EMBO/SourceData huggingface dataset:
Figure 5Heatmap of AM signature-up genes (D), AM signature-down genes (E), and AM-specific transcription factors (F) in the precursors and mature AM described above in A. the data are from one RNA-seq experiment (1 - 2 biological replicates per group).qPCR (n = 3 biological replicates) analysis of Myb, Maf, Mafb, and Pparg expression in different fetal precursors from WT embryos and mature AM from WT adult mice.
Figure 6Oxygen consumption rate (OCR) of pMΦ and fetal Mo from E14.5 WT fetal liver at baseline and after treatment with oligomycin (Oligo), FCCP or rotenone (Rot) and antimycin A (AA).Extracellular acidification rate (ECAR) of pMΦ and fetal Mo from E14.5 WT fetal liver at baseline and after treatment with glucose, Oligo, or 2-Deoxy-D-glucose (2-DG).RNA-seq (C, n = 2 biological replicates) analysis of Eno1, Slc2a6, Txn1, and Txnip gene expression in pMΦ and fetal Mo from E14.5 WT fetal liver.qPCR (D, n = 3 biological replicates) analysis of Eno1, Slc2a6, Txn1, and Txnip gene expression in pMΦ and fetal Mo from E14.5 WT fetal liver.
Figure 1C-E. Western blot analysis for CCNA, CCNB, Cdk1 and GAPDH (loading control) protein expression in lysates from control (si-GFP) and p53-depleted (si-p53; si-p53_1) (C) MDA-MB-231 and CAL27 cell lines. Representative images are shown.C-E. Western blot analysis for CCNA, CCNB, Cdk1 and GAPDH (loading control) protein expression in lysates from control (si-GFP) and YAP-depleted (si-YAP; si-YAP_1) (D) MDA-MB-231 and CAL27 cell lines. Representative images are shown.C-E. Western blot analysis for CCNA, CCNB, Cdk1 and GAPDH (loading control) protein expression in lysates from control (si-GFP) and TAZ-depleted (si-TAZ) (E) MDA-MB-231 and CAL27 cell lines. Representative images are shown.
Figure 2A. Primary human breast cancers of the METABRIC dataset were stratified according to high or low YAP activity signature [47] and by TP53 mutational status, and then the levels of the cycline signature score were determined in the four groups. Cyclin activity is significantly higher in mut-p53 tumors with high levels of the YAP signature, as visualized by box-plot. Signature scores have been obtained summarizing the standardized expression levels of signature genes into a combined score with zero mean [7]. The values shown in graphs are thus adimensional. The bottom and top of the box are the first and third quartiles, and the band inside the box is the median; whiskers represent 1st and 99th percentiles; values lower and greater are shown as circles (p<0.0001, n=701).B. Same as in A) using the YAP/TAZ activity signature of Zhang et al. (2009) (p<0.0001, n=701).C. Kaplan-Meier analysis representing the probability of disease-specific survival in mutant and wild-type p53 breast cancer patients from the METABRIC dataset stratified according high or low YAP/TAZ signature score. The log-rank test p-value reflects the significance of the association between high levels of the YAP/TAZ signature score and shorter survival in in mutant-p53 as compared to wild-type p53 patients (p<0.0001, n=251). All p-values were calculated with two-tailed Student's t-test.
Figure 3A. SKBr3 and MDA-MB-468 cells knocked-down as indicated in the figure, were transiently transfected with pCCAAT-B2LUC (100 ng) or pmutCCAAT-B2LUC (100 ng) luciferase reporter vectors. Values are means + s.d. of three replicates from three independent experiments.B. RT-qPCR of CCNA, CCNB2 and cdk1 mRNA levels in SKBr3 cells upon transduction with si-GFP and si-YAP (left graph) or si-p53 (right graph). Values are means + s.d. of three replicates from three independent experiments.C. H1299 transfected with the indicated siRNAs, were transiently transfected with pCCAAT-B2LUC (100 ng) luciferase reporter vector together with empty pcDNA3 or mutant p53R175H. Representative western blotting to control the transfections presented in the right panel. Values are means + s.d. of three replicates from three independent experiments.D. RT-qPCR of CCNA and cdk1 mRNA levels in H1299 cells upon transfection with si-GFP or si-YAP oligos and empty pCDNA3 or mutp53R280H expression vectors as indicated in the figure. Error bars represent mean + s.d., from three biological replicates. P-values are indicated in the figures; two-tailed Student's t-test was applied throughout.
