EMBO/SourceData huggingface dataset:
figf3a, Western blot analysis of flag-tagged beclin-1-transfected MCF7 clones. C, MCF7.control cells (clone 38); St, MCF7.beclin1stop cells (clone 70). Mr, relative molecular mass.b, Western blot analysis of flag-tagged beclin1stop-transfected MCF7 clones. C, MCF.control cells (clone 38); B, MCF7.beclin1 cells (clone 1).c, Photomicrographs of MCF7.control cells (left panel, clone 38), MCF7. beclin1 cells (middle panel, clone 17) and MCF7.beclin1stop cells (right panel, clone 70) 48 h after seeding at similar densities.d, Proliferation of MCF7.control cells (clones 37 and 38; closed squares and circles, respectively, solid lines), MCF7.beclin1 cells (clones 1, 6, 17; open squares, circles, triangles, respectively, solid lines) and MCF7.beclin1stop cells (clone 70, closed triangles, dotted line). Results represent mean Absorbance (A) (±s.e.m.) for six wells; similar results were obtained in three independent experiments.e, Cell viability of MCF7 clones determined by trypan blue staining. Symbols for each clone are as in df, Clonigenicity in semisolid medium (soft agar) of MCF7 clones. Results are mean (±s.e.m.) for pooled triplicate wells from 3-4 independent experiments.g, Tumour formation in NCR nude mice injected subcutaneously with MCF7 clones. Numbers on top of bar indicate number of autopsy-confirmed tumours at 8 weeks per number of mice injected with each clone.
figf4a, Western blot analysis of human breast carcinoma cell lines. Results are from 8 representative cell lines out of a total of 11 analysed.b, Western blot analysis of matched normal breast and breast carcinoma tissue from patients with sporadic invasive breast carcinoma. Results are from 7 representative cases out of a total of 17 analysed. N, normal breast tissue; T, tumour tissue; Mr, relative molecular mass.c, Immunohistochemical analysis of paraffin-embedded sections of breast tissue from patient 441 shown in b. Adjacent sections are stained with pre-immune 843 rabbit serum (left column), 843 anti-Beclin 1serum (middle column) and anti-cytokeratin (right column). Top row shows low power view of region of breast containing tumour (T) and normal mammary epithelial duct (N). Middle row shows higher power view of normal mammary epithelial duct. Bottom row shows higher power view of tumour. Scale bars, 1 mm.
Figure 1Biological replicates of transcriptional profiles (Tx) measured in separately prepared cell free lysate batches of E. coli.Correlation between transcriptional profiles (Tx) for regulatory sequence libraries from in vitro and in vivo measurements in E. coli. The color scale indicates the density of data points in a given area of the plot.Correlation between primary TSS calls (in bp from ATG start codon) of regulatory sequences from in vitro and in vivo measurements in E. coli.
Figure 2Optimization of transcriptional output using different concentrations of Mg-glutamate and K-glutamate in 10 bacterial cell-free expression systems with Broccoli as a reporter (solid lines, optimal buffer composition shown in red). No DNA template controls are shown as grey dashed lines. 12.5 nM of DNA template was used in all systems, except L. lactis (50 nM used).
Figure 3Transcriptional activities (Tx) of 421 regulatory sequences that were active in all 10 bacterial cell-free expression systems.Pairwise comparison of transcriptional profiles of 421 universally-active regulatory sequences between bacterial species. Example scatter plots with relatively high and low Pearson correlation in the heatmap (marked i and ii) are shown on left.Correlation between evolutionary divergences (16S rRNA percent identity) and pairwise Pearson correlation of transcriptional profiles. Dashed line represents linear regression.
Figure 5Pairwise correlation of RS7003 transcriptional profiles between hybrid lysate and single constituent species lysates for (d) E. coli + B. subtilisPairwise correlation of RS7003 transcriptional profiles between hybrid lysate and single constituent species lysates for (e) E. coli + C. glutamicum hybrids with different mixing ratios.
