Journal: Nature Communications

Article Title: A gain-of-function TPC2 variant R210C increases affinity to PI(3,5)P 2 and causes lysosome acidification and hypopigmentation

doi: 10.1038/s41467-023-35786-9

Figure Lengend Snippet: a A cartoon (drawn using BioRender with permission) depicting the three-step protocol used for whole-endolysosome recording from an enlarged endolysosome. b A cartoon (drawn using BioRender with permission) depicting the whole-endolysosome configuration and solutions used for inhibiting constitutive currents of R210C in endolysosome patches. c A typical TPC2 WT response to 1 µM PI(3,5)P 2 -diC8. d Representative traces of lysosomal TPC2 R210C currents in response to NMDG + and YM201636 (800 nM) as indicated. The outward current at +100 mV carried by K + was eliminated by substituting all cations in the bath solution with NMDG + . The inward current at −100 mV was sensitive to YM201636, a potent PIKfyve inhibitor that blocks PI(3,5)P 2 synthesis. e Analysis of the basal current of TPC2 WT and TPC2 R210C at −100 mV under the resting condition. WT: −49.5 ± 8.7 pA ( n = 11), R210C: −898.1 ± 346.2 pA ( n = 8). Data are presented in mean ± SEM, *** p < 0.001 by Student’s unpaired t -test, two-tailed. Source data are provided as a Source Data file. f Example traces of R210C currents recorded in whole-endolysosome configuration. NMDG + , tetrandrine (2 µM) and Ned-19 (100 nM) were applied as indicated. Similar results were obtained from three patches. g Single-channel activity of TPC2 WT and TPC2 R210C at −100 mV in inside-out patches excised from HEK293 cells that expressed endolysosomal TPC2 WT or TPC2 R210C . The upper and middle traces were obtained from untreated cells, while the lower trace was obtained from a TPC2 WT expressing cell pretreated with 30 µM A1N for 5 min and recorded in the presence of 100 nM PI(3,5)P 2 -diC8. For each trace, the closed and open states are labeled with red dashed lines and the total time is 2 s. h Bar graph illustrating the mean open probability (Po) of single-channel events of TPC2 WT (0, n = 3), TPC2 R210C (0.65 ± 0.09, n = 3), and TPC2 WT + 100 nM PI(3,5)P 2 (0.52 ± 0.13, n = 3). Data are presented in mean ± SEM, * p < 0.05, ** p < 0.01, by one-way ANOVA. Source data are provided as a Source Data file. i Bar graph illustrating the mean amplitude of single-channel currents of TPC2 R210C (8.92 ± 0.36 pA, n = 3) and TPC2 WT (8.79 ± 0.50 pA, n = 3, recorded in the presence of 100 nM PI(3,5)P 2 ). Data are presented in mean ± SEM, N.S., not significant ( p > 0.05), by one-way ANOVA. Source data are provided as a Source Data file.

Article Snippet: LysoTracker Red DND-99 (Thermo Fisher Scientific, L7528), LysoSensor Yellow/Blue Dextran (Invitrogen, L22460), TPC2-A1-N (MCE, HY-131614), trans Ned-19 (MCE, HY-103316A), YM201636 (MCE, HY-13228), Fura2 acetoxymethyl ester (Fura2 AM, F14185, Invitrogen), and Tetrandrine (MCE, HY-13764) were purchased as indicated in parentheses.

Techniques: Two Tailed Test, Activity Assay, Expressing, Labeling

Journal: American Journal of Translational Research

Article Title: Differentiation induction of human breast cancer cells by arsenite in combination with tetrandrine

doi:

Figure Lengend Snippet: Induction of the expression of ICAM-1 in breast cancer cells treated with AsIII and Tetra, alone or in combination. The expression level of ICAM-1 was determined using a FACSCanto flow cytometer after the treatment with various concentrations of AsIII alone (0.125, 0.25, 0.5, 1 and 2 µM), Tetra alone (0.125, 0.25, 0.5, 1 and 2 µg/ml), their combination in constant ratio (0.125 µM AsIII+0.125 µg/ml Tetra, 0.25 µM AsIII+0.25 µg/ml Tetra, 0.5 µM AsIII+0.5 µg/ml Tetra, 1 µM AsIII+1 µg/ml Tetra and 2 µM AsIII+2 µg/ml Tetra) for 96 h. The expression levels of ICAM-1 in breast cancer cells were analyzed and presented as histograms plotted by FACS as described in methods section. Representative FACS histograms of ICAM-1 expression in MDA-MB-231 (A) and MCF-7 (B) are shown. Untreated and treated cells are represented by filled histograms and open histograms, respectively. Relative expression levels of ICAM-1 in MDA-MB-231 cells (C) and MCF-7 cells (D) were calculated, respectively, based on FACS profiles shown in (A) and (B). Experiments were independently repeated at least three times and results are shown as means ± SD. *, P<0.05 vs. control; #, P<0.05 vs. each alone. As, AsIII; Tetra, tetrandrine; ATRA, all-trans retinoic acid. MFI mean fluorescence intensity. ATRA (1 µM) used as a positive control for differentiation.

Article Snippet: Materials Sodium arsenite (NaAsO 2 , As III ) and tetrandrine (Tetra) were purchased from Tri Chemical Laboratories (Yamanashi, Japan) and National Institutes for Food and Drug Control (Beijing, China), respectively.

Techniques: Expressing, Flow Cytometry, Control, Fluorescence, Positive Control

Journal: American Journal of Translational Research

Article Title: Differentiation induction of human breast cancer cells by arsenite in combination with tetrandrine

doi:

Figure Lengend Snippet: Expression profile of differentiation-related proteins in breast cancer cells treated with AsIII and Tetra, alone or in combination. After treatment with indicated concentrations of AsIII and Tetra, alone or in combination, for 96 h, the expression profiles of differentiation-related proteins in MDA-MB-231 cells (A) and MCF-7 cells (B) were analyzed using western blot as described in material and methods. Representative image of the expression profile of each protein is shown from three independent experiments. The densitometry of protein bands was analyzed using a program, NIH ImageJ 1.52a. The values above each image represent the ratios between each key molecule and β-actin protein expression levels, which were further compared with those of control group (untreated cells). p-Erk, phosphorylated Erk; As, AsIII; Tetra, tetrandrine; ATRA, all-trans retinoic acid. ATRA (1 µM) used as a positive control for differentiation.

Article Snippet: Materials Sodium arsenite (NaAsO 2 , As III ) and tetrandrine (Tetra) were purchased from Tri Chemical Laboratories (Yamanashi, Japan) and National Institutes for Food and Drug Control (Beijing, China), respectively.

Techniques: Expressing, Western Blot, Control, Positive Control

Journal: American Journal of Translational Research

Article Title: Differentiation induction of human breast cancer cells by arsenite in combination with tetrandrine

doi:

Figure Lengend Snippet: Upregulation of ICAM-1 and activation of Erk signaling pathway in MDA-MB-231 mouse xenografts treated with AsIII and Tetra, alone or in combination. MDA-MB-231 tumors in the mice xenograft model treated with AsIII and Tetra, alone or in combination, were obtained from our previous study, in which long-term (10 weeks) co-administration of AsIII (2 mg/kg/day) and Tetra (20 mg/kg/day) significantly reduced tumor volume and weight [8]. (A) The expression profiles of ICAM-1, phospho-Erk and total Erk were analyzed using western blot as described in material and methods. Results are representatives of three independent experiments. The relative expression levels of ICAM-1 (B) and phospho-Erk/Erk (C) were expressed as the ratios between each target gene protein and β-actin protein expression levels, and compared with those of untreated control group, respectively. Results are shown as the means ± SD from three independent experiments. p-Erk, phosphorylated Erk; As, AsIII; Tetra, tetrandrine. The procedures for establishment of MDA-MB-231 mouse xenografts, and the following administration of AsIII and Tetra, each alone or in combination, and the preparation of tumor tissues are shown in Supplementary Materials.

Article Snippet: Materials Sodium arsenite (NaAsO 2 , As III ) and tetrandrine (Tetra) were purchased from Tri Chemical Laboratories (Yamanashi, Japan) and National Institutes for Food and Drug Control (Beijing, China), respectively.

Techniques: Activation Assay, Expressing, Western Blot, Control

Journal: American Journal of Translational Research

Article Title: Differentiation induction of human breast cancer cells by arsenite in combination with tetrandrine

doi:

Figure Lengend Snippet: Effect of PD98059 on the expression profile of ICAM-1 induced by AsIII and Tetra, alone or in combination, in breast cancer cells. After treatment with indicated concentrations of AsIII and Tetra, alone or in combination, in the presence or absence of 10 μM PD98059, for 96 h, the expression profiles of ICAM-1 were determined using a FACSCanto flow cytometer as described in material and methods. Relative expression levels of ICAM-1 in MDA-MB-231 (A) and MCF-7 (B) were calculated based on their respective FACS profiles. *, P<0.01, **, P<0.0001 vs. control; †, P<0.05, ††, P<0.001, †††, P<0.0001 group with PD98059 vs. respective group without PD98059; #, P<0.001 vs. each alone. As, AsIII; Tetra, tetrandrine.

Article Snippet: Materials Sodium arsenite (NaAsO 2 , As III ) and tetrandrine (Tetra) were purchased from Tri Chemical Laboratories (Yamanashi, Japan) and National Institutes for Food and Drug Control (Beijing, China), respectively.

Techniques: Expressing, Flow Cytometry, Control

Journal: American Journal of Translational Research

Article Title: Differentiation induction of human breast cancer cells by arsenite in combination with tetrandrine

doi:

Figure Lengend Snippet: Effects of AsIII and Tetra, alone or in combination, on the proliferation of PBMCs. Following the treatment with various concentrations of AsIII alone (0.125, 0.25, 0.5, 1, 2 μM), Tetra alone (0.125, 0.25, 0.5, 1, 2 μg/mL), or their combination (0.125 μM+0.125 μg/mL, 0.25 μM+0.25 μg/mL, 0.5 μM+0.5 μg/mL, 1 μM+1 μg/mL, 2 μM+2 μg/mL) for 72 h, cell viability was determined by XTT assay as described under materials and methods. Relative cell viability was calculated as the ratio of the absorbance at 450 nm of each treatment group against those of the corresponding untreated control group. Data are shown as the means ± SD from more than three independent experiments. *P<0.05, vs control. As, AsIII; Tetra, tetrandrine.

Article Snippet: Materials Sodium arsenite (NaAsO 2 , As III ) and tetrandrine (Tetra) were purchased from Tri Chemical Laboratories (Yamanashi, Japan) and National Institutes for Food and Drug Control (Beijing, China), respectively.

Techniques: XTT Assay, Control

Journal: American Journal of Translational Research

Article Title: Differentiation induction of human breast cancer cells by arsenite in combination with tetrandrine

doi:

Figure Lengend Snippet: Effects of AsIII and Tetra, each alone or in combination, on the population of CD4+ T cells and Treg cells in mitogen-activated human PBMCs. After treatment with 0.5 μM AsIII and 0.5 μg/ml Tetra, alone or in combination, for 72 h, alterations of CD4+ T cell and Treg cell populations in ConA-activated PBMCs were analyzed by a flow cytometer as described in materials and methods. (A) Lymphocyte fraction of PBMCs (a1) were gated for CD4+ T cells (a2), and the percentage of Treg cells in the CD4+ T cells fraction in each treatment were calculated (B). Treg cells (C) and CD4+ T cells (D) in ConA-activated PBMCs treated with 0.5 μM AsIII and 0.5 μg/ml Tetra, alone or in combination, for 72 h were calculated, respectively. Data are presented as means ± SD from more than three independent experiments. As, AsIII; Tetra, tetrandrine.

Article Snippet: Materials Sodium arsenite (NaAsO 2 , As III ) and tetrandrine (Tetra) were purchased from Tri Chemical Laboratories (Yamanashi, Japan) and National Institutes for Food and Drug Control (Beijing, China), respectively.

Techniques: Flow Cytometry

Journal: Cancer Cell International

Article Title: Antitumor activity of arsenite in combination with tetrandrine against human breast cancer cell line MDA-MB-231 in vitro and in vivo

doi: 10.1186/s12935-018-0613-0

Figure Lengend Snippet: Cytotoxic effect of As III and Tetra, alone or in combination, against human breast cancer cell line MDA-MB-231. Cell viability was determined by XTT assay after the treatment with various concentrations of As III alone (0.3, 1, 3, 10, 30 and 100 µM) ( a ), Tetra alone (2, 4, 6, 8, 10 and 12 µg/ml) ( b ), their combination in constant ratio (5 µM As III + 2 µg/ml Tetra, 10 µM As III + 4 µg/ml Tetra, 15 µM As III + 6 µg/ml Tetra, 20 µM As III + 8 µg/ml Tetra, 25 µM As III + 10 µg/ml Tetra, and 30 µM As III + 12 µg/ml Tetra) ( c ), or combination treatment with relatively low concentrations (5 µM As III + 3.5 µg/ml Tetra, 10 µM As III + 4 µg/ml Tetra, 15 µM As III + 4.5 µg/ml Tetra) ( d ) for 48 h. Relative cell viability was calculated as the ratio of the absorbance at 450 nm of each treatment group against those of the corresponding untreated control group. Data are shown as the means and SD from more than three independent experiments. † p < 0.05 vs. each alone. As, As III ; Tetra, tetrandrine; As + Tetra, As III + tetrandrine

Article Snippet: Tetrandrine hydrochloride injection and arsenious acid and sodium chloride injection were obtained from Jiangxi Yintao Pharmaceutical Co., Ltd. (Jiangxi, China) and Heilongjiang Harbin Pharmaceutical Co., Ltd. (Heilongjiang, China), respectively for the in vivo study.

Techniques: XTT Assay, Control

Journal: Cancer Cell International

Article Title: Antitumor activity of arsenite in combination with tetrandrine against human breast cancer cell line MDA-MB-231 in vitro and in vivo

doi: 10.1186/s12935-018-0613-0

Figure Lengend Snippet: Antitumor activity of As III alone and in combination with Tetra in MDA-MB-231 mouse xenografts. The experiment was carried out using human breast cancer nude mice implanted subcutaneously with 1 × 10 7 MDA-MB-231 cells. The administration of As III and Tetra, alone or in combination, was performed as described under “ ”. Tumor volumes ( a ) and body weights ( b ) were measured at indicated time points, respectively. Tumor weights at time of sacrifice ( c ), and photographs of isolated tumors derived from vehicle-control and treated mice ( d ) were presented. * p < 0.05, ‡ p < 0.01 vs. vehicle-control (treated with PBS). As, As III ; Tetra, tetrandrine; As + Tetra, As III + tetrandrine

Article Snippet: Tetrandrine hydrochloride injection and arsenious acid and sodium chloride injection were obtained from Jiangxi Yintao Pharmaceutical Co., Ltd. (Jiangxi, China) and Heilongjiang Harbin Pharmaceutical Co., Ltd. (Heilongjiang, China), respectively for the in vivo study.

Techniques: Activity Assay, Isolation, Derivative Assay, Control

Journal: Cancer Cell International

Article Title: Antitumor activity of arsenite in combination with tetrandrine against human breast cancer cell line MDA-MB-231 in vitro and in vivo

doi: 10.1186/s12935-018-0613-0

Figure Lengend Snippet: Effects of As III and Tetra, alone or in combination, on the cell cycle profiling and the expression level of cell cycle related-proteins in MDA-MB-231 cells. a – d After treatment with various concentrations of As III (5, 10 and 15 µM), and Tetra (3.5, 4 and 4.5 µg/ml), alone or in combination (5 µM As III + 3.5 µg/ml Tetra, 10 µM As III + 4 µg/ml Tetra), for 48 h, cell cycle profiling was performed by FACSCanto flow cytometer as described under “ ”. Analyzed data and profiles for each G 0 /G 1 and G 2 /M phase using Diva software and ModFit LT™ ver. 3.0. are shown in the gray area. Cells at S phase are shown as shaded area. A representative FACS histogram from three separate experiments is shown. Significant difference between control and treatment with As III and Tetra, alone or in combination, are shown (* p < 0.05, ‡ p < 0.01, § p < 0.001, † p < 0.0001 vs. control, # p < 0.05, $ p < 0.01 vs. As III alone; & p < 0.05, ¶ p < 0.01 vs. Tetra alone). e Representative image of the expression profile of each protein is shown from three independent experiments. The densitometry of protein bands was analyzed using a program, NIH ImageJ 1.52a. The values under each image represent the ratios between each key molecule and β-actin protein expression levels, which were further compared with those of control group (untreated cells). As, As III ; Tetra, tetrandrine. Since enough cells cannot be collected in the group treated with 15 µM As III in combination with 4.5 µg/ml Tetra due to its strong cytotoxicity, cell cycle and western blot analysis were not conducted

Article Snippet: Tetrandrine hydrochloride injection and arsenious acid and sodium chloride injection were obtained from Jiangxi Yintao Pharmaceutical Co., Ltd. (Jiangxi, China) and Heilongjiang Harbin Pharmaceutical Co., Ltd. (Heilongjiang, China), respectively for the in vivo study.

