Journal: Science Advances

Article Title: Switch of serotonergic descending inhibition into facilitation by a spinal chloride imbalance in neuropathic pain

doi: 10.1126/sciadv.abo0689

Figure Lengend Snippet: ( A ) GFP (green) and PAX2 (red) immunolabeling in the DH. Scale bar, 100 μm. Inset: high magnification showing contacts between Pax2 and 5-HT fibers. Scale bar, 50 μm. ( B ) GFP (green) and Tlx3 (red) immunolabeling in the DH. Scale bar, 100 μm. Inset: high magnification, low or no contact. Scale bar, 25 μm. ( C ) After 3D reconstruction (see Materials and Methods) quantification of contacts between cell bodies (Pax2 and Tlx3) and 5-HT fibers. ( C ) Contacted cells are significantly higher for Pax2(+) than for Tlx3(+) in both males and females [males (blue): 86.2 ± 4.6% for Pax2 and 15.5 ± 3.8% for Tlx3, P < 0.001, Mann-Whitney; females (red): 76.3 ± 2.9% for Pax2 and 8.3 ± 2.2% for Tlx3, P < 0.001, Mann-Whitney]. ( D ) Immunostaining against TPH2 in slices of lumbar spinal cord of GAD67::GFP mice. Scale bar, 25 μm. GABA neurons [GFP(+) in green, stars] or fibers in DH are contacted by TPH2-positive putative synaptic button (in red, white arrows). ( E ) Expression of Myc-tag in synaptic 5-HT terminals in DH following injections of an AAV-synaptophysin-Myc. Immunoreactivity is present in the deeper lamina of the DHSC. Scale bar, 100 μm. Higher magnification shows 5-HT axonic button (white arrow). Scale bar, 20 μm. ( F ) Myc(+) synaptic buttons are in direct contact with GAD67-positive cells and fibers in epet::cre/ GAD67::GFP mice. Scale bar, 20 μm. Inset (top): Myc(+) button on GAD(+) cell bodies. Inset (bottom): Myc(+) button on GAD(+) on GAD(+) fibers.

Article Snippet: For molecular identifications of the spinal network, spinal cord sections were incubated free-floating in 0.1 M PBS containing Triton X-100 (0.3%), bovine serum albumin (1%; Sigma-Aldrich), guinea pig anti-TLX3 antibody (1:1000; a gift from T. Müller), goat anti-Pax2 antibody (1:300; Bio-techne), rabbit anti-Myc antibody (1:500; Euromedex), rabbit anti-TPH2 antibody (1:1000; Bio-techne), and rabbit anti-GAD65/67 (G5163, Sigma-Aldrich) in combination with chicken anti-GFP antibody (1:1000; Averlabs) overnight at 4°C.

Techniques: Immunolabeling, MANN-WHITNEY, Immunostaining, Expressing

Journal: Phytotherapy Research

Article Title: Calycosin Targets the CYP1B1 ‐ AKT / SP1 ‐ GPX4 Axis to Modulate Ferroptosis in Colorectal Carcinogenesis

doi: 10.1002/ptr.70172

Figure Lengend Snippet: Effects of Calycosin on migration, invasion, EMT, and apoptosis of colorectal cancer cells in vitro. (A) Cell scratch assay to evaluate the impact of Calycosin on the migration of colorectal cancer cells. Scale bar = 100 μm. (B) Transwell assay assessing the influence of Calycosin on the migration and invasion of colorectal cancer cells. Scale bar = 50 μm. (C) Western blot (WB) analysis examining the effect of Calycosin on the expression of EMT‐related proteins in colorectal cancer cells. (D) Flow cytometry detecting the effect of Calycosin on the apoptosis of colorectal cancer cells. Data are presented as mean ± standard error of the mean (Mean ± SEM). p values and significance were determined by Two‐Way ANOVA (C).

Article Snippet: (+)‐Gallocatechin (Cat. HY‐N0521A), Ziyuglycoside I (#HY‐N0331), and Calycosin (#HY‐N0519) were synthesized by MCE Company.

