Journal: Journal for Immunotherapy of Cancer

Article Title: AGPAT3 reshapes tumor cell vulnerability to IFNγ-mediated ferroptosis and enhances immunotherapy efficacy through lipid remodeling

doi: 10.1136/jitc-2025-013305

Figure Lengend Snippet: Prediction of ICI outcomes using the FD.sig model with IFN-γ as a key factor. ( A ) Flowchart of constructing FD.model for ICI prediction response. ( B ) The volcano plot of FD.score between tumor and normal tissues in TCGA data; ( C ) Survival analysis of FD.score in TCGA-SKCM. ( D ) ROC curves of eight machine-learning methods in testing set. ( E ) AUCs of eight methods in training and testing sets. ( F ) Comparison of AUCs between FD.sig and other signatures in training and testing sets. ( G ) Comparison of AUCs between FD.sig and other signatures in each cohort. ( H ) The importance ranking of genes in FD.model. ( I ) Comparison of IFNG expression on T cells between responder, non-responder and treatment-naïve in GSE115978 . ( J–K ) Cell viability of A375 cells and H1299 cells treated with different concentrations of RSL3/RSL3+Fer-1 for 24 hours after IFN-γ incubation for 0–72 hours (n=3), IFN-γ: 100 mg/mL, Fer-1: 1 µM. ( L–M ) Relative ROS level of A375 ( L ) and H1299 cells ( M ) treated with RSL3/RSL3+Fer-1 for 3 hours after IFN-γ incubation for 48 hours (n=3), IFN-γ: 60 mg/mL, RSL3: 500 nM, Fer-1: 1 µM. AUC, area under the receiver operating characteristic curve; BLCA, bladder urothelial carcinoma; BRCA, breast cancer; CRAD, colorectal adenocarcinoma; FD.model, ferroptosis-based machine learning model; FD.sig, ferroptosis-driver signature; HNSC, head and neck squamous carcinoma; ICI, immune checkpoint inhibitor; IFN, interferon; KICH, kidney chromophobe; KIRC, kidney renal clear cell carcinoma; KIRP, kidney renal papillary cell carcinoma; LIHC, liver hepatocellular carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; NR, non-responders; OS, overall survival; PRAD, prostate adenocarcinoma; R, responders; ROC, receiver operating characteristic; ROS, reactive oxygen species; scRNA-seq, single-cell RNA sequencing; SKCM, skin cutaneous melanoma; STAD, stomach adenocarcinoma; TCGA, The Cancer Genome Atlas; THCA, thyroid carcinoma.

Article Snippet: Human melanoma cell line A375 was purchased from FuHeng Biology (Shanghai, China), while the human lung cancer cell line H1299, mouse melanoma cell line B16F10 (B16), and mouse lung cancer cell line lewis lung carcinoma (LLC) were purchased from American Type Culture Collection (ATCC).

Techniques: Comparison, Expressing, Incubation, Single Cell, RNA Sequencing

Journal: Journal for Immunotherapy of Cancer

Article Title: AGPAT3 reshapes tumor cell vulnerability to IFNγ-mediated ferroptosis and enhances immunotherapy efficacy through lipid remodeling

doi: 10.1136/jitc-2025-013305

Figure Lengend Snippet: IFN-γ induces the sensitivity of ferroptosis through lipid remodeling. ( A–B ) Ratios of different lipids in A375 cells and H1299 cells after IFN-γ incubation for different times, IFN-γ: 60 mg/mL. ( C ) Differential analysis of free fatty acids compared the control group with IFN-γ incubation groups in A375 cells and H1299 cells, IFN-γ: 60 mg/mL. ( D ) Ratios of ether lipids and ester lipids in H1299 cells after IFN-γ incubation, IFN-γ: 60 mg/mL. ( E–F ) Heatmap of ether-PC and ether-PE in A375 ( E ) and H1299 cells ( F ) after IFN-γ incubation, IFN-γ: 60 mg/mL. FA, fatty acyls; GL, glycerolipids; GP, glycerophospholipids; IFN, interferon; MUFA, monounsaturated fatty acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PR, prenol lipids; PUFA, polyunsaturated fatty acid; SFA, saturated fatty acid; SP, sphingolipids; ST, sterol lipids.

Article Snippet: Human melanoma cell line A375 was purchased from FuHeng Biology (Shanghai, China), while the human lung cancer cell line H1299, mouse melanoma cell line B16F10 (B16), and mouse lung cancer cell line lewis lung carcinoma (LLC) were purchased from American Type Culture Collection (ATCC).

Techniques: Incubation, Control

Journal: Journal for Immunotherapy of Cancer

Article Title: AGPAT3 reshapes tumor cell vulnerability to IFNγ-mediated ferroptosis and enhances immunotherapy efficacy through lipid remodeling

doi: 10.1136/jitc-2025-013305

Figure Lengend Snippet: IFN-γ induces the sensitivity of ferroptosis through ether lipids. ( A, C ) Volcano plot of ether-PC and ether-PE compared the control group with IFN-γ incubation groups in A375 cells and H1299 cells, IFN-γ: 60 mg/mL. ( B, D ) Intersection Venn diagram of differential lipids in A375 cells and H1299 cells. ( E–J ) Cell viability of A375 and H1299 cells treated with RSL3/RSL3+Fer-1 for 12 hours after incubation with indicated phospholipids, including PC 18_18:1 ( E ), PE 18_18:1 ( F ), PC 18_20:4 ( G ), PE 18_20:4 ( H ), PC 18_22:6 ( I ), PE 18_22:6 ( J ), phospholipids: 25 µM, Fer-1: 1 µm. ( K ) Comparison of lipid metabolism pathways with transcriptome data among groups with different IFN-γ incubation times in A375 cells and H1299 cells, IFN-γ: 60 mg/mL. IFN, interferon; PC, phosphatidylcholine; PE, phosphatidylethanolamine.

