Journal: Scientific Reports

Article Title: Whole-exome sequencing identifies two novel mutations in KCNQ4 in individuals with nonsyndromic hearing loss

doi: 10.1038/s41598-018-34876-9

Figure Lengend Snippet: Surface expression and subunit interactions of WT and mutant KCNQ4 proteins. HEK293T or HeLa cells were transfected with N-terminally Myc- or Flag-tagged WT and mutant KCNQ4 clones. ( a ) Cell surface biotinylation in HEK293T. Proteins on the plasma membrane were labelled with biotin, isolated with avidin beads, and assessed by western blotting. Surface expression of two mutant KCNQ4 proteins was similar to that of WT protein. ( b ) Immunofluorescence of WT and mutant KCNQ4 proteins in HeLa cells. Cells were immunostained with anti-Myc, anti-calnexin, and anti-giantin antibodies. Nuclei were stained with DAPI. Calnexin and giantin are markers for the endoplasmic reticulum and Golgi apparatus, respectively. Both mutant KCNQ4 proteins and WT protein were observed on the plasma membrane. ( c–d ) Subunit interactions between WT and mutant KCNQ4 proteins. Twenty-four hours post-transfection, whole-cell lysates were subjected to immunoprecipitation using anti-Myc ( c ) or anti-FLAG ( d ) antibodies and immunoblotted. Both KCNQ4 mutant proteins interacted with WT protein.

Article Snippet: Human embryonic kidney 293 T (HEK293T) and HeLa cells were cultured in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum and penicillin (50 IU/ml)/streptomycin (50 μg/ml; Invitrogen).

Techniques: Expressing, Mutagenesis, Transfection, Clone Assay, Isolation, Avidin-Biotin Assay, Western Blot, Immunofluorescence, Staining, Immunoprecipitation

Journal: Scientific Reports

Article Title: Whole-exome sequencing identifies two novel mutations in KCNQ4 in individuals with nonsyndromic hearing loss

doi: 10.1038/s41598-018-34876-9

Figure Lengend Snippet: Impaired potassium conductance of homomeric mutant KCNQ4 channels. ( a ) Whole-cell KCNQ4 K + current traces recorded from the HEK293T cells transiently expressing WT, p.D266Y, p.V87_N89del, or GFP. Homomeric p.Asp266Tyr and p.Val87_Asn89del mutant channels produced barely detectable K + currents. ( b,c ) Current-voltage (I-V) relationships of linopirdine (30 μM)-sensitive K + currents ( b ), and current densities measured at +40 mV (c). The I-V curve of the WT protein exhibited a typical outwardly rectifying KCNQ4 channel current, and the current densities of p.D266Y and p.V87_N89del mutants were not significant (NS) compared with GFP. WT, n = 24; p.D266Y, n = 10; p.V87_N89del, n = 10; GFP, n = 22. *P < 0.05 versus mutants and GFP. ( d–g ) Homomeric mutant channels were not activated by known KCNQ openers. Single or combination treatment with retigabine (Ret, 10 μM), ML213 (3 μM), and zinc pyrithione (ZnPy, 10 μM) did not activate mutant channels ( d,f ), and the current densities after treating KCNQ openers were not significantly different from that of GFP ( e,g ). Mean ± SEM, n = 6–9; **P < 0.01 and ***P < 0.005 versus the WT protein.

Article Snippet: Human embryonic kidney 293 T (HEK293T) and HeLa cells were cultured in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum and penicillin (50 IU/ml)/streptomycin (50 μg/ml; Invitrogen).

