 + cell population. A. Sanger sequencing results of bisulfite treated DNA from murine tissues. Arrows point toward CpG sites where cytosines (C) are preserved in methylated samples (Liver, Kidney), or converted to thymines (T) in samples containing demethylated CpGs, leading to a mixed population of C′s and T's (Brain, Spinal Cord). B. Methylation-sensitive DNA digestion was performed on the magnetically spinal cord of liver derived-DNA using the McrBC enzyme. Digested DNA was subjected to semi-quantitative PCR (cycles 32 and 35) and run on agarose gel. UnTx—untreated DNA receiving all reaction components except McrBC enzyme. Pos—positive control using cloned native MOG DNA. NTC—no template control. C and D. O4 + and O4 − cells were separated from four separate murine brains by magnetic beads. FACS analysis showed > 92.6 ± 3.9% enrichment of O4 + cells among four independent preparations when compared to O4 − fractions. E. DNA from murine O4 + cells is differentially methylated in the MOG gene compared to DNA from O4 − cells, the SW10 Schwann cell line, and liver. Sequence analysis was performed on first-step PCR product of each sample, 10 clones from each are shown (○ represent demethylated cytosines; ●, methylated cytosines). Locations in relation to the MOG transcription start site are listed, methylation-specific murine primers incorporate the CpG site at bp + 2752.

Article Snippet: SW10 immortalized neuronal murine Schwann cells were purchased from American Type Culture Collection (Manassas, VA) and cultured using the provided protocols.

Techniques: Methylation, Sequencing, Derivative Assay, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Positive Control, Clone Assay, Control, Magnetic Beads

Journal: EBioMedicine

Article Title: A Minimally-invasive Blood-derived Biomarker of Oligodendrocyte Cell-loss in Multiple Sclerosis

doi: 10.1016/j.ebiom.2016.06.031

Figure Lengend Snippet: Methylation-specific primers display high specificity and sensitivity and can detect demethylated MOG-DNA in murine brain, spinal cord, and O4 + cells. A. A depiction of MOG gene region utilized for mouse methylation-specific qPCR analysis; cytosine at bp + 2752 from MOG transcription start site incorporated into reverse primer sequence. B. Methylation-specific primers tested using plasmids containing methylated and demethylated murine MOG-DNA inserts over a wide range of serial dilutions (R 2 = 0.987, p < 0.0001). C. Methylation-specific primers were used in qPCR with bisulfite treated DNA from murine liver, kidney, brain, and spinal cord. Three independent analyses used to compute DMI averages; Liver vs. Brain/Spinal Cord p < 0.001, Kidney vs. Brain/Spinal Cord p < 0.001. D. Methylation-specific primers were used in qPCR with murine O4 + cells, O4 − cells, and SW10 Schwann cells. Three independent analyses used to compute DMI averages; ANOVA p = 0.0008, O4 + vs. O4 − p < 0.01, O4 + vs. SW10 Schwann cells p < 0.01.

Article Snippet: SW10 immortalized neuronal murine Schwann cells were purchased from American Type Culture Collection (Manassas, VA) and cultured using the provided protocols.

Techniques: Methylation, Sequencing

Journal: BMC Molecular and Cell Biology

Article Title: Genetic and protein interaction studies between the ciliary dyslexia candidate genes DYX1C1 and DCDC2

doi: 10.1186/s12860-023-00483-4

Figure Lengend Snippet: CPAP interacts with DCDC2 and DYX1C1. ( A ) CPAP endogenously interacts with DCDC2 and DYX1C1. Endogenous immunoprecipitations using HeLa cell extracts. CPAP was used as a bait to pull down interactors. CEP350 was used as a negative control and CEP152 was used as a positive control for interaction with CPAP. Beads alone were used as a negative control for the IP´s. ( B ) CPAP and DYX1C1 interaction in brain organoids. CPAP was immunoprecipitated by anti- DYX1C1 in 15 day old brain organoids. Reciprocally, DYX1C1 was pulled down by anti-CPAP. ( C ) Schematic representation of domain structures of DYX1C1, CPAP and the deletion constructs DYX1C1ΔTPR, DYX1C1Δp23, DYX1C1ΔDYX. p23 = p23 domain, TPR = tetratricopeptide repeat domain, DYX = DYX domain. ( D ) CPAP interacts with DYX1C1 via the p23 domain. hTERT-RPE1 cells stably expressing DOX-CPAP-GFP and growing in normal serum conditions were induced with doxycycline and transiently transfected with the indicated constructs. GFP-Trap was used to pull down CPAP-GFP. anti-V5 antibody was used for immunodetection of interactors.

Article Snippet: The human retinal pigmented epithelial cell line immortalized with hTERT (hTERT-RPE1, ATCC, CRL-4000TM) was cultured in DMEM/F12, 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin, 0.01 mg/ml hygromycin B at 5% CO 2 .

Techniques: Negative Control, Positive Control, Immunoprecipitation, Construct, Stable Transfection, Expressing, Transfection, Immunodetection

Journal: BMC Molecular and Cell Biology

Article Title: Genetic and protein interaction studies between the ciliary dyslexia candidate genes DYX1C1 and DCDC2

doi: 10.1186/s12860-023-00483-4

Figure Lengend Snippet: Co-occurrence of DYX1C1 and CPAP at the subcellular level. A ) DYX1C1 and CPAP co-occur around the centrosome. Confocal immunofluorescence images of endogenous protein in hTERT-RPE1 cells grown in normal serum conditions. B )- C ) DCDC2 co-occurs with CPAP depending on cell cycle stage. Nuclei are stained with DRAQ5. Scale bar = 10 μm

Article Snippet: The human retinal pigmented epithelial cell line immortalized with hTERT (hTERT-RPE1, ATCC, CRL-4000TM) was cultured in DMEM/F12, 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin, 0.01 mg/ml hygromycin B at 5% CO 2 .

Techniques: Immunofluorescence, Staining

Journal: BMC Molecular and Cell Biology

Article Title: Genetic and protein interaction studies between the ciliary dyslexia candidate genes DYX1C1 and DCDC2

doi: 10.1186/s12860-023-00483-4

Figure Lengend Snippet: Transcriptional control between DYX1C1/dyx1c1 and DCDC2/dcdc2b. A - D ). hTERT-RPE1 cells were transfected with siDYX1C1, siDCDC2 or siCtrl, respectively, RNA was extracted and qRT-PCR was run (n = 3 experiments with 3 replicates, mean ± sem). A , C ) Knockdown of DYX1C1 and DCDC2 efficiently reduced the respective transcripts (p-value < 0.0001, t-test). B , D ) Expression of DCDC2 and DYX1C1 is reduced after knockdown of siDYX1C1 and siDCDC2, respectively (p-value < 0.05, t-test). E ) dcdc2b expression was unchanged in dyx1c1 morphants: RNA of dyx1c1 morphant zebrafish was collected at 1dpf and 2dpf after morpholino injection at 1-cell stage. RT-qPCR analysis shows that dcdc2b expression is unchanged in dyx1c1 morphant embryos compared to control embryos (n = 3, mean ± sem, p-value (1dpf) = 0.2597, p-value (2dpf) = 0.4521), t-test). dpf = days post fertilization

Article Snippet: The human retinal pigmented epithelial cell line immortalized with hTERT (hTERT-RPE1, ATCC, CRL-4000TM) was cultured in DMEM/F12, 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin, 0.01 mg/ml hygromycin B at 5% CO 2 .