Techniques: Expressing, Flow Cytometry, Software, Control, Western Blot

Journal: Cancer Cell International

Article Title: Antitumor activity of arsenite in combination with tetrandrine against human breast cancer cell line MDA-MB-231 in vitro and in vivo

doi: 10.1186/s12935-018-0613-0

Figure Lengend Snippet: Enhanced LDH release in MDA-MB-231 cells treated with As III combined with Tetra. After treatment with 10 μM As III and 4 μg/ml Tetra, alone or in combination, in the presence or absence of 0.25 μM wortmannin, for 48 h, LDH leakage was measured using the LDH-Cytotoxic test kit as described under “ ”. Significant difference between control and treatment with As III and Tetra, alone or in combination, are shown ( † p < 0.0001 vs. control; ∫ p < 0.0001 vs. As III alone; ♪ p < 0.001 vs. Tetra). As, As III ; Tetra, tetrandrine

Article Snippet: Tetrandrine hydrochloride injection and arsenious acid and sodium chloride injection were obtained from Jiangxi Yintao Pharmaceutical Co., Ltd. (Jiangxi, China) and Heilongjiang Harbin Pharmaceutical Co., Ltd. (Heilongjiang, China), respectively for the in vivo study.

Techniques: Control

Journal: Cancer Cell International

Article Title: Antitumor activity of arsenite in combination with tetrandrine against human breast cancer cell line MDA-MB-231 in vitro and in vivo

doi: 10.1186/s12935-018-0613-0

Figure Lengend Snippet: Effect of wortmannin on the cytotoxicity of the combined treatment of As III and Tetra. After the treatment with 10 μM As III combined with 4 μg/ml Tetra in the presence or absence of 0.25 μM and 1.0 μM wortmannin, respectively, for 24 h ( a ) and 48 h ( b ), cell viability was determined by XTT assay as described under “ ”. Relative cell viability was calculated as the ratio of the absorbance at 450 nm of each treatment group against those of the corresponding untreated control group. † p < 0.0001 vs. control; ∂ p < 0.05, £ p < 0.0001 vs. As III + Tetra. As, As III ; Tetra, tetrandrine

Article Snippet: Tetrandrine hydrochloride injection and arsenious acid and sodium chloride injection were obtained from Jiangxi Yintao Pharmaceutical Co., Ltd. (Jiangxi, China) and Heilongjiang Harbin Pharmaceutical Co., Ltd. (Heilongjiang, China), respectively for the in vivo study.

Techniques: XTT Assay, Control

Journal: Cancer Cell International

Article Title: Antitumor activity of arsenite in combination with tetrandrine against human breast cancer cell line MDA-MB-231 in vitro and in vivo

doi: 10.1186/s12935-018-0613-0

Figure Lengend Snippet: Expression profile of autophagy-related proteins in MDA-MB-231 cells treated with As III and Tetra, alone or in combination. After treatment with various concentrations of As III and Tetra, alone or in combination, for 48 h, the expression profile of autophagy-related proteins was analyzed using western blot as described in “ ”. Representative image of the expression profile of each protein is shown from three independent experiments. The densitometry of protein bands was analyzed using a program, NIH ImageJ 1.52a. The values under each image represent the ratios between each key molecule and β-actin protein expression levels, which were further compared with those of control group (untreated cells). As, As III ; Tetra, tetrandrine. Since enough cells cannot be collected in the group treated with 15 µM As III in combination with 4.5 µg/ml Tetra due to its strong cytotoxicity, western blot analysis were not conducted

Article Snippet: Tetrandrine hydrochloride injection and arsenious acid and sodium chloride injection were obtained from Jiangxi Yintao Pharmaceutical Co., Ltd. (Jiangxi, China) and Heilongjiang Harbin Pharmaceutical Co., Ltd. (Heilongjiang, China), respectively for the in vivo study.

Techniques: Expressing, Western Blot, Control