EMBO/soda-nel huggingface dataset:
NR2F1 regulates regional progenitor dynamics in the mouse neocortex and cortical gyrification in BBSOAS patients (2020)
Figure 5

Figure 5. Nr2f1-mediated molecular control of neural progenitors (NPs).

(A-B') Pax6 (green; aRGs) and Nr2f1 (red) IF of E12.5 WT (A,A') and KO (B,B') lateral pallia. See Appendix Figure S5 for Pax6 and Nr2f1 staining and pixel intensity quantification in HET embryos.

(C) Pax6 and Nr2f1 pixel intensity quantification at E12.5 and E14.5 indicating increased Pax6 levels upon Nr2f1 removal. n ≥ 3 brains.

(D) Real time RT-PCR quantification of Nr2f1 and Pax6 expression in E12.5 cortices. n ≥ 3 cortices.

(E-G'') GFP (green; electroporated cells), Pax6 (red) and Nr2f1 (blue) IF of pCIG2-Nr2f1-IRES-GFP electroporated brains at E13.5 (24-hours after IUE). VZ (F-F'') and CP (G-G'') regions are shown at high magnification. Arrowheads point to GFP+ electroporated cells over-expressing Nr2f1 (blue in F',G'), down-regulating Pax6 (F'') and rapidly migrating out of the VZ (G-G'').

(H) Scatter plot showing Pax6 and Nr2f1 pixel intensity of VZ electroporated progenitors, comparing Nr2f1 overexpressing cells (orange dots) with control pCIG2-GFP electroporated cells (blue dots). Average Nr2f1 and Pax6 pixel intensities of the 2 populations were compared by 2-way ANOVA and resulted significantly different (***= <0.0001). n = 2 electroporated brains.

(I) Real time RT-PCR of cell cycle genes comparing WT (blue line) and KO (orange line) cortices. CyclinD1 and P21 transcripts are down-regulated whereas Dct is up-regulated in mutants. n = 3 cortices.

(J-L) GFP (green; electroporated cells) and P21 (red) IF of E14.5 cortex electroporated 48 hours earlier with control PX458 plasmid (J) or CRISPR/Cas9-expressing plasmid directed against Nr2f1 sequence (PX458-αNr2f1; K). Percentage of P21/GFP double positive cells (L). Arrowheads in (J,K) point to P21+ cells. n = 2 electroporated brains.

(M) Schematic model of Nr2f1 action on NP cell cycle and neural differentiation. Nr2f1 promotes neurogenesis by repressing Pax6 and Dct expression and thus cell cycle progression, and by activating P21-mediated cell cycle exit. Both actions modulate the G1-phase length.

(N) Graph showing the number of differentially expressed genes (DEG; see Methods) up-regulated (UP) or down-regulated (DOWN) in KO compared to WT, as detected by RNA-Seq of E15.5 neocortices.

(O) Gene ontology (GO) categories significantly enriched among DEG genes (DAVID Gene Ontology software; see Methods).

(P) Hierarchical clustering and heatmap of the expression level of DEGs belonging to the "neuron differentiation" GO category in (O). Heat map color scale indicates normalized gene expression from high (red) to low (white) level.

Data information: In (A,B,J,K), nuclei (blue) were stained with DAPI. In (C) the pixel intensity was quantified in 100µm-width boxes, randomly placed across the lateral pallium. Data are represented as means ± SEM. Student t-test (L; *P<0.05) and 2-way ANOVA (C,D,H,I) (*P<0.05, **P<0.01, ***P<0.001). For statistical analysis of RNA-Seq data (N-P), see Methods. Scale bars: 50µm. SVZ: subventricular zone; VZ: ventricular zone.
NR2F1 regulates regional progenitor dynamics in the mouse neocortex and cortical gyrification in BBSOAS patients (2020)
Figure 6

Figure 6. Functional rescue of NP cell cycle dynamics via Pax6 genetic modulation.

(A) Real time RT-PCR analysis of Pax6 expression in neurospheres with different genotypes, as indicated. n = 3 cortices. N KO P HET: Nr2f1 KO, Pax6 HET.

(B) Representative images of Nr2f1 KO Pax6 HET neurospheres obtained from E15.5 neocortices and cultured in vitro.

(C) Graph showing the number of neurospheres per P100 cell plate, at different steps, as indicated. While Nr2f1 KO neurospheres (orange line) proliferate for a longer time compared to WT ones (blue line), the loss of one Pax6 allele (gray line) almost restores normal proliferation rate and exhaustion time. Complete Pax6 loss is not compatible with stem cell renewal (yellow line). n ≥ 3 culture wells from n = 2 independent batches.