Figure 4A and B. Coimmunoprecipitation and western blot analysis from H1299 cell lysates showing endogenous YAP (A) and overexpressed GFP-YAP (B) bound to mutp53R273H (A) or to mutp53R175H (B). In (A) YAP protein was immunoprecipitated with a rabbit polyclonal antibody and rabbit IgG was used as negative control of IP. In (B) p53 protein was immunoprecipitated with a sheep polyclonal antibody and the total lysate from H1299 transfected with empty pCDNA3 vector was used in as negative control of IP.C and D. Immunoprecipitations of YAP protein and western blot analysis for p53 binding are performed from lysates of cancer cell lines expressing different mutant p53 proteins (described in Materials and Methods) (C) YAP protein was immunoprecipitated with a rabbit polyclonal antibody and the same amount of rabbit IgG was used as negative control of IP.C and D. Immunoprecipitations of YAP protein and western blot analysis for p53 binding are performed from lysates of cancer cell lines expressing different mutant p53 proteins (described in Materials and Methods) (C) and from HCT116 cells expressing wild type p53 protein (D). YAP protein was immunoprecipitated with a rabbit polyclonal antibody and the same amount of rabbit IgG was used as negative control of IP.E. Cellular extracts from H1299 cells transiently transfected with 2 μg of each indicated plasmids were immunoprecipitated with rabbit polyclonal YAP antibody. Cell lysates derived from cells transfected with the empty vector were used as negative control of immunoprecipitation.F. Cellular extracts from MDA-MB-468 were immunoprecipitated with rabbit polyclonal YAP, sheep polyclonal p53 and rabbit polyclonal NF-YB antibodies. Rabbit (for YAP and NF-YB IPs) and sheep (for p53 IP) IgGs were used as negative control of IPs.G. Cell lysates from CAL27 cells transfected with si-GFP and si-NF-YB oligonucleotides were immunoprecipitated with rabbit polyclonal YAP. As specificity control, immunoprecipitations were performed with rabbit IgG.H and I. ChIP analysis of mutant p53, YAP and H4 acetylated histone-bound chromatin from MDA-MB-468 cells on transduction with siRNAs oligos targeting endogenous YAP (siYAP) (H), NF-Y (siNF-YB) (I), or siGFP as a negative control (H and I). The experiment was performed in biological triplicates. The CCNA, CCNB2 and Cdk1 promoter occupancy was analyzed by RT-qPCR. Normalization was performed to the amount of input chromatin. The ChIP results were further normalised on the RT-qPCR of a region resulted negative for the recruitment of mutant p53 (Appendix Table S5). P-values were calculated with two tailed Student's t-test. Statistically significant results were referred with p-value<0.05.
Figure 5A and B. Proliferation curves of SKBr3 cell line knocked-down for YAP (A) or p53 (B). si-GFP oligos are used as negative control.Cell viability analysis in A and B was determined by trypan blue dye exclusion staining, in C was determined by ATPlite luminescence analysis. All the values are means + s.d. of six replicates from three independent experiments.C. Viability of CAL27 cell line transfected with si-GFP or si-p53 oligos and empty vector or GFP-YAP construct, as indicated in the legend to the graph.Cell viability analysis in A and B was determined by trypan blue dye exclusion staining, in C was determined by ATPlite luminescence analysis. All the values are means + s.d. of six replicates from three independent experiments.