Techniques: Control, Transfection, Quantitative RT-PCR, Knockdown, Expressing, Injection

Journal: Cells

Article Title: Moderate Elevation of Homocysteine Induces Endothelial Dysfunction through Adaptive UPR Activation and Metabolic Rewiring

doi: 10.3390/cells13030214

Figure Lengend Snippet: Sub-lethally increased Hcy causes endothelial dysfunction. ( A ) Bar graph represents percentage of cell death in HUVEC/TERT2 cells treated with increasing concentration of Hcy for 24 h. A sub-lethal concentration of 2 mM (24 h) was chosen for all the subsequent experiments. ( B ) HPLC mediated quantification of intracellular Hcy concentration in HUVEC/TERT2 cells revealing induction of moderate Hyperhomocysteinemic condition post 2 mM Hcy treatment for 24 h. ( C ) Representative images of tube formation assay showing functional abnormality in Hcy treated HUVEC/TERT2 cells compared with untreated cells. Scale bar, 250 μm. ( D , E ) Respective quantifications showing total tube length and number of branches formed during tube formation assay are drastically reduced upon 2 mM Hcy treatment for 24 h. Data are shown as Mean ± SEM with n ≥ 3. ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001 and ns is non-significant ( p > 0.05).

Article Snippet: Immortalized human umbilical vein endothelial cell line (HUVEC/TERT2) was purchased from ATCC (CRL4053, Rockville, MD, USA) and cultured in vascular cell basal medium (ATCC, PCS100030, USA) which was supplemented with endothelial cell growth kit-BBE (ATCC, PCS100040, USA).

Techniques: Concentration Assay, Tube Formation Assay, Functional Assay

Journal: Cells

Article Title: Moderate Elevation of Homocysteine Induces Endothelial Dysfunction through Adaptive UPR Activation and Metabolic Rewiring

doi: 10.3390/cells13030214

Figure Lengend Snippet: Sub-lethal HHcy reduces endothelial migration and proliferation without suppressing VEGF/VEGFR transcripts and ROS level change. ( A ) Scratch wound assay in presence of Mitomycin C showing less migrated endothelial cells at 24 h post 2 mM Hcy treatment. ( B ) Quantification of migrated cells revealing that endothelial migration is significantly reduced in Hcy treated cells compared with control cells. ( C ) Bar plot of BrdU cell proliferation assay indicating that 2 mM Hcy treatment for 24 h causes proliferation defect in endothelial cells. ( D ) Representing scratch wound assay images depicting that as opposed to Hcy treatment, a similar concentration of Cys did not affect migration of endothelial cells when compared with untreated cells at 24 h. ( E ) Bar plot of measurement of migrated cells in scratch wound assay showing that contrary to Hcy treated cells, fold change in migrated cells is not altered upon 2 mM Cys treatment for 24 h compared with control cells. ( F ) BrdU cell proliferation assay revealing that unlike Hcy treated cells, 2 mM Cys treatment for 24 h did not influence endothelial proliferation. ( G ) Bars showing that compared with untreated cells, exposure to sub-lethal Hcy caused upregulation of mRNA levels of canonical VEGF signaling markers. 18S was used as internal control. ( H ) Bar graph showing that sub-lethal Hcy treatment does not induce ROS production in endothelial cells as determined by the fluorescent probe CM-H2DCFDA. For positive control, H 2 O 2 was used. ( I ) Representative western blots showing protein levels of major antioxidant markers GPX1 and SOD1. Corresponding bar graphs showing densitometric analysis of the protein bands which suggest a non-significant but slight trend of upregulation of both the proteins in 2 mM Hcy (24 h) treated cells. As a loading control β-actin was used. Data are shown as Mean ± SEM with n ≥ 3. * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001 and ns is non-significant ( p > 0.05).

Article Snippet: Immortalized human umbilical vein endothelial cell line (HUVEC/TERT2) was purchased from ATCC (CRL4053, Rockville, MD, USA) and cultured in vascular cell basal medium (ATCC, PCS100030, USA) which was supplemented with endothelial cell growth kit-BBE (ATCC, PCS100040, USA).

Techniques: Migration, Scratch Wound Assay Assay, Control, BrdU Cell Proliferation Assay, Concentration Assay, Positive Control, Western Blot

Journal: Cells

Article Title: Moderate Elevation of Homocysteine Induces Endothelial Dysfunction through Adaptive UPR Activation and Metabolic Rewiring

doi: 10.3390/cells13030214

Figure Lengend Snippet: Sub-lethal HHcy-induced adaptive UPR controls endothelial migration defect. ( A ) Representative western blots showing protein levels of UPR markers GRP78, IRE1p and ATF4 are upregulated upon 2 mM Hcy treatment for 24 h. Terminal UPR marker CHOP remained unaltered post-sub-lethal Hcy treatment. As a loading control β-actin was used. Corresponding bar graph showing densitometric analysis (normalized to β-actin) of the blots. ( B , C ) Respective quantifications of scratch wound assay at 24 h revealing that chemical chaperone 4-PBA (1 mM) and TUDCA (1 mM) can significantly improve sub-lethal HHcy-induced endothelial migration defect. ( D ) Representative confocal images of rhodamine-phalloidin stained endothelial cells showing that sub-lethal Hcy-induced abnormally elongated cell morphology as well as actin stress fiber (white arrows) disappearance are rescued by 4-PBA pre-treatment. Scale bar, 5 μm. ( E ) ImageJ based analysis demonstrating that 4-PBA pre-treatment reversed the aberrant reduction in cellular aspect ratio (major axis/minor axis), induced by 24 h treatment of 2 mM Hcy. ( F ) Quantification by ImageJ suggesting that exposure to sub-lethal Hcy significantly decreased the surface area of endothelial cells which was rescued by 4-PBA. ( G ) Bar plot showing no beneficial effect of chemical chaperone TUDCA on impairment of endothelial proliferation caused by sub-lethal Hcy treatment. Data are shown as Mean ± SEM with n ≥ 3. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, and ns is non-significant ( p > 0.05).

Article Snippet: Immortalized human umbilical vein endothelial cell line (HUVEC/TERT2) was purchased from ATCC (CRL4053, Rockville, MD, USA) and cultured in vascular cell basal medium (ATCC, PCS100030, USA) which was supplemented with endothelial cell growth kit-BBE (ATCC, PCS100040, USA).

Techniques: Migration, Western Blot, Marker, Control, Scratch Wound Assay Assay, Staining

Journal: Cells

Article Title: Moderate Elevation of Homocysteine Induces Endothelial Dysfunction through Adaptive UPR Activation and Metabolic Rewiring

doi: 10.3390/cells13030214

Figure Lengend Snippet: Sub-lethal HHcy linked malfunctional ETC impairs mitochondrial respiration of endothelial cells. ( A ) OCR curves showing drastic reduction in endothelial mitochondrial respiration upon 2 mM Hcy treatment for 24 h as measured by an extracellular flux analyzer. ( B – D ) Respective bar graphs demonstrating that in comparison to untreated cells, there is significant decrease in basal respiration, ATP production and maximal respiration of endothelial cells with sub-lethal HHcy. ( E ) OCR curves showing no restoration of sub-lethal HHcy-induced mitochondrial respiration defect in presence of TUDCA. ( F – H ) Bar plots respectively showing no significant improvement in the reduction in basal respiration, ATP production and maximal respiration upon TUDCA pre-treatment, compared with sub-lethal Hcy treated endothelial cells. ( I ) Targeted metabolomics mediated quantification exhibiting significant elevation of metabolites of TCA cycle in sub-lethal Hcy treated endothelial cells compared with untreated control cells. AUC, area under the curve. ( J ) Bar plot showing that sub-lethal HHcy in endothelial cells causes significant reduction in enzymatic activity of COX, the terminal electron acceptor of ETC. Data are shown as Mean ± SEM with n ≥ 3. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and ns is non-significant ( p > 0.05).