Article Snippet: Human melanoma cell line A375 was purchased from FuHeng Biology (Shanghai, China), while the human lung cancer cell line H1299, mouse melanoma cell line B16F10 (B16), and mouse lung cancer cell line lewis lung carcinoma (LLC) were purchased from American Type Culture Collection (ATCC).

Techniques: Control, Incubation, Comparison

Journal: Journal for Immunotherapy of Cancer

Article Title: AGPAT3 reshapes tumor cell vulnerability to IFNγ-mediated ferroptosis and enhances immunotherapy efficacy through lipid remodeling

doi: 10.1136/jitc-2025-013305

Figure Lengend Snippet: IFN-γ alters the ether lipid metabolism via the IRF1-AGPAT3 axis. ( A ) Schematic summarizing the PUFA-ePLs biosynthesis pathway and their contribution to ferroptosis susceptibility. ( B ) Heatmap of different groups on the expression of specific genes in A375 cells and H1299 cells. ( C ) Representative western blot for AGPS, AGPAT3 and GNPAT in A375 cells and H1299 cells after IFN-γ incubation, IFN-γ: 60 mg/mL. ( D ) Cell viability of different groups treated with different concentrations of RSL3/RSL3+Fer-1 for 24 hours after IFN-γ incubation for 48 hours (n=3), IFN-γ: 100 mg/mL, Fer-1: 1 µM. ( E ) Relative ROS level of different groups treated with RSL3/RSL3+Fer-1 for 3 hours after IFN-γ incubation for 48 hours (n=3), IFN-γ: 60 mg/mL, RSL3: 500 nM, Fer-1: 1 µM. ( F ) Heatmap of different groups on the expression of IFN-γ downstream transcription factors in A375 cells and H1299 cells. ( G–H ) IRF1 binding sites at the AGPAT3 promoter region in lung cancer cell lines from GSE186168 ChIP-seq data. ( I–J ) The peak of IRF1 binding at the Agpat3 promoter region becomes higher after IFN-γ incubation from GSE201881 ( I ) and GSE141606 ( J ) ChIP-seq data. ( K ) IRF1 binding sites at the AGPAT3 promoter region in A375 from CUT&Tag data. ChIP-seq, chromatin Immunoprecipitation sequencing; CUT&Tag, Cleavage Under Targets and Tagmentation; IFN, interferon; PUFA-ePLs, polyunsaturated ether phospholipids; ROS, reactive oxygen species.

Article Snippet: Human melanoma cell line A375 was purchased from FuHeng Biology (Shanghai, China), while the human lung cancer cell line H1299, mouse melanoma cell line B16F10 (B16), and mouse lung cancer cell line lewis lung carcinoma (LLC) were purchased from American Type Culture Collection (ATCC).

Techniques: Expressing, Western Blot, Incubation, Binding Assay, ChIP-sequencing

Journal: Journal for Immunotherapy of Cancer

Article Title: AGPAT3 reshapes tumor cell vulnerability to IFNγ-mediated ferroptosis and enhances immunotherapy efficacy through lipid remodeling

doi: 10.1136/jitc-2025-013305

Figure Lengend Snippet: IFN-γ alters the ether lipid metabolism via the IRF1-AGPAT3 axis. ( A–B ) Changes in ratio of ether lipids and eater lipids in A375 and H1299 cells in NC, sh-AGPAT3 and sh-AGPAT3+IFN-γ group, IFN-γ: 60 mg/mL. ( C–D ) Changes in ratio of different ether lipids in A375 and H1299 cells in NC, sh-AGPAT3 and sh-AGPAT3+IFN-γ group, IFN-γ: 60 mg/mL. ( E–F ) Heatmap of ether-PC and ether-PE in A375 ( E ) and H1299 cells ( F ) in different groups, IFN-γ: 60 mg/mL. IFN, interferon; NC, normal control; PC, phosphatidylcholine; PE, phosphatidylethanolamine.

Article Snippet: Human melanoma cell line A375 was purchased from FuHeng Biology (Shanghai, China), while the human lung cancer cell line H1299, mouse melanoma cell line B16F10 (B16), and mouse lung cancer cell line lewis lung carcinoma (LLC) were purchased from American Type Culture Collection (ATCC).

Techniques: Control

Journal: Antioxidants

Article Title: THC and CBD Induce Heme Oxygenase-1-Dependent Cell Death and Trigger Mitochondrial Dysfunction in Human Melanoma and Cutaneous Squamous Cell Carcinoma Cells

doi: 10.3390/antiox15030286

Figure Lengend Snippet: Effect of THC ( A ), CBD ( B ), mAEA ( C ), and JWH-133 ( D ) on metabolic activity of A375 and A431 cells. Cells were incubated with the respective cannabinoid at the indicated concentrations for the indicated times. The values given are based on WST-1 assays. All percentage values shown refer to the respective time-matched vehicle control, which was set to 100%. The data are mean values ± SEM of n = 8–9 per group from 3 independent experiments. * p ≤ 0.05 vs. corresponding vehicle control; one-way ANOVA with Dunnett’s post hoc test.

Article Snippet: A375 (human melanoma cell line; #300110; RRID:CVCL_0132) and A431 cells (human skin epidermoid carcinoma cell line; #300112; RRID:CVCL_0037) were obtained from CLS Cell Lines Service (Eppelheim, Germany).

Techniques: Activity Assay, Incubation, Control

Journal: Antioxidants

Article Title: THC and CBD Induce Heme Oxygenase-1-Dependent Cell Death and Trigger Mitochondrial Dysfunction in Human Melanoma and Cutaneous Squamous Cell Carcinoma Cells

doi: 10.3390/antiox15030286

Figure Lengend Snippet: Effect of THC ( A ), CBD ( B ), and mAEA ( C ) on activation of caspases-3/7 in A375 and A431 cells. Cells were incubated with the respective cannabinoid at the indicated concentrations for the indicated times. The values given are based on caspase-3/7 activity assays. All percentage values shown refer to the respective time-matched vehicle control, which was set to 100%. The data are mean values ± SEM of n = 9 per group from 3 independent experiments. * p ≤ 0.05 vs. corresponding vehicle control; one-way ANOVA with Dunnett’s post hoc test.