Figure 1A Expression of transcripts coding for PI3K subunit genes in pDCs (n =12 biological replicates). In the boxplot, the middle, the bottom, and the top of the box represent the median, the 1st quantile and the 3rd quantile, respectively; the height of the box is given by the difference between the 3rd and the 1st quantiles (IQR, interquartile range); the whiskers length is 1.5 times the interquartile range. Data points beyond the whiskers represent values more extreme than 1.5 times the interquartile range above or below the 3rd or the 1st quantile, respectively.C Kyn levels in culture supernatants of pDCs incubated for 24 h with medium alone (Ctrl) or IFN-γ after 1−h pretreatment with PI3K inhibitors. D Real-time PCR analysis of Ido1 and Tgfb1 transcripts in pDCs stimulated for 18 h with TGF-β, in the presence or absence (Ctrl, control) of catalytic PI3K inhibitors, normalized to the expression of Gapdh and presented relative to results in untreated cells (dotted line, 1-fold). E In vivo suppression of the activity of HY-pulsed CD8− DCs in combination with a minority fraction (5%) of pDCs conditioned in vitro with TGF-β (or medium) for 24 h in the presence or absence (Ctrl) of catalytic inhibitors as in C. Analysis of skin reactivity of recipient mice to the eliciting peptide at 15 days is presented as change in footpad weight (experimental versus control footpads).F Real-time PCR analysis of Ido1 and Tgfb1 transcripts in pDCs transfected with p110α-, β-, δ-specific, or negative control (nc) siRNA and stimulated for 18 h with TGF-β, normalized to the expression of Gapdh and presented relative to results in unstimulated cells (dotted line, 1-fold). G In vivo suppression of the activity of HY-pulsed CD8− DCs in combination with a minority fraction (5%) of pDCs transfected with siRNA as in (F) and conditioned in vitro with TGF-β (or medium) for 24 h. Analysis of skin reactivity of recipient mice to the eliciting peptide at 15 days is presented as change in footpad weight (experimental versus control footpads).
Figure 2C Pull-down (PD) assay of lysates from P1.HTR cells with unphosphorylated (YENM) and tyrosine-phosphorylated IDO1 peptides (pYENM, pITIM1, and pITIM2), analyzed by sequential immunoblotting with anti−p85, −SHP-1, and -SHP-2 antibodies.D Immunoblotting with anti-IDO1 and -p85 antibodies of IDO1 immunoprecipitates obtained from P1.HTR cells transfected with IDO1.WT or IDO1.Y149F. Mock-transfected P1.HTR cells were used as a control.E Tyrosine phosphatase activity in anti-IDO1 immunoprecipitates from lysates of P1.HTR transfectants as in D.F Kyn release over 24 h by P1.HTR transfectants as in D.
Figure 3A Confocal immunofluorescence microscopy images of P1.HTR cells either untransfected (Untr) or transfected with IDO1.WT (WT) or vector alone (Mock) and stained with antibodies recognizing IDO1 (green). Nuclei were stained with DAPI (blue). Scale bar, 20 µm.B Confocal immunofluorescence microscopy images of P1.HTR cells transfected with IDO1.WT, co-stained with antibodies recognizing IDO1 (green) and markers of intracellular organelles and structures (as indicated; red). Nuclei were stained with DAPI (blue). The merge imagine is shown for all stainings or without DAPI (no DAPI). Scale bar, 10 µm.C Immunoblotting analysis of EE purified from P1.IDO1.WT cells. Mock-transfected cells as in A were used as control.D Distribution of the IDO1.WT protein expressed by P1.HTR transfected cells in isopycnic sucrose gradients. Fractions were immunoblotted using antibodies specific for IDO1 and intracellular organelles or structures as indicated.
Figure 4A Confocal immunofluorescence microscopy images of P1.HTR cells transfected with IDO1.WT or IDO1.Y149F and co-stained with antibodies recognizing IDO1 (green) and EEA1 (red). Nuclei were stained with DAPI (blue). The merge imagine is shown for all stainings or without DAPI (no DAPI). Scale bar, 10 µm.B Pearson's coefficient of IDO1 colocalization with EEA1 in P1.HTR cells transfected as in A, with each point representing colocalization within an image stack. Data represent mean ± SD (n > 24 cells from three independent experiments). Statistical analysis was performed using two-tailed unpaired Student's t-test. ***P < 0.001.C Distribution of IDO1.WT and the IDO1.Y149F mutants in homogenates of P1.HTR cells along the isopycnic sucrose gradient as in Fig 3D. Fractions were immunoblotted using antibodies specific for IDO1.D The distribution of IDO1.WT and IDO1.Y149F proteins along the isopycnic sucrose gradient is shown in term of densitometric analysis in each fraction (mean ± SD of three experiments).
