Fig 1: The action of Prostratin and IDB depends on the phosphorylation of the S606 at the pore region of the hERG channel. a Schematic illumination of putative PKC phosphorylation sites and point mutations in hERG proteins. b Effects of Prostrain and IDB on the current densities of hERGWT-hERGA561V channels with a lack of 17 PKC phosphorylation sites (indicated as 17(A)). c–e Effects of Prostrain and IDB on the current densities of hERGWT-hERGA561V channels with a lack of PKC phosphorylation sites (S26A, T162A, T174A, S179A, S250A, S278A, S354A, and T371A) in the N-terminus (indicated as N(A)), PKC phosphorylation sites (T526A, S606A, S636A, and T670A) in the transmembrane domain (indicated as T(A)), or PKC phosphorylation sites (S890A, T895A, S918A, S960A, and T1133A) in the C-terminus (indicated as C(A)). f Effects of Prostrain and IDB on the current densities of hERGWT-hERGA561V channels with a lack of the S606 PKC phosphorylation site. g Effects of the mutation S606E on the current densities of hERGWT-hERGA561V channels. The ratio of hERGWT/hERGA561V was 2:1. All experiments were performed at least three times. Data shown are mean ± s.e.m. ***P < 0.001, n.s., not significant (one-way ANOVA Dunnett’s test for b, c, d, e, and f; Student’s t tests for g)
Fig 2: Knockdown of hERG1 reduces proliferation of osteosarcoma cells. (A) Efficiency of knockdown by hERG1-siRNA was measured by Western blot. (B-E) Proliferation of MG-63 cells transfected with hERG1-siRNA (30 nM) (B and C), or treated with hERG1 inhibitor E-4031 (D) or activator PD 118057 (E) was measured using CCK-8 or colony formation assay (n = 6). (F) Protein expression of hERG1 in HEK293-wt and HEK293-hERG1 cells was detected by Western blot. (G) 1 × 105 HEK293-wt and HEK293-hERG1 cells were cultured for 48 h and the CCK-8 assay was performed. (H) The effects of E-4031 on the proliferation of HEK293-wt and HEK293-hERG1 cells were determined by CCK-8 assay. * P < 0.05, ** P < 0.01, *** P < 0.001.
Fig 3: The involvement of NF-κB pathway in hERG1-regulated osteosarcoma cell survival. (A and B) Effect of hERG1 inhibition on NF-κB pathway-related gene expression. MG-63 cells were transfected hERG1-siRNA or control-siRNA, or left untreated, and the indicated mRNA and proteins were analyzed by semi-quantitative RT-PCR and Western blot. (C) Effect of hERG1 inhibition on NF-κB mediated transcription. (D) Nuclear localization of NF-κB p65 in MG-63 cells transfected with pcDNA3.1-hERG1 or pcDNA3.1. (E and F) CCK-8 assays were performed in MG-63 cells transiently transfected with NF-κB p65-siRNA or pCDNA3.1 NF-κB p65. (G) Effect of hERG1 knockdown on Akt phosphorylation. (H) Nuclear localization of NF-κB p65 (up panel) and relative cell proliferate rate (down panel) were determined. (I) Nuclear localization of NF-κB p65 in MG-63 cells treated with LY264002. * P < 0.05, ** P < 0.01.
Fig 4: The influence of hERG1 knockdown on osteosarcoma cell apoptosis. (A) MG-63 cells were transfected with hERG1-siRNA and then analyzed by flow cytometry. Cells untreated or transfected with control-siRNA were served as controls. Cells in the right lower quadrant are Annexin-positive, which are early apoptotic cells (n = 3). (B) Caspase-3 and PARP cleavage was determined by Western blot. (C) Cells transfected with hERG1-siRNA had a significant increase in caspase-3 activity compared to control-siRNA transfected and untreated cells. ** P < 0.01, *** P < 0.001.
Fig 5: A small-molecule screen in C. elegans identifies a novel hERG trafficking inhibitor. a Schematic illumination of the screening assay. b Molecular formula of alphitolic acid (ALA, upper) and microscopic images of acs-20;hERGchimera/A536W transgenic worms in the presence of 20 µM ALA in cultivating plates (lower). White arrows indicate eggs. c Representative whole-cell currents of HEK293T cells expressing wild-type hERG channels before or after treated with 1 or 10 µM ALA. n = 6 cells. d, e Representative whole-cell currents and I/V curves of wild-type hERG channels expressed in HEK293T cells treated with DMSO (n = 19) or 20 µM ALA (n = 25) for 24 h. The protocol is shown in the lower right of d, and traces at the time course between two dashed lines are shown. f Dose-dependent effects of long-term ALA treatment on the current densities of wild-type hERG channels recorded at a membrane potential of -140 mV. n = 20 cells per dose were recorded and analyzed. g Western blot analysis of hERG proteins expressed in HEK293T cells treated with DMSO or 20 µM ALA for 24 h (top), and quantitantive analysis of the ratio of fully glycosylated (FG, 155 kD) to total hERG proteins (down). Black and gray arrows indicate 155 kD and 135 kD bands of hERG proteins, respectively. h Immunostaining of hERG and ER marker protein Calnexin in HEK293T cells treated with DMSO or 20 µM ALA for 24 h. Scale bar: 20 µm. Data shown are mean ± s.e.m. **P < 0.01,***P < 0.001 (Student’s t-tests for e, g). All experiments were performed at least three times
Supplier Page from Abcam for Anti-H-ERG antibody