Article Snippet: Immortalized human umbilical vein endothelial cell line (HUVEC/TERT2) was purchased from ATCC (CRL4053, Rockville, MD, USA) and cultured in vascular cell basal medium (ATCC, PCS100030, USA) which was supplemented with endothelial cell growth kit-BBE (ATCC, PCS100040, USA).

Techniques: Comparison, Control, Activity Assay

Journal: Cells

Article Title: Moderate Elevation of Homocysteine Induces Endothelial Dysfunction through Adaptive UPR Activation and Metabolic Rewiring

doi: 10.3390/cells13030214

Figure Lengend Snippet: Glycolysis is elevated upon induction of sub-lethal HHcy in endothelial cells. ( A ) ECAR curves showing drastically upregulated glycolysis of endothelial cells treated by 2 mM Hcy for 24 h as measured by an extracellular flux analyzer. ( B – D ) Respective bar graphs revealing that in comparison to untreated cells, there is a significant enhancement of glycolysis, glycolytic reserve, and glycolytic capacity of sub-lethal Hcy treated endothelial cells. ( E ) Bar graph of targeted metabolomics showing metabolic intermediates of glycolysis are elevated in endothelial cells with sub-lethal HHcy. AUC, area under the curve. ( F ) Glucose uptake assay using fluorescence analog 2-NBDG showing that in comparison to control cells, consumption of extracellular glucose is higher in sub-lethal Hcy treated endothelial cells. Data are shown as Mean ± SEM with n ≥ 3. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001.

Article Snippet: Immortalized human umbilical vein endothelial cell line (HUVEC/TERT2) was purchased from ATCC (CRL4053, Rockville, MD, USA) and cultured in vascular cell basal medium (ATCC, PCS100030, USA) which was supplemented with endothelial cell growth kit-BBE (ATCC, PCS100040, USA).

Techniques: Comparison, Fluorescence, Control

Journal: Cells

Article Title: Moderate Elevation of Homocysteine Induces Endothelial Dysfunction through Adaptive UPR Activation and Metabolic Rewiring

doi: 10.3390/cells13030214

Figure Lengend Snippet: Mechanistic features of pathologically relevant Hcy exposure are conserved in adult endothelial cells. ( A ) Schematic diagram showing experimental model and treatment condition used for microarray profiling of GSE175735 from GEO database, previously published by M Jan et al. ( B ) Heatmap illustrating the trend of downregulation in differentially expressed genes (DEGs) of cell cycle and cellular migration compared between control and Hcy treated (0.5 mM, 48 h) groups. ( C – E ) Respective heatmaps showing pathway specific expression of angiogenesis and antioxidant response genes, UPR and metabolism (TCA cycle and glycolysis), in the same Hcy treated and untreated control cell groups. Statistically significant ( p -value < 0.05) genes were considered to obtain DEGs. Respective color legends representing Z-score ((observed value–mean)/standard deviation) values. Three sets of samples each from control and Hcy treated cells were used for analysis.

Article Snippet: Immortalized human umbilical vein endothelial cell line (HUVEC/TERT2) was purchased from ATCC (CRL4053, Rockville, MD, USA) and cultured in vascular cell basal medium (ATCC, PCS100030, USA) which was supplemented with endothelial cell growth kit-BBE (ATCC, PCS100040, USA).

Techniques: Microarray, Migration, Control, Expressing, Standard Deviation

Journal: American Journal of Cancer Research

Article Title: LMNB1, a potential marker for early prostate cancer progression

doi:

Figure Lengend Snippet: Western blotting to detect the expression levels of LMNA and LMNB1 in cells. (A) The protein signal bands for LMNA, LMNB1, and GAPDH. (B, C) The relative expression levels of LMNB1 and LMNA (normalized to GAPDH expression) in different cell lines. The mean ± SEM of two independent experiments are shown (P = 0.0005 and P < 0.0001 for LMNB1 and LMNA, respectively; ANOVA). The results show that LMNB1-overexpressing EP156T (LMN-EP156T) cells express significantly higher amounts of LMNB1 than the parental and Mock EP156T cells. All three prostate cancer cell lines express high levels of LMNB1. All cells express LMNA, with PC-3 and DU145 expressing higher amounts of LMNA than the other cells.

Article Snippet: EP156T prostate epithelial cells and transfection with lentiviral vectors harboring LMNB1 A human TERT -immortalized EP156T prostatic epithelial cell line was purchased from the American Type Culture Collection (CRL3289TM, ATCC, Manassas, VA, USA) and grown in MCDB-153 medium supplemented with bovine pituitary extract (25 mg/500 ml medium), hEGF (5 ng/ml medium), 1% fetal bovine serum (FBS), and 0.5 μg/ml puromycin.

Techniques: Western Blot, Expressing

Journal: American Journal of Cancer Research

Article Title: LMNB1, a potential marker for early prostate cancer progression

doi:

Figure Lengend Snippet: Immunohistochemical (IHC) staining of LMNB1 expression. Staining results were shown for (A) parental EP156T, (B) Mock EP156T, (C) LMNB1-overexpressing EP156T (LMN-EP156T), and (D) PC-3 cells (positive control). Both LMN-EP156T and PC-3 cells expressed strong nuclear staining of LMNB1, which was higher than either parental EP156T or Mock EP156T cells.

Article Snippet: EP156T prostate epithelial cells and transfection with lentiviral vectors harboring LMNB1 A human TERT -immortalized EP156T prostatic epithelial cell line was purchased from the American Type Culture Collection (CRL3289TM, ATCC, Manassas, VA, USA) and grown in MCDB-153 medium supplemented with bovine pituitary extract (25 mg/500 ml medium), hEGF (5 ng/ml medium), 1% fetal bovine serum (FBS), and 0.5 μg/ml puromycin.

Techniques: Immunohistochemical staining, Immunohistochemistry, Expressing, Staining, Positive Control

Journal: American Journal of Cancer Research

Article Title: LMNB1, a potential marker for early prostate cancer progression

doi:

Figure Lengend Snippet: MTS assay to measure the rate of cell growth. A total of 6.5 × 103 cells in 0.1 ml of complete medium were seeded onto each well of 96 well-plates in triplicate for each cell line. Five repeated plates were prepared for measurements at 5 time points. At each time point, one plate was used for this assay. MTS reagent (20 μl) was added to each well, and the plates were incubated at 37°C for 1 h. The absorbance at 490 nm is shown as the mean ± SEM of three independent experiments. LMNB1-overexpressing EP156T (LMN-EP156T) cells show significantly faster growth compared to the other two cell lines (P < 0.01; ANOVA).

Article Snippet: EP156T prostate epithelial cells and transfection with lentiviral vectors harboring LMNB1 A human TERT -immortalized EP156T prostatic epithelial cell line was purchased from the American Type Culture Collection (CRL3289TM, ATCC, Manassas, VA, USA) and grown in MCDB-153 medium supplemented with bovine pituitary extract (25 mg/500 ml medium), hEGF (5 ng/ml medium), 1% fetal bovine serum (FBS), and 0.5 μg/ml puromycin.

