Journal: American Journal of Translational Research

Article Title: PIAS3/SOCS1-STAT3 axis responses to oxidative stress in hepatocellular cancer cells

doi:

Figure Lengend Snippet: Effect of H2O2 treatment on oxidative stress induction and malignant processes in HCC cells. A. H2O2 (50 μM for 24 h) and/or antioxidant TA treatment. Total antioxidant capacity (TAC) in HepG2 cells was determined. VCEAC: vitamin C equivalent antioxidant capacity (n=3). B. Protein carbonyl content in HepG2 cells was detected (n=3). C. Western blotting detected the expression of Nrf2 protein in cells (n=3). The band density for Nrf2 protein are shown (n=3). D. HepG2 cell proliferation 48 h after different treatments (n=3). E. Cell growth of HepG2 cells 24-72 h post treatment (n=3). F. Cell apoptosis in HepG2 cells. G. Invasion ability of HepG2 cells (n=3). Scale bar, 50 μm. H. Migration capacity of HepG2 cells (n=3). Scale bar, 50 μm. *P<0.05, **P<0.01.

Article Snippet: Immunohistochemical staining Immunohistochemical staining was performed according to the manufacturer’s instructions with the following reagents and instruments: horse serum (RTU Vectastain Kit, PK-7200), 1:200 mouse anti-Nrf2 antibody (AF3925, R&D Systems), ABC reagent (LS-J1026-1, Vector labs), and IHC slide staining system (NanoMtrx 100, BioGenex).

Techniques: Western Blot, Expressing, Migration

Journal: American Journal of Translational Research

Article Title: PIAS3/SOCS1-STAT3 axis responses to oxidative stress in hepatocellular cancer cells

doi:

Figure Lengend Snippet: Effects of PIAS3 and SOCS1 overexpression and colivelin treatment on oxidative stress induction and malignant processes of HCC cells. HepG2 cells were transfected with PIAS3 or SOCS1 overexpression vector for 36 h and then treated with H2O2 (50 μM) and/or 0.5 μM CO for 24 h. A. Total antioxidant capacity (TAC) in HepG2 cells was determined. VCEAC: vitamin C equivalent antioxidant capacity (n=3). B. Protein carbonyl content in HepG2 cells was detected (n=3). C. Western blotting detected the expression of Nrf2 protein in cells (n=3). Quantification data from three independent repeats were showed below the blot. D. HepG2 cell proliferation 48 h after different treatments (n=3). E. MTT assays detected cell growth of HepG2 cells 24-72 h post treatment (n=3). F. Cell apoptosis of HepG2 cells. G. Invasion ability of HepG2 cells (n=3). Scale bar, 50 μm. H. Migration capacity of HepG2 cells (n=3). Scale bar, 50 μm. *P<0.05, **P<0.01.

Article Snippet: Immunohistochemical staining Immunohistochemical staining was performed according to the manufacturer’s instructions with the following reagents and instruments: horse serum (RTU Vectastain Kit, PK-7200), 1:200 mouse anti-Nrf2 antibody (AF3925, R&D Systems), ABC reagent (LS-J1026-1, Vector labs), and IHC slide staining system (NanoMtrx 100, BioGenex).

Techniques: Over Expression, Transfection, Plasmid Preparation, Western Blot, Expressing, Migration

Journal: Journal of Veterinary Science

Article Title: Epigallocatechin-3-gallate suppresses hemin-aggravated colon carcinogenesis through Nrf2-inhibited mitochondrial reactive oxygen species accumulation

doi: 10.4142/jvs.22097

Figure Lengend Snippet: WST, water-soluble tetrazolium salt; EGCG, epigallocatechin-3-gallate; Nrf2, nuclear factor erythroid-2-related factor 2. * p < 0.05.

Article Snippet: Nrf2 (NBP1-32822) and Keap1 (NBP2-03319) antibodies were purchased from Novus Biologicals (Centennial, CO, USA).