Figure 5 A Pull-down assay of lysates from untransfected P1.HTR cells with unphosphorylated (YENM) and tyrosine-phosphorylated (pYENM) IDO1 peptides, analyzed by immunoblotting with anti-p85, -p110α, -p110β, -p110γ, and -p110δ antibodies. Whole cell lysates (WCL) were used as control of lysate amount. B Confocal immunofluorescence microscopy images of P1.HTR cells transfected with IDO1.WT incubated with class IA PI3K p110 isoform inhibitors (indicated) or medium alone (Ctrl) for 24 h and co-stained with antibodies recognizing IDO1 (green) and EEA1 (red). Nuclei were stained with DAPI (blue). The merge imagine is shown for all stainings or without DAPI (no DAPI). Scale bar, 10 µm.C Pearson's coefficient of IDO1 colocalization with EEA1 in P1.HTR cells transfected and treated as in (B), with each point representing colocalization within an image stack. Data represent mean ± SD (n > 20 cells from two independent experiments).D Tyrosine phosphatase activity in anti-IDO1 immunoprecipitates from P1.HTR cells transfected with IDO1.WT cells and treated with PI3K catalytic inhibitors (indicated) or medium alone (ctrl). Data represent the mean ± SD (n = 3 technical replicates).
Figure 6A Confocal immunofluorescence microscopy images of freshly purified pDCs co-stained with antibodies recognizing IDO1 (green) and markers of intracellular organelles and structures (indicated; red). Nuclei were stained with DAPI (blue). The merge imagine is shown for all stainings or without DAPI (no DAPI). Scale bar, 5 µm.B Confocal immunofluorescence microscopy images of pDCs incubated for 24 h with TGF-β or IFN-γ and co-stained with antibodies recognizing IDO1 (green) and EEA1 (red). Nuclei were stained with DAPI (blue). Scale bar, 5 µm. C Pearson's coefficient of IDO1 colocalization with EEA1 in pDCs either untreated or stimulated with TGF-β or IFN-γ (as in B), with each point representing colocalization within an image stack. Data represent mean ± SD (n > 20 cells from three independent experiments).
Figure 1A. Size of adult (9-14 week) CEP250+/+ and CEP250-/- mice. B. Body weight of CEP250+/+ and CEP250-/- mice (9-14 week). CEP250+/+ n=5 mice, CEP250-/- n=5 mice. Data information: In (B , data are presented as mean ± SEM. n.s., not significant, **P < 0.01, ****P < 0.0001 (the unpaired Student's t-test). In (B whiskers represent the range of the data, with bottom and top represent min and max value of the dataset respectively.C. Brains from adult CEP250+/+ and CEP250-/- mice (9-14 week). Scale bars: 1000 µm. D. Brain weight of 9-14-week-old mice. CEP250+/+ n=5 mice, CEP250-/- n=5 mice. Data information: In data are presented as mean ± SEM. n.s., not significant, **P < 0.01, ****P < 0.0001 (the unpaired Student's t-test). In whiskers represent the range of the data, with bottom and top represent min and max value of the dataset respectively.F. Adult testis (9-14 week) of CEP250+/+ and CEP250-/- mice. Scale bars: 1000 µm. G. Adult testis (9-14 week) weight of CEP250+/+ and CEP250-/- mice. CEP250+/+ n=10 mice, CEP250-/- n=10 mice. Data information: In data are presented as mean ± SEM. n.s., not significant, **P < 0.01, ****P < 0.0001 (the unpaired Student's t-test). In G) whiskers represent the range of the data, with bottom and top represent min and max value of the dataset respectively.K. Bright-field image of live epididymal sperm from CEP250+/+ and CEP250-/- mice (9-14 week). Scale bars: 10 µm. Arrow heads highlight the sperm heads. L. Quantification of (K), sperm number per mouse. CEP250+/+ n=3 mice, CEP250-/- n=3 mice. Data information: In data are presented as mean ± SEM. n.s., not significant, **P < 0.01, ****P < 0.0001 (the unpaired Student's t-test).N. Immunofluorescence for GATA4 (green) with DAPI staining (blue) of 10 µm testis cryo-sections from adult CEP250+/+ and CEP250-/- mice (9-14 week). Scale bars: 100 µm. Dashed lines highlight the STs. O. Quantification of (N), CEP250+/+ n=3 mice, CEP250-/- n=3 mice. 50 STs were analyzed for each mouse. See Appendix Fig S2 for ST number per section and ST diameter. Data information: In O), data are presented as mean ± SEM. n.s., not significant, **P < 0.01, ****P < 0.0001 (the unpaired Student's t-test).