Techniques: MTS Assay, Incubation

Journal: American Journal of Cancer Research

Article Title: LMNB1, a potential marker for early prostate cancer progression

doi:

Figure Lengend Snippet: Matrigel-coated transwell assay (37°C for 48 h, 40 × magnification). A total of 5 × 104 cells of each cell line in 0.1 ml medium + 0.1% FBS were seeded onto an insert well (pore size, 8 µm) for a 24-well plate pre-coated with Matrigel. The insert wells were placed on receiver wells with 0.65 ml of medium + 10% FBS per well. After incubation at 37°C for 48 h, the cells on the apical side of the insert wells were removed with swabs. Only cells that had passed the membrane could be visualized after staining in 20% methanol with 0.25% crystal violet for 10 min. The results are shown for (A) EP156T, (B) Mock EP156T, and (C) LMN-EP156T cells. (D) The number of cells that penetrated the wells are shown as the mean ± SEM of three independent experiments. Statistical significance was determined by ANOVA (P < 0.0001). Significantly more LMNB1-overexpressing EP156T (LMN-EP156T) cells passed through the 8-µm pores than parental and Mock EP156T cells.

Article Snippet: EP156T prostate epithelial cells and transfection with lentiviral vectors harboring LMNB1 A human TERT -immortalized EP156T prostatic epithelial cell line was purchased from the American Type Culture Collection (CRL3289TM, ATCC, Manassas, VA, USA) and grown in MCDB-153 medium supplemented with bovine pituitary extract (25 mg/500 ml medium), hEGF (5 ng/ml medium), 1% fetal bovine serum (FBS), and 0.5 μg/ml puromycin.

Techniques: Transwell Assay, Pore Size, Incubation, Membrane, Staining

Journal: American Journal of Cancer Research

Article Title: LMNB1, a potential marker for early prostate cancer progression

doi:

Figure Lengend Snippet: Colony-formation assay. Results are shown for (A) EP156T, (B) Mock EP156T, (C) LMNB1-overexpressing EP156T (LMN-EP156T), and (D) PC-3 cells. A total of 5 × 103 cells of each cell line were mixed in 0.3% soft agar with complete growth medium, and plated onto a culture dish (diameter, 3.5 cm). Numerous colonies can be seen in the plates inoculated with PC-3 after 2 weeks of incubation (magnification, 100 ×) at 37°C. However, no colonies have formed in the plates inoculated with the other cell lines. White arrows indicate PC-3 cell colonies.

Article Snippet: EP156T prostate epithelial cells and transfection with lentiviral vectors harboring LMNB1 A human TERT -immortalized EP156T prostatic epithelial cell line was purchased from the American Type Culture Collection (CRL3289TM, ATCC, Manassas, VA, USA) and grown in MCDB-153 medium supplemented with bovine pituitary extract (25 mg/500 ml medium), hEGF (5 ng/ml medium), 1% fetal bovine serum (FBS), and 0.5 μg/ml puromycin.

Techniques: Colony Assay, Incubation

Journal: American Journal of Cancer Research

Article Title: LMNB1, a potential marker for early prostate cancer progression

doi:

Figure Lengend Snippet: Xenograft mouse model showing evident subcutaneous tumors in the right chest 7 weeks after the injection of PC-3 cells. (A-D) Four of the five mice injected with PC-3 cells formed subcutaneous tumors (red arrows). The tumor sizes range between 13 mm and 19 mm in diameter. No tumors were seen in mice injected with EP156T, Mock EP156T, or LMNB1-overexpressing EP156T cells (photos not shown).

Article Snippet: EP156T prostate epithelial cells and transfection with lentiviral vectors harboring LMNB1 A human TERT -immortalized EP156T prostatic epithelial cell line was purchased from the American Type Culture Collection (CRL3289TM, ATCC, Manassas, VA, USA) and grown in MCDB-153 medium supplemented with bovine pituitary extract (25 mg/500 ml medium), hEGF (5 ng/ml medium), 1% fetal bovine serum (FBS), and 0.5 μg/ml puromycin.

Techniques: Injection

Journal: American Journal of Cancer Research

Article Title: LMNB1, a potential marker for early prostate cancer progression

doi:

Figure Lengend Snippet: Hematoxylin and eosin-stained tissue sections from xenografted mice. (A) An enlarged and metastasized lymph node from the PC-3-injected mouse. (B-E) Lung sections from mice injected with PC-3, EP156T, Mock EP156T, and LMNB1-overexpressing EP156T cells, respectively. Each row shows the same section with increasing magnification. Red arrows indicate PC-3 metastatic cell nests with clear nucleoli in either the lymph node (A) or lung (B). There are no subcutaneous growths or metastatic lesions in mice injected with the parental, Mock, or LMNB1-overexpressing EP156T cells.

Article Snippet: EP156T prostate epithelial cells and transfection with lentiviral vectors harboring LMNB1 A human TERT -immortalized EP156T prostatic epithelial cell line was purchased from the American Type Culture Collection (CRL3289TM, ATCC, Manassas, VA, USA) and grown in MCDB-153 medium supplemented with bovine pituitary extract (25 mg/500 ml medium), hEGF (5 ng/ml medium), 1% fetal bovine serum (FBS), and 0.5 μg/ml puromycin.

Techniques: Staining, Injection

Journal: PLoS ONE

Article Title: Biochemical properties of thyroid peroxidase (TPO) expressed in human breast and mammary-derived cell lines

doi: 10.1371/journal.pone.0193624

Figure Lengend Snippet: The biochemical properties of TPO protein expressed in breast tissues (A and B) and breast-derived cell lines (C). (A) N-linked glycan content in TPO expressed in breast tissues. Total cell extract was digested with PNGase F, then subjected to 8% SDS-PAGE, followed by Western blotting, and probing with TPO-specific mAb 47 monoclonal antibody. Controls were processed under the same conditions as the samples except that no enzyme was added. One representative immunoblot out of at least three independent experiments is shown. (B) Enzymatic activity of TPO expressed in breast tissues. Tissue lysate was incubated with TPO-specific mAb A4, then protein A agarose was added to precipitate immune complexes. TPO-antibody complexes bound to agarose were incubated with luminol in the presence of hydrogen peroxide. The intensity of luminescencent signal was measured and results were expressed as relative light units (RLU). As positive control, TPO immunoprecipitated from human thyroid tissue lysate (Graves’ disease case) was used to measure luminol oxidation. Agarose A incubated with mAb A4 alone (lysate omitted) was used as negative control. One representative of three independent experiments is shown. (C) TPO protein expression in breast epithelial normal (184A1) and cancer cell lines (MCF-7 and MDA-MB-231). Western blotting was used to detect TPO protein presence. The specificity of the reaction was verified by preabsorption of ab76935 antibody with the excess of highly purified human TPO. NTHY was used as a positive control. β-actin-specific Ab was used as a loading control. BN: peri-tumoral breast tissue; BC: breast cancer tissue; G-B: Graves’ disease thyroid tissue; NTHY: NTHY-ori 3–1 cell line; PNGase F: Peptide-N-Glycosidase F; RLU: relative light units.

Article Snippet: Chemically immortalized normal human breast epithelial cell line, 184A1, used as a non-cancerous control, and breast cancer cell lines, MCF-7 and MDA-MB-231, were obtained from American Type Culture Collection (ATCC).