Techniques:

Journal: Cell Biology and Toxicology

Article Title: Polystyrene Nanoplastics induced placental toxicology by activating Keap1-mediated ferroptosis via METTL3-dependent m6A methylation

doi: 10.1007/s10565-026-10152-9

Figure Lengend Snippet: PS-NPs activated ferroptosis in vivo. ( A ) The coimmunoprecipitation between Keap1 (Red) and p62 (green) in placenta following PS-NPs treatment was shown (n = 4). ( B ) The image showed the coimmunoprecipitation between Keap1 (Red) and Nrf2 (green) in placenta following PS-NPs treatment for 24 h (n = 4). ( C - D ) METTL3, Keap1, Nrf2, p62, NQO1, HO-1, FTH1, GPX4 and ASCL4 expression in placenta were determined (n = 4). ( E ) IHC staining assay was used to evaluate METTL3 , Keap1, Nrf2, HO-1, FTH1 and GPX4 expression(n = 4). * P < 0.05, compared with controls

Article Snippet: The sections were incubated with the following primary antibodies: METTL3, GPX4, FTH1, Nrf2, Keap1, ACSL4 and HO-1, which were all used at a 1:300 dilution and were purchased from Proteintech.

Techniques: In Vivo, Expressing, Immunohistochemistry

Journal: Cell Biology and Toxicology

Article Title: Polystyrene Nanoplastics induced placental toxicology by activating Keap1-mediated ferroptosis via METTL3-dependent m6A methylation

doi: 10.1007/s10565-026-10152-9

Figure Lengend Snippet: PS-NPs induced ferroptosis through regulating p62-Keap1-Nrf2 pathway. ( A and B ) Western blotting analysis Ferroptosis-associated proteins (GPX4 and ACSL4), autophagy-associated proteins (p62) and lipid peroxidation-associated proteins (FTH1, NQO1, and HO-1) were measured. ( C ) METTL3 expression was measured by RT-qPCR (n = 3). ( D - E ) The green fluorescence intensity of METTL3, Nrf2, Keap1, p62 and GPX4 was measured with LSCM

Article Snippet: The sections were incubated with the following primary antibodies: METTL3, GPX4, FTH1, Nrf2, Keap1, ACSL4 and HO-1, which were all used at a 1:300 dilution and were purchased from Proteintech.

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Fluorescence

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting of a novel interplay between MET tyrosine kinase and NRF2 enhances sensitivity to Paclitaxel in triple negative breast cancer

doi: 10.1186/s13046-025-03625-y

Figure Lengend Snippet: High expression levels of RTKs and NRF2 correlate with worst prognosis in TNBC patients. A Volcano plot for Spearman correlation between NRF2 expression and indicated RTKs in BC samples from TCGA-BRCA dataset. Significant positive correlations are shown in red, and negative correlations in blue. Common RTKs are highlighted. B Expression levels of MET and EGFR across the four different BC subtypes from the TCGA dataset. Kaplan–Meier curves showing the probability of overall survival in TNBC patients with different expression levels of MET/NRF2 (left) and EGFR/NRF2 (right) ( C ), AXL , TGFBR1 , PDGFRA , and NRF2 ( D ). Statistical analysis: ( B ) One-way ANOVA followed by Tukey’s multiple comparison statistical test was performed. C-D Survival data derived from GSE31519 dataset for TNBC patients. P values were computed using the Logrank (Mantel Cox) test. ns: not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: For transient silencing of MET expression (MGT-13 and BT-549) and NRF2 expression (MGT-13), cells were seeded the day before and transfected for 24 h with LipofectamineTM RNAiMAX Transfection Reagent (Invitrogen) by using murine and human MET siRNA (Santa Cruz Biotechnology; sc-35924, sc-29397) or NRF2 siRNA (Santa Cruz Biotechnology; sc-37049) according to the manufacturer’s instructions.

