EMBO/soda-nel huggingface dataset:
Genome‐wide screen identifies signaling pathways that regulate autophagy during Caenorhabditis elegans development (2014)
Figure 4

ATFS‐1‐mediated mitochondrial stress suppresses the rpl‐43 phenotype

A Phsp‐60::GFP is weakly expressed in wild‐type animals.
B egl‐46(RNAi) causes upregulation of Phsp‐60::GFP.
C Percentage of indicated mutant animals with different levels of SQST‐1::GFP aggregates. S: strong. M: medium. N: none. >30 animals were examined in each group.
D Levels of SQST‐1::GFP in various genetic mutants. 200 young adult animals for each genotype were collected for analysis.
E, F Loss of function of atfs‐1 suppresses the upregulation of Phsp‐60::GFP in egl‐46(n1126) mutants. (E): DIC image of (F).
G, H The dramatic decrease in the number of SQST‐1::GFP aggregates in the intestine in rpl‐43(bp399); egl‐46(RNAi) mutants (G) is restored by simultaneous loss of function of atfs‐1(gk3094) (H).
I, J GFP::LGG‐1 forms numerous puncta in the intestine in egl‐46(RNAi) (I), gfi‐1(RNAi) (J) animals.

Data information: Scale bars: 100 μm (A, B, E, F); 20 μm (G, H); 10 μm (I, J).
Autophagy proteins regulate ERK phosphorylation (2013)
f1

(a) EGF enhances colocalization of phosphorylated (P)-bRAF, P-MEK and P-ERK with LC3. Immunofluorescence (IF) showing colocalization of P-bRAF (green), P-MEK (green) and P-ERK (green) with LC3 (red) in NIH/3T3 cells in presence or absence of EGF (10 min as shown in scheme). The bars represent mean±s.e.m. **P0.01, ***P0.001; Student’s t-test, 60 cells analysed from two experiments. Scale bars, 10 μm. (b) P-ERK colocalizes with pre-autophagosomal and autophagosomal structures. IF depicting colocalization of P-ERK (green or red) with LC3 (red), ATG5–ATG12 (red), ATG16 (green), vps34 (red), WIPI1 (red), WIPI2 (red) and P-ULK1 (red) in EGF-treated NIH/3T3 cells. Scale bars, 10 μm. The bars represent mean±s.e.m. 50 cells analysed from two experiments. (c) Growth factors in serum maintain P-ERK/LC3 colocalization. IF depicting colocalization of P-ERK (green) with LC3 (red) in 2 h serum-fed NIH/3T3 cells. (a–c) Native merged images or images with colocalization highlighted in white pixels are shown. Nuclei are blue (DAPI). Scale bar, 10 μm.
Autophagy proteins regulate ERK phosphorylation (2013)
f2

(a) P- and total MEK and ERK are enriched in APh in vivo. Immunoblots for indicated proteins in homogenate (Hom), APh, APL and Lys fractions from livers of fed mice, n=4. (b) EGF enhances enrichment of P-MEK and ERK in APh fractions in vitro. Immunoblots for indicated proteins in Hom, APh and APL fractions from NIH/3T3 cells in presence or absence of EGF (10 min). The bars represent mean±s.e.m. *P0.05, **P0.01; Student’s t-test, n=3. (c) P- and total MEK and ERK localize on to the cytoplasmic/extra-luminal face of APh in vivo. Left; model depicting localization of MEK and ERK on the extra-luminal face of autophagic vesicles and the ability of trypsin to degrade extra-luminal MEK and ERK, and right; immunoblots for indicated proteins in APh fractions from mice livers untreated (−) or treated with increasing amounts of trypsin for 15 min. The bars represent mean±s.e.m. *P0.05, **P0.01, ***P0.001 compared with corresponding trypsin-untreated value; ANOVA–Bonferroni post hoc test, n=3. The p44, p42 forms of ERK, LC3-I and membrane-associated LC3-II, and 37 kDa and 25 kDa forms of cathepsin (Cath) B are indicated.
Autophagy proteins regulate ERK phosphorylation (2013)
f3