(D-E) Paired cell analysis after neurosphere dissociation and 24-hour culture at clonal density. Couples of dividing cells were identified by Map2 (red; N, neurons) and BLBP (green; P, progenitors) IF. Pie charts in (E) show the proportion of P-P (proliferative; D), P-N (asymmetric differentiative; D') or N-N (symmetric differentiative; D'') couples in WT, Nr2f1KO and Nr2f1KO Pax6HET animals. n ≥ 3 samples from n = 2 culture batches.

(F-I) Pax6 (green) and Nr2f1 (red) IF in the posterior-most region of E12.5 cortex, showing Pax6 upregulation in Nr2f1 KO animals (G,G') compared to WT (F',F''). Normal Pax6 levels can be restored by loss of one Pax6 allele (Nr2f1 KO, Pax6 HET; H,H'). Pixel intensity quantification of Nr2f1 (blue) and Pax6 (orange) is shown in (I). n ≥ 3 brains. N KO: Nr2f1 KO; P HET: Pax6 HET.

(J) Cell cycle duration at E12.5 in different mutants as indicated, quantified in posterior regions with double EdU/BrdU injection protocol (see Methods). n ≥ 6 sections from n = 2 brains.

(K) Insets showing representative γ-Tubulin (green) and Phospho-Vimentin (red) IF of the cortical VZ surface to evaluate the orientation of the cleavage plane of dividing aRGs. Pie charts show the percentage of vertical (green), oblique (yellow) and horizontal (red) division planes of mitotic figures in the E12.5 lateral pallium of animals with different genotypes, as indicated. n ≥ 2 brains per genotype.

Data information: Nuclei (blue) were stained with DAPI. In (I,J) the pixel intensity or the number of positive cells was quantified in 100µm-width boxes, randomly placed across the LP. Data are represented as means ± SEM. 2-way ANOVA (A,C,E,I,J,K; *P<0.05, **P<0.01, ***P<0.001). For statistical analysis of (E,K), see Appendix Table S5. Scale bars: 50µm.
NR2F1 regulates regional progenitor dynamics in the mouse neocortex and cortical gyrification in BBSOAS patients (2020)
Figure 7

Figure 7. Delayed neurogenesis upon NR2F1 downregulation in human brain organoids. (A-C) NR2F1 expression in cerebral organoids during neural induction and differentiation in vitro (day 40 and day 70, respectively), together with the NP markers PAX6 (green in A), SOX2 (green in B,B') and DCX as a neural marker (blue in A,B,B''). Quantification of NR2F1 levels in NPs is shown in (C). n ≥ 4 organoids from n = 2 batches.

(D-G) NR2F1 (red) and PAX6 (green) IF in day 40 organoids. Neuroepithelia with high NR2F1 levels show weak PAX6 expression (E,E'), and vice versa (F,F'). Quantification by pixel intensity analysis (G). n ≥ 4 organoids from n = 2 batches.

(H) Schematic representation of an organoid electroporation. PX458 plasmids were injected in ventricular-like cavities, organoids electroporated and processed after 7 days of in vitro culture.

(I-J) NR2F1 (red) and GFP (green) IF in day 40 human brain organoids upon electroporation of the PX458-αNR2F1 plasmid. In (I,I'), GFP+NR2F1- cells (empty arrowheads) and a GFP+NR2F1+ cell (white arrowhead) are shown; quantification in (J). n ≥ 6 organoids from n = 2 batches.

(K-M) TUJ1 (red; differentiating neurons) and GFP (green) IF 7 days after electroporation of control (K) and PX458-αNr2f1 plasmids (L). Co-expression of GFP with SOX2 or TUJ1 distinguishes neural progenitors (NPs) from neurons (Ns), as quantified in (M). Arrowheads in (K,L) point to TUJ1+ GFP+ cells. n ≥ 6 organoids from n = 2 batches.

(N) Red and green arrowheads in (N) point to a SOX2+ (red) progenitor and a TUJ1+ (blue) neuron, respectively.