Article Snippet: A375 (human melanoma cell line; #300110; RRID:CVCL_0132) and A431 cells (human skin epidermoid carcinoma cell line; #300112; RRID:CVCL_0037) were obtained from CLS Cell Lines Service (Eppelheim, Germany).

Techniques: Activation Assay, Incubation, Activity Assay, Control

Journal: Antioxidants

Article Title: THC and CBD Induce Heme Oxygenase-1-Dependent Cell Death and Trigger Mitochondrial Dysfunction in Human Melanoma and Cutaneous Squamous Cell Carcinoma Cells

doi: 10.3390/antiox15030286

Figure Lengend Snippet: Effect of THC and CBD on activation of caspase-8 ( A – D ) and caspase-9 ( E – H ) in A375 and A431 cells. Cells were incubated with THC or CBD at the indicated concentrations for 6 h. The values given are based on caspase activity assays. All percentage values shown refer to the respective time-matched vehicle control, which was set to 100%. The data are mean values ± SEM of n = 9 per group from 3 independent experiments. * p ≤ 0.05 vs. corresponding vehicle control; one-way ANOVA with Dunnett’s post hoc test.

Article Snippet: A375 (human melanoma cell line; #300110; RRID:CVCL_0132) and A431 cells (human skin epidermoid carcinoma cell line; #300112; RRID:CVCL_0037) were obtained from CLS Cell Lines Service (Eppelheim, Germany).

Techniques: Activation Assay, Incubation, Activity Assay, Control

Journal: Antioxidants

Article Title: THC and CBD Induce Heme Oxygenase-1-Dependent Cell Death and Trigger Mitochondrial Dysfunction in Human Melanoma and Cutaneous Squamous Cell Carcinoma Cells

doi: 10.3390/antiox15030286

Figure Lengend Snippet: Effect of THC and CBD on PARP cleavage ( A – D ), LC3-I to LC3-II conversion ( E – H ), and GPX4 expression ( I – L ) in A375 and A431 cells. The cells were incubated with THC or CBD at the indicated concentrations for 24 h. The values given in the bar charts are based on densitometric analyses of the blots. PARP, cleaved PARP (cl. PARP), LC3A/B-I, LC3A/B-II and GPX4 levels were normalized to β-actin. All percentage values shown refer to the respective vehicle control, which was set to 100%. The blots shown are representative. The data are mean values ± SEM of n = 3 ( I – L ), n = 4 ( D , F , G ), n = 5 ( A , B , E , H ) or n = 6 ( C ) independent experiments. * p ≤ 0.05 vs. corresponding vehicle control; one-way ANOVA with Dunnett’s post hoc test.

Article Snippet: A375 (human melanoma cell line; #300110; RRID:CVCL_0132) and A431 cells (human skin epidermoid carcinoma cell line; #300112; RRID:CVCL_0037) were obtained from CLS Cell Lines Service (Eppelheim, Germany).

Techniques: Expressing, Incubation, Control

Journal: Antioxidants

Article Title: THC and CBD Induce Heme Oxygenase-1-Dependent Cell Death and Trigger Mitochondrial Dysfunction in Human Melanoma and Cutaneous Squamous Cell Carcinoma Cells

doi: 10.3390/antiox15030286

Figure Lengend Snippet: Effect of THC and CBD on the expression of HO-1 and HO-2 at the mRNA ( A – D ) and protein ( E – H ) level in A375 and A431 cells. Cells were incubated with THC or CBD at the indicated concentrations for 6 h ( A – D ) or 24 h ( E – H ). The values given in the bar charts are based on quantitative RT-PCR ( A – D ) or densitometric analyses of the blots ( E – H ). HO mRNA levels were normalized to PPIA mRNA levels and HO protein levels to β-actin. All percentage values shown refer to the respective vehicle control, which was set to 100%. The blots shown are representative. The mRNA and protein data are mean values ± SEM of n = 3 ( A – D ), n = 7 ( E ), n = 5 ( F ) or n = 6 ( G , H ) independent experiments. * p ≤ 0.05 vs. corresponding vehicle control; one-way ANOVA with Dunnett’s post hoc test.

Article Snippet: A375 (human melanoma cell line; #300110; RRID:CVCL_0132) and A431 cells (human skin epidermoid carcinoma cell line; #300112; RRID:CVCL_0037) were obtained from CLS Cell Lines Service (Eppelheim, Germany).

Techniques: Expressing, Incubation, Quantitative RT-PCR, Control

Journal: Antioxidants

Article Title: THC and CBD Induce Heme Oxygenase-1-Dependent Cell Death and Trigger Mitochondrial Dysfunction in Human Melanoma and Cutaneous Squamous Cell Carcinoma Cells

doi: 10.3390/antiox15030286

Figure Lengend Snippet: Influence of the HO-1 inhibitor SnPPIX on the decrease in metabolic activity ( A , D , G , J ) and cell number ( B , E , H , K ) as well as on the increase in caspase-3/7 activity ( C , F , I , L ) mediated by THC or CBD in A375 and A431 cells. Cells were pre-treated with SnPPIX (25 µM) or its vehicle for 1 h, followed by a 48-h (A375 cells) or 24-h (A431 cells) co-incubation with the indicated concentrations of THC or CBD or its vehicle. The data are mean values ± SEM of n = 9 from 3 independent experiments ( A – J ) or n = 12 from 4 independent experiments ( K , L ). * p ≤ 0.05 vs. corresponding vehicle control; # p ≤ 0.05 vs. corresponding THC- or CBD-treated group; one-way ANOVA with Bonferroni’s post hoc test.