Techniques: Derivative Assay, Glycoproteomics, SDS Page, Western Blot, Activity Assay, Incubation, Positive Control, Immunoprecipitation, Negative Control, Expressing, Purification, Control

Journal: PLoS ONE

Article Title: Biochemical properties of thyroid peroxidase (TPO) expressed in human breast and mammary-derived cell lines

doi: 10.1371/journal.pone.0193624

Figure Lengend Snippet: Representative TPO immunostaining results obtained with a panel of monoclonal antibodies (mAbs) against human thyroid peroxidase (TPO) (A) and human serum pools (B) in breast epithelial normal (184A1) and cancer cell lines (MCF-7 and MDA-MB-231). A normal human thyroid cell line, NTHY, was used as a positive control. (A) Positive signal (red) was detected with all mAbs except the isotype control (negative control). In the cells incubated with TPOAbs obtained from autoimmune thyroid disease (AITD) patients (TPOAbs(+)), a positive signal (green) was observed but this staining was not observed when TPOAb-free serum (TPOAbs(-)) was used (negative control). Nuclei (blue) were counterstained with DAPI. Magnification: 630×. IIAb: secondary antibody; DAPI: 4′,6-diamino-2-phenylindole; NTHY: NTHY-ori 3–1 cell line.

Article Snippet: Chemically immortalized normal human breast epithelial cell line, 184A1, used as a non-cancerous control, and breast cancer cell lines, MCF-7 and MDA-MB-231, were obtained from American Type Culture Collection (ATCC).

Techniques: Immunostaining, Bioprocessing, Positive Control, Control, Negative Control, Incubation, Staining

Journal: The Journal of Biological Chemistry

Article Title: Toll-like Receptor 3 (TLR3) Induces Apoptosis via Death Receptors and Mitochondria by Up-regulating the Transactivating p63 Isoform α (TAP63α) *

doi: 10.1074/jbc.M110.178798

Figure Lengend Snippet: Poly(I-C) induces endothelial cell apoptosis. A, poly(I-C) induced the detachment of primary HUVECs. Cells, pretreated with 1 μg/ml poly(I-C) for 24 h, were re-stimulated with or without 10 μg/ml poly(I-C) for 24 h, and the cells in representative fields were photographed. Cells, treated with 2 μm staurosporine, were photographed as a positive control. B, poly(I-C) induced apoptosis in primary HUVECs. Cells, pretreated with 1 μg/ml poly(I-C) for 24 h, were re-stimulated with the indicated concentrations of poly(I-C) for 24 h. Unfixed cells were stained with FITC-annexin V/PI. Cell apoptosis was measured by flow cytometry analysis. C, poly(I-C) induced the dose-dependent cell apoptosis in immortalized HUVECs. Cells were treated with the indicated concentrations of poly(I-C) for 24 h. Cell apoptosis was measured as in B. Data are shown as percentages and represented as mean ± S.D. of triplicates. Cells treated with 1 μm staurosporine were used as a positive control. *, p < 0.05 compared with the control group. D, poly(I-C) (10 μg/ml) induced time-dependent cell apoptosis in immortalized HUVECs. *, p < 0.05 compared with the control group.

Article Snippet: Cell Lines and Reagents The immortalized human umbilical vein endothelial cells (HUVECs) expressing endothelial cell characteristic markers, endothelial nitric-oxide synthase, CD31, and Ve-cadherin ( 21 ) were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Positive Control, Staining, Flow Cytometry, Control

Journal: The Journal of Biological Chemistry

Article Title: Toll-like Receptor 3 (TLR3) Induces Apoptosis via Death Receptors and Mitochondria by Up-regulating the Transactivating p63 Isoform α (TAP63α) *

doi: 10.1074/jbc.M110.178798

Figure Lengend Snippet: Poly(I-C) induces TLR3-dependent apoptosis. A, poly(I-C) up-regulated gene expression of TLR3. Immortalized HUVECs, treated with the indicated concentrations of poly(I-C) for 24 h, were harvested, and the gene expression of TLR3 was measured by RT-PCR. GAPDH transcript was measured as a loading control. B, immortalized HUVECs expressed TLR3 protein. Cells, cultured in the presence or absence of 10 μg/ml poly(I-C) for 24 h, were harvested. The expression of TLR3 protein was measured by flow cytometry. Mouse isotype Ig was used as a control. C, poly(I-C) up-regulated the expression of TLR3 protein in primary HUVECs. Cells, treated with the indicated concentrations of poly(I-C) for 24 h, were harvested and lysed with sample buffer. Equal amounts of total proteins were electrophoresed and blotted for the detection of TLR3 protein expression. β-Actin protein was detected as a loading control. D, poly(I-C) significantly up-regulated the gene expression of TLR3 in primary HUVECs. Cells were treated as in Fig. 1B. The gene transcript of TLR3 was measured by qRT-PCR. GAPDH gene expression was detected as an endogenous control. *, p < 0.05 compared with the control group. E, poly(I-C) induced the phosphorylation of the NF-κB p65 subunit. Immortalized HUVECs, starved overnight with serum-free medium, were cultured in the presence of 2 μg/ml poly(I-C) at 37 °C and lysed at the indicated time points. Phospho-p65 (P-p65) and total p65 (T-p65) were detected by Western blot. F, RT-PCR results show that poly(I-C) up-regulated the gene expression of cytokines in immortalized HUVECs. G, TLR3 neutralization abrogated poly(I-C)-induced cell apoptosis. Immortalized HUVECs (iHUVEC) and 1 μg/ml poly(I-C) pretreated (37 °C for 24 h) primary HUVECs (pHUVEC) were cultured in the absence or presence of mouse anti-human TLR3 antibody at 37 °C for 1 h and then treated with or without 2 μg/ml poly(I-C) for 24 h. Cell apoptosis was tested as in Fig. 1B. Mouse isotype Ig was used as a control. Data are represented as mean ± S.D. of triplicates. *, p < 0.05 compared with the poly(I-C) treatment group. H, TLR3 down-regulation by RNA interference abrogated poly(I-C)-induced cell apoptosis in immortalized HUVECs. Wild-type (Wt), nonspecific shRNA (NSsi), and TLR3 shRNA (TLR3si)-transfected cells were treated with 2 μg/ml poly(I-C) for 24 h. Cell apoptosis was detected as in Fig. 1B. *, p < 0.05 compared with the control group.

Article Snippet: Cell Lines and Reagents The immortalized human umbilical vein endothelial cells (HUVECs) expressing endothelial cell characteristic markers, endothelial nitric-oxide synthase, CD31, and Ve-cadherin ( 21 ) were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Gene Expression, Reverse Transcription Polymerase Chain Reaction, Control, Cell Culture, Expressing, Flow Cytometry, Quantitative RT-PCR, Phospho-proteomics, Western Blot, Neutralization, shRNA, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Toll-like Receptor 3 (TLR3) Induces Apoptosis via Death Receptors and Mitochondria by Up-regulating the Transactivating p63 Isoform α (TAP63α) *

doi: 10.1074/jbc.M110.178798

Figure Lengend Snippet: Poly(I-C) activates both the intrinsic and extrinsic pathways. A, poly(I-C) activated both caspases 8 and 9. Immortalized HUVECs, treated with the indicated concentrations of poly(I-C) for 24 h, were lysed with sample buffer. The cleaved fragments from caspases 8 and 9 were detected by Western blot. 2 μm staurosporine (SP) was used as a control. B, caspase inhibitors down-regulated the cell apoptosis induced by poly(I-C). Immortalized HUVECs were cultured in the absence or presence of caspase 8 inhibitor (Z-IETD-FMK), caspase 9 inhibitor (Z-LEHD-FMK), or pan-caspase inhibitor (Z-VAD-FMK) at 37 °C for 1 h, followed by the treatment with or without 2 μg/ml poly(I-C) for 24 h. Cell apoptosis was tested as in Fig. 1B. Data are represented as mean ± S.D. of triplicates. *, p < 0.05 compared with the poly(I-C) treatment group. C, Western blot results show that poly(I-C) activated the caspase downstream molecule PARP. D, PARP inhibitors down-regulated the cell apoptosis induced by poly(I-C). *, p < 0.05 compared with the poly(I-C) treatment group.

