Journal: Nature Communications

Article Title: TRPV1 inhibition overcomes cisplatin resistance by blocking autophagy-mediated hyperactivation of EGFR signaling pathway

doi: 10.1038/s41467-023-38318-7

Figure Lengend Snippet: a Kaplan–Meier analysis of overall survival (calculated as months to death or months to last follow-up) in cisplatin-treated CESC or cisplatin-untreated CESC. The 30th and 70th percentiles were used as cutoffs values for the EGFR activity scores (EGFR activity score high > 70th; EGFR activity score low < 30th). b Comparison of the level of EGFR activity scores in responder (R, n = 55) and non-respo n der (NR, n = 12) of cisplatin-treated CESC cohort. c Western blot analysis of pEGFR, EGFR and MCL1 expression. d , e CaSki P and CR cells were treated with gefitinib as the indicated dose. f , g CaSki P and CR cells were transfected with siRNA targeting GFP or EGFR . d , f The protein levels of pEGFR, EGFR and MCL1 were determined by western blots. β-actin was used as an internal loading control. Numbers below the blot images indicate the expression as measured by fold-change. e , g Flow cytometry analysis of the frequency of apoptotic (active caspase 3 + ) cells after incubation with or without cisplatin for 24 h. All experiments were performed in triplicate. In the box plots, the top and bottom edges of boxes indicate the first and third quartiles, respectively; the center lines indicate the medians; and the ends of whiskers indicate the maximum and minimum values, respectively. The p values by Gehan–Breslow–Wilcoxon test ( a ), two-tailed Student’s t test ( b ) or two-way ANOVA ( e , g ) are indicated. The data represent the mean ± SD. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies against pEGFR (1:3000; Cell Signaling Technology, Danvers, MA, USA, 3777 S), EGFR (1:3000; Cell Signaling Technology, 4267 S), MCL1 (1:3000; Santa Cruz Biotechnology, Paso Robles, CA, USA, sc-819), ATG7 (1:3000; Cell Signaling Technology, 8558 S), LC3B (1:3000; Cell Signaling Technology, 2775 S), EGF (1:1000; Abcam, Cambridge, UK, ab206106), NANOG (1:3000; Bethyl Laboratories, Montgomery, TX, USA, A300-379A), TRPV1 (1:3000; Abcam, ab6166), FLAG (1:5000; Medical & Biological Laboratories, Nagoya, JPN, M185-3L), pAKT (1:3000; Cell Signaling Technology, 9271), AKT1 (1:3000; Cell Signaling Technology, 9272), and β-actin (1:5000; Medical & Biological Laboratories, M177-3) were used.

Techniques: Activity Assay, Western Blot, Expressing, Transfection, Flow Cytometry, Incubation, Two Tailed Test

Journal: Nature Communications

Article Title: TRPV1 inhibition overcomes cisplatin resistance by blocking autophagy-mediated hyperactivation of EGFR signaling pathway

doi: 10.1038/s41467-023-38318-7

Figure Lengend Snippet: a The cells were stained with anti-LC3B (red) antibodies and then visualized by confocal microscopy. DAPI was used to stain the nuclei. The images are representative of three separate experiments. Scale bar, 20 μm. The graph depicts the experimental quantitation of puncta. b The protein levels of LC3B were determined by western blot analysis. c CaSki P and CR cells starved for 24 hr in medium supplemented with 0.1% FBS were fixed and imaged by TEM. d – f CaSki CR cells were transfected with siRNA targeting GFP or ATG7 . d The protein levels of secreted EGF and internal ATG7, LC3B, pEGFR, EGFR, and β-actin were confirmed by western blots. e The amount of EGF secreted into the media was measured by ELISA. f Flow cytometry analysis of the frequency of apoptotic (active caspase 3 + ) cells after incubation with or without cisplatin for 24 h. g In the cells treated with or without spautin-1, the protein levels of secreted EGF and internal LC3B, pEGFR, EGFR, and β-actin were determined by western blots. h Flow cytometry analysis of the frequency of apoptotic (active caspase 3 + ) cells after incubation with or without cisplatin for 24 h. b , c , g β-actin was used as the internal loading control. Numbers below the blot images indicate the expression as measured by fold-change. All experiments were performed in triplicate. The p values by two-tailed Student’s t test ( a – c ), one-way ANOVA ( e ), or two-way ANOVA ( f , h ) are indicated. The data represent the mean ± SD. Source data are provided as a Source Data file. (NS, not significant).

Article Snippet: Primary antibodies against pEGFR (1:3000; Cell Signaling Technology, Danvers, MA, USA, 3777 S), EGFR (1:3000; Cell Signaling Technology, 4267 S), MCL1 (1:3000; Santa Cruz Biotechnology, Paso Robles, CA, USA, sc-819), ATG7 (1:3000; Cell Signaling Technology, 8558 S), LC3B (1:3000; Cell Signaling Technology, 2775 S), EGF (1:1000; Abcam, Cambridge, UK, ab206106), NANOG (1:3000; Bethyl Laboratories, Montgomery, TX, USA, A300-379A), TRPV1 (1:3000; Abcam, ab6166), FLAG (1:5000; Medical & Biological Laboratories, Nagoya, JPN, M185-3L), pAKT (1:3000; Cell Signaling Technology, 9271), AKT1 (1:3000; Cell Signaling Technology, 9272), and β-actin (1:5000; Medical & Biological Laboratories, M177-3) were used.

Techniques: Staining, Confocal Microscopy, Quantitation Assay, Western Blot, Transfection, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Incubation, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: TRPV1 inhibition overcomes cisplatin resistance by blocking autophagy-mediated hyperactivation of EGFR signaling pathway

doi: 10.1038/s41467-023-38318-7

Figure Lengend Snippet: a Top, quantification of NANOG expression in tumor cells at different stages of selection with cisplatin (P to CR). Parallel stages without selection are labeled as N1 to N3. Bottom, representative Western blot images. b Top, representative images of flow cytometry analysis of NANOG + tumor cells. Bottom, quantification of the frequency of NANOG + tumor cells. c , d CaSki CR cells were transfected with siRNA targeting GFP or NANOG . e , f CaSki P cells were transfected with empty vector ( no insert ) or NANOG wild-type. c , e The protein levels of secreted EGF and internal NANOG, LC3B, pEGFR, and EGFR were confirmed by western blots. β-actin was used as the internal loading control. Numbers below the blot images indicate the expression as measured by fold-change. d , f Flow cytometry analysis of the frequency of apoptotic (active caspase 3 + ) cells after incubation with or without cisplatin for 24 h. g , h In the cells treated with or without anti-EGF or EGF, the frequency of apoptotic (active caspase 3 + ) cells after incubation with or without cisplatin for 24 h were confirmed by flow cytometry. All experiments were performed in triplicate. The p values by one-way ANOVA ( a , b ) or two-way ANOVA ( d , f , g , h ) are indicated. The data represent the mean ± SD. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies against pEGFR (1:3000; Cell Signaling Technology, Danvers, MA, USA, 3777 S), EGFR (1:3000; Cell Signaling Technology, 4267 S), MCL1 (1:3000; Santa Cruz Biotechnology, Paso Robles, CA, USA, sc-819), ATG7 (1:3000; Cell Signaling Technology, 8558 S), LC3B (1:3000; Cell Signaling Technology, 2775 S), EGF (1:1000; Abcam, Cambridge, UK, ab206106), NANOG (1:3000; Bethyl Laboratories, Montgomery, TX, USA, A300-379A), TRPV1 (1:3000; Abcam, ab6166), FLAG (1:5000; Medical & Biological Laboratories, Nagoya, JPN, M185-3L), pAKT (1:3000; Cell Signaling Technology, 9271), AKT1 (1:3000; Cell Signaling Technology, 9272), and β-actin (1:5000; Medical & Biological Laboratories, M177-3) were used.

Techniques: Expressing, Selection, Labeling, Western Blot, Flow Cytometry, Transfection, Plasmid Preparation, Incubation

Journal: Nature Communications

Article Title: TRPV1 inhibition overcomes cisplatin resistance by blocking autophagy-mediated hyperactivation of EGFR signaling pathway

doi: 10.1038/s41467-023-38318-7

Figure Lengend Snippet: a Venn diagram showing the overlap of genes upregulated in high versus low chemotherapy-resistant cervical cancer patients (red), CaSki NANOG versus CaSki no insert (blue), and cervical cancer patients from the TCGA cohort classified as having high or low EGFR activity score (dark). b The cells were stained with anti-TRPV1 (red) antibodies and then visualized by confocal microscopy. DAPI was used to stain the nuclei. Scale bar, 10 μm. The graph depicts the experimental quantitation of TRPV1. The protein levels of TRPV1 and β-actin were confirmed by western blots. c Current-voltage relationships from capsaicin (Cap, red line) or AMG9810 (AMG, blue line)-induced whole-cell current in CaSki P versus CR cells. Summary of average outward current at +60 mV during Cap exposure in CaSki P versus CR cells. d The protein levels of secreted EGF and internal LC3B, pEGFR, and EGFR were confirmed by western blots. e Flow cytometry analysis of the frequency of apoptotic (active caspase 3 + ) cells after incubation with or without cisplatin for 24 h. f mRNA expression of TRPV1 was analyzed by qRT-PCR. g TRPV1 mRNA levels were measured by qRT-PCR. h Levels of TRPV1 and FLAG-NANOG proteins were confirmed by western blot. i Diagram of the human TRPV1 promoter region containing the NANOG-binding site. The arrows indicate the qChIP-PCR amplicon. j The cross-linked chromatin from HEK293 cells transfected with empty vector or FLAG- NANOG was immunoprecipitated with rabbit IgG or anti-FLAG antibodies. Relative enrichment of FLAG-NANOG in the TRPV1 promoter region was assessed by qChIP-PCR analysis with primers that amplified the genomic region indicated above. The ChIP data values represent the relative ratio to the input. Numbers below the blot images indicate the expression as measured by fold-change. All experiments were performed in triplicate. The p values by two-tailed Student’s t test ( b ), two-way ANOVA ( c , e ), or one-way ANOVA ( f , g , j ) are indicated. The data represent the mean ± SD. Source data are provided as a Source Data file. (NS, not significant).

Article Snippet: Primary antibodies against pEGFR (1:3000; Cell Signaling Technology, Danvers, MA, USA, 3777 S), EGFR (1:3000; Cell Signaling Technology, 4267 S), MCL1 (1:3000; Santa Cruz Biotechnology, Paso Robles, CA, USA, sc-819), ATG7 (1:3000; Cell Signaling Technology, 8558 S), LC3B (1:3000; Cell Signaling Technology, 2775 S), EGF (1:1000; Abcam, Cambridge, UK, ab206106), NANOG (1:3000; Bethyl Laboratories, Montgomery, TX, USA, A300-379A), TRPV1 (1:3000; Abcam, ab6166), FLAG (1:5000; Medical & Biological Laboratories, Nagoya, JPN, M185-3L), pAKT (1:3000; Cell Signaling Technology, 9271), AKT1 (1:3000; Cell Signaling Technology, 9272), and β-actin (1:5000; Medical & Biological Laboratories, M177-3) were used.

Techniques: Activity Assay, Staining, Confocal Microscopy, Quantitation Assay, Western Blot, Flow Cytometry, Incubation, Expressing, Quantitative RT-PCR, Binding Assay, Amplification, Transfection, Plasmid Preparation, Immunoprecipitation, Two Tailed Test

Journal: Nature Communications

Article Title: TRPV1 inhibition overcomes cisplatin resistance by blocking autophagy-mediated hyperactivation of EGFR signaling pathway

doi: 10.1038/s41467-023-38318-7

Figure Lengend Snippet: a , b CaSki NANOG cells were transfected with siRNA targeting GFP or TRPV1 . c , d CaSki NANOG cells were treated with DMSO or AMG9810. a , c The levels of TRPV1, LC3B, pEGFR, EGFR, pAKT, and AKT proteins were confirmed by western blot analysis. b , d The frequency of apoptotic (active caspase 3 + ) cells were analyzed by flow cytometry. e Current-voltage relationships in capsaicin (Cap, red line) or AMG9810 (AMG, blue line)-induced whole-cell current in CaSki no insert versus CaSki NANOG cells. f Fluorescence calcium imaging by TRPV1 activity in tumor cells. Top, representative calcium images of FURA-2-AM-loaded cells. Bottom, changes in intracellular Ca 2+ current over time in tumor cells. g , h CaSki NANOG cells were transfected with siRNA targeting GFP or CaMKKβ . The expression levels of indicated protein were confirmed by western blots. a , c , g , h β-actin was used as the internal loading control. Numbers below the blot images indicate the expression as measured by fold-change. All experiments were performed in triplicate. The p values by two-way ANOVA ( b , d ) are indicated. The data represent the mean ± SD. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies against pEGFR (1:3000; Cell Signaling Technology, Danvers, MA, USA, 3777 S), EGFR (1:3000; Cell Signaling Technology, 4267 S), MCL1 (1:3000; Santa Cruz Biotechnology, Paso Robles, CA, USA, sc-819), ATG7 (1:3000; Cell Signaling Technology, 8558 S), LC3B (1:3000; Cell Signaling Technology, 2775 S), EGF (1:1000; Abcam, Cambridge, UK, ab206106), NANOG (1:3000; Bethyl Laboratories, Montgomery, TX, USA, A300-379A), TRPV1 (1:3000; Abcam, ab6166), FLAG (1:5000; Medical & Biological Laboratories, Nagoya, JPN, M185-3L), pAKT (1:3000; Cell Signaling Technology, 9271), AKT1 (1:3000; Cell Signaling Technology, 9272), and β-actin (1:5000; Medical & Biological Laboratories, M177-3) were used.

Techniques: Transfection, Western Blot, Flow Cytometry, Fluorescence, Imaging, Activity Assay, Expressing

Journal: Nature Communications

Article Title: TRPV1 inhibition overcomes cisplatin resistance by blocking autophagy-mediated hyperactivation of EGFR signaling pathway

doi: 10.1038/s41467-023-38318-7

Figure Lengend Snippet: a – c CaSki no insert and TRPV1 cells were treated with DMSO or AMG9810, as indicated. a The protein levels of TRPV1, LC3B, pEGFR, and EGFR were confirmed by western blots. b The frequency of apoptotic (active caspase 3 + ) cells were analyzed by flow cytometry. c The amount of EGF secreted into the media was measured by ELISA. d CaSki TRPV1 cells were treated with IgG or anti-EGF. Levels of pEGFR and EGFR were confirmed by western blots. e and f CaSki TRPV1 cells were transfected with siRNA targeting GFP or EGFR . e The protein levels of pEGFR and EGFR were confirmed by western blot analysis. f Flow cytometry analysis of the frequency of apoptotic (active caspase 3 + ) cells after incubation with or without cisplatin for 24 h. a , d , e β-actin was used as the internal loading control. Numbers below the blot images indicate the expression as measured by fold-change. All experiments were performed in triplicate. The p values by two-way ANOVA ( b , f ) or one-way ANOVA ( c ) are indicated. The data represent the mean ± SD. Source data are provided as a Source Data file. (NS, not significant).

Article Snippet: Primary antibodies against pEGFR (1:3000; Cell Signaling Technology, Danvers, MA, USA, 3777 S), EGFR (1:3000; Cell Signaling Technology, 4267 S), MCL1 (1:3000; Santa Cruz Biotechnology, Paso Robles, CA, USA, sc-819), ATG7 (1:3000; Cell Signaling Technology, 8558 S), LC3B (1:3000; Cell Signaling Technology, 2775 S), EGF (1:1000; Abcam, Cambridge, UK, ab206106), NANOG (1:3000; Bethyl Laboratories, Montgomery, TX, USA, A300-379A), TRPV1 (1:3000; Abcam, ab6166), FLAG (1:5000; Medical & Biological Laboratories, Nagoya, JPN, M185-3L), pAKT (1:3000; Cell Signaling Technology, 9271), AKT1 (1:3000; Cell Signaling Technology, 9272), and β-actin (1:5000; Medical & Biological Laboratories, M177-3) were used.

Techniques: Western Blot, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Transfection, Incubation, Expressing

Journal: Nature Communications

Article Title: TRPV1 inhibition overcomes cisplatin resistance by blocking autophagy-mediated hyperactivation of EGFR signaling pathway

doi: 10.1038/s41467-023-38318-7

Figure Lengend Snippet: a CaSki CR cells were treated with cisplatin and AMG9810, as indicated. The frequency of apoptotic (active caspase 3 + ) cells was analyzed by flow cytometry. b The combination score was calculated based on changes in the percentage of apoptotic cells in cisplatin-treated tumor cells with or without AMG9810. Combination score = (% of active caspase 3 + tumor cells by cisplatin and AMG9810)/((% of active caspase 3 + tumor cells by cisplatin). c Schematic of the therapeutic regimen in mice implanted with CaSki CR cells. d tumor growth, e mass (at 19 d after challenge), and f survival of mice inoculated with CaSki CR cells treated with the indicated reagents. g The expression levels of LC3B, pEGFR, EGFR, pAKT, AKT, and MCL1 protein were confirmed by western blot analysis. β-actin was used as the internal loading control. Numbers below the blot images indicate the expression as measured by fold-change. h The frequency of apoptotic (active caspase 3 + ) cells was analyzed by flow cytometry in mice administrated DMSO or cisplatin, with or without AMG9810. For the in vivo experiments, five mice from each group were used. All experiments were performed in triplicate. The p values by two-way ANOVA ( a , b , d ) or one-way ANOVA ( e , h ) are indicated. The data represent the mean ± SD. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies against pEGFR (1:3000; Cell Signaling Technology, Danvers, MA, USA, 3777 S), EGFR (1:3000; Cell Signaling Technology, 4267 S), MCL1 (1:3000; Santa Cruz Biotechnology, Paso Robles, CA, USA, sc-819), ATG7 (1:3000; Cell Signaling Technology, 8558 S), LC3B (1:3000; Cell Signaling Technology, 2775 S), EGF (1:1000; Abcam, Cambridge, UK, ab206106), NANOG (1:3000; Bethyl Laboratories, Montgomery, TX, USA, A300-379A), TRPV1 (1:3000; Abcam, ab6166), FLAG (1:5000; Medical & Biological Laboratories, Nagoya, JPN, M185-3L), pAKT (1:3000; Cell Signaling Technology, 9271), AKT1 (1:3000; Cell Signaling Technology, 9272), and β-actin (1:5000; Medical & Biological Laboratories, M177-3) were used.

Techniques: Flow Cytometry, Expressing, Western Blot, In Vivo