Article Snippet: A375 (human melanoma cell line; #300110; RRID:CVCL_0132) and A431 cells (human skin epidermoid carcinoma cell line; #300112; RRID:CVCL_0037) were obtained from CLS Cell Lines Service (Eppelheim, Germany).

Techniques: Activity Assay, Incubation, Control

Journal: Antioxidants

Article Title: THC and CBD Induce Heme Oxygenase-1-Dependent Cell Death and Trigger Mitochondrial Dysfunction in Human Melanoma and Cutaneous Squamous Cell Carcinoma Cells

doi: 10.3390/antiox15030286

Figure Lengend Snippet: Effect of THC and CBD on mitochondrial membrane potential (ΔΨm) of A375 ( A ) and A431 cells ( B ). The cells were incubated with THC or CBD at the indicated concentrations for 24 h. ΔΨm was assessed using the JC-10 fluorescence ratio (orange to green) as described in and expressed relative to the vehicle control (set to 100%). Data are mean values ± SEM of n = 9 from 3 independent experiments. All percentage values shown refer to the respective vehicle control, which was set to 100%. * p ≤ 0.05 vs. corresponding vehicle control; one-way ANOVA with Dunnett’s post hoc test.

Article Snippet: A375 (human melanoma cell line; #300110; RRID:CVCL_0132) and A431 cells (human skin epidermoid carcinoma cell line; #300112; RRID:CVCL_0037) were obtained from CLS Cell Lines Service (Eppelheim, Germany).

Techniques: Membrane, Incubation, Fluorescence, Control

Journal: Antioxidants

Article Title: THC and CBD Induce Heme Oxygenase-1-Dependent Cell Death and Trigger Mitochondrial Dysfunction in Human Melanoma and Cutaneous Squamous Cell Carcinoma Cells

doi: 10.3390/antiox15030286

Figure Lengend Snippet: Effect of THC and CBD on mitochondrial HO-1 protein levels in A375 ( A , B ) and A431 cells ( C , D ). The cells were treated with the indicated concentrations of THC or CBD for 24 h. Thereafter, the corresponding proteins in the mitochondrial fractions were determined using Western blot analysis. The values given in the bar charts are based on densitometric analyses of the blots. Mitochondrial HO-1 was normalized to VDAC. All percentage values shown refer to the respective vehicle control, which was set to 100%. The blots shown are representative. In ( B ) the same VDAC blot is shown as in D, and in ( C ) as in C, as the same membranes were stripped and reprobed for different target proteins. The data are mean values ± SEM of n = 4 ( A – C ) or n = 3 ( D ) independent experiments. * p ≤ 0.05 vs. corresponding vehicle control; one-way ANOVA with Dunnett’s post hoc test.

Article Snippet: A375 (human melanoma cell line; #300110; RRID:CVCL_0132) and A431 cells (human skin epidermoid carcinoma cell line; #300112; RRID:CVCL_0037) were obtained from CLS Cell Lines Service (Eppelheim, Germany).

Techniques: Western Blot, Control

Journal: Antioxidants

Article Title: THC and CBD Induce Heme Oxygenase-1-Dependent Cell Death and Trigger Mitochondrial Dysfunction in Human Melanoma and Cutaneous Squamous Cell Carcinoma Cells

doi: 10.3390/antiox15030286

Figure Lengend Snippet: Effect of THC and CBD on the concentrations of subunits of mitochondrial respiratory chain complexes of A375 ( A – D ) and A431 cells ( E – H ). The cells were treated with the indicated concentrations of THC or CBD for 24 h. Thereafter, the corresponding proteins in the mitochondrial fractions were determined using Western blot analysis. The values given in the bar charts are based on densitometric analyses of the blots. Mitochondrial proteins were normalized to VDAC. All percentage values shown refer to the respective vehicle control, which was set to 100%. The blots shown are representative. In ( B ) the same VDAC blot is shown as in A, in ( D ) as in B, and in ( H ) as in D, as the same membranes were stripped and reprobed for different target proteines. The data are mean values ± SEM of n = 4 independent experiments. * p ≤ 0.05 vs. corresponding vehicle control; one-way ANOVA with Dunnett’s post hoc test.

Article Snippet: A375 (human melanoma cell line; #300110; RRID:CVCL_0132) and A431 cells (human skin epidermoid carcinoma cell line; #300112; RRID:CVCL_0037) were obtained from CLS Cell Lines Service (Eppelheim, Germany).

Techniques: Western Blot, Control

Journal: Antioxidants

Article Title: THC and CBD Induce Heme Oxygenase-1-Dependent Cell Death and Trigger Mitochondrial Dysfunction in Human Melanoma and Cutaneous Squamous Cell Carcinoma Cells

doi: 10.3390/antiox15030286

Figure Lengend Snippet: Effect of THC and CBD on the release of mitochondrial cytochrome c (Cyt c) into the cytosol of A375 ( A , B ) and A431 cells ( C , D ). The cells were incubated with THC or CBD at the indicated concentrations for 24 h. The values given in the bar charts are based on densitometric analyses of the blots. Cyt c levels were normalized on VDAC in mitochondrial fractions and on GAPDH in cytosolic fractions. The blots shown are representative. In ( A ) the same VDAC blot is shown as in B, in ( C ) as in C, and in ( D ) as in H, as the same membranes were stripped and reprobed for different target proteines. The data are mean values ± SEM of n = 4 (( A , B , D ) left graph each; ( C )) or n = 3 (( A , B , D ) right graph each) independent experiments. All percentage values shown refer to the respective vehicle control, which was set to 100%. * p ≤ 0.05 vs. corresponding vehicle control with Dunnett’s post hoc test.

Article Snippet: A375 (human melanoma cell line; #300110; RRID:CVCL_0132) and A431 cells (human skin epidermoid carcinoma cell line; #300112; RRID:CVCL_0037) were obtained from CLS Cell Lines Service (Eppelheim, Germany).