Article Snippet: Cell Lines and Reagents The immortalized human umbilical vein endothelial cells (HUVECs) expressing endothelial cell characteristic markers, endothelial nitric-oxide synthase, CD31, and Ve-cadherin ( 21 ) were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Western Blot, Control, Cell Culture

Journal: The Journal of Biological Chemistry

Article Title: Toll-like Receptor 3 (TLR3) Induces Apoptosis via Death Receptors and Mitochondria by Up-regulating the Transactivating p63 Isoform α (TAP63α) *

doi: 10.1074/jbc.M110.178798

Figure Lengend Snippet: TNFα and IFNβ do not trigger cell apoptosis. A, poly(I-C) up-regulated the expression of TNFα in immortalized HUVECs. Cells were treated with the indicated concentrations of poly(I-C) for 24 h. The supernatant was harvested to detect the TNFα secretion by ELISA. Data are represented as mean ± S.D. of triplicates. *, p < 0.05 compared with the medium control. B, TNFα (37 °C for 24 h) did not induce cell apoptosis in immortalized HUVECs. C, TNFα (50 ng/ml) induced NF-κB signaling in immortalized HUVECs. D, TNFα neutralization did not inhibit poly(I-C)-induced cell apoptosis in immortalized HUVECs. E, IFNβ (37 °C for 24 h) did not induce cell apoptosis in immortalized HUVECs. F, IFNβ neutralization did not inhibit poly(I-C)-induced cell apoptosis in immortalized HUVECs.

Article Snippet: Cell Lines and Reagents The immortalized human umbilical vein endothelial cells (HUVECs) expressing endothelial cell characteristic markers, endothelial nitric-oxide synthase, CD31, and Ve-cadherin ( 21 ) were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Control, Neutralization

Journal: The Journal of Biological Chemistry

Article Title: Toll-like Receptor 3 (TLR3) Induces Apoptosis via Death Receptors and Mitochondria by Up-regulating the Transactivating p63 Isoform α (TAP63α) *

doi: 10.1074/jbc.M110.178798

Figure Lengend Snippet: TRAIL-DR4/5 and Noxa trigger the extrinsic and intrinsic pathways, respectively. A and B, RT-PCR results show that poly(I-C) (37 °C for 24 h) up-regulated the gene expression of TRAIL, DR4, and DR5 in immortalized (A) and 1 μg/ml poly(I-C) pretreated primary (B) HUVECs. C, poly(I-C) up-regulated the protein expression of TRAIL in primary HUVECs. Cells, pretreated with 1 μg/ml poly(I-C), were re-treated with the indicated concentrations of poly(I-C) for 24 h. TRAIL in the cell lysates was assayed by ELISA. *, p < 0.05 compared with the control. D, TRAIL neutralization repressed the cell apoptosis induced by poly(I-C) in immortalized HUVECs. *, p < 0.05 compared with the poly(I-C) treatment group. E and F, RT-PCR results show the effect of poly(I-C) (37 °C for 24 h) on the gene expression of Bcl-2 and Noxa in immortalized (E) and primary (F) HUVECs. G and H, Western blot results show the effect of poly(I-C) on the protein expression of Bcl-2 and Noxa in immortalized (G) and primary (H) HUVECs. I, inhibition of TLR3 repressed the poly(I-C)-induced down-regulation of Bcl-2 and up-regulation of Noxa in immortalized HUVECs. Cells were transiently transfected with human TLR3 shRNA plasmid and then treated with 2 μg/ml poly(I-C) for 24 h. The protein expression of TLR3, Bcl-2 and Noxa was detected by Western blot.

Article Snippet: Cell Lines and Reagents The immortalized human umbilical vein endothelial cells (HUVECs) expressing endothelial cell characteristic markers, endothelial nitric-oxide synthase, CD31, and Ve-cadherin ( 21 ) were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Gene Expression, Expressing, Enzyme-linked Immunosorbent Assay, Control, Neutralization, Western Blot, Inhibition, Transfection, shRNA, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Toll-like Receptor 3 (TLR3) Induces Apoptosis via Death Receptors and Mitochondria by Up-regulating the Transactivating p63 Isoform α (TAP63α) *

doi: 10.1074/jbc.M110.178798

Figure Lengend Snippet: Roles of p53 and p63 in poly(I-C)-induced cell apoptosis. A, RT-PCR results show that poly(I-C) up-regulated p21 but neither MDM2 nor p53 gene expression. B, quantitative expression of p21, MDM2, and p53 in A by qRT-PCR. *, p < 0.05 compared with the control group. C, effect of p53 inhibitor pifithrin-α (PFT) on poly(I-C)-induced cell apoptosis in immortalized HUVECs. D, p53 expression was down-regulated by RNA interference in immortalized HUVECs. Cells were transiently transfected with p53 siRNA. p53 gene expression was detected 48 h after the transfection. E, effect of p53 down-regulation on poly(I-C)-induced cell apoptosis in immortalized HUVECs. Cells were transiently transfected with p53 siRNA and cultured for 24 h and then were treated with poly(I-C) for 24 h for the detection of cell apoptosis. F and G, poly(I-C) up-regulated the gene expression of TAp63α. Immortalized (F) and 1 μg/ml poly(I-C) pretreated primary (G) HUVECs were treated with the indicated concentrations of poly(I-C) for 24 h. The gene expression of p63 was measured by RT-PCR using a primer pair in the N terminus to detect the transactivation domain and a primer pair in the C terminus to detect p63α, -β, and -γ splices. H and I, Western blot results show that poly(I-C) up-regulated the protein expression of TAp63α in immortalized (H) and primary (I) HUVECs. J, Western blot results show that the TLR3 down-regulation by TLR3 shRNA transient transfection inhibited the expression of TAp63α in primary HUVECs.

Article Snippet: Cell Lines and Reagents The immortalized human umbilical vein endothelial cells (HUVECs) expressing endothelial cell characteristic markers, endothelial nitric-oxide synthase, CD31, and Ve-cadherin ( 21 ) were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Gene Expression, Expressing, Quantitative RT-PCR, Control, Transfection, Cell Culture, Western Blot, shRNA

Journal: The Journal of Biological Chemistry

Article Title: Toll-like Receptor 3 (TLR3) Induces Apoptosis via Death Receptors and Mitochondria by Up-regulating the Transactivating p63 Isoform α (TAP63α) *

doi: 10.1074/jbc.M110.178798

Figure Lengend Snippet: Modulation of p63 expression represses poly(I-C)-induced cell apoptosis. A, p63 was down-regulated by RNA interference in immortalized HUVECs. Wild-type (Wt), nonspecific shRNA (NSsi), and p63 shRNA (p63si) stably transfected cells were treated with 2 μg/ml poly(I-C) for 24 h. TAp63α protein was measured by Western blot. B, p63 down-regulation repressed poly(I-C)-induced cell apoptosis in immortalized HUVECs. *, p < 0.05 compared with the Wt group. C, Western blot results show that p63 RNA interference inhibited the down-regulation of Bcl-2 and the up-regulation of Noxa in immortalized HUVECs. D, Western blot results show that p63 RNA interference inhibited the activation of caspases 8 and 9 and PARP in immortalized HUVECs. E, RT-PCR results show that p63 RNA interference inhibited the expression of TRAIL, DR4, and DR5 in immortalized HUVECs. F, ΔNp63α was up-regulated by gene transfection in immortalized HUVECs. Cells were transiently transfected with pCDNA3+ (Mock) or pCDNA3+/ΔNp63α and harvested at 48 h post-transfection. The gene expression of ΔNp63α was detected by RT-PCR. G, ΔNp63α overexpression inhibited poly(I-C)-induced cell apoptosis in immortalized HUVECs. Cells were transfected as in F and cultured for 24 h, treated with 2 μg/ml poly(I-C) for another 24 h, and then assayed for cell apoptosis. *, p < 0.05 compared with control groups.