Data information: Nuclei (blue in D,I,K,L) stained with DAPI. In (C,G,J) the pixel intensity or the number of positive cells were quantified in 100µm-width boxes, randomly placed across the cortex/organoid neuroepithelia, while in (M) the number of positive cells was normalized over the total number of GFP+ cells. In graphs, data are represented as means ± SEM. 2-way ANOVA test (*P<0.05, **P<0.01, ***P<0.001). Scale bars: 50µm.
A dual role for autophagy in a murine model of lung cancer (2014)
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(a) The Kaplan–Meier survival curves for KRas;Atg5+/+ (n=12), KRas;Atg5fl/+ (n=19) and KRas;Atg5fl/fl (n=18) littermate mice injected intranasally with AdCre (2.5 × 107 p.f.u.). P0.01 (log-rank test) between the KRas;Atg5fl/fl cohort and controls. (b) Western blot analysis for Atg5, LC3I and LC3II and p62 in purified primary lung tumour cells. Representative data are shown for tumour cells isolated from KRas;Atg5fl/+ and KRas;Atg5fl/fl mice 18 weeks after AdCre inhalation. β-actin is shown as a loading control. (c) Immunohistochemistry to detect LC3I, p62 and Atg5 in tumours from KRas;Atg5fl/+ and KRas;Atg5fl/fl mice 18 weeks after AdCre inhalation. Note highly elevated levels of LC3I and p62, both indicative of defective autophagy, in KRas;Atg5fl/fl tumours. Scale bars, 50 μm. (d,e) Impaired formation of autophagosomes (arrows) in KRas;Atg5fl/fl tumour cells. (d) Representative electron microscopy images are shown for KRas;Atg5fl/+ and KRas;Atg5fl/fl tumours 18 weeks after AdCre inhalation. Scale bars, 5 μm for upper and 2 μm for lower panels. M, mitochondria; *, lamellar bodies (Corpuscula lamellariae), rare cell organelles containing surfactant lipoproteins characteristic for type II pneumocytes. (e) Mean percentages (±s.e.m.) of tumour cells with detectable autophagosomes or autolysosomes, counted on electron microscopy images. n>=8 mice per genotype and at least 20 intact cells for each electron microscopy section were counted. ***P0.001 (χ2-test of a generalized linear model with logit link assessing the genotype effect).
A dual role for autophagy in a murine model of lung cancer (2014)
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(a) Representative electron microscopy images of autolysosomes (left panels), autophagosomes (middle panels) and mitophagy (right panels), all indicated by arrows, in lung tumour cells from control KRas;Atg5fl/+ mice 18 weeks after AdCre inhalation. Note the near-to-complete absence of such structures in the lung tumour cells from KRas;Atg5fl/fl littermates. Very rarely, we found cells with one autolysosome (left panels) or one single autophagosome (middle panels; arrows) in KRas;Atg5fl/fl tumours, whereas such structures can frequently be seen within single KRas;Atg5fl/+ tumour cells. Of note, the presence of a single autophagosome or a single autolysosome was scored as a positive event in Fig. 1e. Also note aberrant mitochondrial morphologies and increased mitochondrial numbers in KRas;Atg5fl/fl tumour cells. * indicates lamellar bodies. Scale bars, 2 μm. (b) Mean numbers (±s.e.m.) of mitochondria per lung tumour cell from KRas;Atg5fl/+ and KRas;Atg5fl/fl mice 18 weeks after tumour induction. Numbers of mitochondria were determined by electron microscopy. n≥8 mice per genotype and a minimum of 20 intact cells were counted per sample. ***P0.001 (χ2-test assessing the genotype effect in a generalized linear model with log link). (c) Mitochondria (arrows) in KRas;Atg5fl/+ and KRas;Atg5fl/fl lung tumour cells 18 weeks after AdCre inhalation. Note markedly altered mitochondrial morphology, that is, disorganized matrix structures, rounding and enlargement in the autophagy-defective KRas;Atg5fl/fl cells. Au, autophagosomes; Au-L, autolysosome; *, lamellar bodies. Scale bars, 2 μm. (d–g) Bioenergetics profiling of purified tumour cells from KRas;Atg5fl/+ and KRas;Atg5fl/fl mice (e) 6 weeks and (g) 18 weeks after tumour induction. Representative experimental data are shown. Oxygen consumption rates (OCR) were used to detect the bioenergetics profiles of purified tumour cells using a Seahorse Bioscience 24XF extracellular flux analyser. OCR±s.e.m. were recorded to construct bioenergetics profiles for basal respiration (BR), ATP turnover (AT), respiratory capacity (RC), spare respiratory capacity (SRC) and proton leakage (PL). A minimum of seven different samples were analysed for each group. *P0.05; **P0.01(unpaired, two-sided t-test).