(a) EGF enhances nuclear LC3-II content in hepatocytes. Immunofluorescence (IF) depicting nuclear LC3-II in RALA hepatocytes in presence or absence of EGF (10 min). Native/inverted images are shown. Scale bar, 5 μm. Bars represent mean±s.e.m. ***P0.001 compared with control (Con); Student’s t-test, 50 cells from two experiments. (b) EGF enhances nuclear P-ERK/LC3-II colocalization. IF depicting P-ERK (green)/LC3-II (red) colocalization in untreated (Con) or EGF-treated NIH/3T3 cells. Native images (top)/images with colocalization in white pixels (bottom) are shown. Scale bar, 5 μm. Bars represent mean±s.e.m. ***P0.001 compared with Con; Student’s t-test, 50 cells from two experiments. (c) Adipogenic differentiation increases nuclear LC3-II. Images depict nuclear LC3-II in 3T3-L1 preadipocytes in maintenance or differentiation medium (2 h). Scale bar, 5 μm. Bars represent mean±s.e.m. **P0.01 compared with Con; Student’s t-test, 50 cells from two experiments. (d) Nuclear P-ERK/LC3-II colocalization in serum-fed cells. IF showing P-ERK (green)/LC3 (red) colocalization in 2 h serum-fed NIH/3T3 cells. Native images (top)/images with colocalization in white pixels (bottom) are shown. Scale bar, 5 μm. (e) LC3 interacts with ERK in vivo. Immunoblots showing co-immunoprecipitation of LC3 with ERK, MEK and bRAF in homogenate (Hom) (e, top), and of LC3 with P- and total ERK in nuclear fractions from mouse livers (e, bottom). (f) Blocking nuclear transport decreases EGF-induced increase in nuclear LC3-II. LC3 IF (red) in EGF-treated NIH/3T3 cells pre-exposed (30 min) or not to WGA. Bars represent mean±s.e.m. ***P0.001 compared to with; Student’s t-test, 60 cells from n=3. Scale bar, 10 μm. (g) Blocking nuclear transport decreases nuclear ERK content. ERK IF (green) in EGF-treated NIH/3T3 cells pre-exposed (30 min) or not to WGA. Bars represent mean±s.e.m. ***P0.001 compared with Con; Student’s t-test. (h) Blocking nuclear transport does not modify P-ERK/LC3-II colocalization. IF depicting nuclear P-ERK (green)/LC3 (red) colocalization in EGF-treated NIH/3T3 cells pre-exposed or not to WGA. For (g) and (h): Scale bar, 10 μm, bars are mean±s.e.m. 50 cells from n=2. Nuclei are blue (DAPI). Arrows indicate LC3 puncta, ERK or colocalization.
Autophagy proteins regulate ERK phosphorylation (2013)
f4

(a) Atg7−/− livers display decreased ERK phosphorylation. Immunoblots for the indicated proteins in liver homogenate (Hom) and cytosolic (Cyt) fractions of control (Con) and liver-specific Atg7−/− mice are shown. The bars represent mean±s.e.m. *P0.05, **P0.01, ***P0.001 compared with Con; Student’s t-test, n=4. (b) Atg7−/− livers display decreased ERK2 dimers. Immunoblots show the indicated proteins in Hom from Con and Atg7−/− livers. The bars represent mean±s.e.m. *P0.05, ***P0.001 compared to Con; Student’s t-test, n=4–5. Arrows indicate p42 monomers and dimers. (c) Atg7−/− brown adipose tissues (BAT) display decreased ERK phosphorylation. Immunoblots for indicated proteins in Hom from Con and Atg7−/−BAT. The bars represent mean±s.e.m. **P0.01 compared with Con; Student’s t-test, n=4–6. (d) Atg7−/− livers exhibit decreased nuclear ERK phosphorylation. Immunoblots show the indicated proteins in homogenate (Hom, lane 1) and nuclear fractions (lanes 2–5) from Con and Atg7−/− livers. The bars represent mean±s.e.m. ***P0.001 compared with Con; Student’s t-test, n=4–5. (e) LC3 C terminus glycine-deleted (ΔG) mutants exhibit decreased ERK phosphorylation. Immunoblots for indicated proteins in total lysates from CFP-LC3- and CFP-LC3ΔG-transfected NIH/3T3 cells exposed or not to EGF (10 min). The bars represent mean±s.e.m. ***P0.001 compared with Con; Student’s t-test, n=8.
Autophagy proteins regulate ERK phosphorylation (2013)
f5

