EMBO/SourceData huggingface dataset:
Figure 5(G) SMAD3 depletion (siRNA#1 & #2) decreased cell density and increased BRAFi effect (vemurafenib) on BRAFi-resistant cells (SKMel28R). CTR for non-targeting siRNA. DMSO for dimethylsulfoxide (solvent of vemurafenib; BRAFi). n=3 biologically independent experiments. Each histogram represents the mean + s.d. (H) SMAD3 depletion (siRNA#1 & #2) decreased cell density and increased BRAFi effect (vemurafenib, BRAFi) on BRAFi-resistant cells (Me1402). CTR for non-targeting siRNA. DMSO for dimethylsulfoxide (solvent of vemurafenib). n=3 biologically independent experiments. Each histogram represents the mean + s.d.(L) SMAD3, Phospho-SMAD3 Ser423/425 and BIRC3 expression levels in melanoma cell lines. Four subtypes of melanoma cells (U, undifferentiated; NC, neural crest-like; T, transitory; M, melanocytic) have been compared. 501Mel and M249 cells are melanocytic cells in contrast to dedifferentiated BRAFi-resistant cells (R). Three couples of melanoma cell lines (Sensitive (S) and (R)) have been used to illustrate the SMAD3 and BIRC3 increases in BRAFi-resistant cell lines. HEK293 cells are used as control (kidney). HSC70 serves as loading control for western-blot experiments. Each antibody has been evaluated on individual membrane (explaining the three HSC70, loading controls).(O) The chemical inhibition of SMAD3 by SIS3 (SMAD3i) improved current therapy effect (BRAFi+MEKi; Vem 5µM and Cobi 1µM) on BRAFi-resistant cells (SKMel28R, M229R & M238R). Cells have been treated
Figure 6(B) AhR activation by exogenous and endogenous ligands promotes SMAD3 induction. 501Mel cells AhR wild-type or knock-out have been exposed to exogenous and endogenous AhR ligands; TCDD (5nM) or ITE (10µM) or the solvent (DMSO) during 10 days. n=5 biologically independent experiments for AhR WT cells and n=3 for AhR KO cells. Each histogram represents the mean + s.d. (C) AhR activation by exogenous and endogenous AhR ligands promotes TIPARP induction. 501Mel cells have been treated as described in B. n=5 biologically independent experiments for AhR WT cells and n=3 for AhR KO cells. Each histogram represents the mean + s.d.;(E) Loss of AhR reduces SMAD3 expression levels. SMAD3 expression has been investigated in SKMel28 cells AhR wild-type (WT) or knock-out (KO). SKMel28R, have been obtained from SKMel28S by chronic exposure to non-lethal doses of BRAFi R for BRAFi-resistant SKMel28 cells and S for sensitive.
Figure 7(E) Heatmap depicting mRNA levels of SMAD3-signature in BRAF(V600E) non-treated melanoma patients (dataset from TCGA; SKCM, BRAF(V600E) mutated: n=118). Three pigmentation genes (MITF, MLANA and TYR) have been added to highlight the differentiation states of tumors. ~20% of tumors are considered as dedifferentiated tumors (red box) with a high SMAD3-signature.(H) SMAD3 depletion decreases expression of SMAD3-regulated genes. SMAD3 knock-down by siRNA decreased mRNA expression of BRAFi-resistance genes in SKMel28R and Me1402. NRP1 mRNA was not detected in our experimental conditions.