Techniques: Migration, In Vitro, Wound Healing Assay, Transwell Assay, Western Blot, Expressing, Flow Cytometry

Journal: Phytotherapy Research

Article Title: Calycosin Targets the CYP1B1 ‐ AKT / SP1 ‐ GPX4 Axis to Modulate Ferroptosis in Colorectal Carcinogenesis

doi: 10.1002/ptr.70172

Figure Lengend Snippet: The influence of Calycosin on the expression of genes related to colorectal cancer cell death and its identification. (A) Results of whole‐genome transcriptome sequencing after treating SW620 cells ( n = 3) with DMSO and Calycosin (100 μM) for 24 h. The volcano plot of differentially expressed genes is shown on the right, with upregulated genes in red and downregulated genes in blue. (B) Volcano plot depicting the expression of genes related to apoptosis, necrosis, and ferroptosis in SW620 cells. (C) Bubble chart showing GO and KEGG pathway enrichment analysis of genes related to apoptosis, necrosis, and ferroptosis. (D) Protein expression levels of GPX4, FTH1, NOX1, and ACSL4 after treating HCT116, HT29, and SW620 cells with DMSO and Calycosin (100 μM) for 24 h. (E) Immunohistochemical detection of GPX4 and ACSL4 expression in three types of subcutaneous colorectal cancer mouse tumor tissues. Scale bar = 100 μm.

Article Snippet: (+)‐Gallocatechin (Cat. HY‐N0521A), Ziyuglycoside I (#HY‐N0331), and Calycosin (#HY‐N0519) were synthesized by MCE Company.

Techniques: Expressing, Sequencing, Immunohistochemical staining

Journal: Phytotherapy Research

Article Title: Calycosin Targets the CYP1B1 ‐ AKT / SP1 ‐ GPX4 Axis to Modulate Ferroptosis in Colorectal Carcinogenesis

doi: 10.1002/ptr.70172

Figure Lengend Snippet: Identification of potential binding targets and sites of Calycosin. (A) Structural formula of Calycosin and its potential binding targets. (B) Prognostic curves for CYP1B1 gene in CRC from the GEPIA database. (C) Expression of CYP1B1 protein in normal and tumor tissues of the colon and rectum from the HPA database. Scale bar = 50 μm. (D) CYP1B1 protein expression levels in HCT116, HT29, and SW620 cells. (E) CETSA assessment of the thermal stability of CYP1B1 after treatment with Calycosin (100 μM) for 24 h in HT29 and SW620 cells. (F) Molecular docking calculation of the binding energy between Calycosin and CYP1B1. (G) Amino acid sequence of CYP1B1 protein from the UniProt database. (H) Effect of Calycosin on the thermal stability of wild‐type (WT) H_CYP1B1 and overexpressed mutant H_CYP1B1 (N265T) Mut1, H_CYP1B1 (G329A) Mut2 in HEK‐293 cells.

Article Snippet: (+)‐Gallocatechin (Cat. HY‐N0521A), Ziyuglycoside I (#HY‐N0331), and Calycosin (#HY‐N0519) were synthesized by MCE Company.

Techniques: Binding Assay, Expressing, Sequencing, Mutagenesis

Journal: Phytotherapy Research

Article Title: Calycosin Targets the CYP1B1 ‐ AKT / SP1 ‐ GPX4 Axis to Modulate Ferroptosis in Colorectal Carcinogenesis

doi: 10.1002/ptr.70172

Figure Lengend Snippet: The impact of CYP1B1 on subcutaneous tumor growth in colorectal cancer mice. (A) Establishment of subcutaneous mouse models of human colorectal cancer (HT29, SW620) with stable CYP1B1 knockdown (sh_CYP1B1) and subsequent treatment with Calycosin. (B) Immunofluorescence detection of CYP1B1 expression in subcutaneous tumor tissues of human colorectal cancer mice in three groups (NC, sh_CYP1B1, sh_CYP1B1 + Calycosin), Scale bar = 100 μm. (C, D) Line charts of tumor volume and bar charts of tumor weight in subcutaneous tumor mice with stable CYP1B1 knockdown in HT29 (C) and SW620 (D), with each point representing an individual mouse. (E, F) Immunofluorescence detection of GPX4 (E) and ACSL4 (F) expression levels in tumor tissues of subcutaneous tumor mice with stable CYP1B1 knockdown in HT29 and SW620, Scale bar = 50 μm. Data are presented as Mean ± SEM, and p values and significance were determined by two‐tailed Student's t ‐test (C, D) and One‐Way ANOVA (C, D).

Article Snippet: (+)‐Gallocatechin (Cat. HY‐N0521A), Ziyuglycoside I (#HY‐N0331), and Calycosin (#HY‐N0519) were synthesized by MCE Company.