Techniques: Mutagenesis, Expressing, Produced

Journal: bioRxiv

Article Title: Ephrin receptor A4 (EphA4) is a new Kaposi’s sarcoma-associated herpesvirus virus entry receptor

doi: 10.1101/510651

Figure Lengend Snippet: A , CHO-K1 cells were transfected with T7 luciferase plasmid and either control plasmid, or EBV gH/gL, EBV gB, or KSHV gH/gL. Transfected CHO-K1 cells were overlaid with HEK293T cells transfected with pcDNA3.1, EphA2 or EphA4 together with T7 polymerase. Fusion activity was standardized to EBV gB, KSHV gH/gL fusion with HEK293T cells transfected with control pcDNA3.1 which was set to 100%. ***P<0.001 (ANOVA followed by post-hoc Tukey’s multiple comparison test), compared to pcDNA 3.1. B . 2.5 x 10 5 CHO-K1 cells transfected with Rluc8 1-7 luciferase plasmid together with either control plasmid, EBV gH/gL, EBV gB, or KSHV gH/gL, EBV gB, were overlaid with 2.5 x 10 5 CHO-K1 cells transfected with pcDNA3.1, EphA2, or EphA4 together with Rluc8 8-11. Green cells, indicative of fusion, were visualized and captured with an EVOS fluorescence microscope. C , HEK293T cells were transfected with pcDNA3.1, EphA2, or EphA4. 24 hours post-transfection, 5×10 4 cells were seeded into a 48-well plate. 24 hours later, the cells were infected with concentrated KSHV. After an additional 24 hours, the infected cells were analyzed by flow cytometry (C) or visualized by microscopy and images captured with an EVOS fluorescence microscope ( D ).

Article Snippet: Human Embryonic Kidney 293 T (HEK293T) cells (ATCC CRL-3216) or HEK293-T14 cells derived from HEK293T stably expressing T7 RNA polymerase ( ) were grown in DMEM (Corning) with 100μg/mL zeocin (Invitrogen, for HEK293T cells expressing T7 RNA polymerase only), containing 10% heat-inactivated FBS and 1% penicillin-streptomycin, respectively. iSLK.219 KSHV cells ( ) were kindly provided by Eva Gottwein and were grown in DMEM (Corning) containing 10% Tetracycline-free fetal bovine serum complex (Clontech) and 1% penicillin-streptomycin (100 U penicillin/mL, 100 μg streptomycin/mL; Sigma).

Techniques: Transfection, Luciferase, Plasmid Preparation, Activity Assay, Fluorescence, Microscopy, Infection, Flow Cytometry

Journal: bioRxiv

Article Title: Ephrin receptor A4 (EphA4) is a new Kaposi’s sarcoma-associated herpesvirus virus entry receptor

doi: 10.1101/510651

Figure Lengend Snippet: A. EphA2 cell surface expression in EphA2 and EphA4 single or double knockout (DKO) HEK293T cells by flow cytometry. The X axis represents the relative number of cells analyzed by flow cytometry with a particular level of EphA2 expression. The y-axis represents the level of expression within the analyzed cell population on a log scale. B , EphA4 expression in EphA2 and EphA4 single or double knockout HEK293T cells by Western blotting. GAPDH was used as loading control. C , For fusion function of knockout cell lines, CHO-K1 cells transfected with T7 luciferase and either a control plasmid or KSHV gH/gL, EBV gB were overlaid with EphA2 and EphA4 single or double knockout cells ( C ), or EphA2/A4 double knockout cells overexpressing EphA2, EphA4 or EphA2/EphA4 ( D ), that were transfected with T7 polymerase. E , WT or EphA2 and EphA4 double knockout cells were transfected with EphA2 or EphA4, and infected with KSHV as described in the material and methods. 24 hours later, cells were analyzed by flow cytometry for GFP expression to identify infected cells ( E ).

Article Snippet: Human Embryonic Kidney 293 T (HEK293T) cells (ATCC CRL-3216) or HEK293-T14 cells derived from HEK293T stably expressing T7 RNA polymerase ( ) were grown in DMEM (Corning) with 100μg/mL zeocin (Invitrogen, for HEK293T cells expressing T7 RNA polymerase only), containing 10% heat-inactivated FBS and 1% penicillin-streptomycin, respectively. iSLK.219 KSHV cells ( ) were kindly provided by Eva Gottwein and were grown in DMEM (Corning) containing 10% Tetracycline-free fetal bovine serum complex (Clontech) and 1% penicillin-streptomycin (100 U penicillin/mL, 100 μg streptomycin/mL; Sigma).