Techniques: Incubation, Control

Journal: Antioxidants

Article Title: THC and CBD Induce Heme Oxygenase-1-Dependent Cell Death and Trigger Mitochondrial Dysfunction in Human Melanoma and Cutaneous Squamous Cell Carcinoma Cells

doi: 10.3390/antiox15030286

Figure Lengend Snippet: Influence of THC and CBD on the mitochondrial structure of A375 and A431 cells. Representative images from the transmission electron microscopy of A375 ( A – C ) and A431 cells ( D – F ) treated for 24 h with vehicle ( A , D ), 6 µM THC ( B , E ) or 6 µM CBD ( C , F ). The images on the right of a treatment group (scale bar: 500 nm) are enlarged views of the areas outlined by white boxes in the corresponding left image (scale bar: 2 µm). The structures highlighted with white arrows in the enlarged views represent intact mitochondria (vehicle) vs. damaged mitochondria (THC, CBD), while white arrowheads mark altered mitochondria-associated ER membranes (CBD).

Article Snippet: A375 (human melanoma cell line; #300110; RRID:CVCL_0132) and A431 cells (human skin epidermoid carcinoma cell line; #300112; RRID:CVCL_0037) were obtained from CLS Cell Lines Service (Eppelheim, Germany).

Techniques: Transmission Assay, Electron Microscopy

Journal: Antioxidants

Article Title: THC and CBD Induce Heme Oxygenase-1-Dependent Cell Death and Trigger Mitochondrial Dysfunction in Human Melanoma and Cutaneous Squamous Cell Carcinoma Cells

doi: 10.3390/antiox15030286

Figure Lengend Snippet: Effect of THC and CBD on oxygen consumption rate (OCR) of A375 ( A – D ) and A431 cells ( E – H ). The cells were incubated with THC or CBD at the indicated concentrations for 24 h. A mitochondrial stress test was then carried out and OCR values were determined using the Seahorse XFe24 Analyzer. Therefore, oligomycin (port A), FCCP (port B) and antimycin A/rotenone (port C) were loaded into the respective ports of the sensor cartridges and released into the wells at the specified times, as marked by enclosed uppercase letters with arrows in the time-course panels. From this assay, time courses of OCR in both cell lines treated with THC ( A , E ) or CBD ( C , G ) and calculations of basal respiration (basal), ATP-linked respiration (ATP), spare respiratory capacity and proton leak ( B , D , F , H ) are shown. The data represent mean values ± SEM of n = 3 per group from 3 independent experiments. * p ≤ 0.05 vs. corresponding vehicle control; one-way ANOVA with Dunnett’s post hoc test.

Article Snippet: A375 (human melanoma cell line; #300110; RRID:CVCL_0132) and A431 cells (human skin epidermoid carcinoma cell line; #300112; RRID:CVCL_0037) were obtained from CLS Cell Lines Service (Eppelheim, Germany).

Techniques: Incubation, Control

Journal: Cancers

Article Title: Targeting p53 for Melanoma Treatment: Counteracting Tumour Proliferation, Dissemination and Therapeutic Resistance

doi: 10.3390/cancers13071648

Figure Lengend Snippet: SLMP53-2 inhibits melanoma cell growth through induction of cell cycle arrest and apoptosis. ( A ) IC 50 values of SLMP53-2 in A375, G361, MEWO and SK-MEL-5 melanoma cells obtained by colony formation assay for 11 days; data were normalized to DMSO and correspond to mean ± SEM, n = 5 (two replicates each). ( B ) Colony formation assay for A375, G361, MEWO and SK-MEL-5 melanoma cells treated with SLMP53-2 for the indicated concentrations. Images are representative of five independent experiments. ( C ) Effect of SLMP53-2 on growth and morphology of A375 cells for the indicated time points; images are representative of five independent experiments (scale bar = 100 μm, magnification = ×100). ( D ) Apoptosis (Annexin V-positive cells) was evaluated in A375 cells after 24, 48 and 72 h of treatment with 12 μM SLMP53-2. ( E ) Cell cycle analysis in A375 cells was determined after 24, 48 and 72 h of treatment with 12 μM SLMP53-2. In ( D , E ), data are mean ± SEM, n = 5; values are significantly different from DMSO: * p < 0.05, one-way ANOVA followed by Tukey’s test. ( F , G ) Effect of SLMP53-2 on three-day-old A375 spheroids, for up to 8 days of treatment. In G , data are mean ± SEM, n = 5; values are significantly different from DMSO: * p < 0.05, one-way ANOVA followed by Tukey’s test. ( H , I ) Evaluation of spheroid formation after 10 days of treatment with SLMP53-2; treatment was performed at the seeding time of A375 cells. In I , data are mean ± SEM, n = 5; values are significantly different from DMSO: * p < 0.05, one-way ANOVA followed by Tukey’s test. In ( F , H ), images are representative of five independent experiments; scale bar = 100 μm; magnification = 100×.

Article Snippet: A total of 5.0 × 10 5 /flask A375 cells were treated with 12 and 18 μM SLMP53-2 for 4 h; after cell lysis and protein lysate separation, 300 μg of total protein was incubated with 10 μL of mouse monoclonal anti-p53 (DO-1) or mouse immunoglobulin G (IgG, negative control) from Santa Cruz Biotechnology (Frilabo, Porto, Portugal), overnight at 4 °C.