Figure 6A. CCNA and CCNB2 expression transcripts were analyzed by RT-qPCR in cDNA derived from tumours of control (saline) or zoledronic acid (ZA)-treated mice (ref. 31). N=3 mice per group. P-values were calculated with two-tailed t-test.B. ChIP analysis of mutant p53 -bound chromatin from MDA-MB-231 cells treated or not with 1 µM cerivastatin (Cer) for 24h. The CCNB2 promoter occupancy was analyzed by RT-qPCR. Normalization was performed to the amount of input chromatin. The experiment was performed in biological triplicate. P-values were calculated with two-tailed t-test.C. MDA-MB-231 cells treated with the indicated concentrations of Cer for 24h were transfected with pCCAAT-B2LUC (100 ng) luciferase reporter vector. Error bars represent mean + s.d., from three biological replicates. P-values are indicated in the figures;p-values were calculated with two-tailed t-test.D. CCNA, CCNB2 and Cdk1 expression transcripts were analyzed by RT-qPCR in cDNA derived from MDA-MB-231 (left panel) and SKBr3 (right panel) cell lines treated or not with 1 µM Cer for 48h. Error bars represent mean + s.d., from three biological replicates. P-values were calculated with two-tailed t-test. P-value<0.01.E. Western blot analysis for CCNA, CCNB and Actin (loading control) expression in lysates from MDA-MB-231 cells treated or not with 1 µM Cer.F. CCNA, CCNB2 and Cdk1 expression transcripts were analyzed by RT-qPCR in cDNA derived from MDA-MB-231cell line treated or not with 30µM ZA for 48h. Error bars represent mean + s.d., from three biological replicates. P-values were calculated with two-tailed t-test. P-value<0.01.G. Viability assay of siGFP and sip53 -MDA-MB-231 cells after treatment with increasing amounts of cerivastatin (0, 0.01, 0.1, 1 and 10 µM) for 48 h. Data are normalized to untreated. Error bars represent mean + s.d., from three biological replicates.
Figure 1Mouse YUMM1.7 melanoma cells were subcutaneously inoculated into C57BL/6 mice and when tumors reached 100 mm3 mice were treated with vehicle (Ctrl), Nintedanib/BIBF1120 (BIBF), MAPKi (BRAFi, Vemurafenib and MEKi, Trametinib) or BRAFi/MEKi plus BIBF (n=6). (C) Kaplan-Meier survival curves of mice treated with the indicated therapies (n = 6). Log rank (Mantel-Cox) statistical test was used for MAPKi vs MAPKi/BIBF1120. ****P≤0.0001.Mouse YUMM1.7 melanoma cells were subcutaneously inoculated into C57BL/6 mice and when tumors reached 100 mm3 mice were treated with vehicle (Ctrl), Nintedanib/BIBF1120 (BIBF), MAPKi (BRAFi, Vemurafenib and MEKi, Trametinib) or BRAFi/MEKi plus BIBF (n=6). (F) Quantification of collagen fibers thickness (n = 6 for control, BIBF and BRAFi/MEKi groups and n = 5 for BRAFi/MEKi + BIBF group). Two-way ANOVA statistical test was used for statistical analysis of mature collagen fibers thickness quantification. **P≤0.01, ***P≤0.001, ****P≤0.0001. Significance was calculated against the control group. Statistical significance of BIBF vs BIBF + BRAFi/MEKi was also calculated.Mouse YUMM1.7 melanoma cells were subcutaneously inoculated into C57BL/6 mice and when tumors reached 100 mm3 mice were treated with vehicle (Ctrl), Nintedanib/BIBF1120 (BIBF), MAPKi (BRAFi, Vemurafenib and MEKi, Trametinib) or BRAFi/MEKi plus BIBF (n=6). (G) Heatmap showing the differential expression of ECM and myofibroblast/CAF genes in mice treated with MAPK-targeted therapies with or without BIBF compared to control mice (log2 ratio, n = 5).(H) Heatmap and Western Blot showing the expression of ECM, myofibroblast/CAF and phenotype switch markers in human M238R cells compared to the parental M238P cells. Heatmap represents the mean of expression of 3 independent experiments by RT-qPCR. (I) Heatmap showing the expression of ECM, myofibroblast/CAF and phenotype switch markers in human M238R cells treated with BIBF (2 µM, 72 h) or vehicle alone by RT-qPCR (n = 3).