A dual role for autophagy in a murine model of lung cancer (2014)
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(a) Increased tumour initiation in KRas;Atg5fl/fl mice as compared with KRas;Atg5fl/+ littermates. Representative histological sections at 2 weeks after AdCre inhalation are shown. Insets show typical lung lesions. Haematoxylin and eosin staining. Scale bars, 2 mm for whole lung section; 50 μm for insets. (b) Quantification of numbers of hyperplastic lesions/per lung (mean values±s.e.m.) in KRas;Atg5fl/+ and KRas;Atg5fl/fl mice 2 weeks after AdCre inhalation. n=6 per group. **P0.01 (χ2-test assessing the genotype effect in a generalized linear model with log link). (c) Tumour growth curves based on mean tumour-to-lung area ratios (±s.e.m.) determined in KRas;Atg5fl/+ and KRas;Atg5fl/fl littermates at the indicated time points. At least three different planes from each lung were analysed. n≥6 per cohort for each time point. *P0.05, **P0.01 (unpaired, two-sided Wicoxon test). (d) MicroCT analysis of lung tumours of KRas;Atg5fl/+ and KRas;Atg5fl/fl littermate mice 18 weeks after AdCre inhalation. Representative data from individual mice are shown. Scale bars, 5 mm.
A dual role for autophagy in a murine model of lung cancer (2014)
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(a) Distribution and (b) absolute numbers of hyperplastic lesions, adenomas and adenocarcinomas in KRas;Atg5fl/+ and KRas;Atg5fl/fl littermate mice 6, 12 and 18 weeks post AdCre infection. Data are shown as means±s.e.m.; n=6 per group. Combining the tumour progression data from 6, 12 and 18 weeks, a cumulative linked mixed model (clmm) analysis showed also a significant dependency of tumour progression on the genotype; P0.05 (χ2-test of clmms assessing the genotype effect). Applying generalized linear-mixed effects (glmer) models with logit link to individual time points, shows a significant dependency of the transition from hyperplastic lesions to adenomas (12 weeks, P0.05) and from adenomas to adenocarcinomas (12 weeks, P0.01 and 18 weeks, P0.01) on the mouse genotype (the last three P-values are from χ2-tests of a generalized linear models with logit link assessing the genotype effect). (c,d) Histological analyses (haematoxylin and eosin staining) of lung tumours in KRas;Atg5fl/+ and KRas;Atg5fl/fl littermates 18 weeks after AdCre inhalation. (c) Nuclear and cellular polymorphy, more dense and sometimes irregular chromatin as well as more nucleoli in tumour cells of KRas;Atg5fl/+ mice as compared with KRas;Atg5fl/fl tumours. Sections were analysed 18 weeks after AdCre inhalation. Scale bars, 50 μm. (d) KRas;Atg5fl/fl tumours exhibited markedly increased apoptotic figures and necrotic areas in the centre of larger tumours (asterisk).
A dual role for autophagy in a murine model of lung cancer (2014)
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(a,b) Induction of Atg5 deletion in pneumocytes isolated from KRas;Atg5fl/+ and KRas;Atg5fl/fl mice. (a) Primary pneumocytes were infected with AdCre for 96 h and Atg5 protein expression determined by western blot. β-actin is shown as a loading control. (b) Atg5 mRNA expression was determined by quantitative PCR. Data are shown as mean relative mRNA expression±s.e.m. in KRas;Atg5fl/fl tumours as compared with tumours from KRas;Atg5fl/+ mice (set to 1). Values were normalized to β-actin mRNA expression. n=3 per group. (c) GO terms showed enrichment of genes annotated for locomotion and leukocyte migration, cell adhesion, blood vessel remodelling, immune responses, oxidative stress or hypoxia in KRas;Atg5fl/fl as compared with control KRas;Atg5fl/+ pneumocytes. Enrichment and vizualization analysis was performed using GOrilla and REViGO, with colour depth indicating the enrichment significance (P-value). Differentially expressed genes between KRas;Atg5fl/fl and KRas;Atg5fl/+ pneumocytes were selected using DESeq (‘blind’ variance estimation, fold-change>2, adjP0.05, false discovery rate adjusted for multiple testing).
A dual role for autophagy in a murine model of lung cancer (2014)
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(a) Ingenuity analysis of genes differentially expressed between KRas;Atg5fl/+ and KRas;Atg5fl/fl pneumocytes determined by RNAseq (set selection using DESeq with adjP0.05, fold-change>2 cutoffs). Biological functions overrepresented in the differentially expressed gene sets are shown with the respective Bonferroni–Hochberg P-value indicating enrichment significance. (b) Representative immunohistochemistry and quantitative assessment of γH2AX and phospho-Chk2 in lung tumours from KRas;Atg5fl/+ and KRas;Atg5fl/fl littermates 18 weeks after AdCre inhalation. For γH2AX and phospho-Chk2 staining, sections were scored independently in a double-blinded manner. Data are shown as mean percentages (±s.e.m.) of positive cells per total cell numbers. n=6 mice per genotype. Scale bars, 50 μm. ***P0.001 (χ2-test of a generalized linear model with logit link assessing the genotype effect). (c) Representative histological lung sections of KRas;Atg5fl/+;p53fl/fl and KRas;Atg5fl/fl;p53fl/fl littermate mice 16 weeks after AdCre inhalation. Scale bars, 2 mm. (d) Distribution of hyperplastic lesions, adenomas and adenocarcinomas (±s.e.m.) in KRas;Atg5fl/+;p53fl/fl and KRas;Atg5fl/fl;p53fl/fl littermates 16 weeks post AdCre infection (n=4 per genotype). (e) The Kaplan–Meier survival curves for KRas;Atg5fl/+;p53fl/fl (n=8) and KRas;Atg5fl/fl;p53fl/fl (n=9) littermate mice injected intranasally with AdCre (2.5 × 107 p.f.u.). There was no statistical difference between the cohorts (log-rank test).
A dual role for autophagy in a murine model of lung cancer (2014)
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(a,b) Representative immunohistochemistry and quantitative assessment of (a) tumour-infiltrating CD3ε-positive T cells and (b) FoxP3+ Tregs in lung tumours from KRas;Atg5fl/+ and KRas;Atg5fl/fl littermates 6 weeks after AdCre inhalation. Data are shown as mean percentages (±s.e.m.) of the indicated cell types per total cell numbers in the tumour areas. Only cells within tumours were scored. At least six mice per genotype were analysed. Scale bars, 50 μm. ***P0.001(χ2-test assessing the genotype effect in a generalized linear model with logit link). (c) Representative immunohistochemistry and quantitative assessment of FoxP3+ Tregs in lung tumours from KRas;Atg5fl/+ and KRas;Atg5fl/fl littermates 2 weeks after AdCre inhalation. Data are shown as mean percentages (±s.e.m.) of FoxP3+ cells among total cell numbers in the tumour areas. Only cells within tumours were scored. At least six mice per genotype were analysed. Scale bars, 50 μm. **P0.01(χ2-test assessing the genotype effect in a generalized linear model with logit link). (d) FACS blots to detect CD25+FoxP3+ Treg cells in lungs 2 weeks after AdCre inhalation. Cells were gated on CD4+ populations. Numbers indicate percentages among total CD4+ cells in the lungs. (e) Mediastinal lymph node and lung cells were isolated from KRas;Atg5fl/+ and KRas;Atg5fl/fl littermates 2 weeks after AdCre inhalation and stained for flow cytometric analysis as described in Methods. The left panel shows the percentage of CD4+ T cells among all lung-infiltrating CD45+ haematopoietic cells (±s.e.m.). The right panel shows the percentage of FoxP3+ cells among CD4+ T cells in mediastinal lymph nodes and lungs (±s.e.m.). n=4 per genotype; *P0.05 (unpaired, two-sided t-test).
A dual role for autophagy in a murine model of lung cancer (2014)
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(a) Quantification of numbers of hyperplastic lesions/per lung (mean values±s.e.m.) in KRas;Atg5fl/+ and KRas;Atg5fl/fl mice 2 weeks after AdCre inhalation treated either with an isotype control Ab or a depleting anti-CD25 Ab. n=4 per group. **P0.01 (χ2-test assessing the genotype effect in a generalized linear model with log link). (b) Histological lung sections of KRas;Atg5fl/+ and KRas;Atg5fl/fl littermate mice treated with an isotype control Ab or anti-CD25-immunodepleting Abs. Mice were killed 2 weeks after AdCre inhalation and their lungs were processed for histopathology. Representative lung images (haematoxylin and eosin (HE) staining) are shown for each genotype and treatment. Scale bars, 2 mm. (c) Quantification of numbers of hyperplastic lesions/per lung (mean values±s.e.m.) in KRas;Atg5fl/+ and KRas;Atg5fl/fl mice 2 weeks after AdCre inhalation treated either with an isotype control Ab or an Ab specific to FR4. n=4 per group. ***P0.001 (χ2-test assessing the genotype effect in a generalized linear model with log link). (d) Histological lung sections of KRas;Atg5fl/+ and KRas;Atg5fl/fl littermate mice treated with isotype control Abs or Abs specific to FR4. Mice were killed 2 weeks after AdCre inhalation and their lungs were processed for histopathology. Representative lung images (HE staining) are shown for each genotype and treatment. Scale bars, 2 mm.