Techniques: Colony Assay, Cell Cycle Assay

Journal: Cancers

Article Title: Targeting p53 for Melanoma Treatment: Counteracting Tumour Proliferation, Dissemination and Therapeutic Resistance

doi: 10.3390/cancers13071648

Figure Lengend Snippet: SLMP53-2 has p53-dependent growth inhibitory effect in melanoma cells with enhancement of p53 transcriptional activity. ( A – C ) Colony formation assay for silenced p53 (sip53) and control (CTRL) A375 cells treated with SLMP53-2, allowed to grow for 11 days. In ( A ), silencing efficacy of p53 by siRNA is shown; immunoblots are representative of five independent experiments and GAPDH was used as loading control; data plotted were normalized to CTRL and correspond to mean ± SEM, n = 5; values are significantly different from CTRL: * p < 0.05, unpaired Student’s t -test. In ( B ), images are representative of five independent experiments. In ( C ), data are normalized to DMSO and correspond to mean ± SEM, n = 5; values of sip53 cells significantly different from CTRL cells: * p < 0.05, two-way ANOVA followed by Sidak’s test. ( D , E ) Protein levels of p53 transcriptional targets in A375 cells treated with SLMP53-2 for 24 h (p53, MDM2, PTEN, Cyclin D1, p21 and KILLER) or 48 h (GADD45, PUMA, BCL-2, BCL-xL and BAX). In ( D ), immunoblots are representative of five independent experiments; GAPDH was used as loading control. In ( E ), quantification of protein expression levels is shown; values with DMSO were set as 1; data are mean ± SEM, n = 5. ( F ) mRNA levels of p53 target genes were determined by RT-qPCR in A375 cells after 24 h treatment with SLMP53-2; fold change is relative to DMSO; data are mean ± SEM, n = 5; values are significantly different from DMSO: * p < 0.05, two-way ANOVA with Dunnett’s multiple comparison test.

Article Snippet: A total of 5.0 × 10 5 /flask A375 cells were treated with 12 and 18 μM SLMP53-2 for 4 h; after cell lysis and protein lysate separation, 300 μg of total protein was incubated with 10 μL of mouse monoclonal anti-p53 (DO-1) or mouse immunoglobulin G (IgG, negative control) from Santa Cruz Biotechnology (Frilabo, Porto, Portugal), overnight at 4 °C.

Techniques: Activity Assay, Colony Assay, Control, Western Blot, Expressing, Quantitative RT-PCR, Comparison

Journal: Cancers

Article Title: Targeting p53 for Melanoma Treatment: Counteracting Tumour Proliferation, Dissemination and Therapeutic Resistance

doi: 10.3390/cancers13071648

Figure Lengend Snippet: SLMP53-2 enhances p53 stabilization by disrupting the p53–MDM2 interaction and interferes with the miRNA network in melanoma cells. ( A ) p53 protein levels in A375 melanoma cells treated for 24 h with 12 µM SLMP53-2 or solvent followed by cycloheximide treatment from 0 to 2 h (CHX; 150 μg/mL). ( B ) Quantification of p53 protein expression levels; immunoblots are representative of five independent experiments; GAPDH was used as loading control. Values for cells nontreated with cycloheximide (0 h) were set as 1; data are mean ± SEM, n = 5. ( C , D ) Coimmunoprecipitation (Co-IP) was performed in A375 cells treated with SLMP53-2 for 4 h. In C, representative immunoblots of five independent experiments are shown—whole-cell lysate (Input). p53 from IP was used as loading control. In D, quantification of protein expression levels relative to DMSO is shown (set as 1). Data shown are mean ± SEM, n = 5. ( E ) Expression levels of miR-145 and miR-23a in A375 cells after 24 h of treatment with SLMP53-2 were determined by RT-qPCR; fold of change is relative to DMSO; data are mean ± SEM, n = 5; values are significantly different from DMSO: * p < 0.05, one-way ANOVA followed by Tukey’s test. ( F , G ) Protein levels of miR-145 target genes, in A375 cells treated with SLMP53-2 for 24 h. In ( F ), immunoblots are representative of five independent experiments; GAPDH was used as loading control. In ( G ), quantification of protein expression levels is shown; values with DMSO were set as 1; data are mean ± SEM, n = 5.

Article Snippet: A total of 5.0 × 10 5 /flask A375 cells were treated with 12 and 18 μM SLMP53-2 for 4 h; after cell lysis and protein lysate separation, 300 μg of total protein was incubated with 10 μL of mouse monoclonal anti-p53 (DO-1) or mouse immunoglobulin G (IgG, negative control) from Santa Cruz Biotechnology (Frilabo, Porto, Portugal), overnight at 4 °C.

Techniques: Solvent, Expressing, Western Blot, Control, Co-Immunoprecipitation Assay, Quantitative RT-PCR

Journal: Cancers

Article Title: Targeting p53 for Melanoma Treatment: Counteracting Tumour Proliferation, Dissemination and Therapeutic Resistance

doi: 10.3390/cancers13071648

Figure Lengend Snippet: SLMP53-2 inhibits melanoma cell migration and invasion. ( A ) A375 and SK-MEL-5 confluent cells were treated with 2 or 4 μM SLMP53-2, respectively; cells were observed at 24 and 32 h (A375) and 30 and 48 h (SK-MEL-5) in the wound-healing assay. Images are representative of five independent experiments; scale bar = 100 μM; magnification = 100×. ( B ) Quantification of wound closure using randomly selected microscopic fields (six fields per sample). Data are mean ± SEM, n = 5; values are significantly different from DMSO: * p < 0.05, two-way ANOVA followed by Sidak’s test. ( C ) Effect of 2 μM SLMP53-2 on migration of A375 and SK-MEL-5 cells after 24 h of treatment. The relative number of migratory cells was determined by analysis of fluorescence signal intensity; values with DMSO were set as 1. Data are mean ± SEM, n = 5 (two replicates each); values are significantly different from DMSO: * p < 0.05, Student’s t -test. ( D ) Effect of 2 μM SLMP53-2 on the invasion of A375 and SK-MEL-5 cells after 24 h of treatment. Cells able to invade through an ECMatrix layer were quantified by fluorescence signal; values with DMSO were set as 1. Data are mean ± SEM, n = 5 (two replicates each); values are significantly different from DMSO: * p < 0.05, Student’s t -test. ( E ) Effect of SLMP53-2 on lactate secretion by A375 and SK-MEL-5 cells after 8 h of treatment. Cell density for each sample was used to normalize relative luminescence units (RLU) signal. Data are mean ± SEM, n = 5 (two replicates each); values are significantly different from DMSO: * p < 0.05; unpaired Student’s t -test.

