Journal: Scientific reports

Article Title: Curcumin synergistically enhances the efficacy of gemcitabine against gemcitabine-resistant cholangiocarcinoma via the targeting LAT2/glutamine pathway.

doi: 10.1038/s41598-024-66945-7

Figure Lengend Snippet: Figure 5. Overexpression of LAT2 protein in gemcitabine-resistant CCA cells. (a) Volcano plot of differentially expressed genes of KKU-213B (parental) vs. KKU-213BGemR. Green dots indicate downregulated genes, orange dots indicate upregulated genes, and brown dots indicate stable genes. (b) The fold change of SLC7A8 expression in KKU-213B compared to KKU-213BGemR. (c + d) LAT2 expression and intensity in parental CCA cells and gemcitabine-resistant CCA cells and (e–h) LAT2, glutaminase (GLS) and glutamine synthetase (GS) expression and intensity after treatment with gemcitabine, curcumin and the combination of curcumin and gemcitabine. Original blots are presented in Supplementary Table S2. Data are expressed as mean ± SD of three replicate experiments; ns = not significant, *p < 0.01, **p < 0.01 and ***p < 0.001 compared between groups; Log2FC: Log2 fold-change.

Article Snippet: Membranes were block with 1 min protein-free blocking buffer (BlockPROTM, Energenesis biomedical co., ltd, Taipei, Taiwan) for 1 h and then incubated overnight at 4 °C with primary antibodies (1:1000) including SLC7A8 (LAT2) (A14861/A24043), GS (A5437) and GLS (A11043), which were purchased from abclonal in Wuhan, China, and GAPDH (sc-25778), which was purchased from Santa Cruz Biotechnology in Dallas, TX, USA.

Techniques: Over Expression, Expressing

Journal: Scientific reports

Article Title: Curcumin synergistically enhances the efficacy of gemcitabine against gemcitabine-resistant cholangiocarcinoma via the targeting LAT2/glutamine pathway.

doi: 10.1038/s41598-024-66945-7

Figure Lengend Snippet: Figure 6. LAT2 promotes chemotherapeutic sensitivity against gemcitabine-resistant cell. Western blot analyses of (a–d) LAT2, glutaminase (GLS) and glutamine synthetase (GS) in KKU-213BGemR knockdown SLC7A8. (e) Cell morphology and confluence of cells before and after transfection at 48h (10X magnification). (f) Cell proliferation using MTT assay after transfection with siSLC7A8 at 48 h and 72 h (OD 540), treatment with (g) gemcitabine 0–2000 μM at 48 h and 72 h and (h) the combination of curcumin and gemcitabine at 24–72 h. Original blots are presented in Supplementary Table S3. Western blot data are expressed as mean ± SD of biological tripicates and cell proliferation are expressed as mean ± SD of biological and technical replicate experiments; ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001 and #p < 0.0001 compared to control and between groups. G: gemcitabine, C: curcumin.

Article Snippet: Membranes were block with 1 min protein-free blocking buffer (BlockPROTM, Energenesis biomedical co., ltd, Taipei, Taiwan) for 1 h and then incubated overnight at 4 °C with primary antibodies (1:1000) including SLC7A8 (LAT2) (A14861/A24043), GS (A5437) and GLS (A11043), which were purchased from abclonal in Wuhan, China, and GAPDH (sc-25778), which was purchased from Santa Cruz Biotechnology in Dallas, TX, USA.