A dual role for autophagy in a murine model of lung cancer (2014)
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(a) Ingenuity analysis identifies Hif1α as a key upstream regulator (activation z-score=2.003; P=9.43e−08) that connects to a set of upregulated (red node) or downregulated (green nodes) genes in isolated KRas;Atg5fl/fl, non-transformed pneumocytes 48 h after infection with AdCre. Expression was determined by RNAseq. Orange edge, leads to activation; yellow edge, inconsistent state; grey edge, effect not predicted. (b) Protein expression of Hif1α, Atg5 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in isolated KRas;Atg5fl/+ and KRas;Atg5fl/fl pneumocytes (analysed at 4 days after AdCre infection) and tumours purified from KRas;Atg5fl/+ and KRas;Atg5fl/fl littermate mice at week 2 after AdCre inhalation. Quantifications of the hypoxia-inducible factor (HIF)1α/GAPDH ratios are shown for each lane; two independent blots are quantified. (c) CD39, Atg5 and as a control β-actin protein expression in isolated Atg5fl/+, Atg5fl/fl, KRas;Atg5fl/+ and KRas;Atg5fl/fl pneumocytes. Protein lysated were analysed at 4 days after AdCre infections by western blot. Quantifications of the CD39/β-actin ratios are shown for each lane, normalized to the Atg5fl/+ samples. Two independent blots are quantified. (d,e) CD39, Atg5 and as a control β-actin protein expression in isolated KRas;Atg5fl/+ and KRas;Atg5fl/fl pneumocytes treated with the (d) HIF1α inhibitor 3-(2-(4-adamantan-1-yl-phenoxy)-acetylamino)-4-hydroxybenzoic acid methyl ester or left untreated and (e) the Nrf2 inhibitor Brusatol (100 nM) or the ROS scavenger N-acetyl-L-cysteine (5 mM). Protein lysates were analysed on day 4 by western blot. Quantifications of the CD39/β-actin ratios are shown for each lane. The inhibitors were present during the entire 4day culture period.
A dual role for autophagy in a murine model of lung cancer (2014)
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(a) Quantification of numbers of hyperplastic lesions/per lung (mean values±s.e.m.) and (b) representative histological lung sections (haematoxylin and eosin (HE) staining) of KRas;Atg5fl/+ and KRas;Atg5fl/fl mice 2 weeks after AdCre inhalation treated in vivo either with either vehicle control or POM1 (intraperitoneally; i.p. 10 mg kg−1 daily) to inhibit CD39 activity. n=4 per group. ***P0.001 (χ2-test assessing the genotype effect in a generalized linear model with log link). Scale bars, 2 mm. (c) Quantification of numbers of hyperplastic lesions/per lung (mean values±s.e.m.) and (d) representative histological lung sections (HE staining) of KRas;Atg5fl/+ and KRas;Atg5fl/fl mice 2 weeks after AdCre inhalation treated either with either vehicle control or the adenosine receptor blocker PSB1115 (i.p. 10 mg kg−1 daily). n=4 per group. **P0.01 (χ2-test assessing the genotype effect in a generalized linear model with log link). Scale bars, 2 mm. (e,f) Representative immunohistochemistry and quantitative assessment of FoxP3+ Tregs in lung tumours from KRas;Atg5fl/+ and KRas;Atg5fl/fl littermates treated with vehicle control or the CD39 blocker polyoxometalate-1 (POM1) and treated with vehicle or the adenosine receptor inhibitor PSB1115 (4-(2,3,6,7-tetrahydro-2,6-dioxo-1-propyl-1H-purin-8-yl)-benzenesulphonic acid potassium salt). Mice were killed 2 weeks after AdCre inhalation and their lungs were processed for immunohistopathology. Data are shown as mean percentages (±s.e.m.) of FoxP3+ cells among total cell numbers in the tumour areas. Only cells within tumours were scored. At least four mice per genotype were analysed. Scale bars, 50 μm. **P0.01. NS, not significant; (χ2-test assessing the genotype effect in a generalized linear model with logit link).