Techniques: Knockdown, Immunofluorescence, Expressing, Two Tailed Test

Journal: Phytotherapy Research

Article Title: Calycosin Targets the CYP1B1 ‐ AKT / SP1 ‐ GPX4 Axis to Modulate Ferroptosis in Colorectal Carcinogenesis

doi: 10.1002/ptr.70172

Figure Lengend Snippet: Molecular mechanism of CYP1B1 regulating GPX4 expression. (A) Expression of key transcription factors regulating GPX4 in transcriptome sequencing data from SW620 cells treated with DMSO and Calycosin. (B) Protein expression of CYP1B1 in the cytoplasm and p‐SP1 and TFAP2A in the nucleus of HT29 and SW620 cells after CYP1B1 knockdown and Calycosin treatment. (C) Expression of key kinases AKTs, JAKs, and MAPKs regulating GPX4 in transcriptome sequencing data from SW620 cells treated with DMSO and Calycosin. (D) Protein expression of CYP1B1, p‐AKT1, p‐ERK1/2 in the cytoplasm, and p‐SP1 in the nucleus of HT29 and SW620 cells after CYP1B1 knockdown and Calycosin treatment. (E) Three potential binding sites of SP1 on the GPX4 promoter. (F) Protein expression levels of SP‐1 after transfection of wild‐type and h‐SP1 overexpressing plasmids into HEK‐293 T cells. (G) Luciferase activity detected after transfection of wild‐type/h‐SP1 overexpressing plasmids into HEK‐293 T cells expressing the full‐length/mutant GPX4 promoter. Data are presented as mean ± standard error of the mean (Mean ± SEM). p values and significance were determined by two‐tailed t ‐test (F, G).

Article Snippet: (+)‐Gallocatechin (Cat. HY‐N0521A), Ziyuglycoside I (#HY‐N0331), and Calycosin (#HY‐N0519) were synthesized by MCE Company.

Techniques: Expressing, Sequencing, Knockdown, Binding Assay, Transfection, Luciferase, Activity Assay, Mutagenesis, Two Tailed Test

Journal: BMC Complementary Medicine and Therapies

Article Title: Toxicity and toxicokinetics of the ethanol extract of Zuojin formula

doi: 10.1186/s12906-022-03684-0

Figure Lengend Snippet: Acquisition parameters in MRM mode

Article Snippet: Carbamazepine (CAS No.: 298–46-4), berberine (CAS No.: 2086-83-1), coptisine (CAS No.: 6020-18-4), epiberberine (CAS No.: 6873-09-2), palmatine (CAS No.: 3486-67-7), jatrorrhizine (CAS No.: 3621-38-3), columbamine (CAS No.: 483–34-1), evodiamine (CAS No.: 518–17-2) and rutaecarpine (CAS No.: 20575–76-2), all > 98% purity, were purchased from Shanghai Yuanye Bio-Technology Company.

Techniques:

Journal: American Journal of Cancer Research

Article Title: MUC1 is responsible for the pro-metastatic potential of calycosin in pancreatic ductal adenocarcinoma

doi:

Figure Lengend Snippet: Effects of calycosin on the expression of MUC1 (with or without MUC1 gene silencing). Immunofluorescence staining of MUC1 (green) in MIA-PaCa2 cells treated with 100 μM calycosin for 24 h using (A) 2D or (B) 3D cell culture. The nuclei were stained with DAPI (blue). Magnifications: 60× for 2D cell culture (scale bar is 5 μm); 20× for 3D cell culture (scale bar is 50 μm). (C) Gene expression of MUC1 in MIA PaCa-2 cells after treatment with calycosin (50 or 100 μM) for 24 h was detected by quantitative reverse transcription PCR. *P<0.01, **P<0.0001 versus the corresponding negative control (NCtrl) group. #P<0.001, ##P<0.0001 versus corresponding group without calycosin treatment (0 μM). (D) Protein expression of MUC1 in MIA PaCa-2 cells after treatment with calycosin (50 or 100 μM) for 24 h was detected by Western immunoblotting. *P<0.05, **P<0.01, ***P<0.001 versus the corresponding negative control (NCtrl) group. #P<0.05 versus corresponding group without calycosin treatment (0 μM). Cells treated with DMSO were used as negative control. Data are expressed as mean ± SD from 3 independent experiments.

Article Snippet: Calycosin (CAS no. 20575-57-9; >98% purity by HPLC) was purchased from Chengdu Herbpurify Co., Ltd. (Chengdu, Sichuan, China).