Techniques: Western Blot, Knockdown, Transfection, MTT Assay, Control

Journal: Scientific reports

Article Title: Curcumin synergistically enhances the efficacy of gemcitabine against gemcitabine-resistant cholangiocarcinoma via the targeting LAT2/glutamine pathway.

doi: 10.1038/s41598-024-66945-7

Figure Lengend Snippet: Figure 7. Anti-tumor effects of curcumin, gemcitabine and their combination in gemcitabine- resistant xenograft mouse model. (a) Tumor growth rates and images of tumor tissue (N = 5 in each group), (b) LAT2 immunohistochemical staining in the untreated group, curcumin- and combination-treated groups (the gemcitabine-treated group was not included due to fluid accumulation in the tumor mass) and (c) the positive- staining area (%) of LAT2. (d–g) Protein intensities of LAT2, GLS and GS. Original blots are presented in Supplementary Table S4. All data are expressed as means ± SD of three biological independent experiments; ns = not significant, ** p < 0.01, ***p < 0.001 and #p < 0.0001 compared between groups (N = 3 in each group). CUR: Curcumin, GEM: Gemcitabine.

Article Snippet: Membranes were block with 1 min protein-free blocking buffer (BlockPROTM, Energenesis biomedical co., ltd, Taipei, Taiwan) for 1 h and then incubated overnight at 4 °C with primary antibodies (1:1000) including SLC7A8 (LAT2) (A14861/A24043), GS (A5437) and GLS (A11043), which were purchased from abclonal in Wuhan, China, and GAPDH (sc-25778), which was purchased from Santa Cruz Biotechnology in Dallas, TX, USA.

Techniques: Immunohistochemical staining, Staining

Journal: Scientific reports

Article Title: Curcumin synergistically enhances the efficacy of gemcitabine against gemcitabine-resistant cholangiocarcinoma via the targeting LAT2/glutamine pathway.

doi: 10.1038/s41598-024-66945-7

Figure Lengend Snippet: Figure 8. This summary schematic demonstrates that curcumin enhances gemcitabine’s ability to (I) suppress the SLC7A8 (LAT2)-regulated glutamine pathway, (II) inhibit GLS, and (III) inhibit GS. This leads to reduced ability of glutamine (Gln) to participate in synthesis of nucleotides, and of glutamate (Glu) being unable to enter the tricarboxylic acid (TCA) cycle and synthesize glutathione and amino acids19. These mechanisms result in inhibited cell proliferation, induced cell cycle arrest, and apoptosis, ultimately inhibiting cancer progression in gemcitabine-resistant cholangiocarcinoma.

Article Snippet: Membranes were block with 1 min protein-free blocking buffer (BlockPROTM, Energenesis biomedical co., ltd, Taipei, Taiwan) for 1 h and then incubated overnight at 4 °C with primary antibodies (1:1000) including SLC7A8 (LAT2) (A14861/A24043), GS (A5437) and GLS (A11043), which were purchased from abclonal in Wuhan, China, and GAPDH (sc-25778), which was purchased from Santa Cruz Biotechnology in Dallas, TX, USA.

Techniques:

Journal: Oncology Letters

Article Title: Monascin accelerates anoikis in circulating tumor cells and prevents breast cancer metastasis

doi: 10.3892/ol.2020.12029

Figure Lengend Snippet: MS inhibits 4T1 breast cancer cell aggregation under the suspension condition. (A) Phase-contrast images of 4T1 cells on the polyHEMA-coated plate treated with or without 20 µM MS for 48 h. Quantitative counts of clustered 4T1 cells reveal a significant decrease in MS treatment. (B) Western blot analysis of E-cadherin and β-catenin expression to determine the effect of MS treatment for 48 h on the floating cells. *P<0.05 vs. control. MS, monascin; Ctrl, control.

Article Snippet: After blocking with BlockPROTM Protein-Free Blocking Buffer (Visual Protein; cat. no. BF01) for 1 h at room temperature, membranes were incubated with primary antibodies against PARP (1:1,000; Cell Signaling Technology, Inc.; cat. no. 9542), caspase-3 (1:1,000; Cell Signaling Technology, Inc.; cat. no. 9665), E-cadherin (1:1,000; Cell Signaling Technology, Inc.; cat. no. 3195), β-catenin (1:1,000; Cell Signaling Technology, Inc.; cat. no. 9562) and β-actin (1:2,000; Cell Signaling Technology, Inc.; cat. no. 4970) primary antibodies overnight at 4°C.