Article Snippet: A total of 5.0 × 10 5 /flask A375 cells were treated with 12 and 18 μM SLMP53-2 for 4 h; after cell lysis and protein lysate separation, 300 μg of total protein was incubated with 10 μL of mouse monoclonal anti-p53 (DO-1) or mouse immunoglobulin G (IgG, negative control) from Santa Cruz Biotechnology (Frilabo, Porto, Portugal), overnight at 4 °C.

Techniques: Migration, Wound Healing Assay, Fluorescence

Journal: Cancers

Article Title: Targeting p53 for Melanoma Treatment: Counteracting Tumour Proliferation, Dissemination and Therapeutic Resistance

doi: 10.3390/cancers13071648

Figure Lengend Snippet: SLMP53-2 interferes with key molecular players in epithelial-to-mesenchymal transition (EMT) and angiogenesis. ( A – D ) Protein expression levels of crucial regulators of EMT and angiogenesis in A375 ( A , B ) and SK-MEL-5 ( C , D ) melanoma cells after 48 h of treatment with SLMP53-2 (in A375 cells, β-catenin was detected for 8 h and E-cadherin and TWIST for 24 h of treatment). Immunoblots are representative of five independent experiments; GAPDH was used as a loading control. In ( B , D ), quantification of protein expression levels is shown; values with DMSO were set as 1; data are means ± SEM, n = 5.

Article Snippet: A total of 5.0 × 10 5 /flask A375 cells were treated with 12 and 18 μM SLMP53-2 for 4 h; after cell lysis and protein lysate separation, 300 μg of total protein was incubated with 10 μL of mouse monoclonal anti-p53 (DO-1) or mouse immunoglobulin G (IgG, negative control) from Santa Cruz Biotechnology (Frilabo, Porto, Portugal), overnight at 4 °C.

Techniques: Expressing, Western Blot, Control

Journal: Cancers

Article Title: Targeting p53 for Melanoma Treatment: Counteracting Tumour Proliferation, Dissemination and Therapeutic Resistance

doi: 10.3390/cancers13071648

Figure Lengend Snippet: SLMP53-2 sensitizes melanoma cells to clinically used chemotherapeutic agents. ( A – C ) Cells were treated with a concentration range of vemurafenib ( A ), dacarbazine ( B ) and cisplatin ( C ) alone and in combination with 2 μM SLMP53-2, for 48 h, and the growth was analysed by SBR assay. Growth with DMSO was set as 100%. For each combination, the combination index (C.I.) and dose reduction index (D.R.I.) values were obtained. Data are mean ± SEM, n = 5 (two replicates each); values are significantly different from chemotherapeutic drug alone: * p < 0.05; two-way ANOVA followed by Sidak’s test. ( D ) Apoptosis (Annexin V-positive cells) was evaluated in A375 cells after 48 h of treatment with 2 μM SLMP53-2 (SLMP) and 5 µM cisplatin and 0.03 µM vemurafenib. Data are mean ± SEM, n = 5; values are significantly different from drug alone: * p < 0.05, one-way ANOVA followed by Tukey’s test. ( E , F ) Protein expression levels of BCL-2 after 48 h of treatment of SLMP53-2 with cisplatin (cisp) and with vemurafenib (vem). Immunoblots are representative of five independent experiments; GAPDH was used as a loading control. In F , quantification of protein expression levels; values with DMSO were set as 1; data are mean ± SEM, n = 5. ( G ) Cell cycle analysis in A375 cells was determined after 48 h of treatment with 2 μM SLMP53-2 and 2 μM dacarbazine (Dac). Data are mean ± SEM, n = 5; values are significantly different from drug alone: * p < 0.05, one-way ANOVA followed by Tukey’s test. ( H , I ) Protein expression levels of p21 after 48 h treatment of SLMP53-2 with dacarbazine (Dac). Immunoblots are representative of five independent experiments; GAPDH was used as a loading control. In I , quantification of protein expression levels is shown; values with DMSO were set as 1; data are mean ± SEM, n = 5. ( J , K ) Effect of 2 μM SLMP53-2 in combination with 0.027 μM Vemurafenib (Vem) on three-day-old A375 spheroids for up to 8 days of treatment. For the combination, the C.I. value was obtained. Images are representative of five independent experiments; scale bar = 100 μm; magnification = 100×. In ( K ), data are mean ± SEM, n = 5; values are significantly different from DMSO: * p < 0.05, one-way ANOVA followed by Tukey’s test.

Article Snippet: A total of 5.0 × 10 5 /flask A375 cells were treated with 12 and 18 μM SLMP53-2 for 4 h; after cell lysis and protein lysate separation, 300 μg of total protein was incubated with 10 μL of mouse monoclonal anti-p53 (DO-1) or mouse immunoglobulin G (IgG, negative control) from Santa Cruz Biotechnology (Frilabo, Porto, Portugal), overnight at 4 °C.

Techniques: Concentration Assay, Expressing, Western Blot, Control, Cell Cycle Assay

Journal: Cancers

Article Title: Targeting p53 for Melanoma Treatment: Counteracting Tumour Proliferation, Dissemination and Therapeutic Resistance

doi: 10.3390/cancers13071648

Figure Lengend Snippet: Melanoma cells do not develop resistance to SLMP53-2: vemurafenib-resistant melanoma cells show no cross-resistance to SLMP53-2 and are resensitized to vemurafenib by SLMP53-2. ( A ) A375 cells were exposed to six rounds of treatment with 6, 9, 12, 18, 24 and 30 μM of SLMP53-2. IC 50 values were determined at the end of each round by SRB assay after 48 h of treatment. Data were normalized to DMSO and correspond to mean ± SEM, n = 5 (two replicates each); values not significantly different from parental cells: p > 0.05, two-way ANOVA followed by Sidak’s test. ( B ) Representative images of parental, vemurafenib-resistant (Vem-res) A375 cells; scale bar = 100 μm; magnification = 100×. ( C ) Concentration–response curves for vemurafenib in parental and Vem-res A375 cells after 48 h of treatment. Data were normalized to DMSO and correspond to mean ± SEM, n = 6 (two replicates each); values of Vem-res cells significantly different from parental cells: * p < 0.05; two-way ANOVA followed by Sidak’s test. ( D , E ) Protein levels of p-AKT/AKT, p-ERK/ERK, PTEN and MDR-1 in untreated parental and Vem-res A375 cells. In D, immunoblots are representative of five independent experiments; GAPDH was used as loading control. In E, quantification of protein expression levels is shown; values with DMSO were set as 1; data are mean ± SEM, n = 5. ( F ) Concentration–response curves for SLMP53-2 in parental and Vem-res A375 cells after 48 h of treatment. Data were normalized to DMSO and correspond to mean ± SEM, n = 6 (two replicates each); values of Vem-res cells are not significantly different from parental cells: two-way ANOVA followed by Sidak’s test. ( G ) Vem-res A375 cells were treated with a concentration range of vemurafenib alone and in combination with 2 μM of SLMP53-2. Cell growth was evaluated for 48 h of treatment; growth obtained with DMSO was set as 100%. For each combination, the C.I. and D.R.I. values were obtained. Data are mean ± SEM, n = 6 (two replicates each); values are significantly different from vemurafenib alone: * p < 0.05, two-way ANOVA followed by Sidak’s test. ( H ) Representative images of Vem-res A375 cells treated with DMSO, 2 μM SLMP53-2, 1.3 μM vemurafenib (Vem) and the combination (SLMP53-2 + Vem) for 48 h; images are representative of five treatments; scale bar = 100 μm; magnificatio n = 100×. ( I , J ) Protein levels of PTEN, BCL-2, MDR-1 and p-AKT/AKT, in Vem-res cells after 48 h of treatment with 2 µM SLMP53-2. In I, immunoblots are representative of five independent experiments; GAPDH was used as loading control. In J, quantification of protein expression levels is shown; values with DMSO were set as 1; data are mean ± SEM, n = 5.

Article Snippet: A total of 5.0 × 10 5 /flask A375 cells were treated with 12 and 18 μM SLMP53-2 for 4 h; after cell lysis and protein lysate separation, 300 μg of total protein was incubated with 10 μL of mouse monoclonal anti-p53 (DO-1) or mouse immunoglobulin G (IgG, negative control) from Santa Cruz Biotechnology (Frilabo, Porto, Portugal), overnight at 4 °C.

Techniques: Sulforhodamine B Assay, Concentration Assay, Western Blot, Control, Expressing

Journal: Cancers

Article Title: Targeting p53 for Melanoma Treatment: Counteracting Tumour Proliferation, Dissemination and Therapeutic Resistance

doi: 10.3390/cancers13071648

Figure Lengend Snippet: In vivo melanoma antitumour activity of SLMP53-2. C57BL/6-Rag2 −/− IL2rg −/− mice carrying A375 xenografts were treated with 50 mg∙kg −1 SLMP53-2 or vehicle by intraperitoneal injection twice a week for a total of six administrations. ( A ) Tumour volume curves of mice carrying A375 xenografts treated with SLMP53-2 or vehicle. Fold change is relative to the start of treatments; data are mean ± SEM, n = 7; values are significantly different from vehicle: * p < 0.05, two-way ANOVA followed by Sidak’s test. ( B ) Tumour weights measured at the end of the in vivo experiment; data are mean ± SEM, n = 7; values are significantly different from vehicle: * p < 0.05, unpaired Student’s t -test. Representative images of the tumours treated with SLMP53-2 or vehicle at the end of the experiment. ( C ) Body weight of the mice registered during the course of the experiment. Data are mean ± SEM, n = 7; values are not significantly different from vehicle: p > 0.05, two-way ANOVA followed by Sidak’s test. ( D ) Weight of heart, spleen, kidney and livers from animals treated with SLMP53-2 or vehicle. Data are mean ± SEM, n = 7; values are not significantly different from vehicle: p > 0.05, two-way ANOVA followed by Sidak’s test. ( E ) Representative images of p53, Ki-67, BAX, BCL-2, TUNEL, β-catenin, Vimentin, and Slug detection in tumour tissues of A375 xenografts treated with SLMP53-2 or vehicle, collected at the end of treatment (scale bar = 5 μm; magnificatio n = 200×); haematoxylin and eosin (H&E). ( F – H ) Quantification of immunohistochemistry of A375 xenograft tumour tissues treated with SLMP53-2 or vehicle. In F, quantification of the number of Ki-67-positive and -negative cells; values are significantly different from vehicle: * p < 0.05, two-way ANOVA followed by Sidak’s test. In G, quantification of the percentage of positive-staining cells with TUNEL, n = 5; values are significantly different from vehicle: * p < 0.05, unpaired Student’s t -test. In H, quantification of the p53, Vimentin, BAX, BCL-2, β-catenin and Slug staining by evaluation of 3,3′-diaminobenzidine (DAB) intensity is shown, n = 5; values are significantly different from vehicle: * p < 0.05, unpaired Student’s t -test.

Article Snippet: A total of 5.0 × 10 5 /flask A375 cells were treated with 12 and 18 μM SLMP53-2 for 4 h; after cell lysis and protein lysate separation, 300 μg of total protein was incubated with 10 μL of mouse monoclonal anti-p53 (DO-1) or mouse immunoglobulin G (IgG, negative control) from Santa Cruz Biotechnology (Frilabo, Porto, Portugal), overnight at 4 °C.

Techniques: In Vivo, Activity Assay, Injection, TUNEL Assay, Immunohistochemistry, Staining