Figure 2D. Immunofluorescence for MVH (green) with DAPI staining (blue) of 10 µm testis cryo-sections from neonatal mice (P5, P10 and P14) CEP250+/+ and CEP250-/-. Scale bars: 20 µm.E. Quantification of CEP250+/+ n=3 mice, CEP250-/- n=3 mice. Data information: E), data are presented as mean ± SEM. n.s., not significant, **P < 0.01, ****P < 0.0001 (the unpaired Student's t-test).F. Quantification of C-CASP3 positive cells normalized to total cell number per seminiferous tubule (ST). Dots (colors reflect STs from different mice) represent value per ST, CEP250+/+ n=3 mice, CEP250-/- n=3 mice. 30 STs were analyzed for each mouse. Data information: In (F data are presented as mean ± SEM. n.s., not significant, ***P < 0.005 , ****P < 0.0001 (the Mann-Whitney test).G. C-CASP3 (green) and MVH (magenta) staining with DAPI (blue) of 10 µm testis cryo-sections from neonatal mice (P5, P10 and P14) CEP250+/+ and CEP250-/-, scale bars: 20 µm. H. Quantification of (G). Percentage of C-CASP3 and MVH double positive cells in relation to total C-CASP3 positive cells per ST from P5, P10 and P14 mice. Dots (colors reflect STs from different mice) represent value per ST, CEP250+/+ n=3 mice, CEP250-/- n=3 mice. 30 STs were analyzed for each mouse. I. Quantification of (G). Percentage of C-CASP3 and MVH double positive cells in relation to total MVH positive cells (germ cells) per ST from P5, P10 and P14 mice. Dots (colors reflect STs from different mice) represent value per ST, CEP250+/+ n=3 mice, CEP250-/- n=3 mice. 30 STs were analyzed for each mouse. Data information In H, I), data are presented as mean ± SEM. n.s., not significant, ***P < 0.005 , ****P < 0.0001 (the Mann-Whitney test).
Figure 3A. Immunofluorescence staining for PLZF (red) with DAPI staining (blue) of 10 µm cryo-sections of testes from CEP250+/+ and CEP250-/- mice at P5, P10 and P14. Scale bars: 20 μm.B. Quantification of PLZF+ cells per seminiferous (ST) section of CEP250+/+ and CEP250-/-, testes at P5, P10 and P14. Dots (colors reflect STs from different mice) represent value per ST, CEP250+/+ (P5) n=3 mice, CEP250-/- (P5) n=3 mice, 50 STs were analyzed for each mouse; CEP250+/+ (P10) n=3 mice, CEP250-/- (P10) n=3 mice, at least 30 STs were analyzed for each mouse; CEP250+/+ (P14) n=3 mice, CEP250-/- (P14) n=3 mice, at least 50 ST were analyzed for each mouse. Data information: In (B data are presented as mean ± SEM. n.s., not significant, *P<0.05, ***P < 0.005, ****P < 0.0001 (the Mann-Whitney test).C. Immunofluorescence staining for SCP1 (green), PLZF (red) and DAPI (blue) of 10 µm cryo-sections of testes from CEP250+/+ and CEP250-/- mice at P2, P3, P5 and P7. Arrowheads highlight PLZF- SCP1+ (green) cells, which represent differentiated spermatogonia. Scale bars: 20 μm.Quantification of The number of PLZF+ SCP1-, PLZF+ SCP1+, PLZF- SCP1+ cells normalized to total cell number per ST section in CEP250+/+ and CEP250-/- testes at P2, P3 respectively. Dots (colors reflect STs from different mice) represent value per ST, CEP250+/+ n=3 mice, CEP250-/- n=3 mice, at least 40 STs were analyzed for each mouse. Data information: data are presented as mean ± SEM. n.s., not significant, *P<0.05, ***P < 0.005, ****P < 0.0001 (the Mann-Whitney test).Quantification of The number of PLZF+ SCP1-, PLZF+ SCP1+, PLZF- SCP1+ cells normalized to total cell number per ST section in CEP250+/+ and CEP250-/- testes at , P5 and P7 respectively. Dots (colors reflect STs from different mice) represent value per ST, CEP250+/+ n=3 mice, CEP250-/- n=3 mice, at least 40 STs were analyzed for each mouse. Data information: data are presented as mean ± SEM. n.s., not significant, *P<0.05, ***P < 0.005, ****P < 0.0001 (the Mann-Whitney test).