Techniques: Suspension, Western Blot, Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: Atractylodin Suppresses TGF-β-Mediated Epithelial-Mesenchymal Transition in Alveolar Epithelial Cells and Attenuates Bleomycin-Induced Pulmonary Fibrosis in Mice

doi: 10.3390/ijms222011152

Figure Lengend Snippet: Effect of atractylodin stymied on TGF-β1-induced EMT-associated protein expressions in A549 cells. A549 cells were pretreated with ATL for 1 h followed by TGF-β1 (2 ng/mL) stimulation for an additional 24 h. Cells treated with DMSO were set up as the control groups. ( A ) Protein expression levels of N-cadherin, E-cadherin, α-SMA, and vimentin were measured by Western blot assay. ( B ) Quantitation of Western blot signal intensities by ImageJ software. ( C ) The transcriptional expressions of type I collagen and ( D ) type III collagen were conducted by RT-qPCR. Values represent the mean ± SEM from triplicate samples for each treatment. (*) p < 0.05 versus TGF-β1 + 0.1% DMSO-treated control, as determined by non-parametric Kruskal–Wallis test and all pairwise multiple comparison procedures (Dunn’s Method).

Article Snippet: The membranes were blocked with BlockPROTM Protein-Free Blocking Buffer for 1.5 h at room temperature, and then incubated with specific primary antibodies, E-cadherin (clone EP700Y, 1:1000, Epitomics, Burlingame, CA, USA), α-SMA (Cat#ab5694, 1:1000, Abcam, Cambridge, MA, USA), vimentin (Cat# GTX100619, 1:1000, GeneTex Inc, Texas, USA), p-p38 (clone 3D7, 1:1000, Cell Signaling Technology, Danvers, MA, USA), p38 (clone D13EE1, 1:1000, Cell Signaling Technology, Danvers, MA, USA), p-JNK (clone 81E11, 1:1000, Cell Signaling Technology, Danvers, MA, USA), JNK (Cat# 9252, 1:1000, Cell Signaling Technology, Danvers, MA, USA), p-ERK (clone E-4, 1:1000, Santa Cruz Biotechnology, CA, USA), ERK (clone H-72, 1:1000, Santa Cruz Biotechnology, CA, USA), p-AKT (Ser473) (clone D9E, 1:1000, Cell Signaling Technology, Danvers, MA, USA), AKT (clone C67E7, 1:1000, Cell Signaling Technology, Danvers, MA, USA), p-SMAD2 (Ser465/467) (clone 138D4, 1:1000, Cell Signaling Technology, Danvers, MA, USA), SMAD2 (clone D43B4, 1:1000, Cell Signaling Technology, Danvers, MA, USA), p-SMAD3 (Ser465/Ser467) (clone E8F3R, 1:1000, Cell Signaling Technology, Danvers, MA, USA), SMAD3 (clone C67H9, 1:1000, Cell Signaling Technology, Danvers, MA, USA), and GAPDH (Cat# ab8245, 1:5000, Abcam, Cambridge, MA, USA), at 4 °C.

Techniques: Control, Expressing, Western Blot, Quantitation Assay, Software, Quantitative RT-PCR, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Atractylodin Suppresses TGF-β-Mediated Epithelial-Mesenchymal Transition in Alveolar Epithelial Cells and Attenuates Bleomycin-Induced Pulmonary Fibrosis in Mice

doi: 10.3390/ijms222011152

Figure Lengend Snippet: Atractylodin reduced BLM-induced EMT in mice pulmonary tissues. Lung tissue homogenates were collected on day 21 from each group of mice. ( A ) Relative mRNA expression levels of E-cadherin, α-SMA, and vimentin were measured with real-time PCR. ( B ) Protein expression levels of N-cadherin, E-cadherin, α-SMA, and vimentin were assessed with Western blot assay. ( C ) Quantitation of Western blot signal intensities by ImageJ software. Data are expressed as mean ± SEM of five mice in each group. (*) p < 0.05, and (**) p < 0.01 versus vehicle-treated BLM model group (as control group), as determined by non-parametric Kruskal–Wallis test and all pairwise multiple comparison procedures (Dunn’s Method).