Techniques: Expressing, Double Knockout, Flow Cytometry, Western Blot, Knock-Out, Transfection, Luciferase, Plasmid Preparation, Infection

Journal: bioRxiv

Article Title: Ephrin receptor A4 (EphA4) is a new Kaposi’s sarcoma-associated herpesvirus virus entry receptor

doi: 10.1101/510651

Figure Lengend Snippet: ( A ) CHO-K1 cells were transiently transfected with control pcDNA3.1, EBV gH/gL, or KSHV gH/gL plasmids. Soluble EphA2-Fc or EphA4-Fc was prepared by transfecting CHO-K1 cells with EphA2-Fc or EphA4-Fc plasmid constructs and media supernatants containing EphA2-Fc or EphA4-Fc were overlaid on HEK293T cells expressing EBV gH/gL or KSHV gH/gL in 96-well plates in triplicate for 2 hours at 4°C and bound protein was detected using anti-human IgG which recognizes the Fc portion of the Eph2-Fc and EphA4-Fc by CELISA. B . CHO-K1 cells seeded in 6-well plates were transfected with control plasmid pcDNA3.1, EBV gH/gL, or KSHV gH/gL with gL containing a His-tagged as indicated. After 24 hours, cells were washed twice with ice-cold PBS and incubated with supernatants from pcDNA3.1 (control plasmid), EphA2-Fc, or EphA4-Fc transfected cells isolated 24 hours post-transfection for 2 hours at 4°C. The cells were then washed with ice-cold PBS three times and lysed with 200 ul of 1X SDS lysis buffer. Proteins bound to the cells expressing EBV gH/gL or KSHV gH/gL were then analyzed using antibodies to the Fc region of the EphA2 and EphA4 fusions by Western blotting. GAPDH was used as a loading control. Expression of the KSHV gH/L or EBV gH/gL complex was monitored by analyzing KSHV gL or EBV gL expression using anti-His antibodies directed against a His-tagged added to KSHV gL or polyclonal antibodies directed against EBV gL. C and D , CHO-K1 cells seeded in 6-well plates were transfected with control plasmid pcDNA3.1, EBV gH/gL, or KSHV gH/gL together with EphA2-Fc or EphA4-Fc. Transfected cells were seeded in 96-well plates in triplicate post-transfection. The cells were then washed with ice-cold PBS three times and gH/gL-associated EphA2 ( C ) or EphA4 ( D ) was determined by CELISA using anti-human IgG antibodies.

Article Snippet: Human Embryonic Kidney 293 T (HEK293T) cells (ATCC CRL-3216) or HEK293-T14 cells derived from HEK293T stably expressing T7 RNA polymerase ( ) were grown in DMEM (Corning) with 100μg/mL zeocin (Invitrogen, for HEK293T cells expressing T7 RNA polymerase only), containing 10% heat-inactivated FBS and 1% penicillin-streptomycin, respectively. iSLK.219 KSHV cells ( ) were kindly provided by Eva Gottwein and were grown in DMEM (Corning) containing 10% Tetracycline-free fetal bovine serum complex (Clontech) and 1% penicillin-streptomycin (100 U penicillin/mL, 100 μg streptomycin/mL; Sigma).

Techniques: Transfection, Plasmid Preparation, Construct, Expressing, Incubation, Isolation, Lysis, Western Blot

Journal: bioRxiv

Article Title: Ephrin receptor A4 (EphA4) is a new Kaposi’s sarcoma-associated herpesvirus virus entry receptor

doi: 10.1101/510651

Figure Lengend Snippet: A. Schematic drawing of the EphA2, EphA4, and, EphA2/EphA4 chimeras including the ligand binding domain (LBD). B. KSHV fusion with HEK293T cells transfected with control plasmid pcDNA3.1, EphA2, EphA4 or EphA2 and EphA4 chimeras as indicated. C , KSHV fusion with HEK293T cells transfected with control plasmid pcDNA3.1, EphA4, or EphA4 kinase-dead mutants as indicated. Fusion activity of HEK293T cells transfected with pcDNA 3.1 was set to 100% ( B and C ).