Figure 2(B) Relative miRNA expression levels were quantified in M238P cells treated for 72 h with BIBF1120 (BIBF, 2 μM), MAPKi (BRAFi, Vemurafenib and MEKi, Trametinib) (1µM), or with MAPKi (1 μM) plus BIBF (2 µM) by RT-qPCR and normalized to miR-16-5p. Data is represented as mean ± SEM from a triplicate representative of 3 independent experiments. One-way ANOVA was used for statistical analysis. **P≤0.01, ***P≤0.001. Significance was calculated against the control group. Statistical significance of BRAFi/MEKi vs BRAFi/MEKi + BIBF was also calculated. (C) Expression of miR-143-3p and miR-145-5p in control mice and mice treated with the indicated therapies was quantified by RT-qPCR. Data is represented as mean ± SEM from 2 independent experiments performed on 6 mice, with 2 sites of injections. One-way ANOVA was used for statistical analysis. *P≤0.05, ****P≤0.0001. Significance was calculated against the control group. Statistical significance of BRAFi/MEKi vs BRAFi/MEKi + BIBF was also calculated.(D) Relative miRNA expression levels was quantified in M238P cells stimulated for 48 h with TGF-β (10 ng/mL) or PDGF-BB (20 ng/mL) by RT-qPCR and normalized to miR-16-5p. Data is represented as mean ± SEM from a triplicate representative of 3 independent experiments. P-values were calculated using Paired Student t-test. *P≤0.05, ***P≤0.001. (E) Relative miRNA expression levels was quantified in M238R cells treated for 48 h with the triple kinase inhibitor Nintedanib/BIBF1120 (BIBF, 2 μM), the TGF-β receptor kinase inhibitor SB431542 (SB, 10 µM), and the pan-AKT inhibitor GSK690693 (GSK, 10 µM) by RT-qPCR. Data is represented as mean ± SEM from a triplicate representative of 3 independent experiments. P-values were calculated using Paired Student t-test. *P≤0.05, **P≤0.01, ***P≤0.001.(F, Phenotype switch/invasive/ECM markers were quantified in therapy-naïve (N) and resistant (R) PDX samples. The log2 fold change or the ratio of the fold change R vs N is shown for each couple of samples.
Figure 3(B) M238P cells were treated 72 h with BRAFi (Vemurafenib, 3 μM) in the presence or the absence of LNA-based anti-miR-143 (LNA-143) or anti-miR-145 (LNA-145) (50 nM) or a combination of the two. ECM markers RT-qPCR data is represented as mean ± SD from a triplicate representative of at least 3 independent experiments. One-way ANOVA was used for statistical analysis. *P≤0.05, **P≤0.01, ***P≤0.001.(C) Western Blot analysis of ECM remodeling markers on total cell lysates from M238P cells transfected as in (A). (D) Western Blot analysis of ECM remodeling markers on total cell lysates from cells treated with or without BRAFi and the indicated combination of inhibitors(F) Migration assay performed in Boyden chambers. Representative images showing migrating cells in the different conditions. The histogram represents the quantitative determination of mean ± SD from 6 independent fields representative of 3 independent experiments, using ImageJ software. Paired Student t-test was used for statistical analysis. * P≤0.05, ***P≤0.001.
Figure 4(F) Luciferase assay in HEK cells overexpressing miR-143 or miR-145 transfected with a plasmid harboring the WT or muted sequence of the miR-143 and miR-145 binding sites present in FSCN1 3'UTR. Each bar represents the mean ± SE of experiments performed at least in triplicate. ***P≤0.001, ****P≤0.0001. P-values were calculated using Paired Student t-test.(G) RT-qPCR analysis of FSCN1 expression in M238P cells transfected with the indicated mimics. Data is represented as mean ± SE from a triplicate representative of at least 3 independent experiments. Paired Student t-test was used for statistical analysis. ** P≤0.01, ***P≤0.001.