Article Snippet: The membranes were blocked with BlockPROTM Protein-Free Blocking Buffer for 1.5 h at room temperature, and then incubated with specific primary antibodies, E-cadherin (clone EP700Y, 1:1000, Epitomics, Burlingame, CA, USA), α-SMA (Cat#ab5694, 1:1000, Abcam, Cambridge, MA, USA), vimentin (Cat# GTX100619, 1:1000, GeneTex Inc, Texas, USA), p-p38 (clone 3D7, 1:1000, Cell Signaling Technology, Danvers, MA, USA), p38 (clone D13EE1, 1:1000, Cell Signaling Technology, Danvers, MA, USA), p-JNK (clone 81E11, 1:1000, Cell Signaling Technology, Danvers, MA, USA), JNK (Cat# 9252, 1:1000, Cell Signaling Technology, Danvers, MA, USA), p-ERK (clone E-4, 1:1000, Santa Cruz Biotechnology, CA, USA), ERK (clone H-72, 1:1000, Santa Cruz Biotechnology, CA, USA), p-AKT (Ser473) (clone D9E, 1:1000, Cell Signaling Technology, Danvers, MA, USA), AKT (clone C67E7, 1:1000, Cell Signaling Technology, Danvers, MA, USA), p-SMAD2 (Ser465/467) (clone 138D4, 1:1000, Cell Signaling Technology, Danvers, MA, USA), SMAD2 (clone D43B4, 1:1000, Cell Signaling Technology, Danvers, MA, USA), p-SMAD3 (Ser465/Ser467) (clone E8F3R, 1:1000, Cell Signaling Technology, Danvers, MA, USA), SMAD3 (clone C67H9, 1:1000, Cell Signaling Technology, Danvers, MA, USA), and GAPDH (Cat# ab8245, 1:5000, Abcam, Cambridge, MA, USA), at 4 °C.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Quantitation Assay, Software, Control, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Atractylodin Suppresses TGF-β-Mediated Epithelial-Mesenchymal Transition in Alveolar Epithelial Cells and Attenuates Bleomycin-Induced Pulmonary Fibrosis in Mice

doi: 10.3390/ijms222011152

Figure Lengend Snippet: Atractylodin reduced Smad-independent pathway activated by TGF-β1 in A549 cells. A549 cells were pretreated with ATL for 1 h followed by TGF-β1 (2 ng/mL) stimulation for an additional 6 h. Cells treated with DMSO were set up as the control group. ( A ) Protein expression levels of phospho- and non-phospho- p38, JNK, ERK, and AKT were measured by Western blot assay. ( B ) Quantitation of Western blot signal intensities by ImageJ software. Values represent the mean ± SEM from triplicate samples for each treatment. (*) p < 0.05 versus TGF-β1 + 0.1% DMSO-treated control, as determined by non-parametric Kruskal–Wallis test and all pairwise multiple comparison procedures (Dunn’s Method).