(a) Atg5−/− MEFs display reduced ERK phosphorylation during nutrient deprivation. Quantification of P-ERK levels normalized to total ERK (detected by immunoblotting) in WT and Atg5−/− MEFs cultured in the absence of serum for indicated times. The bars represent mean±s.e.m. *P0.05 compared with corresponding WT value; ***P0.001, ****P0.0001 compared with 10 min serum-starved Atg5−/− MEFs; ANOVA-Bonferroni post hoc test, n=3. (b) Serum-deprived Atg5−/− MEFs display decreased ERK and p90RSK phosphorylation. Immunoblots for the indicated proteins in total lysates from 2 h serum-deprived WT and Atg5−/− MEFs. The bars represent mean±s.e.m. *P0.05, ***P0.001 compared with Con; Student’s t-test, n=3. (c) Atg5−/− MEFs display decreased nuclear ERK activity. Immunoblots for phosphorylated HA (haemagglutinin)-tagged Elk1 in total lysates from 2 h serum-deprived WT and Atg5−/− MEFs cells exposed or not to EGF (10 min). (d) Atg5−/− MEFs display decreased Elk1-driven gene expression. Elk1-driven ZFP36 mRNA levels from 2 h serum-deprived WT and Atg5−/− MEFs cells exposed or not to EGF (10 min). The bars represent mean±s.e.m. ***P0.001 compared with Con; Student’s t-test, n=3. (e) Decreased ERK phosphorylation in Atg5−/− MEFs occurs independently of changes in ERK phosphatases. Immunoblots for the indicated proteins in 2 h serum-deprived WT MEFs transfected with scrambled siRNA (scr), and Atg5−/− MEFs transfected with scr or siRNAs against MKP3 or PP2A in the presence or absence of EGF. The bars represent mean±s.e.m., n=3.
Autophagy proteins regulate ERK phosphorylation (2013)
f6

(a) Mutations in FRS on ERK2 decrease colocalization of ERK2 with ATG5–ATG12, LC3 and WIPI1. Immunofluorescence (IF) showing colocalization (depicted as white pixels by ‘colocalization finder application’) of WT-ERK2-HA, FRS ERK2 mutants (L198A-, L232A-, L198A/L232A-, Y261A-ERK2-HA) or common docking (CD) mutant (D319N-ERK2-HA) with ATG5–ATG12 (panels 1–6), LC3 (panels 7–12) or WIPI1 (panels 13–18) in EGF-treated NIH/3T3 cells. ERK2 is stained in red, and autophagy proteins are stained in green. Scale bar, 10 μm. The bars represent mean±s.e.m. **P0.01, ****P0.0001 compared with WT-ERK2-transfected cells; Student’s t-test, 50 cells analysed from n=2. (b) Mutations in FRS on ERK2 decrease colocalization of ERK2 with nuclear ATG5–ATG12 or LC3. IF showing colocalization (white pixels) of WT-ERK2-HA (red), FRS ERK2 mutants (L198A-, L232A-, L198A/L232A-, Y261A-ERK2-HA) or common docking (CD) mutant (D319N-ERK2-HA) with ATG5–ATG12 (panels 1–6) or LC3-II (panels 7–12) in EGF-treated NIH/3T3 cells. Scale bar, 5 μm. The bars represent mean±s.e.m. **P0.01, ***P0.001, ****P0.0001 compared with WT-ERK2-transfected cells; Student’s t-test, 50 cells analysed from n=2. Arrows depict colocalization. (c) ATG5–ATG12 conjugation is required for ERK phosphorylation. Immunoblots for P-ERK, total ERK and β-actin in NIH/3T3 cells transfected with WT ATG5 or the conjugation-defective ATG5 K130R mutant and treated with EGF (10 min). The bars represent mean±s.e.m. *P0.05 compared with WT ATG5-transfected cells; Student’s t-test, n=3.
Autophagy proteins regulate ERK phosphorylation (2013)
f7