Figure 5(B) FSCN1 immunofluorescent staining and quantification of fluorescence intensity in M238P cells treated or not with BRAFi (Vemurafenib, 3 μM) in the presence or the absence of LNA-based anti-miR-143 (LNA-143) or anti-miR-145 (LNA-145) (50 nM) or a combination of the two. Data are represented as scatter plots with mean ± SD from 10 independent fields representative of 3 independent experiments, Mann-Whitney U test was used for statistical analysis. ****P≤0.0001. Scale bar 40 μm.M238P cells were transfected with two different sequences of siRNAs vs FSCN1 or with a control siRNA (72 h, 100 nM). (D) Migration assay performed in Boyden chambers. Representative images showing migration of M238P cells treated with the indicated siRNAs. The bar graph represents the quantitative determination of data obtained of mean ± SD from 5 independent fields representative of 3 independent experiments, using ImageJ software. Paired Student t-test was used for statistical analysis. *P≤0.05, ***P≤0.001M238P cells were transfected with two different sequences of siRNAs vs FSCN1 or with a control siRNA (72 h, 100 nM). (E) Western Blot analysis of phenotype-switch markers on cell lysates from M238P cells transfected with the indicated siRNAs.BRAFi-resistant M238R cells overexpressing FSCN1 were obtained after transduction with a FSCN1 lentiviral construct. M238R transduced with a Ctrl lentivirus were used as control. (G) Western Blot analysis of FSCN1, phenotype switch markers and ECM remodeling markers on cell lysates from control and FSCN1 overexpressing cells.BRAFi-resistant M238R cells overexpressing FSCN1 were obtained after transduction with a FSCN1 lentiviral construct. M238R transduced with a Ctrl lentivirus were used as control. (H) Crystal violet viability assay of M238R cells stably overexpressing FSCN1 during 6 days with the indicated doses of the BRAFi Vemurafenib. Data is represented as mean ± SD from a triplicate representative of at least 3 independent experiments. Paired Student t-test was used for statistical analysis. ****P≤0.0001.
Figure 6(A, M238P cells were transfected with miR-143-3p, miR-145-5p or a control mimic (miR-neg) (72 h, 30 nM). (B, M238P cells were treated 72 h with BRAFi (Vemurafenib, 3 µM) or a combination of BRAFi (Vemurafenib, 0.5 µM) and MEKi (Trametinib, 1 µM). (A-B) Images and quantification of cell area in cells stained for F-actin (red) and nuclei (blue). Data is represented as scatter plot with mean ± SD (n≥30 cells per condition). Mann-Whitney U test was used for statistical analysis. ****P≤0.0001. Scale bar 40 μm.C) M238P cells were transfected with miR-143-3p, miR-145-5p or a control mimic (miR-neg) (72 h, 30 nM). D) M238P cells were treated 72 h with BRAFi (Vemurafenib, 3 µM) or a combination of BRAFi (Vemurafenib, 0.5 µM) and MEKi (Trametinib, 1 µM). (C-D) Images and quantification of focal adhesions number in cells stained for pPaxillin (green) and nuclei (blue). Focal adhesions (FA) number is represented as mean ± SD (n≥30 cells per condition). Each point represents the average number of focal adhesions per cell calculated for each field. Paired Student t-test has been used for statistical analysis. *P≤0.01, ***P≤0.001, ****P. ≤0.0001. Scale bar 40 μm.(E-F) M238P cells were transfected with two different sequences of siRNAs vs FSCN1 or with a control siRNA (72 h, 100 nM). (E) Images and quantification of cell area in cells stained for F-actin (red) and nuclei (blue). Data is represented as scatter plot with mean ± SD (n≥30 cells per condition). Mann-Whitney U test was used for statistical analysis. ****P≤0.0001. Scale bar 40 μm. (F) Images and quantification of focal adhesions (FA) number in cells stained for pPaxillin (green) and nuclei (blue). Focal adhesions number is represented as mean ± SD (n≥30 cells per condition). Each point represents the average number of focal adhesions per cell calculated for each field. Paired Student t-test was used for statistical analysis. ****P≤0.0001. Scale bar 40 μm.