Article Snippet: The membranes were blocked with BlockPROTM Protein-Free Blocking Buffer for 1.5 h at room temperature, and then incubated with specific primary antibodies, E-cadherin (clone EP700Y, 1:1000, Epitomics, Burlingame, CA, USA), α-SMA (Cat#ab5694, 1:1000, Abcam, Cambridge, MA, USA), vimentin (Cat# GTX100619, 1:1000, GeneTex Inc, Texas, USA), p-p38 (clone 3D7, 1:1000, Cell Signaling Technology, Danvers, MA, USA), p38 (clone D13EE1, 1:1000, Cell Signaling Technology, Danvers, MA, USA), p-JNK (clone 81E11, 1:1000, Cell Signaling Technology, Danvers, MA, USA), JNK (Cat# 9252, 1:1000, Cell Signaling Technology, Danvers, MA, USA), p-ERK (clone E-4, 1:1000, Santa Cruz Biotechnology, CA, USA), ERK (clone H-72, 1:1000, Santa Cruz Biotechnology, CA, USA), p-AKT (Ser473) (clone D9E, 1:1000, Cell Signaling Technology, Danvers, MA, USA), AKT (clone C67E7, 1:1000, Cell Signaling Technology, Danvers, MA, USA), p-SMAD2 (Ser465/467) (clone 138D4, 1:1000, Cell Signaling Technology, Danvers, MA, USA), SMAD2 (clone D43B4, 1:1000, Cell Signaling Technology, Danvers, MA, USA), p-SMAD3 (Ser465/Ser467) (clone E8F3R, 1:1000, Cell Signaling Technology, Danvers, MA, USA), SMAD3 (clone C67H9, 1:1000, Cell Signaling Technology, Danvers, MA, USA), and GAPDH (Cat# ab8245, 1:5000, Abcam, Cambridge, MA, USA), at 4 °C.

Techniques: Control, Expressing, Western Blot, Quantitation Assay, Software, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Atractylodin Suppresses TGF-β-Mediated Epithelial-Mesenchymal Transition in Alveolar Epithelial Cells and Attenuates Bleomycin-Induced Pulmonary Fibrosis in Mice

doi: 10.3390/ijms222011152

Figure Lengend Snippet: Atractylodin reduced Smad-independent pathway activated by TGF-β1 in A549 cells. A549 cells were pretreated with ATL for 1 h followed by TGF-β1 (2 ng/mL) stimulation for an additional 6 h. Cells treated with DMSO were set up as the control group. ( A ) Protein expression levels of phospho- and non-phospho- p38, JNK, ERK, and AKT were measured by Western blot assay. ( B ) Quantitation of Western blot signal intensities by ImageJ software. Values represent the mean ± SEM from triplicate samples for each treatment. (*) p < 0.05 versus TGF-β1 + 0.1% DMSO-treated control, as determined by non-parametric Kruskal–Wallis test and all pairwise multiple comparison procedures (Dunn’s Method).

Article Snippet: The membranes were blocked with BlockPROTM Protein-Free Blocking Buffer for 1.5 h at room temperature, and then incubated with specific primary antibodies, E-cadherin (clone EP700Y, 1:1000, Epitomics, Burlingame, CA, USA), α-SMA (Cat#ab5694, 1:1000, Abcam, Cambridge, MA, USA), vimentin (Cat# GTX100619, 1:1000, GeneTex Inc, Texas, USA), p-p38 (clone 3D7, 1:1000, Cell Signaling Technology, Danvers, MA, USA), p38 (clone D13EE1, 1:1000, Cell Signaling Technology, Danvers, MA, USA), p-JNK (clone 81E11, 1:1000, Cell Signaling Technology, Danvers, MA, USA), JNK (Cat# 9252, 1:1000, Cell Signaling Technology, Danvers, MA, USA), p-ERK (clone E-4, 1:1000, Santa Cruz Biotechnology, CA, USA), ERK (clone H-72, 1:1000, Santa Cruz Biotechnology, CA, USA), p-AKT (Ser473) (clone D9E, 1:1000, Cell Signaling Technology, Danvers, MA, USA), AKT (clone C67E7, 1:1000, Cell Signaling Technology, Danvers, MA, USA), p-SMAD2 (Ser465/467) (clone 138D4, 1:1000, Cell Signaling Technology, Danvers, MA, USA), SMAD2 (clone D43B4, 1:1000, Cell Signaling Technology, Danvers, MA, USA), p-SMAD3 (Ser465/Ser467) (clone E8F3R, 1:1000, Cell Signaling Technology, Danvers, MA, USA), SMAD3 (clone C67H9, 1:1000, Cell Signaling Technology, Danvers, MA, USA), and GAPDH (Cat# ab8245, 1:5000, Abcam, Cambridge, MA, USA), at 4 °C.

Techniques: Control, Expressing, Western Blot, Quantitation Assay, Software, Comparison