Article Snippet: Human Embryonic Kidney 293 T (HEK293T) cells (ATCC CRL-3216) or HEK293-T14 cells derived from HEK293T stably expressing T7 RNA polymerase ( ) were grown in DMEM (Corning) with 100μg/mL zeocin (Invitrogen, for HEK293T cells expressing T7 RNA polymerase only), containing 10% heat-inactivated FBS and 1% penicillin-streptomycin, respectively. iSLK.219 KSHV cells ( ) were kindly provided by Eva Gottwein and were grown in DMEM (Corning) containing 10% Tetracycline-free fetal bovine serum complex (Clontech) and 1% penicillin-streptomycin (100 U penicillin/mL, 100 μg streptomycin/mL; Sigma).

Techniques: Ligand Binding Assay, Transfection, Plasmid Preparation, Activity Assay

Journal: Scientific Reports

Article Title: Significant modulations of linc001128 and linc0938 with miR-24-3p and miR-30c-5p in Parkinson disease

doi: 10.1038/s41598-022-06539-3

Figure Lengend Snippet: Transfection of constructed plasmids in the HEK293T cells and dual luciferase assay. ( a – c ) Fluorescent imaging of HEК293T cells 48 h after transfection using fluorescent microscopy. Magnification × 10. ( d ) The luciferase activity of linc00938-WT, linc00938-MT and psiCHECK (empty vector) in the HEK293T cells treated with hsa-mir-30c. ( e ) Relative mRNA levels of LRRK2 determined by real-time PCR analysis after transfection with psiCHECK-linc00938 (WT) + pBud-mir-30c, psiCHECK-linc00938 (MT) + pBud-mir-30c, NC, and cells without any vector (Blank). Error bars represent SD. (**p ≤ 0.01, and ns p > 0.05).

Article Snippet: HEK293T (human embryonic kidney 293 T) cells were purchased from Pasteur Institute, Iran.

Techniques: Transfection, Construct, Luciferase, Imaging, Microscopy, Activity Assay, Plasmid Preparation, Real-time Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Ephrin receptor A4 (EphA4) is a new Kaposi’s sarcoma-associated herpesvirus virus entry receptor

doi: 10.1101/510651

Figure Lengend Snippet: A , CHO-K1 cells were transfected with T7 luciferase plasmid and either control plasmid, or EBV gH/gL, EBV gB, or KSHV gH/gL. Transfected CHO-K1 cells were overlaid with HEK293T cells transfected with pcDNA3.1, EphA2 or EphA4 together with T7 polymerase. Fusion activity was standardized to EBV gB, KSHV gH/gL fusion with HEK293T cells transfected with control pcDNA3.1 which was set to 100%. ***P<0.001 (ANOVA followed by post-hoc Tukey’s multiple comparison test), compared to pcDNA 3.1. B . 2.5 x 10 5 CHO-K1 cells transfected with Rluc8 1-7 luciferase plasmid together with either control plasmid, EBV gH/gL, EBV gB, or KSHV gH/gL, EBV gB, were overlaid with 2.5 x 10 5 CHO-K1 cells transfected with pcDNA3.1, EphA2, or EphA4 together with Rluc8 8-11. Green cells, indicative of fusion, were visualized and captured with an EVOS fluorescence microscope. C , HEK293T cells were transfected with pcDNA3.1, EphA2, or EphA4. 24 hours post-transfection, 5×10 4 cells were seeded into a 48-well plate. 24 hours later, the cells were infected with concentrated KSHV. After an additional 24 hours, the infected cells were analyzed by flow cytometry (C) or visualized by microscopy and images captured with an EVOS fluorescence microscope ( D ).

Article Snippet: Human Embryonic Kidney 293 T (HEK293T) cells (ATCC CRL-3216) or HEK293-T14 cells derived from HEK293T stably expressing T7 RNA polymerase ( ) were grown in DMEM (Corning) with 100μg/mL zeocin (Invitrogen, for HEK293T cells expressing T7 RNA polymerase only), containing 10% heat-inactivated FBS and 1% penicillin-streptomycin, respectively. iSLK.219 KSHV cells ( ) were kindly provided by Eva Gottwein and were grown in DMEM (Corning) containing 10% Tetracycline-free fetal bovine serum complex (Clontech) and 1% penicillin-streptomycin (100 U penicillin/mL, 100 μg streptomycin/mL; Sigma).