Techniques: Expressing, Comparison, Derivative Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting of a novel interplay between MET tyrosine kinase and NRF2 enhances sensitivity to Paclitaxel in triple negative breast cancer

doi: 10.1186/s13046-025-03625-y

Figure Lengend Snippet: Inhibition of the MET receptor reduces NRF2 nuclear localization and activity in TNBC MMTV-R26 Met cell lines. A Immunoblotting (left) of pY 1234/35 MET, MET, NRF2, and relative densitometric analysis (right) of NRF2 protein levels in all MGT cell lines derived from the MMTV-R26 Met tumours. Actin was used as a loading control. B Immunoblotting (left) of pY 1234/35 MET, MET, NRF2, and relative densitometric (right) analysis of NRF2 protein levels in the MGT-13 cell line upon 16 h of PHA treatment. Actin was used as a loading control. C Immunofluorescence (left) and relative quantification (right) of NRF2 (red) nuclear intensity in MGT-13 cells upon 16 h of PHA treatment. DNA (Hoechst, blue). D Immunoblotting (top) and relative densitometric analysis (bottom) of NRF2 cytosolic and nuclear fractions in MGT-13 cells upon 16 h of PHA treatment. Vinculin and Lamin A/C were used as loading and quality controls. ( E ) RT-qPCR of NRF2 target genes in MGT-13 cells after 16 h of PHA treatment. Actin was used as housekeeping gene. F Workflow of RNA-seq experiments. p-value ≤ 0.5 and Log 2 foldchange (log₂FC) > 0.7 were used as threshold for analysis. G Activation z-score for transcription factors (CTRL vs. PHA). Values lower than − 2 (blue bars) suggest an inhibition of the pathway related to the corresponding transcription factor in the PHA treatment. Values over 2 (red bars) suggest an activation of the pathway in PHA samples. NFE2L2 (NRF2) is indicated in bold. Data from IPA. H Heatmap of z-scores for genes involved in the NRF2 pathway (according to IPA) for CTRL vs. PHA (3 samples for each condition, see x axis). Positive values of z-score (red) indicate upregulation within the sample, negative values (blue) downregulation. Results represent the mean of at least three independent experiments (± SEM or ± SD). Statistical analysis: ( A-B-D ) Unpaired t -test. C Mann-Whitney test according to the normal distribution. E Multiple t- test. PHA: PHA-665752 1 µM, MET inhibitor. ns: not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: For transient silencing of MET expression (MGT-13 and BT-549) and NRF2 expression (MGT-13), cells were seeded the day before and transfected for 24 h with LipofectamineTM RNAiMAX Transfection Reagent (Invitrogen) by using murine and human MET siRNA (Santa Cruz Biotechnology; sc-35924, sc-29397) or NRF2 siRNA (Santa Cruz Biotechnology; sc-37049) according to the manufacturer’s instructions.

Techniques: Inhibition, Activity Assay, Western Blot, Derivative Assay, Control, Immunofluorescence, Quantitative Proteomics, Quantitative RT-PCR, RNA Sequencing, Activation Assay, MANN-WHITNEY

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting of a novel interplay between MET tyrosine kinase and NRF2 enhances sensitivity to Paclitaxel in triple negative breast cancer

doi: 10.1186/s13046-025-03625-y

Figure Lengend Snippet: Targeting of the MET receptor reduces NRF2 activity in human TNBC cell lines. A Immunoblotting analysis of pY 1234/35 MET, MET, and NRF2 in human BC cell lines. Ponceau was used as a loading control. B Immunofluorescence (left) and relative quantification analysis (right) of NRF2 (red) nuclear intensity in human TNBC cells upon 16 h of PHA treatment. DNA (Hoechst, blue). C RT-qPCR of NRF2 target genes in human TNBC cells after 16 h of PHA treatment. Actin was used as housekeeping. D Immunoblotting (top) analysis of pY 1234/35 MET, MET, and NRF2 and relative densitometric analysis (bottom) of NRF2 in human BC cell lines after 4 h of serum-free media and 10 min of HGF stimulation (50ng/mL). Vinculin was used as a loading control. E Clonogenic assays (top) and relative quantification of the number of colonies (bottom) on MDA-MB-231, BT-549 and MCF10-A cells treated with PHA and ML-385 for 72 h. Results represent the mean of at least three independent experiments (± SEM or ± SD). Statistical analysis: ( B ) Mann-Whitney test according to the normal distribution. C Multiple t -test. D Unpaired t -test. E One-way ANOVA statistical test. PHA: PHA-665752 1 or 2 µM, MET inhibitor. ML-385: 5 µM, NRF2 inhibitor. ns: not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: For transient silencing of MET expression (MGT-13 and BT-549) and NRF2 expression (MGT-13), cells were seeded the day before and transfected for 24 h with LipofectamineTM RNAiMAX Transfection Reagent (Invitrogen) by using murine and human MET siRNA (Santa Cruz Biotechnology; sc-35924, sc-29397) or NRF2 siRNA (Santa Cruz Biotechnology; sc-37049) according to the manufacturer’s instructions.

Techniques: Activity Assay, Western Blot, Control, Immunofluorescence, Quantitative Proteomics, Quantitative RT-PCR, MANN-WHITNEY

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting of a novel interplay between MET tyrosine kinase and NRF2 enhances sensitivity to Paclitaxel in triple negative breast cancer

doi: 10.1186/s13046-025-03625-y

Figure Lengend Snippet: MET or NRF2 targeting enhances Paclitaxel sensitivity of TNBC cells. Cell viability of MGT-13 ( A ) and BT-549 ( B ) cells exposed to PTX in combination with PHA or ML-385. Percentage of cell viability in presence of drugs compared to controls (untreated cells) is indicated using a colour scale (from green to red). C-D Top panel: matrix synergy plot representing the synergy/antagonism score of each combination and its statistical significance calculated by Loewe model. Bottom panel: mapped to d-r surface showing the synergy distribution of drug combinations. The synergistic analysis was performed on MGT-13 ( C ) and BT-549 ( D ) cell lines. E-F Clonogenic assays on MGT-13 cells exposed to PTX in combination with PHA ( E ) or ML-385 ( F ) and treated as in Fig. 4A. G-H Histograms of flow cytometry experiments representing the percentage of dead cells upon ANNEXIN V/DAPI + staining of MGT-13 cells exposed for 72 h to PHA and PTX ( G ) or ML-385 and PTX ( H ) treatments alone or in combination. Results represent the mean of at least three independent experiments (± SEM). Statistical analysis: ( A-B-G-H ) One-way ANOVA statistical test was performed for each combination compared to the drug alone. (In A-B, * indicate the significance respect to PTX treatment). C-D Loewe models were used for synergy score calculation by Combenefit software. PHA: PHA-665752: MET inhibitor; ML-385: NRF2 inhibitor. PTX: Paclitaxel. ns: not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: For transient silencing of MET expression (MGT-13 and BT-549) and NRF2 expression (MGT-13), cells were seeded the day before and transfected for 24 h with LipofectamineTM RNAiMAX Transfection Reagent (Invitrogen) by using murine and human MET siRNA (Santa Cruz Biotechnology; sc-35924, sc-29397) or NRF2 siRNA (Santa Cruz Biotechnology; sc-37049) according to the manufacturer’s instructions.

Techniques: Flow Cytometry, Staining, Software

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting of a novel interplay between MET tyrosine kinase and NRF2 enhances sensitivity to Paclitaxel in triple negative breast cancer

doi: 10.1186/s13046-025-03625-y

Figure Lengend Snippet: MET-NRF2 axis inhibition increases ROS levels and DNA damage accumulation. Immunoblotting ( A ) and relative densitometric analysis ( B ) of p62 and HO-1 in MGT-13 and BT-549 cells exposed for 24 h to PTX in combination with PHA. Actin was used as a loading control. C-F Cytofluorimetric analyses (top) and relative histograms (bottom) of reactive oxygen species (ROS) upon DHE staining on MGT-13 ( C-D ) and BT-549 ( E-F ) cells exposed for 24 h to PTX in combination with PHA or ML-385. Confocal microscopy analysis ( G ) and relative quantification ( H ) of 8-oxo-DG (red) foci/cell in MGT-13 cells treated alone or in combination with PTX and PHA. DNA (Hoechst, blue). Confocal microscopy analysis ( I ) and relative quantification ( J ) of γH2AX (green) positive cells in MGT-13 cells treated alone or in combination with PTX and PHA. K Immunoblotting analysis of γH2AX in MGT-13 and BT-549 treated as in ( I ). Vinculin and GAPDH were used as loading control. Results represent the mean of at least three independent experiments (± SEM or ± SD). Statistical analysis: ( B-D-F-H-J ) One-way ANOVA statistical test was performed for each combination compared to the drug alone. PHA: PHA-665752 1 or 3 µM, MET inhibitor. ML-385: 5 µM, NRF2 inhibitor. PTX: Paclitaxel 3nM or 30nM. ns: not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: For transient silencing of MET expression (MGT-13 and BT-549) and NRF2 expression (MGT-13), cells were seeded the day before and transfected for 24 h with LipofectamineTM RNAiMAX Transfection Reagent (Invitrogen) by using murine and human MET siRNA (Santa Cruz Biotechnology; sc-35924, sc-29397) or NRF2 siRNA (Santa Cruz Biotechnology; sc-37049) according to the manufacturer’s instructions.

Techniques: Inhibition, Western Blot, Control, Staining, Confocal Microscopy, Quantitative Proteomics

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting of a novel interplay between MET tyrosine kinase and NRF2 enhances sensitivity to Paclitaxel in triple negative breast cancer

doi: 10.1186/s13046-025-03625-y

Figure Lengend Snippet: MET modulates NRF2 signalling via SRC kinase-dependent p62/KEAP1 pathway. Immunoblotting ( A ) of pY 1234/35 MET, MET, pY 416 SRC, SRC and relative densitometric analyses ( B ) of pY 416 SRC normalized on total SRC in TNBC cell lines after 16 h of PHA treatment. GAPDH was used as loading control. C Immunoblotting pY 1234/35 MET, MET, pY 416 SRC, SRC, and NRF2 in MGT-13 cells transiently transfected with empty vector (pSGT) and active SRC (SRC Y527F ) after 16 h of PHA treatment. Actin was used as loading control. Immunoblotting ( D ) and relative densitometric analyses ( E ) of KEAP1, pS 349 p62 and p62 in human TNBC cell lines after 16 h of PHA treatment. Actin was used as loading control. F Confocal microscopy analyses and relative quantification of colocalizing dots of KEAP1 (red) and p62 (green) in human TNBC cell lines upon 16 h of PHA treatment. DNA (Hoechst, blue). 3X digital magnification showing merged signals. Results represent the mean of at least three independent experiments (± SEM or ± SD). Statistical analysis: ( B-E ) Unpaired t -test. F Mann-Whitney test according to the normal distribution. PHA: PHA-665752 1 µM, MET inhibitor. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: For transient silencing of MET expression (MGT-13 and BT-549) and NRF2 expression (MGT-13), cells were seeded the day before and transfected for 24 h with LipofectamineTM RNAiMAX Transfection Reagent (Invitrogen) by using murine and human MET siRNA (Santa Cruz Biotechnology; sc-35924, sc-29397) or NRF2 siRNA (Santa Cruz Biotechnology; sc-37049) according to the manufacturer’s instructions.

Techniques: Western Blot, Control, Transfection, Plasmid Preparation, Confocal Microscopy, Quantitative Proteomics, MANN-WHITNEY

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting of a novel interplay between MET tyrosine kinase and NRF2 enhances sensitivity to Paclitaxel in triple negative breast cancer

doi: 10.1186/s13046-025-03625-y

Figure Lengend Snippet: SRC expression sustains NRF2 nuclear accumulation and activity in TNBC. A Immunoblotting (top) of NRF2, pY 416 SRC, SRC and relative densitometric analysis (bottom) of NRF2 protein levels in MDA-MB-231 cells upon 16 h of DAS treatment. GAPDH was used as a loading control. B Immunofluorescence (left) and relative quantification (right) of NRF2 (red) nuclear intensity in MDA-MB-231 cells upon 16 h of DAS treatment. DNA (Hoechst, blue). C Immunoblotting (top) and relative densitometric analysis (bottom) of NRF2 cytosolic and nuclear fractions in MDA-MB-231 cells upon 16 h of DAS treatment. Vinculin and Lamin A/C were used as loading and quality controls. D RT-qPCR of NRF2 target genes in MDA-MB-231 cells after 16 h of DAS treatment. Actin was used as housekeeping gene. E Expression levels of SRC in log 2 across the four different breast cancer subtypes from the TCGA dataset. F-G Kaplan–Meier curves show the overall survival probability of TNBC ( n = 579; F ) and non-TNBC ( n = 544; G ) patients with different expression levels of SRC and NRF2 . Results represent the mean of at least three independent experiments (± SEM or ± SD). Statistical analysis: ( A-C ) Unpaired t- test. B Mann-Whitney test according to the normal distribution. D Multiple t -test. E One-way ANOVA followed by Tukey’s multiple comparison statistical test was performed. F-G Survival data obtained from Gene Expression Omninbus (GEO) Id GSE31519 for TNBC patients and TCGA dataset for non-TNBC patients. P values were computed using the Logrank (Mantel Cox). DAS: Dasatinib 50 nM; SRC inhibitor. ns: not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: For transient silencing of MET expression (MGT-13 and BT-549) and NRF2 expression (MGT-13), cells were seeded the day before and transfected for 24 h with LipofectamineTM RNAiMAX Transfection Reagent (Invitrogen) by using murine and human MET siRNA (Santa Cruz Biotechnology; sc-35924, sc-29397) or NRF2 siRNA (Santa Cruz Biotechnology; sc-37049) according to the manufacturer’s instructions.

Techniques: Expressing, Activity Assay, Western Blot, Control, Immunofluorescence, Quantitative Proteomics, Quantitative RT-PCR, MANN-WHITNEY, Comparison, Gene Expression

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting of a novel interplay between MET tyrosine kinase and NRF2 enhances sensitivity to Paclitaxel in triple negative breast cancer

doi: 10.1186/s13046-025-03625-y

Figure Lengend Snippet: MET, SRC or NRF2 targeting improves Paclitaxel efficacy in TNBC patient-derived organoids. Organoid viability assays of PDO-21, PDO-43, and PDO-46 after PHA and PTX ( A ), ML-385 and PTX ( B ), DAS and PTX ( C ) combination treatments. D 4X digital magnification of bright-field images and relative quantification of organoids size of PDO-21 culture treated as described in ( A-B-C ). Scale bar: 400 μm. E Percentage of small, medium, and large PDO-21 treated as in ( A - B - C ). F Working model. Results represent the mean of at least three independent experiments (± SEM). Statistical analysis: ( A-B-C ) One-way ANOVA statistical test was performed for each combination compared to the drug alone. E Two-way ANOVA followed by Tukey’s multiple comparison test (* respect to CTR, $ respect to inhibitors and # respect to PTX). PHA: PHA-665752, MET inhibitor. DAS: Dasatinib. ML-385: NRF2 inhibitor. PTX: Paclitaxel. ns: not significant; *, $, # p < 0.05; **, $$, ## p < 0.01; ***, $$$, ### p < 0.001; ****, $$$$, #### p < 0.0001

Article Snippet: For transient silencing of MET expression (MGT-13 and BT-549) and NRF2 expression (MGT-13), cells were seeded the day before and transfected for 24 h with LipofectamineTM RNAiMAX Transfection Reagent (Invitrogen) by using murine and human MET siRNA (Santa Cruz Biotechnology; sc-35924, sc-29397) or NRF2 siRNA (Santa Cruz Biotechnology; sc-37049) according to the manufacturer’s instructions.

Techniques: Derivative Assay, Quantitative Proteomics, Comparison