Figure 7E) M238P cells were transfected with miR-143-3p, miR-145-5p or a control mimic (miR-neg) (72 h, 30 nM). (E) Effect of miR-143-3p or miR-145-5p overexpression on the expression of YAP/MRTF target genes assessed by RT-qPCR. Data are normalized to the expression in control cells. Data is represented as mean ± SD from a triplicate representative of at least 3 independent experiments. Paired Student t-test was used for statistical analysis. *P≤0.05, **P≤0.01, ****P≤0.0001.M238P cells were transfected with two different sequences of siRNAs vs FSCN1 or with a control siRNA (72 h, 100 nM). Effect of FSCN1 downregulation on MRTFA (F) and YAP1 (G) nuclear translocation assessed by immunofluorescence in M238P. Data are represented as scatter plot with mean ± SD (n≥30 cells per condition). Each point represents the nuclear/cytoplasm ratio. Mann-Whitney U test was used for statistical analysis. ***P≤0.001, ****P≤0.0001. Scale bar 40 μm.M238P cells were transfected with two different sequences of siRNAs vs FSCN1 or with a control siRNA (72 h, 100 nM). (H) RT-qPCR analysis for the expression of MRTFA/YAP target genes in M238P cells transfected with the indicated siRNAs. Data are normalized to the expression in parental cells. Data is represented as mean ± SE from a triplicate representative of at least 3 independent experiments. Paired Student t-test was used for statistical analysis. *P≤0.05, **P≤0.01, ***P≤0.001. Scale bar 40 μm.
Figure 1(B-C) Coimmunoprecipitation of Ams1 (B) and Ape4 (C) with Nbr1. Endogenously TAP-tagged Nbr1 was immunoprecipitated (IP) using IgG Sepharose beads. Cell lysates and immunoprecipitates were examined by immunoblotting (IB).(D-E) Subcellular localization of Ams1 (D) and Ape4 (E). Wild-type (WT), nbr1Δ, sst4Δ, and atg1Δ cells were grown to mid-log phase in EMM medium and examined by live cell imaging. In (D), Ams1 was endogenously tagged at its C-terminus with mCherry. In (E), Ape4 was endogenously tagged at its C-terminus with GFP and in the same cells GFP-binding-protein (GBP) fused 3xUb was expressed under the P41nmt1 promoter. Cpy1 is a vacuole lumen marker. Scale bar, 3 μm.
Figure 2Localization of Ams1 in WT, lap2Δ, ape2Δ, and ape4Δ cells. Scale bar, 3 μm.   Localization of Ape4 in WT, lap2Δ, ape2Δ, and ams1Δ cells. Scale bar, 3 μm.   Ams1, Lap2, and Ape2 did not co-immunoprecipitate with Ape4. Endogenously TAP-tagged Ape4 was immunoprecipitated. Ams1 was endogenously tagged with an mCherry tag.
Figure 3The ZZ1-L1 fragment of Nbr1 co-immunoprecipitated with Ams1.   The ZZ1-L1 fragment of Nbr1 co-immunoprecipitated with Ape4.   The ZZ1 region but not the ZZ2-ZZ3 region in Nbr1 is necessary for vacuolar targeting of Ams1. Scale bar, 3 μm.   The ZZ1 region but not the ZZ2-ZZ3 region in Nbr1 is necessary for vacuolar targeting of Ape4. Scale bar, 3 μm.   The ZZ2-ZZ3 region but not the ZZ1 region in Nbr1 is necessary for vacuolar targeting of Ape2. Scale bar, 3 μm.   The ZZ2-ZZ3 region but not the ZZ1 region in Nbr1 is necessary for vacuolar targeting of Lap2. Scale bar, 3 μm.
Figure 6Surface plasmon resonance (SPR) analysis of the Nbr1-Ams1 interaction. GST-tagged ZZ1-L1 fragment of Nbr1 purified from E. coli was captured on a CM5 sensor chip using the GST Capture Kit. Ams1 purified from S. pombe (two-fold serial dilutions from 1000 nM to 62.5 nM) was flowed through the chip surface.SPR analysis of the Nbr1-Ape4 interaction. GST-tagged ZZ1-L1 fragment of Nbr1 purified from E. coli was captured on a CM5 sensor chip using the GST Capture Kit. Ape4 purified from S. pombe (two-fold serial dilutions from 1000 nM to 62.5 nM) was flowed through the chip surface.
Figure 7The effects of individually mutating the first two residues of Ams1 on its Nbr1-binding ability. Nbr1-Ams1 interactions were examined by the Pil1 co-tethering assay. Scale bar, 3 μm.The effects of individually mutating the third residue and other residues in the U-shaped loop of Ape4 on its Nbr1-binding ability. Nbr1-Ape4 interactions were examined by the Pil1 co-tethering assay. Scale bar, 3 μm.