Techniques: Transfection, Luciferase, Plasmid Preparation, Activity Assay, Fluorescence, Microscopy, Infection, Flow Cytometry

Journal: bioRxiv

Article Title: Ephrin receptor A4 (EphA4) is a new Kaposi’s sarcoma-associated herpesvirus virus entry receptor

doi: 10.1101/510651

Figure Lengend Snippet: A. EphA2 cell surface expression in EphA2 and EphA4 single or double knockout (DKO) HEK293T cells by flow cytometry. The X axis represents the relative number of cells analyzed by flow cytometry with a particular level of EphA2 expression. The y-axis represents the level of expression within the analyzed cell population on a log scale. B , EphA4 expression in EphA2 and EphA4 single or double knockout HEK293T cells by Western blotting. GAPDH was used as loading control. C , For fusion function of knockout cell lines, CHO-K1 cells transfected with T7 luciferase and either a control plasmid or KSHV gH/gL, EBV gB were overlaid with EphA2 and EphA4 single or double knockout cells ( C ), or EphA2/A4 double knockout cells overexpressing EphA2, EphA4 or EphA2/EphA4 ( D ), that were transfected with T7 polymerase. E , WT or EphA2 and EphA4 double knockout cells were transfected with EphA2 or EphA4, and infected with KSHV as described in the material and methods. 24 hours later, cells were analyzed by flow cytometry for GFP expression to identify infected cells ( E ).

Article Snippet: Human Embryonic Kidney 293 T (HEK293T) cells (ATCC CRL-3216) or HEK293-T14 cells derived from HEK293T stably expressing T7 RNA polymerase ( ) were grown in DMEM (Corning) with 100μg/mL zeocin (Invitrogen, for HEK293T cells expressing T7 RNA polymerase only), containing 10% heat-inactivated FBS and 1% penicillin-streptomycin, respectively. iSLK.219 KSHV cells ( ) were kindly provided by Eva Gottwein and were grown in DMEM (Corning) containing 10% Tetracycline-free fetal bovine serum complex (Clontech) and 1% penicillin-streptomycin (100 U penicillin/mL, 100 μg streptomycin/mL; Sigma).

Techniques: Expressing, Double Knockout, Flow Cytometry, Western Blot, Knock-Out, Transfection, Luciferase, Plasmid Preparation, Infection

Journal: bioRxiv

Article Title: Ephrin receptor A4 (EphA4) is a new Kaposi’s sarcoma-associated herpesvirus virus entry receptor

doi: 10.1101/510651

Figure Lengend Snippet: ( A ) CHO-K1 cells were transiently transfected with control pcDNA3.1, EBV gH/gL, or KSHV gH/gL plasmids. Soluble EphA2-Fc or EphA4-Fc was prepared by transfecting CHO-K1 cells with EphA2-Fc or EphA4-Fc plasmid constructs and media supernatants containing EphA2-Fc or EphA4-Fc were overlaid on HEK293T cells expressing EBV gH/gL or KSHV gH/gL in 96-well plates in triplicate for 2 hours at 4°C and bound protein was detected using anti-human IgG which recognizes the Fc portion of the Eph2-Fc and EphA4-Fc by CELISA. B . CHO-K1 cells seeded in 6-well plates were transfected with control plasmid pcDNA3.1, EBV gH/gL, or KSHV gH/gL with gL containing a His-tagged as indicated. After 24 hours, cells were washed twice with ice-cold PBS and incubated with supernatants from pcDNA3.1 (control plasmid), EphA2-Fc, or EphA4-Fc transfected cells isolated 24 hours post-transfection for 2 hours at 4°C. The cells were then washed with ice-cold PBS three times and lysed with 200 ul of 1X SDS lysis buffer. Proteins bound to the cells expressing EBV gH/gL or KSHV gH/gL were then analyzed using antibodies to the Fc region of the EphA2 and EphA4 fusions by Western blotting. GAPDH was used as a loading control. Expression of the KSHV gH/L or EBV gH/gL complex was monitored by analyzing KSHV gL or EBV gL expression using anti-His antibodies directed against a His-tagged added to KSHV gL or polyclonal antibodies directed against EBV gL. C and D , CHO-K1 cells seeded in 6-well plates were transfected with control plasmid pcDNA3.1, EBV gH/gL, or KSHV gH/gL together with EphA2-Fc or EphA4-Fc. Transfected cells were seeded in 96-well plates in triplicate post-transfection. The cells were then washed with ice-cold PBS three times and gH/gL-associated EphA2 ( C ) or EphA4 ( D ) was determined by CELISA using anti-human IgG antibodies.