Techniques: Expressing, Immunofluorescence, Staining, Cell Culture, Gene Expression, Reverse Transcription, Negative Control, Western Blot

Journal: American Journal of Cancer Research

Article Title: MUC1 is responsible for the pro-metastatic potential of calycosin in pancreatic ductal adenocarcinoma

doi:

Figure Lengend Snippet: Effects of calycosin on the expression of TGF-β1 (with or without MUC1 gene silencing). Immunofluorescence staining of TGF-β1 (green) in MIA-PaCa2 cells treated with 100 μM calycosin for 24 h using (A) 3D cell culture (scale bar is 50 μm). The nuclei were stained with DAPI (blue). Magnifications: 20× for 3D cell culture. (B) Gene expression of TGF-β1 in MIA PaCa-2 cells after treatment with calycosin (50 or 100 μM) for 24 h was detected by quantitative reverse transcription PCR. #P<0.05 versus corresponding group without calycosin treatment (0 μM). (C) Protein expression of TGF-β1 in MIA PaCa-2 cells after treatment with calycosin (50 or 100 μM) for 24 h was detected by Western immunoblotting. *P<0.05, **P<0.01, ***P<0.001 versus the corresponding negative control (NCtrl) group. (D) The protein interaction of MUC1 and TGF-β in MIA PaCa-2 cells after treatment with calycosin (100 μM) for 24 h was detected by co-immunoprecipitation and Western immunoblotting. *P<0.05, **P<0.01, ***P<0.001 versus the corresponding negative control (NCtrl) group. #P<0.05, ##P<0.01, ###P<0.0001 versus corresponding group without calycosin treatment (0 μM). Cells treated with DMSO were used as negative control. Data are expressed as mean ± SD from 3 independent experiments.

Article Snippet: Calycosin (CAS no. 20575-57-9; >98% purity by HPLC) was purchased from Chengdu Herbpurify Co., Ltd. (Chengdu, Sichuan, China).

Techniques: Expressing, Immunofluorescence, Staining, Cell Culture, Gene Expression, Reverse Transcription, Western Blot, Negative Control, Immunoprecipitation

Journal: American Journal of Cancer Research

Article Title: MUC1 is responsible for the pro-metastatic potential of calycosin in pancreatic ductal adenocarcinoma

doi:

Figure Lengend Snippet: Effects of calycosin on the migration of MIA PaCa-2 cells (with or without MUC1 gene silencing). A. Wound healing assay was used in MIA-PaCa2 cells treated with 50 μM calycosin with or without MUC1 siRNA for 0, 24, 48 h or 72 h. Area between the inner margins of migrating cells during wound closure was quantified. Three independent experiments were conducted each in triplicate. Data are presented as mean ± SD. *P<0.05, ***P<0.001 versus the corresponding group without calycosin treatment. B. Gene expression of Snail in MIA PaCa-2 cells after treatment with calycosin (50 or 100 μM) for 24 h was detected by quantitative reverse transcription PCR. *P<0.01, **P<0.001 versus the corresponding negative control (NCtrl) group. C. Protein expression of Snail in MIA PaCa-2 cells after treatment with calycosin (50 or 100 μM) for 24 h was detected by Western immunoblotting. *P<0.01, **P<0.001 versus the corresponding negative control (NCtrl) group. #P<0.01 versus corresponding group without calycosin treatment (0 μM). Cells treated with DMSO were used as negative control. Data are expressed as mean ± SD from 3 independent experiments.

Article Snippet: Calycosin (CAS no. 20575-57-9; >98% purity by HPLC) was purchased from Chengdu Herbpurify Co., Ltd. (Chengdu, Sichuan, China).

Techniques: Migration, Wound Healing Assay, Gene Expression, Reverse Transcription, Negative Control, Expressing, Western Blot

Journal: American Journal of Cancer Research

Article Title: MUC1 is responsible for the pro-metastatic potential of calycosin in pancreatic ductal adenocarcinoma

doi:

Figure Lengend Snippet: Effects of calycosin on the apoptosis of MIA-PaCa2 cells (with or without MUC1 gene silencing). A. FITC/PI staining was performed in MIA-PaCa2 cells treated with 50 μM calycosin with or without MUC1 siRNA for 24 h. Three independent experiments were performed each in triplicate. *P<0.01, **P<0.001 versus the corresponding group without calycosin treatment (No drug). #P<0.01 versus corresponding group without calycosin treatment (0 μM). B. Protein expression of Cleaved-PARP and Atg5 in MIA PaCa-2 cells after treatment with calycosin (50 or 100 μM) for 24 h was detected by Western immunoblotting. *P<0.05, **P<0.01 versus the corresponding negative control (NCtrl) group. #P<0.05 versus corresponding group without calycosin treatment (0 μM). Cells treated with DMSO were used as negative control. Data are expressed as mean ± SD from 3 independent experiments.