Figure 1IncuCyte cell proliferation curves of PC3 cells treated with the indicated concentrations of ASNase. Data information: For IncuCyte proliferation assays, images were taken every 4 hours and the cell confluence was calculated by averaging three mapped images per well. All results were calculated from three replicates and presented as mean ± SD, unless otherwise stated. The p-value was calculated by two-tailed unpaired t test by Prism7. **p<0.01, ***p<0.001.IncuCyte cell proliferation curves for ASNS knockout (sgASNS) and control (sgNon-targeting) PC3 cells in the absence and presence of ASNase. Data information: For IncuCyte proliferation assays, images were taken every 4 hours and the cell confluence was calculated by averaging three mapped images per well. All results were calculated from three replicates and presented as mean ± SD, unless otherwise stated. The p-value was calculated by two-tailed unpaired t test by Prism7. **p<0.01, ***p<0.001. Volcano plots for the MAGeCK pipeline analysis of the sgRNA abundance from the screen. Green dots indicate positive controls and red dots indicate candidates with a fold discovery rate (FDR) <0.003.IncuCyte cell proliferation curves of SLC1A3 knockout (sgSLC1A3) and control (sgNon-targeting) PC3 cells in the absence and presence of ASNase treatment. #3 and #4 represent 2 different sgRNAs targeting SLC1A3. Data information: For IncuCyte proliferation assays, images were taken every 4 hours and the cell confluence was calculated by averaging three mapped images per well. All results were calculated from three replicates and presented as mean ± SD, unless otherwise stated. The p-value was calculated by two-tailed unpaired t test by Prism7. **p<0.01, ***p<0.001. Radioactive labeled aspartate and glutamate uptake measurement in control (sgNon-targeting) and SLC1A3 knockout (sgSLC1A3) PC3 cells. #3 and #4 represent two different sgRNAs targeting SLC1A3. Radioactive labeled leucine uptake was used as a control. Data was normalized to the reads of control PC3 cells.Endogenous levels of aspartate, asparagine, glutamate and glutamine in control (sgNon-targeting) and SLC1A3 knockout (sgSLC1A3) PC3 cells with or without ASNase for 3 days. Median peak intensity was used for the read normalization.IncuCyte cell proliferation curves of SLC1A3 knockout (sgSLC1A3#3) PC3 cells treated with ASNase and supplemented with either esterified-aspartate (6mM) or esterified-glutamate (6mM), and esterified-leucine (6mM) as a control. Data information: For IncuCyte proliferation assays, images were taken every 4 hours and the cell confluence was calculated by averaging three mapped images per well. All results were calculated from three replicates and presented as mean ± SD, unless otherwise stated. The p-value was calculated by two-tailed unpaired t test by Prism7. **p<0.01, ***p<0.001.
Figure 2RT-qPCR analysis was used to determine the relative SLC1A3 mRNA expression (to GAPDH) in different prostate and breast cancer cell lines, as indicated. Data information: Results were calculated based on three replicates (except for SUM159 and BT549 in B, n=2) and presented as mean ± SD. The p-value was calculated by two-tailed unpaired t test in Prism7. **p<0.01, ***p<0.001. a.u. indicates arbitrary unit.cell lines were transduced with either control (sgNon-targeting) or sgSLC1A3. Aspartate uptake levels were determined and compared between control and SLC1A3 KO in these cell lines. Leucine uptake level was used for normalization. The numbers above the control column denote the basal aspartate uptake capacity. Data information: Results were calculated based on three replicates (except for SUM159 and BT549 in B, n=2) and presented as mean ± SD. The p-value was calculated by two-tailed unpaired t test in Prism7. **p<0.01, ***p<0.001. a.u. indicates arbitrary unit.RT-qPCR was used to determine the relative mRNA levels (to GAPDH) of aspartate/glutamate transporter genes (SLC1A1, SLC1A2, SLC1A3, SLC1A6 and SLC1A7) in LNCaP, BT549 and SUM159PT cells. Data information: Results were calculated based on three replicates (except for SUM159 and BT549 in B, n=2) and presented as mean ± SD. The p-value was calculated by two-tailed unpaired t test in Prism7. **p<0.01, ***p<0.001. a.u. indicates arbitrary unit.batch of cancer cells subjected to IncuCyte cell proliferation assays in the absence or presence of ASNase at indicated concentrations. 'ns' indicates no significant difference. Data information: Results were calculated based on three replicates (except for SUM159 and BT549 in B, n=2) and presented as mean ± SD. The p-value was calculated by two-tailed unpaired t test in Prism7. **p<0.01, ***p<0.001. a.u. indicates arbitrary unit.MCF7 and DU145 cells were transduced with either lentiviral empty vector (control) or lentiviral vector containing a V5-tagged SLC1A3 coding sequence (V5-SLC1A3). Relative SLC1A3 mRNA levels (to GAPDH) were determined by RT-qPCR.Immunofluorescence staining of the V5-tagged SLC1A3 in MCF7 and DU145 cells using anti-V5 antibody. Green staining indicates the plasma membrane localization of V5-SLC1A3 and blue DAPI staining marks the nuclei. Scale bar stands for 5 μm.Relative aspartate uptake levels in control and V5-SLC1A3-expressed MCF7 and DU145 cells. Leucine uptake level was used for normalization.Control and V5-SLC1A3 expressed MCF7 and DU145 cells were subjected to IncuCyte cell proliferation assays with or without ASNase at indicated concentrations.DU145 cells were supplemented with cell-permeable aspartate (6mM, esterified) or glutamate (3mM, esterified) following ASNase treatment, with esterified leucine (6mM or 3mM) as control.
Figure 3PC3, DU145 and V5-SLC1A3-DU145 cells were subjected to ASNase and TFB-TBOA treatment at indicated concentrations and cell proliferation was measured by IncuCyte assay.PC3 cells were treated under indicated conditions for 9 days and subjected to BrdU assays to determine cell cycle distributions. ASNase (0.3 U/ml), TFB-TBOA (5 μM).DU145 and V5-SLC1A3-DU145 cells were treated under indicated conditions with ASNase (0.2 U/ml) or TFB-TBOA (20μM) or both, and subjected to IncuCyte analysis for apoptotic cell counts.PC3 cells were treated under ASNase (0.3 U/ml), or TFB-TBOA (5 μM) conditions for 3 days and cell lysates were extracted and intracellular contents of aspartate, asparagine, glutamate and glutamine were determined by liquid-chromatography mass spectrometry (LC-MS). Data information: Median peak intensity was used for raw data normalization Results were calculated based on three replicates and presented as mean ± SD (unless otherwise stated). The p-value was calculated by two-tailed unpaired t test from Prism7. *p<0.05, **p<0.01, ***p<0.001.key metabolites involved in urea cycle, pyrimidine synthesis, TCA cycle, oxidation, glycolysis and carnitines metabolism were determined. The NAD+/NADH ratio of the indicated conditions was calculated and normalized to control (mean± SEM). Dash line indicates indirect effect. TCA cycle: tricarboxylic acid cycle; OAA: oxaloacetic acid; UMP: uridine monophosphate; CMP: cytidine monophosphate; PEP: phosphoenolpyruvate; NADH: nicotinamide adenine dinucleotide (reduced form); NAD+: nicotinamide adenine dinucleotide (oxidized form); NADPH: nicotinamide adenine dinucleotide phosphate (reduced form); NADP+: nicotinamide adenine dinucleotide phosphate (oxidized form); FAD: flavin adenine dinucleotide; GSSG: glutathione disulfide; HMG-CoA: 3-hydroxy-3-methylglutaryl-CoA. Data information: Median peak intensity was used for raw data normalization Results were calculated based on three replicates and presented as mean ± SD (unless otherwise stated). The p-value was calculated by two-tailed unpaired t test from Prism7. *p<0.05, **p<0.01, ***p<0.001.
Figure 5SUM159PT human breast cancer cells were orthotopically injected into the mammary glands of NSG mice. Once SUM159PT tumors reached 250mm3 volume, mice were treated with mock or ASNase (60U per day) for 5 consecutive days (n=3). Following treatment, mice were sacrificed, and blood, mammary glands and tumors were collected and subjected to mass spectrometry to determine the asparagine level. Essential amino acids were used for the raw data normalization. Data are presented as mean ± SD. Data information: The pretreatment started 2 days before the injection of cancer cells. And mice were either injected with 60 U ASNase or saline per day. The p-value was calculated by two-tailed unpaired t test in Prism7, unless otherwise stated. *p<0.05, **p<0.01, ***p<0.001.The mouse breast cancer cell lines 4T1 and 4T1-V5-SLC1A3 were orthotopically implanted into the mammary glands of pretreated NSG mice. Presented is the volume measurements of arising tumors at day 9. (n=13 mice for each group, except for 4T1+ASNase, n=12). Data are presented as mean ± SEM. Data information: The pretreatment started 2 days before the injection of cancer cells. And mice were either injected with 60 U ASNase or saline per day. The p-value was calculated by two-tailed unpaired t test in Prism7, unless otherwise stated. *p<0.05, **p<0.01, ***p<0.001.tumors were surgically removed once reached a volume of ~500mm3 and collected and subjected to LC-MS to determine the levels of asparagine, aspartate, glutamine and glutamate. Leucine level was used as a control. Results are based on five tumor samples and presented as mean ± SEM. Data information: The pretreatment started 2 days before the injection of cancer cells. And mice were either injected with 60 U ASNase or saline per day. The p-value was calculated by two-tailed unpaired t test in Prism7, unless otherwise stated. *p<0.05, **p<0.01, ***p<0.001.The human breast cancer cell lines MDA-MB-231 and MDA-MB-231-V5-SLC1A3 cells were intravenously injected into pretreated NSG mice. Once mice showed breathing problems, they were sacrificed, and lung and liver were collected and blindly scored for metastasis lesions. The p-value was calculated by one-tailed unpaired t test in Prism7. Data are presented as mean ± SEM (n=8). Data information: The pretreatment started 2 days before the injection of cancer cells. And mice were either injected with 60 U ASNase or saline per day. The p-value was calculated by two-tailed unpaired t test in Prism7, unless otherwise stated. *p<0.05, **p<0.01, ***p<0.001.
f1(a) Kaplan-Meier survival curves of combined male and female WT and Atg5Tg mice no. 25 (P0.001; n=65 for WT mice and n=70 for Atg5Tg mice). (b,c) Lifespan extension in male Atg5Tg mice no. 25 (P0.001; n=32 for WT mice and n=36 for Atg5Tg mice no. 25) (b) and female Atg5Tg mice no. 25 (P0.001; n=33 for WT mice and n=34 for Atg5Tg mice no. 25) (c). (d-f) Kaplan-Meier survival curves of combined male and female WT and Atg5Tg mice no. 43 (d), no. 47 (e) and no. 471 (f) (P0.001; n=25 for WT mice and n=30 for Atg5Tg mice). P-values were calculated using the log-rank test.(g) Expression levels of Atg5 and Atg12-Atg5 conjugate, the conversion of LC3 I into LC3 II, and p62 level in the hearts of Atg5 Tg mice. Whole-tissue extracts were prepared from 3-month-old Atg5 Tg mice and WT littermates, and analysed by western blotting using the indicated antibodies. β-Actin served as a control. (h) Densitometric analysis of the signals on the blots shown in (g). The bars represent the mean±s.d. (n=3).
f2(a) Western blot analysis showing enhanced levels of Atg5, Atg12-Atg5 conjugate and LC3 conversion, and reduced p62 levels in the indicated tissues of 18-month-old Atg5 Tg mice no. 25 (T) and WT mice (W). (b) Densitometric analysis of the signals on the blots shown in (a).(c) Electron microscopy images of autophagosomes (arrows) and late-autophagic compartments (arrowheads) in the liver tissues of 18-month-old Atg5 Tg mice no. 25 and WT mice. Scale bars are indicated. (d) The numbers of autophagosomes and autolysosomes per cell (n=50) in the electron microscopic images shown in (c) were counted. The bars represent the mean±s.d. ***P0.0001 versus control; Student's t-test.(e) Fluorogenic comparison of basal autophagy activity between GFP-LC3 Tg mice and Atg5/GFP-LC3 double Tg mice. Whole bodies of 8-week-old male (left) and female (right) GFP-LC3 Tg (LC3), and LC3/LC3-LC3Tg mice (Atg5/LC3) were visualized using a Kodak Image Station 4000MM equipped with a filter set for FITC (Kodak Molecular Imaging Software, Version 4.0).(f) Western blot analysis showing autophagy markers in the liver of GFP-LC3 mice. GFP-LC3 mice were injected for 5 days with siRNA-Atg5 or Atg5-HA cDNA through the tail vein. Whole-liver tissue lysates were prepared and subjected to immunoblotting using the indicated antibodies. β-Actin served as a control.
f3(a) Representative physical pictures of 18-month-old WT and Atg5 Tg mice fed with a regular diet. (b) Body weight chart of ageing-induced obesity in WT and Atg5 Tg mice. The mice were fed with a regular diet for 24 months (n=180--250) and their body weights were monitored monthly. The bars represent the mean±s.d. ***P0.0001 versus control; Student's t-test.. (c) Average daily oral food intake normalized to body weight, measured on regular diet. The food intake of mice (n=18-24) was measured for 2 weeks at 3-, 6-, 12- and 18-month-old age.d) Quantification of gonadal fat pads from 18-month-old mice (n=11). ***P0.0001 versus control; Student's t-test. (e,f) Quantification of visceral (e) and subcutaneous (f) fat pads of 18-month-old mice (n=6). ***P0.0001 versus control; Student's t-test.(g) Enhanced glucose tolerance of Atg5 Tg mice. WT and Atg5 Tg mice were starved overnight and then given with an i.p injection of glucose (1 mg g−1 body weight). The results are the mean±s.d. of 12-week-old WT and Atg5 Tg mice (n=12). *P0.01, **P0.001, ***P0.0001 versus control; Student's t-test.(h) Enhanced insulin sensitivity of Atg5 Tg mice. WT and Atg5 Tg mice were starved for 6 h and then i.p injected with 0.75 IU soluble insulin. *P0.01, **P0.001 versus control; Student's t-test.(i) Plasma levels of triglycerol (TG) in WT and Atg5 Tg mice. The mice were fasted for 3 h before measurement. Young mice (4-week old, n=8); old mice (18-month old, n=8). ***P0.0001 versus control; Student's t-test(j) Plasma leptin levels in WT and Atg5 Tg mice (n=8-12). ***P0.0001 versus control; Student's t-test. ((k) GSH/GSSG ratio in the liver (n=4 males, three times trial). **P0.001, ***P0.0001 versus control; Student's t-test.(l) Respiration rates of mitochondria in WT and Atg5 Tg MEFs (n=2).(m) Wire-hang endurance test of Atg5 Tg and WT mice (n=10). All the bars represent the mean±s.d. ***P0.0001 versus control; Student's t-test.
f4(a) Comparison of Atg12-Atg5 conjugate levels in WT and Atg5 Tg MEFs by western blot analysis. MEFs from WT and Atg5 Tg mice were cultured at embryonic day 13, after which cell extracts were examined by western blot analysis using anti-Atg5 and anti-β-actin antibodies.(b) Increased conversion of LC3 and reduced expression of p62 in Atg5 Tg MEFs no. 6 by rapamycin and/or Baf.A1. WT MEFs no. 1 and Atg5 Tg MEFs no. 6 in passage number 3 were incubated for 48 h with 10 μM rapamycin in the absence or presence of Baf.A1, and cell extracts were then analysed by western blotting. The signals on the blot were quantified by densitometric analysis and represented as the ratio of p62 to β-actin.(c,d) Increased resistance of Atg5 Tg MEFs to oxidative stress. WT MEFs no. 1 and Atg5 Tg MEFs no. 6 were treated for 24 h with 50, 100, 200 and 300 μM H2O2 (c) or 300 μM H2O2 in the absence or presence of 5 mM 3-MA or 20 nM Baf.A1 (Baf.A1) (d). Cell viability was assessed by propidium iodide staining. The values are the mean±s.e.m. (n=3). **P0.001, ***P0.0001 versus control; Student's t-test.(e) Representative photographs showing the resistance of Atg5 Tg MEFs to oxidative stress. Primary cultured WT and Atg5 Tg MEFs were treated with 300 μM H2O2 for 24 h and then observed under a light microscope. Scale bars, 50 μm.(f) Effect of oxidative stress on the autophagy activity in Atg5 Tg MEFs. WT MEFs no. 1 and Atg5 Tg MEFs no. 6 were treated with 100 μM H2O2 for 24 h in the presence or absence of 20 nM Baf.A1, and cell extracts were analysed by western blotting. About 10 and 30 μg of proteins were used for LC3 (upper panel) and p62 (bottom panel) detection, respectively. β-Actin served as a control.
Figure 1B-C. Wound healing assays were performed using IBIDI® chambers to evaluate the role of the AhR on cell migration. Images of the wound were captured using an Axio Vert.A1 inverted microscope (Carl Zeiss®) at 5x magnification. The histogram represents the mean ± s.d. Wound closure was determined by measuring the distance between the edges of the wound at time 0 and 15 h (n = 3 independent technical experiments for each cell lines or conditions) and compared using unpaired t-tests with the Sidak-Bonferroni method. B. Results obtained with BRAFi-sensitive or resistant SK28 cells KO for the AhR in the absence of treatment. (n = 3 independent technical experiments for each cell lines). C. Results obtained for the migration assay (0 to 15 h) for SK28 R cells KO or not for the AhR after treatment or not with 10 μM CH-223191. (n = 3 independent technical experiments for each cell lines or conditions, mean ± s.d.). Statistical analysis using unpaired t-tests with the Sidak-Bonferroni method has been performed between the mean of the 3 independent experiments.D. Three-dimensional spheroid growth of BRAFi-sensitive or resistant SK28 cells KO before or after knockout of AhR by CRISPR/Cas9 in the absence of treatment. Images were captured four days after implantation of the spheroids into collagen gel. (n = 4 independent technical experiments, mean ± s.d. E. Three-dimensional spheroid growth of BRAFi-resistant SK28 cells KO before or after knockout of AhR by CRISPR/Cas9 after daily treatment with the specific AhR inhibitor CH-223191 (5 μM) for one week or in the absence of treatment. Images were captured four days after implantation of the spheroids into collagen gel. (n = 3 independent technical experiments, mean ± s.d.). Statistical analysis using unpaired t-tests method has been performed with the Sidak-Bonferroni method, (p<0.01 ##, **; p<0.001 ###,***).
Figure 2A. AhR Protein levels in 501Mel CTR cells and those transduced with sgRNA targeting AhR (#1, #2) were analyzed by western blotting.C. Three-dimensional spheroid growth of 501Mel cells. Images were captured seven days after spheroid implantation (n = 3 independent technical experiments, mean ± s.d.). Each histogram represents the mean ± s.d. Comparisons with control were performed using unpaired t-tests with the Sidak-Bonferroni method, p<0.001 ***.D. AhR Protein levels in SK28R WT, KO, and KO CA-AhR cells were analyzed by western blotting.F. Three-dimensional spheroid growth of SK28 cells KO or not for AhR by CRISPR/Cas9 and rescued by the constitutive active form of AhR (KO CA-AhR). Images were captured four days after spheroid implantation (n = 3 independent technical experiments, mean ± s.d.). Comparisons were performed using unpaired t-tests with the Sidak-Bonferroni method.
Figure 4A. At the basal level, AhR is located at the membrane layer in a protein complex that includes HSP90, p23, XAP, and SRC. Immunoprecipitation of SRC was performed with specific antibodies and SRC/AhR interaction analyzed by western blotting.B. Protein levels of the AhR, p-SRC (Y416), SRC, p-FAK (Y576/577), and FAK in the four different melanoma cell lines (n=3). The level of BRAFi resistance corresponds to our measure of IC50 (Vemurafenib) for the different cell lines 501Mel (0,23uM), SKMel28 (0,29uM), M229 (0,89uM) and M238 (2,16uM). These cell lines correspond to the sensitive parental cells.C. Protein levels of the AhR, p-SRC (Y416), SRC, p-FAK (Y576/577), and FAK in the SK28R cell line KO or not for AhR by CRISPR/Cas9 or after rescue with the activated-form of the AhR (CA-AhR).D. Protein levels of AhR, p-SRC (Y416), and SRC, in the SK28R cell line after treatment with different AhR ligands for 24h (5 μM BaP, 5 μM indirubin, 5 μM ITE, 10 nM TCDD, and 5 μM FICZ).E. Protein levels of AhR, p-SRC (Y416), and SRC, in the SK28R cell line after 24 h of treatment with increasing doses of ITE.