Article Snippet: Cell Lines and Reagents The immortalized human umbilical vein endothelial cells (HUVECs) expressing endothelial cell characteristic markers, endothelial nitric-oxide synthase, CD31, and Ve-cadherin ( 21 ) were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Expressing, shRNA, Stable Transfection, Transfection, Western Blot, Activation Assay, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Over Expression, Cell Culture, Control

Journal: bioRxiv

Article Title: A primitive type of renin-expressing lymphocyte protects the organism against infections

doi: 10.1101/770511

Figure Lengend Snippet: a. A dot membrane immunoassay shows that peritoneal cells derived from the renin lineage appear as dark blue dots, indicating that these cells actively manufacture and release renin (A). A similar pattern is obtained with As4.1 cells, a mouse tumoral cell line that secretes renin constitutively (C). By the contrary, no spots were detected in C2C12 cells, skeletal muscle cells that do not normally synthesize renin (E). Further, no spots are detected in any of the cells when the membrane immunoassay is performed in the absence of the primary renin antibody (B, D, F) (scale bars: A-F 200 μm). b. Semi-quantitative RT-PCR was performed on wildtype peritoneal cells and peritoneal cells from a renin KO animal. Kidney RNA was used as a positive control. c. Peritoneal cells from Ren1 c-YFP reporter mice, where YFP marks active renin expression were grown in culture. YFP was demonstrated by immunofluorescence.

Article Snippet: C2C12 cells are an immortalized mouse myoblast cell line (ATCC, CRL-1772).

Techniques: Membrane, Derivative Assay, Quantitative RT-PCR, Positive Control, Expressing, Immunofluorescence

Journal: PLOS Genetics

Article Title: Multimodal CRISPR perturbations of GWAS loci associated with coronary artery disease in vascular endothelial cells

doi: 10.1371/journal.pgen.1010680

Figure Lengend Snippet: ( A ) From 92 loci associated with coronary artery disease (CAD) risk by genome-wide association studies (GWAS), we identified 2893 sentinel and linkage disequilibrium proxy variants for testing. For each of these variants, we attempted to design a maximum of five high-quality guide RNAs (sgRNAs) within a 100-bp window. In the design of the library, we also included sgRNAs that target genes essential for cell viability, as well as sgRNAs that target the coding sequence and promoter of genes that control endothelial cell functions (known genes, positive controls). ( B ) Number of sgRNAs per targeted variant that passed stringent quality-control filters. In total, we designed 7393 sgRNAs against 1998 CAD-associated variants (mean and median number of sgRNA per variant are 3.7 and 5, respectively). ( C ) Distribution of the absolute distance of the sgRNA cut-site relative to the targeted variant in base pairs (the vertical dashed line indicates mean sgRNA distance). ( D ) Fraction of variants at each locus that are successfully targeted by our pooled CRISPR screens. Each row represents one of the CAD loci that we tested. In green is the fraction of variants—including sentinel and LD proxies—for which we designed high-quality sgRNAs and obtained results for the endothelial function phenotypes. On average, 76% of variants at any given CAD locus are captured in the screens (vertical dashed line). ( E ) Most severe annotation for the 1998 CAD variants targeted by the lentiviral sgRNA libraries using ENSEMBL’s Variant Effect Predictor (VEP) module. ( F ) As a control step, we sequenced the plasmid library to ensure even representation of sgRNAs in the pool. Then, we produced four independent batches of lentiviruses which we used to infect teloHAEC cells that stably express Cas9, dCas9-KRAB (CRISPRi) or dCas9-VP64 (CRISPRa). Following antibiotic selection and TNFα treatment (for Cas9 and CRISPRi), we stained teloHAEC for cell surface markers (E-selectin, ICAM-1, VCAM-1) or intracellular signaling molecules (reactive oxygen species (ROS), nitric oxide (NO), calcium (Ca 2+ )). By flow cytometry, we sorted cells from the bottom and top 10 percentiles of the marker distributions, and sequenced sgRNAs found in each fraction.

Article Snippet: TeloHAEC are immortalized human aortic endothelial cells obtained by over-expressing telomerase (ATCC CRL-4052).

Techniques: GWAS, Sequencing, Control, Variant Assay, CRISPR, Plasmid Preparation, Produced, Stable Transfection, Selection, Staining, Flow Cytometry, Marker

Journal: PLOS Genetics

Article Title: Multimodal CRISPR perturbations of GWAS loci associated with coronary artery disease in vascular endothelial cells

doi: 10.1371/journal.pgen.1010680

Figure Lengend Snippet: ( A ) Heatmap of CAD-associated variants that are significant (false discovery rate (FDR) ≤10%) for at least one of six endothelial phenotypes tested in the teloHAEC pooled CRISPR screens. Each row corresponds to a combination of Cas9 variant and cellular readout, and each column corresponds to a CAD variant. For each variant, we added the name of a nearby gene to simplify locus identification, although we do not imply that these genes are causal. Dendrograms of rows and columns represent hierarchical clustering based on euclidean distance. The FDR is capped at 0.1%. ( B ) Validation by flow cytometry of six hits from the pooled CRISPR screens. For each validation, we used the top sgRNA from the pooled CRISPR screens to target the variant/locus with the corresponding Cas9 variant. We compared the distribution of the fluorescence intensity of the cellular markers ( x -axis) between the sgRNA identified in the screens and a safe harbor negative control sgRNA. We assessed statistical significance using the Kolmogorov-Smirnov (KS) test, all validations shown are significant (KS P-value <2.2x10 -16 ). Validations were performed in at least three independent experiments for each sgRNA . For E-selectin and ICAM1, the fluorochrome is PE; for ROS, the fluorochrome is FITC.

Article Snippet: TeloHAEC are immortalized human aortic endothelial cells obtained by over-expressing telomerase (ATCC CRL-4052).