Article Snippet: Calycosin (CAS no. 20575-57-9; >98% purity by HPLC) was purchased from Chengdu Herbpurify Co., Ltd. (Chengdu, Sichuan, China).

Techniques: Staining, Expressing, Western Blot, Negative Control

Journal: American Journal of Cancer Research

Article Title: MUC1 is responsible for the pro-metastatic potential of calycosin in pancreatic ductal adenocarcinoma

doi:

Figure Lengend Snippet: Effects of calycosin on cell cycle modulation in MIA-PaCa2 cells (with or without MUC1 gene silencing). (A) Cell cycle analysis was performed in MIA-PaCa2 cells treated with 100 μM calycosin with or without MUC1 siRNA for 24 h. Gene expression of (B) p27 and (C) p21 in MIA PaCa-2 cells after treatment with calycosin (50 or 100 μM) for 24 h was detected by quantitative reverse transcription PCR. Cells treated with DMSO were used as negative control. Data are expressed as mean ± SD from 3 independent experiments. *P<0.05 versus the corresponding negative control (NCtrl) group. #P<0.01, ##P<0.001, ###P<0.0001 versus corresponding group without calycosin treatment (0 μM).

Article Snippet: Calycosin (CAS no. 20575-57-9; >98% purity by HPLC) was purchased from Chengdu Herbpurify Co., Ltd. (Chengdu, Sichuan, China).

Techniques: Cell Cycle Assay, Gene Expression, Reverse Transcription, Negative Control

Journal: American Journal of Cancer Research

Article Title: MUC1 is responsible for the pro-metastatic potential of calycosin in pancreatic ductal adenocarcinoma

doi:

Figure Lengend Snippet: Effects of calycosin on metabolic regulation in MIA PaCa2 cells (with or without MUC1 gene silencing). Immunofluorescence staining of AMPKα (red) in MIA-PaCa2 cells treated with 100 μM calycosin for 24 h using (A) 2D or (B) 3D cell culture. The nuclei were stained with DAPI (blue). Magnifications: 60× for 2D cell culture (The scale bar is 5 μm); 20× for 3D cell culture (The scale bar is 50 μm). (C) Gene expression of AMPKα, Sirt1, FGF21 and β-klotho in MIA PaCa-2 cells after treatment with calycosin (50 or 100 μM) for 24 h was detected by quantitative reverse transcription PCR. Cells treated with DMSO were used as negative control. Data are expressed as mean ± SD from 3 independent experiments. *P<0.05, **P<0.01, ***P<0.0001 versus the corresponding negative control (NCtrl) group. #P<0.05, ##P<0.01, ###P<0.0001 versus corresponding group without calycosin treatment (0 μM).

Article Snippet: Calycosin (CAS no. 20575-57-9; >98% purity by HPLC) was purchased from Chengdu Herbpurify Co., Ltd. (Chengdu, Sichuan, China).

Techniques: Immunofluorescence, Staining, Cell Culture, Gene Expression, Reverse Transcription, Negative Control

Journal: American Journal of Cancer Research

Article Title: MUC1 is responsible for the pro-metastatic potential of calycosin in pancreatic ductal adenocarcinoma

doi:

Figure Lengend Snippet: The effect of calycosin in PDAC tumor-bearing C57/BL6 mice. A. Calycosin exerted anti-tumor effect in Pan02 tumor-bearing C57/BL6 mice. Ctrl: PBS, PsCtrl: gemcitabine 30 mg/kg i.p. every other day, calcyosin: 30 mg/kg once daily, calycosin + Gun: 100V electroporation for 12 days. More than 80% reduction in tumor mass. B. Daily change of tumor volume over 12 days. Tumor volume of PsCtrl and calycosin groups were decreased, with increase in calycosin + Gun group. C, D. IHC assays of tissues for MUC1, TGF-β, MMP2, MMP9 and CD31 in different treatment groups (magnification =20×).

Article Snippet: Calycosin (CAS no. 20575-57-9; >98% purity by HPLC) was purchased from Chengdu Herbpurify Co., Ltd. (Chengdu, Sichuan, China).

Techniques: Electroporation

Journal: American Journal of Cancer Research

Article Title: MUC1 is responsible for the pro-metastatic potential of calycosin in pancreatic ductal adenocarcinoma

doi:

Figure Lengend Snippet: The mechanism of MUC1 in treating PDAC by calycosin.

Article Snippet: Calycosin (CAS no. 20575-57-9; >98% purity by HPLC) was purchased from Chengdu Herbpurify Co., Ltd. (Chengdu, Sichuan, China).

Techniques: