EMBO/SourceData huggingface dataset:
Figure 5(a) Knockdown of eIF4A and TSC2 do not elevate TORC1 activity in an additive manner, indicating they act in the same, and not parallel pathways. Whereas knockdown of eIF4A increases TORC1 activity in control Kc167 cells (lanes 1-2 and 5-6), eIF4A knockdown cannot further increase TORC1 activity in TSC2 knockdown cells (lanes 3-4 and 7-8). Error bars: std dev. n=3 biological replicates.(b) Activation of TSC2 with the p90RSK inhibitor BI-D1870 rescues the elevated TORC1 activity caused by eIF4A knockdown in Kc167 cells, consistent with TSC2 acting downstream of eIF4A. BI-D1870 leads to inhibition of TORC1 (lanes 1-4) in a TSC2-dependent manner (see also Appendix Figure S5A). Representative of two biological replicates.(c) FLAG-eIF4A and TSC2-V5 co-immunoprecipitate in Kc167 cells in the absence of chemical cross-linker. Cells were transfected with FLAG-eIF4A and TSC2-V5 expression vectors and treated with media containing or lacking amino acids for indicated timepoints prior to lysis and anti-FLAG immunoprecipitation. The experiment was performed in absence of the chemical cross-linker DSP. Representative of 3 biological replicates.(d) Expression of TSC2-insensitive (S15H or Q63L), but not wild-type (WT) Rheb causes TORC1 activity to remain high upon amino acid removal. Kc167 cells were transfected to express either wild-type or mutant Rheb and then incubated with Schneider's medium either containing or lacking amino acids for 30 minutes. Elevated TORC1 activity can be observed either by looking at phosphorylation of endogenous S6K, or phosphorylation of an HA-tagged S6K that was co-transfected with the Rheb constructs to assay specifically the transfected cells. Representative of two biological replicates.
Figure 6(a) Mass spectrometry analysis of proteins co-immunoprecipitating with eIF4A reveals NAT1 as one of the top interacting proteins. eIF4A immunoprecipitations were performed in triplicate, from cells treated with medium either containing (+aa) or lacking amino acids (-aa), and average values are shown. Peptide counts for each protein in each replicate were normalized to eIF4A peptide counts. Raw peptide counts shown in parentheses.(a') Schematic diagram of eIF4G and NAT1 primary protein structures. Binding sites for other initiation factors are shown.(b) Binding between eIF4A and NAT1 is regulated by amino acid availability in a TORC1-independent fashion. Co-immunoprecipitation of tagged eIF4A and NAT1 in control Kc167 cells, or cells treated with medium lacking amino acids or supplemented with 20 nM rapamycin for indicated times. Representative of 3 biological replicates.(c) Knockdown of NAT1 using 4 independent, non-overlapping dsRNAs leads to reduced TORC1 activity. Kc167 cells, treated with indicated dsRNAs for 4 days and then incubated with medium containing or lacking amino acids for 30 min. Error bars: std. dev. n=3 biological replicates.(d) eIF4A is epistatic to NAT1 for TORC1 regulation. In the absence of amino acids, eIF4A knockdown cells have elevated TORC1 activity, NAT1 knockdown cells have reduced TORC1 activity, and eIF4A NAT1 double-knockdown cells have elevated TORC1 activity compared to control cells. Cells were treated with indicated dsRNAs for 4 days and then treated with medium containing or lacking the indicated amino acids for 30 min. Error bars: std. dev. n=3 biological replicates.(e) TSC2 is epistatic to NAT1 for TORC1 regulation. As in (d), knockdown of TSC2 rescues the reduced TORC1 activity caused by NAT1 knockdown.
Figure 3(B-D) Micrographs of H9C2 cells corresponding to (B) expressions of single species: MuRF1-mCherry (red), titin EGFP-A168-TK (green), Venus-Nbr1 (yellow) or Cerulean-p62 (cyan); (C) Co-expression of titin EGFP-A168-TK with mCherry-MuRF1. Neither Nbr1 nor p62 were co-transfected. The titin fragment and MuRF1 co-localize forming filamentous formations, which is in agreement with their known complexation; (D) Co-expression of MuRF1-mCherry (red), titin EGFP-A168-TK (green) and Venus-Nbr1 (yellow) or Cerulean-p62 (cyan) or respective truncated variants of the latter lacking the ubiquitin binding domain (∆UBA). (Throughout, cell nuclei are stained with DAPI; blue). (Scale bar is 10 μm in all cases).
Figure 1Colonic organoids grown from Lgr5-EGFP-IRES-CreERT2 mice were treated with an AhR agonist (10 nM TCDD) or antagonist (10 µM CH223191) for 3 d. (A) Induced Cyp1a1 mRNA expression in response to different treatments (n=3 per group).Colonic organoids grown from Lgr5-EGFP-IRES-CreERT2 mice were treated with an AhR agonist (10 nM TCDD) or antagonist (10 µM CH223191) for 3 d. (B) Assessment of GFP+ stem cell percentages (n=3 replicates per treatment). Bars with different letters are significantly different (p<0.05).(D) Flow cytometry analysis of GFP+Tomato+ and Tomato+ cells in the large intestine of AhR wild type (Lgr5-GFPCreERT2 X Tomatof/f, GT) and AhR knock out (AhRf/f X Lgr5-GFPCreERT2 X Tomatof/f, HGT) mice. Top left panel shows representative cells from WT negative control animal (no GFP or Tomato expression). Top right panel shows Tomato only control (CDX2P-CreERT2 X Tomatof/f). Middle panels represent DMSO or TCDD treatment in AhR WT mice, respectively. Mice were gavaged every other day, 4 times total, with vehicle or TCDD (25 µg/kg bw) and terminated 1 d later. Bottom left panel is from a representative AhR WT mouse and bottom right panel is from a representative AhR KO mouse.(E) Induced Cyp1a1 mRNA expression in stem cells after oral gavage with TCDD (n=4).(F) Percentage of Lgr5+ stem cells (n=4 mice per group) in (D).Flow cytometry analysis of GFP+Tomato+ and Tomato+ cells in the large intestine of AhR wild type and AhR knock out mice. (G) Quantitative analysis of the percentage of Lgr5+ stem cells in (D), n=7-10 mice per group.Representative immunohistochemistry of GFP+ stem cells per crypt, n=60~70 crypts from n=3 mice per group. Scale 50 µm.Representative violin plots of GFP+ stem cells per crypt, n=60~70 crypts from n=3 mice per group. Scale 50 µm. Solid line indicates median, and dotted lines indicate 25th/75th quartiles.
Figure 2(A) Representative flow cytometry gating and fluorescence microscopy images of sorted GFPhigh stem cells and GFPlow progenitor cells from Lgr5-EGFP-IRES-CreERT2 mice.(B) Representative brightfield images of organoids generated from GFPhigh stem cells isolated from tamoxifen treated AhR WT mice (Lgr5-GFPCreERT2 X CDX2P-CreERT2 [GC]) and KO mice (AhRf/f X Lgr5-GFPCreERT2 X CDX2P-CreERT2 [HGC]), treated with DMSO (control) or TCDD for 5 d. Data are from ≥4 mice, scale bar 200 µm.(C, D) The organoid forming efficiency (C) and diameter (D) from sorted GFPhigh stem cells in AhR WT or KO mice treated for 5 d with DMSO or TCDD (10 nM). n=4-5 mice per group in (C). Each dot represents an individual organoid and grey line indicates mean in (D). Bars with different letters are significantly different (p<0.05).Organoid growth from GFPlow progenitor cells. (E) Representative brightfield images of organoids.(F) Organoid forming efficiency and (G) diameter. n=4-8 mice per group in (F). Each dot represents an individual organoid and grey line indicates mean in (G). Scale bar 200 μm. Bars with different letters are significantly different (p<0.05).(H, I) (H) Lgr5 expression in organoids and (I) sorted stem cells in the absence or presence of TCDD treatment (n≥3 independent samples per group).(J) Representative brightfield images for secondary organoid formation pretreated with DMSO or 10 nM TCDD for 2d. Scale 200 µm.
Figure 3(C) Heatmap comparison of the top 25 upstream regulators between AhR KO (KO/WT) and TCDD (TCDD/vehicle in WT mice) treated cells.
Figure 4(A) FoxM1, Plk1, and Cdc25B mRNA expression in stem and progenitor cells with and without AhR, n=4 mice per group.(B) FoxM1, Plk1, and Cdc25B mRNA expression in WT stem and progenitor cells with and without TCDD, n=3-4 mice per group.Chromatin immunoprecipitation (ChIP) analysis of AhR and FoxM1 promoter interaction using WT colonic organoids treated with DMSO or TCDD, n=3 or 4 per group. A DNA fragment amplified in a gene desert on mouse chromosome 6 was used as a negative control.Chromatin immunoprecipitation (ChIP) analysis of AhR and FoxM1 promoter interaction using WT and KO crypts (E) treated with DMSO or TCDD, n=3 or 4 per group.(F) FoxM1 luciferase activity determination for young adult mouse colonocytes (YAMCs) treated with DMSO or 10 nM TCDD for 1 d, n=3 per group.(G Expression of FoxM1 and PLK1 at mRNA level, , n>=3 independent organoids per treatment.H) Expression of FoxM1 and PLK1 at protein level, n>=3 independent organoids per treatment.(I) Representative brightfield images of mouse colonic organoids generated from WT Lgr5+-GFPhi stem cells treated with DMSO or 5µM FDI-6. Scale bar 200 µm.(J, K) Quantification of organoid forming efficiency (J) and organoid area (K) from WT colonic stem cells treated with DMSO or 5µM FDI-6. Data are represented as mean ± SEM in (K).(L) Organoid expression of Lgr5 in response to FDI-6. Lgr5 expression level was dramatically suppressed by FDI-6 in a dose-dependent manner, n>=3 per treatment.(M) Representative brightfield images for secondary organoid formation following treatment with DMSO or 5 µM FDI-6 for 2 d. Scale 200 µm.
Figure 5(A and B) Proliferating Lgr5+ stem cells marked by EdU in the distal colon from AhR WT (GT) and KO (HGT) mice. Arrowhead and star symbols denote proliferating stem cells (GFP+EdU+) and static stem cells (GFP+EdU-), respectively, during a 2 h EdU pulse, n=3 per treatment.(C) Total number of EdU+ cells per crypt in the distal colon of AhR WT and KO (HGT) mice, n=3 mice per treatment. Dashed line indicates median and dotted lines indicate 25th/75th quartiles.(D&E) Crypt cell proliferative zone analysis in AhR WT and KO (HGT) mice, presented as the distance from the crypt base to the highest EdU+ cell divided by total crypt length. Red lines between crosses show the measurement of distance. Each symbol represents one crypt, n=3 mice per treatment, scale bar 20 µm. Grey line represents mean.(F& AhR KO promoted cell proliferation 5 d post DSS treatment. AhR WT (AhRf/f [H], n=4) and AhR KO (AhRf/f X Villin-Cre [HV], n=4) mice were used.G) AhR KO promoted cell proliferation 5 d post DSS treatment. Data represent mean ± SEM, quantified as the ratio of the EdU+ area relative to the DAPI+ area using KeyenceTM software.(H) Increased cell proliferation (EdU+ cells) observed in AhR KO vs WT organoids derived from HGC and GC mice, respectively (n=6 from 3 mice per group).(I) Effect of AhR KO on cell cycle S and G2-M phases (n=3 mice per group).(J). FoxM1 inhibition reduced the number of colonic stem cells and cell proliferation. Mice were intraperitoneally injected with the FoxM1 inhibitor Thiostrepton (50 mg/kg) for 2 consecutive days, n=80~100 crypts from 4 mice per group. Scale bar, 50 µm. Dashed line indicates median and dotted lines indicate 25th/75th quartiles. Bars with different letters are significantly different (p<0.05).
Figure 6(B) Representative colon images from AhR WT and AhR KO mice following AOM/DSS treatment. Scale bar, 9 mm.(C) H&E staining of representative colon tumor sections from AhR WT and KO mice, scale bar 200 μm.(E, F) (E) Adenoma and (F) adenocarcinoma multiplicity in AhR WT and KO mice.(G) Tumor volume in AhR KO and WT mice. Grey line indicates mean.(H) FoxM1 mRNA expression in normal (uninvolved) crypts adjacent to colonic tumors. Images were quantified and expressed as FoxM1 positive area per crypt (n=5 mice per group). Scale 200 µm. Dashed line indicates median and dotted lines indicate 25th/75th quartiles.(I) FoxM1 mRNA expression in colon tumors. FoxM1 mRNA ISH area was averaged per tumor on each slide (n=5 mice per group). Scale 200 µm. Dashed line indicates median and dotted lines indicate 25th/75th quartiles.
Figure 7(A-B) mRNA expression of human colonic stem cell markers LGR5 and OLFM4, n=5 or 6 independent human samples. Paired student t-test.(C) Representative images of LGR5 ISH in human organoids treated with DMSO or TCDD. Scale bar 50 µm.mRNA expression of target genes, n=5 or 6 independent human samples. Paired student t-test.mRNA expression of target genes, n=5 or 6 independent human samples. Paired student t-test.(H) Representative immunoblots for FoxM1 and PLK1 protein derived from organoids treated with DMSO or TCDD from n=6 independent samples. Total protein was used as a loading control.(I) Determination of luciferase reporter activity of human FoxM1 in SW48 and Caco2 cell lines treated with DMSO or TCDD. n=3-4 per group.(J) FOXM1 and CYP1A1 mRNA expression in different AhR WT and CRISPR KO clones treated with TCDD for 1 d. C56 and C61 designate different human subjects.(K) Validation of CRISPR KO clones was assessed by PCR amplification using primers flanking the targeted AhR exon. Subsequent sequencing revealed indels at the expected locations.(L) The association of FoxM1 and AhR mRNA expression in human normal colon biopsies (n=304). AhR low (n=152) is defined as below its median expression; AhR high (n=152) is defined as above its median expression. Dashed line indicates median and dotted lines indicate 25th/75th quartiles.(M) Correlation between FoxM1 and Cyp1b1 mRNA expression in human normal colon biopsies (n=303) analyzed by Spearman rank correlation.(N) Representative brightfield images of human colonic organoids from sorted live cells at day 6, scale bar 200 μm.(O, P) (O) Human organoid forming efficiency, and (P) organoid diameter from sorted individual cells from human colonic organoids in the presence of DMSO, TCDD and CH223191 (10 μM) for 6 d. n=5 per group, 3 independent experiments. Grey line indicates mean in (P). Treatments not sharing a common letter are significantly different, p<0.05.(Q) Representative brightfield images of human colonic organoids treated with DMSO or FDI-6. FoxM1 inhibition suppressed human colonic organoid growth in a dose-dependent manner, scale bar 200 µm.
Figure 1Survival, gain of weight and mobility plots for Armadillidium vulgare during 75 days post-injection.There were no significant differences between animals injected with wVulC, wDil or control treatment for all these life history traits: Survival (A), Gain of weight (B) and Mobility (C).
Figure 2Animals were injected by pure suspension of wVulC, diluted wVulC inoculum (only for females), wDil or control treatment and monitored during 75 days post-injection. (A) Gain of weight: Both Wolbachia strains negatively impact this parameter compared to the control treatment. However, wVulC (i.e pure or 1/100 diluted inoculum) induces a lower growth rate than the native strain wDil(B) Mobility: this parameter is dramatically reduced for animals injected by wVulC compared to those from other treatments. On the opposite, injection of wDil has no significant effect on the host's mobility.(C) Burying behaviour: The level 0 corresponds to the surface of the substrate, negative values correspond to the number of buried animals while positive values correspond to the number of animals found at the surface of the substrate. For control treatment animals, not a single individual rose up at the surface and for individuals injected by wDil only few animals were spotted at the surface. When inoculated by wVulC, all animals rose up to the substrate after 60 days.(D) Survival (a) data for females and (b) data for males: The injection of wVulC kills all the individuals. However, the males die quicker than the females. No extra-mortalities were reported for injection with wDil compared to control treatment.
Figure 3Wolbachia loads in CNS, gonads and hemocytes of Porcellio d. dilatatus and Armadillidium vulgare.(A) Comparison between (i) P. d. dilatatus in dark grey and A. vulgare in white injected with wVulC and (ii) naturally infected A. vulgare in light grey revealed no significant differences for the Wolbachia loads in CNS cells, gonads and hemocytes (i.e immune cells), at both 30 and 60 days post-injection. (B) Comparison between (i) P. d. dilatatus in dark grey and A. vulgare in white injected with wDil and (ii) naturally infected P. d. dilatatus in dim grey revealed no significant differences for the Wolbachia loads in CNS cells at both 30 and 60 days PI. However, at 60 days post-injection, for gonads and hemocytes wDil loads were significantly higher in the native host P. d. dilatatus than in A. vulgare. A global comparison between wDil and wVulC loads showed that wVulC (A) always exhibited higher bacterial loads than wDil (B) in both host species.
Figure 4Micrograph of nerve cells of Porcellio d. dilatatus injected by wVulC.These cells are localised in the CNS ganglion. (A) At 30 days post-injection, some autolysosomes and bacteria are visible: autophagic process is already quite high compared to control [N: Nucleus, V: Vacuole, Ap: Autophagosomes (Autophagic hallmark), Al: Autolysosomes, Wo: Wolbachia, *: a Wolbachia which seems to be surrounded by an autolysosome, Arrow head: magnified area]. (B) At 60 days post-injection, the nerve cells are severely damaged by the accumulation of autophagic vesicles (N: Nucleus, V: Vacuole, Ap: Autophagosomes (Autophagic hallmark), Al: Autolysosomes, Arrow head: magnified area and probably a Wolbachia in autophagic degradation).
Figure 5Micrograph and LC3B antibody labelling of adipocytes neighboring the nerve cells of Porcellio d. dilatatus.These adipocytes are localised around the nerve cells of the CNS and constitutes the sheathing of this organ. (A) At 30 days post-injection, autolysosomes, autophagosomes but also bacteria were observed: autophagic process was already higher compared to the control (Ad: adipocyte, Ap: Autophagosomes (Autophagic hallmark), Al: Autolysosomes, Wo: Wolbachia, Arrow head: magnified area). (B) At 60 days post-injection, the adipocytes were severely damaged by the accumulation of autophagic vesicles (Ad: adipocyte, Ap: Autophagosomes, Al: Autolysosomes, Wo: Wolbachia, Arrow head: magnified area). (C) With the antibody labelling anti-LC3B, the protein LC3B implicated in autophagic processes was detected in adipocytes of P. d. dilatatus injected with wVulC at 45 days post-injection. The highly labeled spherical structures would reflect the incorporation of the LC3B protein in the phagophores. (D) With adipocytes of P. d. dilatatus injected by wDil but also in other tested situations, only really few of these structures were detected by epiflorescent microscopy.
Figure 1(A) Western analysis for on-target effects for Everolimus (EV, P-RPS6) and CX-5461 (p53). Each lane represents equal amounts of protein from lymph node tissue isolated from a single mouse that received drug vehicles (everolimus vehicle: 1% methylcellulose; CX-5461 vehicle: 25 mM NaH2PO­­­4) (V/V; mouse #1-6), 5 mg/kg everolimus (EV; mouse #7-12), 35 mg/kg CX-5461 (mouse #13-18) or both drugs (CX-5461+EV; mouse #19-24) for 2 hours (n=6 per treatment group). Actin was used as a loading control.(C) Enrichment analysis by MetaCore® GeneGO of genes in "translation up" (red) and "translation down" (blue) categories identified by anota2seq analysis comparing lymph node cells isolated from mice in CX-5461+EV treatment group with the V/V group (n=6). Ratio" values were obtained by dividing the number of significant genes in our data assigned to a molecular process by the total number of genes in the process in MetaCore®'s database. (D) Genes implicated in "Translation: initiation" and "Translation: Elongation-Termination" processes based on Fig 1C
Figure 2(B) Cell viability analysis of the indicated early-passage Eµ-Myc lymphoma cells treated with indicated compound(s) for 48 hours as determined by Beckman® Coulter counter.(E) Intracellular ATP levels determined using the liquid chromatography-mass spectrometry in CTRL, EV, CX and CMB early-passage cell lines.(F) Basal extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) in the early-passage cell lines determined using the Seahorse XF96 Extracellular Flux Analyzer; graphs represent mean ± SEM of 6-8 technical replicates for each biological replicate (n=3).(G) Propidium iodide (PI) exclusion analysis of early-passage CX-5461-everolimus combination therapy-resistant (CMB), CX-5461-resistant (CX) and drug-naïve (CTRL) lymphoma cells treated with indicated concentrations of metformin for 48 hours.(H) CellTiterGLO®-based assay measuring cellular ATP levels of the CX-5461-resistant (CX) and CX-5461-everolimus combination therapy-resistant (CMB) cells treated with metformin as indicated for 48 hours.(I) PI analysis of CTRL and CMB cells treated with CX-5461 and everolimus in the presence and absence of metformin for 48 hours as indicated.(J) PI analysis of CTRL and CX cells treated with CX-5461 in the presence and absence of metformin for 48 hours as indicated.
Figure 3(B) Abundance of intracellular 3'5'-cyclic AMP as measured by a liquid chromatography (LC)-mass spectrometry (MS) analysis. Graphs represent mean ± SEM of n=3 (5-6 technical replicates each).(C) Western analysis of EPAC1 and EPAC2 abundance, as well as active GTP-bound RAP1 levels in the indicated early-passage cells during their log-phase growth period (n=3). Actin and total RAP1 were used as loading controls.(D) Propidium iodide (PI) exclusion assays of CMB cells treated with CX-5461 and everolimus as indicated in the presence or absence of a selective PKA inhibitor H89 for 48 hours.(E) PI exclusion analysis of early-passage drug-naïve (CTRL) lymphoma cells treated with CX-5461 and everolimus in the presence or absence of selective PKA activator 6-Bnz-cAMP for 48 hours.(F) PI exclusion analysis of the CMB cells treated with CX-5461 and everolimus as indicated in the presence or absence of EPAC1 inhibitor CE3F4 or EPAC2 inhibitor ESI-05 for 48 hours.(G) PI exclusion analysis of early-passage drug-naïve (CTRL) cells treated with CX-5461 and everolimus in the presence or absence of the selective EPAC activator 8-pCPT-2-O-Me-cAMP for 48 hours.
Figure 4(A) A heatmap illustrating the abundances of polysome-associated mRNAs that are associated with the mRNA translation/protein synthesis based on the Gene Ontology Resource Database in drug-naïve (CTRL) or CX-5461-everolimus combination therapy-resistant (CMB) cell lines (n=3). The colours illustrate the expression values of indicated mRNAs that were normalized using voom (red: high expression; blue: low expression).(B) Polysome profiles demonstrating increased ribosome abundance in CMB cells as compared to CTRL and CX-5461-resistant (CX) cells (representatives of n=3). (C) Quantitation of polysome:monosome ratio in the indicated early-passage Eµ-Myc lymphoma cells as determined by measuring the area under the curve using ImageJ software. Graph represents mean ± SEM of n=3. (F) Polysomal abundance of mRNAs (selected based on a nominal p-value < 0.01 cut-off in either the CMB vs. CTRL or CX vs. CTRL as analysed by limma) encoding components of mitochondrial oxidative phosphorylation based on polysome profiling data. Red denotes upregulation, blue denotes downregulation (n=3; genes with an adjusted P value (false discovery rate; FDR) ≤ 0.05 were considered to be significant, and are denoted with a black border).
Figure 5(A) Western analysis demonstrating the effects of metformin treatment for 48 hours on the levels of active GTP-bound RAP1 in CX and CMB cells (n=3) and its quantitation.(B, C) Proportion of green fluorescent protein (GFP)-positive CMB (clone #8) cells in (B) lymph node and (C) spleen of transplanted C57BL/6 mice treated as indicated for 6 hours on day 12 post-transplant.(D) Kaplan-Meier curve of C57BL/6 mice transplanted with CX-5461-everolimus-resistant (CMB #8) early-passage Eµ-Myc lymphoma cells treated with vehicles (everolimus vehicle: 1% methylcellulose; CX-5461/metformin vehicle: 25 mM NaH2PO­­­4) (n=6); CX-5461 (35 mg/kg every twice weekly) and everolimus (5 mg/kg daily) (n=8), metformin (400 mg/kg twice daily) (n=6), or CX-5461, everolimus and metformin (35 mg/kg twice weekly, 5 mg/kg daily and 400 mg/kg twice daily, respectively) (n=8). Light grey: 5-day metformin pre-treatment period, dark grey: treatment period. Data were analyzed by a log-rank (Mantel-Cox) test. Vehicle vs. CX-5461-everolimus: P = 0.0006, Vehicle vs. CX-5461-everolimus-metformin: P = 0.0001. CX-5461-everolimus vs. CX-5461-everolimus-metformin: P = 0.0003.