Article Snippet: Human Embryonic Kidney 293 T (HEK293T) cells (ATCC CRL-3216) or HEK293-T14 cells derived from HEK293T stably expressing T7 RNA polymerase ( ) were grown in DMEM (Corning) with 100μg/mL zeocin (Invitrogen, for HEK293T cells expressing T7 RNA polymerase only), containing 10% heat-inactivated FBS and 1% penicillin-streptomycin, respectively. iSLK.219 KSHV cells ( ) were kindly provided by Eva Gottwein and were grown in DMEM (Corning) containing 10% Tetracycline-free fetal bovine serum complex (Clontech) and 1% penicillin-streptomycin (100 U penicillin/mL, 100 μg streptomycin/mL; Sigma).

Techniques: Transfection, Plasmid Preparation, Construct, Expressing, Incubation, Isolation, Lysis, Western Blot

Journal: bioRxiv

Article Title: Ephrin receptor A4 (EphA4) is a new Kaposi’s sarcoma-associated herpesvirus virus entry receptor

doi: 10.1101/510651

Figure Lengend Snippet: A. Schematic drawing of the EphA2, EphA4, and, EphA2/EphA4 chimeras including the ligand binding domain (LBD). B. KSHV fusion with HEK293T cells transfected with control plasmid pcDNA3.1, EphA2, EphA4 or EphA2 and EphA4 chimeras as indicated. C , KSHV fusion with HEK293T cells transfected with control plasmid pcDNA3.1, EphA4, or EphA4 kinase-dead mutants as indicated. Fusion activity of HEK293T cells transfected with pcDNA 3.1 was set to 100% ( B and C ).

Article Snippet: Human Embryonic Kidney 293 T (HEK293T) cells (ATCC CRL-3216) or HEK293-T14 cells derived from HEK293T stably expressing T7 RNA polymerase ( ) were grown in DMEM (Corning) with 100μg/mL zeocin (Invitrogen, for HEK293T cells expressing T7 RNA polymerase only), containing 10% heat-inactivated FBS and 1% penicillin-streptomycin, respectively. iSLK.219 KSHV cells ( ) were kindly provided by Eva Gottwein and were grown in DMEM (Corning) containing 10% Tetracycline-free fetal bovine serum complex (Clontech) and 1% penicillin-streptomycin (100 U penicillin/mL, 100 μg streptomycin/mL; Sigma).

Techniques: Ligand Binding Assay, Transfection, Plasmid Preparation, Activity Assay

Journal: eLife

Article Title: Interplay between PML NBs and HIRA for H3.3 dynamics following type I interferon stimulus

doi: 10.7554/eLife.80156

Figure Lengend Snippet:

Article Snippet: Human BJ primary foreskin fibroblasts (ATCC, CRL-2522), human IMR90 fetal lung fibroblasts (ATCC, CCL-186), human HEK 293T embryonic kidney cells (Intercell, AG) and mouse MEFs embryonic fibroblasts Pml +/+ or Pml -/- (from Dr. Lallemand-Breitenbach, and whose cell identity was authenticated by STR profiling) were cultivated in DMEM medium (Sigma-Aldrich, D6429) containing 10% of fetal calf serum (FCS) (Sigma-Aldrich, F7524), 1% of penicillin/streptomycin (Sigma-Aldrich, P4458), at 37 °C under 5% CO2 and humid atmosphere.

Techniques: Transduction, Recombinant, Plasmid Preparation, Clone Assay, Mutagenesis, Sequencing, Quantitative RT-PCR, Concentration Assay, In Situ, Software, ChIP-sequencing