Techniques: CRISPR, Variant Assay, Biomarker Discovery, Flow Cytometry, Fluorescence, Negative Control

Journal: PLOS Genetics

Article Title: Multimodal CRISPR perturbations of GWAS loci associated with coronary artery disease in vascular endothelial cells

doi: 10.1371/journal.pgen.1010680

Figure Lengend Snippet: ( A ) Perturbations with the Cas9 nuclease highlighted two synonymous variants (rs2074626, rs2240243) in the DHX38 gene for several endothelial phenotypes. DHX38 is located downstream of the HP and HPR genes, which have previously been associated with LDL-C levels. However, the CAD and LDL-C GWAS signals are distinct based on co-localization analyses (posterior probability for two independent association signals (H3) = 80.9%). ( B ) Gene-set enrichment analysis results for differentially expressed genes identified by RNA-seq in teloHAEC between a sgRNA targeting a DHX38 coding exon and a safe harbor negative control sgRNA. Only pathways with a Benjamini-Hochberg-corrected P-value <0.05 and normalized enrichment scores (NES) <-1 or >1 are shown. ( C ) Experimental design for the characterization of DHX38 using the fluorescent marker CRIMSON in place of an antibiotic resistance gene. We did all experiments in teloHAEC that stably express Cas9. We monitored the impact of a DHX38 sgRNA on cell proliferation, indel induction, gene expression and senescence-associated β-galactosidase (SA-βGal) activity. ( D ) Comparison of endothelial cell proliferation between teloHAEC with a DHX38 sgRNA or a safe harbor negative control sgRNA. The differences in the number of CRIMSON + cells were not significant two or four days post-infection. However, there were 27% less CRIMSON + cells with DHX38 sgRNA relative to the safe harbor control at seven days post-infection (Student’s t -test P -value = 7.3x10 -8 ). Results are mean ± standard deviation for 6 replicates for safe harbor and three replicates for two DHX38 targeting sgRNA. ( E ) Quantification of DHX38 indels by tracking of indel by decomposition (TIDE) analysis. As expected, we found no indels in the CRIMSON - cells . However, in CRIMSON + cells that received a DHX38 sgRNA, we found indels with an average frequency of 15%, 42% and 40% at day 2, 4 and 7, respectively. Results are mean ± standard deviation for 6 replicates for safe harbor and three replicates for two DHX38 targeting sgRNA. ( F ) Expression levels of DHX38 and CDKN1A in CRIMSON - and CRIMSON + teloHAEC that have received a sgRNA that targets DHX38 or a safe harbor region (negative control). There were no significant differences in DHX38 expression levels at day 2. However, at day 4 and 7, DHX38 was significantly down-regulated and CDKN1A was significantly up-regulated in CRIMSON + cells that received the DHX38 sgRNA. N.S., not significant. We provide Student’s t -test P-values when P <0.05. Bars are mean normalized expression and error bars represent one standard deviation. ( G ) Quantification of senescent teloHAEC by flow cytometry using senescence-associated β-galactosidase (SA-βGal) staining. At day 4 and 7 post-infection, there were significantly more senescent cells in the CRIMSON + DHX38 sgRNA experiment than in the CRIMSON - cells or in the CRIMSON + cells that received the safe harbor sgRNA. We used the DNA damaging agent etoposide as a positive control to induce senescence. N.S., not significant. We provide Student’s t -test P-values when P <0.05. Results are mean percentage SA-βGal + teloHAEC and error bars represent one standard deviation.

Article Snippet: TeloHAEC are immortalized human aortic endothelial cells obtained by over-expressing telomerase (ATCC CRL-4052).

Techniques: RNA Sequencing, Negative Control, Marker, Stable Transfection, Gene Expression, Activity Assay, Comparison, Infection, Control, Standard Deviation, Expressing, Flow Cytometry, Staining, Positive Control

Journal: PLOS Genetics

Article Title: Multimodal CRISPR perturbations of GWAS loci associated with coronary artery disease in vascular endothelial cells

doi: 10.1371/journal.pgen.1010680

Figure Lengend Snippet: (A) Locus view for the CAD locus with nearby gene CNNM2 . We provide the position of the sentinel CAD variant (rs11191416) and the putative functional variant identified in the pooled CRISPR screen (rs78260931). The LD proxies and sgRNAs tested are also shown. ATAC-seq and RNA-seq data in resting teloHAEC are from ref. . ( B ) Locus view for the CAD locus with nearby genes ZNF664 and CCDC92 . We provide the position of the sentinel CAD variant (rs11057401) and the functional variant identified in the pooled CRISPR screen (rs12311848). The LD proxies and sgRNAs tested are also shown. ATAC-seq and RNA-seq data in resting teloHAEC are from ref. . ( C ) Uniform manifold approximation projection (UMAP) for 11,756 cells from human right coronary arteries analyzed by single-cell RNA-sequencing . We color-coded cells based on the level of expression of candidate causal CAD genes identified and characterized in this study. We used the expression of the endothelial cell marker gene CDH5 (encoding VE-Cadherin) to identify endothelial cells (circle in top left panel). All five candidate genes are expressed in human vascular endothelial cells from coronary arteries.

Article Snippet: TeloHAEC are immortalized human aortic endothelial cells obtained by over-expressing telomerase (ATCC CRL-4052).

Techniques: Variant Assay, Functional Assay, CRISPR, RNA Sequencing, Expressing, Marker

Journal: Frontiers in Neuroscience

Article Title: A Putative Role of Teneurin-2 and Its Related Proteins in Astrocytes

doi: 10.3389/fnins.2019.00655

Figure Lengend Snippet: In vitro characterization of Ten/TCAP gene expressions and TCAP-1 induced activation in mouse cerebellar astrocytes. Gene expression was determined using RT-PCR with RNA extracted from C8D1A mouse cerebellar astrocytes. (A) teneurins 1, 3 and 4 were expressed ( n = 4) and teneurin 2 was not expressed ( n = 4). TCAPs 1-4 were expressed (TCAP-1, n = 3; TCAP-2, n = 5; TCAP-3, n = 3; TCAP-4, n = 3). β-actin served as positive control ( n = 5). (B) fluo-4 fluorescence shown prior to 100 nM TCAP-1 administration (non-stimulated). (C) fluo-4 fluorescence shown 3 min after 100 nM TCAP-1 administration (TCAP-1 treatment). (D) differential interference contrast image (DIC) C8D1A astrocytes. Arrows are showing discreet band. (E) normalized fluo-4 fluorescence comparing vehicle (aCSF) and 100 nM TCAP-1 treated cells ( n = 3 for each treatment, where each n is an average of five cells per coverslip). Mean (± SEM) values from each group were submitted to two-way ANOVA and Bonferroni post hoc test. ** p < 0.01; *** p < 0.001.

Article Snippet: For this, C8D1A mouse cerebellar immortalized astrocytes (#CRL-2541, ATCC, VA, USA) were used.

Techniques: In Vitro, Activation Assay, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Positive Control, Fluorescence

Journal: Frontiers in Oral Health

Article Title: Novel small molecule targeting PgQC reduces Porphyromonas gingivalis virulence

doi: 10.3389/froh.2026.1716188

Figure Lengend Snippet: S-0636 exhibits a low potential of cytotoxicity in human cell lines. Human liver cells (HepG2, black bars), human embryonic kidney cells (HEK293, grey bars), human gingival keratinocytes (hTERT-TIGK, dark grey bars), and human gingival fibroblasts (hTERT-TIGF, light gray bars) were seeded and cultivated as described above. Once the cells reached ∼80% confluency, the medium was replaced with fresh medium containing various concentrations of S-0636, followed by an additional 24-hour incubation under the same conditions. The WST-8 assay determined the viability of cells. Cells treated with 0.1% Triton X-100 served as a negative control), while cells treated with the S-0636 compound diluent served as a positive control. Cell viability was quantified by measuring the absorbance of WST-8 formazan at 450 nM using a Tecan Sunrise microplate reader. All experiments were performed in at least three independent biological replicates.

Article Snippet: The cell lines hTERT-TIGK (human telomerase immortalized gingival keratinocytes) and hTERT-TIGF (human telomerase immortalized gingival fibroblasts) were obtained from American Type Culture Collection (ATCC,CRL-3397). hTERT-TIGK cells were cultured in GibcoTM Medium 154 supplemented with the Human Keratinocyte Growth Kit (Gibco). hTERT-TIGF cells were maintained in Dulbecco's Modified Eagle Medium (DMEM/F12) supplemented with 10% fetal bovine serum (FBS) and GlutaMAXTM (Gibco).

Techniques: Incubation, Negative Control, Positive Control