Figure 4B. Representative images of mouse seminiferous tubule (ST) sections stained for PLZF (red), SCP1 (green) and DAPI (blue) at P4, P6 and P7. Scale bars: 20 μm. Arrowheads highlight PLZF- SCP1+ (green) cells localize to the middle of the ST lumen, which represent differentiated spermatogonia that translocated to the middle of the ST lumen. C. Quantification of (B). Shown is the number of PLZF- SCP1+ cells which translocated inwards the seminiferous tubule lumen per seminiferous tubule section, as normalized to the total cell number per ST section. Dots (colors reflect STs from different mice) represent value per ST, CEP250+/+ n=3 mice, CEP250-/- n=3 mice, at least 30 STs were analyzed for each mouse. D. Quantification of (B). Shown is the total number of SCP1+ cells which translocated inwards the seminiferous tubule lumen per ST section, as normalized to the total cell number per seminiferous tubule section. Dots (colors reflect STs from different mice) represent value per ST, CEP250+/+ n=3 mice, CEP250-/- n=3 mice, at least 30 STs were analyzed for each mouse. E. Quantification of (B). Shown is the number of PLZF+ SCP1+ cells which translocated inwards the seminiferous tubule lumen per seminiferous tubule section, as normalized to the total cell number per seminiferous tubule section. Dots (colors reflect STs from different mice) represent value per ST, CEP250+/+ n=3 mice, CEP250-/- n=3 mice, at least 30 STs were analyzed for each mouse. Data information: In (C-E), data are presented as mean ± SEM. n.s., not significant, *P<0.05, **P < 0.01, ***P < 0.005 (the Mann-Whitney test).
Figure 5A. Representative images of P4 mouse seminiferous tubule (ST) 10 µm cryo-sections stained for PLZF (red), γ-Tubulin (green), PH3 (magenta) and DAPI (blue). Arrowheads highlight the centrosomes. Dashed yellow line highlights the basement membrane. Semi-transparent white line, which bisects both spindle poles was used to determine spindle angle Ø orientation in relation to the basement membrane. Anaphase cells were analyzed. Scale bars: 5 µm. B. Representative images of P5 mouse seminiferous tubule 10 µm cryo-sections stained for PLZF (red), γ-Tubulin (green), PH3 (magenta) and DAPI (blue). Arrowheads highlight the centrosomes. Dashed yellow line highlights the basement membrane. Semi-transparent white line, which bisects both spindle poles was used to determine spindle angle Ø orientation in relation to the basement membrane. Anaphase cells were analyzed. Scale bars: 5 µm. (C Quantitation of spindle angle of anaphase GSCs in P4 ST sections (from A). Spindle angle relative to basement membrane for CEP250+/+ and CEP250-/-). In (C), dots (colors reflect different mice) represent spindle angle of each GSC. CEP250+/+ n=3 mice, CEP250-/- n=3 mice. At least 10 cells were analyzed for each mouse. (D Quantitation of spindle angle of anaphase GSCs in P5 ST sections (from B). for P5. In (D), dots (colors reflect different mice) represent spindle angle of each GSC. CEP250+/+ n=3 mice, CEP250-/- n=3 mice. At least 10 cells were analyzed in each mouse.E) Quantitation of spindle angle of anaphase GSCs in P4 ST sections Spindle angle relative to basement membrane for CEP250+/+ and CEP250-/-). In (E), the three dots represent averaged spindle angle per mouse. CEP250+/+ n=3 mice, CEP250-/- n=3 mice. At least 10 cells were analyzed for each mouse. F) Quantitation of spindle angle of anaphase GSCs in P5 ST sections for P5. In (F), the three dots represent averaged spindle angle per mouse. CEP250+/+ n=3 mice, CEP250-/- n=3 mice. At least 10 cells were analyzed in each mouse. Data information: In (E, F), data are presented as mean ± SEM. n.s., not significant, *P<0.05, ***P < 0.005 (the unpaired Student's t-test).
