Journal: Scientific reports

Article Title: Melatonin suppresses PD-L1 expression and exerts antitumor activity in hepatocellular carcinoma.

doi: 10.1038/s41598-025-93486-4

Figure Lengend Snippet: Fig. 1. Effect of melatonin on the Huh7 cell. (A) Huh7 cells were treated with various concentrations of melatonin (Mel: 0, 0.5, 1, 2, 3, 4, 6 mM) for 24 and 48 h, and cell viability was measured with CCK8 assay. Data are expressed as a percentage of the control. Values are expressed as the mean ± standard deviation. (B , C) Colony formation of huh7 cells in the presence of melatonin (Mel: 0, 0.5, 1 mM) for 14 days. (D , E) Transwell migration assay. After incubating with melatonin (Mel: 0, 0.5, 1 mM) for 24 h, observe Huh7 cells under a microscope and count the migration rate. (F , G) Wound healing experiment: After treatment with melatonin (Mel: 0, 0.5, 1 mM) for 24 and 48 h, observe the degree of scratch closure and count the relative mobility. (H , I) Transwell invasion assay: After incubating with melatonin (Mel: 0, 0.5, 1 mM) for 24 h, observe the invasion of Huh7 cells under a microscope and calculate the relative invasion rate. (J) Western blot and quantitative analysis of the expression of Cleaved-Caspase-3, and BCL2 proteins after incubation with melatonin (Mel: 0, 0.5, 1 mM) for 24 h. (K) RT-qPCR was performed to detect the expression levels of BAX, BAD and BCL2 mRNA after incubation with melatonin (Mel: 0, 0.5, 1 mM) for 24 h. The images presented in figures were representative results of three independent experiments.

Article Snippet: Specific primary antibodies used were as follows: Antibody Source Identifier β-ACTIN Abclonal AC026 PD-L1 Abclonal A1645 P38 Abclonal A14401 P-P38 Cell signaling technology 4511 P-JNK Cell signaling technology 4668 JNK Cell signaling technology 9252 HIF-1α Cell signaling technology 14,179 Cleaved-Ccaspase-3 Proteintech 19677-1-AP BCL2 Biolight MA00081HuM10-C2F Fluorescent quantitative PCR detection technique (1) RNA Extraction: Cell samples were collected using Trizol reagent.

Techniques: CCK-8 Assay, Control, Standard Deviation, Transwell Migration Assay, Microscopy, Migration, Transwell Invasion Assay, Western Blot, Expressing, Incubation, Quantitative RT-PCR

Journal: Scientific reports

Article Title: Melatonin suppresses PD-L1 expression and exerts antitumor activity in hepatocellular carcinoma.

doi: 10.1038/s41598-025-93486-4

Figure Lengend Snippet: Fig. 2. Effect of melatonin on the HepG2 cells. (A) HepG2 cells were treated with various concentrations of melatonin (Mel: 0, 0.5,1, 2, 3, 4, 6 mM) for 24 and 48 h, and cell viability was measured with a CCK8 assay. Data are expressed as a percentage of the control. (B, C) Colony formation of HepG2 cells in the presence of melatonin (Mel: 0, 0.5, 1 mM) for 14 days. (D, E) Transwell migration assay. After incubating with melatonin (Mel: 0, 0.5, 1 mM) for 24 h, observe HepG2 cells under a microscope and count the migration rate. (F, G) Wound healing experiment: After treatment with melatonin (Mel: 0, 0.5, 1 mM) for 24 and 48 h, observe the degree of scratch closure and count the relative mobility. (H, I) Transwell invasion assay: After incubating with melatonin (Mel: 0, 0.5, 1 mM) for 24 h, observe the invasion of HepG2 cells under a microscope and calculate the relative invasion rate. (J) Western blot and quantitative analysis of the expression of Cleaved-Caspase-3,and BCL2 proteins after incubation with melatonin (Mel: 0, 0.5, 1 mM) for 24 h. (K) RT-qPCR was performed to detect the expression levels of BAD, BAX, and BCL2 mRNA after incubation with melatonin (Mel: 0, 0.5, 1 mM) for 24 h. The images presented in figures were representative results of three independent experiments.

Article Snippet: Specific primary antibodies used were as follows: Antibody Source Identifier β-ACTIN Abclonal AC026 PD-L1 Abclonal A1645 P38 Abclonal A14401 P-P38 Cell signaling technology 4511 P-JNK Cell signaling technology 4668 JNK Cell signaling technology 9252 HIF-1α Cell signaling technology 14,179 Cleaved-Ccaspase-3 Proteintech 19677-1-AP BCL2 Biolight MA00081HuM10-C2F Fluorescent quantitative PCR detection technique (1) RNA Extraction: Cell samples were collected using Trizol reagent.

Techniques: CCK-8 Assay, Control, Transwell Migration Assay, Microscopy, Migration, Transwell Invasion Assay, Western Blot, Expressing, Incubation, Quantitative RT-PCR

Journal: Scientific reports

Article Title: Melatonin suppresses PD-L1 expression and exerts antitumor activity in hepatocellular carcinoma.

doi: 10.1038/s41598-025-93486-4

Figure Lengend Snippet: Fig. 5. The effect of melatonin on PD-L1 expression and other protein/mRNA levels in tumor tissue, and its impact on lymphocyte activity. (A) PD-L1 expression and quantitative analysis in tumor tissues (n = 3). (B) CCK8 assay measured lymphocyte activity and RT-qPCR analyzed immune cytokines level (n = 8). (C) RT-qPCR analysis was performed to quantify the mRNA expression levels of BAX, BAD, BCL2, and BCL2-L1 in the tumor tissues of both groups of mice (n = 6). (D) Western blot and quantitative analysis was conducted to assess the protein expression levels of BCL2 and Cleaved-Caspase-3 in the tumor tissues of both groups of mice (n = 3). (E) Immunohistochemical detection of the expression levels of E-cad, caspase-3, BCL2 and CD8 in cancer tissue (n = 6). (F) Western blot detection and quantitative analysis of the expression levels of HIF-1α, JNK and P38 proteins and their phosphorylation status in the cancer tissues of both groups of mice (n = 3).

Article Snippet: Specific primary antibodies used were as follows: Antibody Source Identifier β-ACTIN Abclonal AC026 PD-L1 Abclonal A1645 P38 Abclonal A14401 P-P38 Cell signaling technology 4511 P-JNK Cell signaling technology 4668 JNK Cell signaling technology 9252 HIF-1α Cell signaling technology 14,179 Cleaved-Ccaspase-3 Proteintech 19677-1-AP BCL2 Biolight MA00081HuM10-C2F Fluorescent quantitative PCR detection technique (1) RNA Extraction: Cell samples were collected using Trizol reagent.

Techniques: Expressing, Activity Assay, CCK-8 Assay, Quantitative RT-PCR, Western Blot, Immunohistochemical staining, Phospho-proteomics