Figure 6B. Representative images of P5 mouse ST. 10 µm cryo-sections stained for PLZF (red), E-cadherin (green) and DAPI (blue) showing GSCs in interphase. Scale bars: 5 μm. C. Quantification of (B). Percentage of GSCs displaying different polarity status are indicated. CEP250+/+ n=5 mice, CEP250-/- n=4 mice. At least 100 cells were analyzed in each mouse and the average was calculated for each mouse. Data information: In white arrowheads highlight basement membrane; yellow arrowhead highlights cortical enrichment of E-cadherin towards the basement membrane. In (C data are presented as mean ± SEM. n.s., not significant, *P<0.05, **P < 0.01, ***P < 0.005 (the unpaired Student's t-test).D. Representative images of P5 mouse ST. 10 µm cryo-sections stained for PLZF (red), E-cadherin (green) and PH3 (magenta) showing GSCs in prophase. PH3 staining marks the mitotic chromatin. Scale bars: 5 μm. E. Quantification of (D). Percentage of GSCs displaying different polarity status are indicated. CEP250+/+ n=5 mice, CEP250-/- n=4 mice. At least 10 cells were analyzed in each mouse and the average was calculated for each mouse. Data information: In white arrowheads highlight basement membrane; yellow arrowhead highlights cortical enrichment of E-cadherin towards the basement membrane. In data are presented as mean ± SEM. n.s., not significant, *P<0.05, **P < 0.01, ***P < 0.005 (the unpaired Student's t-test).F. Representative images of P5 mouse ST 10 µm cryo-sections stained for PLZF (red), E-cadherin (green) and PH3 (magenta) showing GSCs in prometaphase. PH3 staining marks the mitotic chromosome condensation. Scale bars: 5 μm. G. Quantification of (F), percentage of GSCs displaying different polarity status are indicated. CEP250+/+ n=5 mice, CEP250-/- n=4 mice. At least 30 cells were analyzed in each mouse and the average was calculated for each mouse. Data information: In F, white arrowheads highlight basement membrane; yellow arrowhead highlights cortical enrichment of E-cadherin towards the basement membrane. In G), data are presented as mean ± SEM. n.s., not significant, *P<0.05, **P < 0.01, ***P < 0.005 (the unpaired Student's t-test).
Figure 7A. Representative images of P5 mouse seminiferous tubule (ST) 10 µm cryo-sections stained for PLZF (red), ODF2 (green), NIN (magenta) and PH3 (blue) showing GSCs in prophase. Scale bars: 2 μm. Yellow arrowheads highlight the two centrosomes.B. Quantification of Centrosome distance in GSCs in prophase at P5. CEP250+/+ n=3 mice, CEP250-/- n=3 mice. At least 50 cells were analyzed in each mouse (color of dot indicates mouse). Dashed line indicates 4 μm as the threshold of well separated centrosomes. Data information: In (B), data are presented as mean ± SEM. ****P < 0.001 (the Mann-Whitney test).D. Representative images of P5 mouse seminiferous tubule 10 µm cryo-sections stained for PLZF (red), ODF2 (green), NIN (magenta) and PH3 (blue), focusing on PLZF+ germ stem cells (GSCs) in prometaphase. Scale bars: 2 μm. Yellow lines highlight basement membrane. Yellow arrowheads highlight the mother centrosomes and white arrowheads highlight the daughter centrosomes.E. Quantification of Percentage of prometaphase GSCs which preferentially position their mother centrosome close to the basement membrane in CEP250+/+ and CEP250-/- testis at P5. CEP250+/+ n=3 mice, CEP250-/- n=3 mice. At least 50 cells were analyzed in each mouse and the average was calculated for each mouse. Data information: In (E), data are presented as mean ± SEM. ****P < 0.001 (the unpaired Student's t-test).