(a) ATG4B depletion increases steady-state LC3-II levels. Immunoblots for indicated proteins in NIH/3T3 cells transfected with scrambled siRNAs (scr) or siRNAs against ATG4B (siATG4B) in presence/absence of EGF. Bars represent mean±s.e.m. *P0.05, ***P0.001 compared with scr; Student’s t-test, n=4. (b) siATG4B cells exhibit increased autophagic vesicles. Representative electron micrographs depicting autophagic vesicles (indicated by black arrows) in scr and siATG4B NIH/3T3 cells. (c) ATG4B depletion does not increase autophagic flux in serum-fed cells. Immunoblots for indicated proteins in lysates from scr or siATG4B NIH/3T3 cells in presence/absence of lysosomal inhibitors, ammonium chloride and leupeptin (Inh) for 2 h. Scale bars, 1 μm. Bars represent mean±s.e.m., n=3. (d) siATG4B cells display increased nuclear LC3-II. Images for LC3-II (red) in scr or siATG4B NIH/3T3 cells in presence/absence of EGF (10 min). Scale bar, 5 μm. Bars represent mean±s.e.m. ***P0.001 compared with scr; Student’s t-test, 60 cells from n=2. (e) ATG4B deficiency increases P-ERK/LC3-II colocalization. Immunofluorescence (IF) for P-ERK (green)/LC3 (red) colocalization in scr or siATG4B NIH/3T3 cells in presence/absence of EGF (10 min). Scale bars, 10 μm. Bars represent mean±s.e.m. **P0.01, ***P0.001 compared with scr; Student’s t-test, 60 cells from n=2. (f) ATG4B-deficient cells display increased nuclear P-ERK/LC3 colocalization. IF depicting P-ERK (green)/LC3 (red) colocalization in nuclei of scr or siATG4B NIH/3T3 cells in presence/absence of EGF. Scale bars, 5 μm. Bars represent mean±s.e.m. **P0.01, ***P0.001 compared with scr; Student’s t-test, 60 cells from n=2. (g) ATG4B deficiency augments EGF-induced MEK and ERK phosphorylation. Immunoblots for P- and total MEK and ERK, LC3 and GAPDH in total lysates from scr or siATG4B NIH/3T3 cells in presence/absence of EGF. Bars represent mean±s.e.m., n=3. (h) ATG4B deficiency increases nuclear P-ERK levels. Native (top)/inverted images (bottom) showing nuclear P-ERK content. P-ERK fluorescence in untreated (Con)/EGF-treated scr or siATG4B NIH/3T3 cells is shown. Scale bars, 5 μm. Bars represent mean±s.e.m. **P0.01, ***P0.001 compared with scr; Student’s t-test, 60 cells from n=2. Nuclei are blue (DAPI).
Pancreatic Cancer Intrinsic PI3Ka Activity accelerates Metastasis and rewires Macrophage Component (2021)
Figure 2

Figure 2: Full annihilation of PI3Kα activity is required to prevent pancreatic cancer cell migration.

A, In vitro IC50 of PI3K inhibitors, obtained on recombinant proteins and based on the literature. The Greek letter on the x axis indicates the most potently targeted isoform.

B-D B, Murine pancreatic tumour cells R211 were treated for 15min with the vehicle, α, β, β/δ, γ-specific or pan-PI3K inhibitors at 1 or 10 µM in the presence of 10% FBS and the protein levels of pAkt (Ser473 and Thr308), total Akt and β Actin were observed by western blot. P-Akt on C, Ser473 and D, Thr308 were quantified and normalised with β Actin. n = 2 in each group.

E, Murine pancreatic tumour cells R211 were treated with the vehicle, α, β, β/δ, γ-specific or pan-PI3K inhibitors at 0.1 or 1 µM and simultaneously subjected to a Boyden chamber migration assay. Migrating cells were quantified after 24h. Representative image of filter after Crystal violet staining is shown. Scale=200µm. n = 3 in each group.

F, Murine pancreatic tumour cells R211 were treated with vehicle, BYL-719 1 µM, AZD4547 2 µM or both treatments and subjected to a Boyden chamber migration assay. Migrating cells were quantified after 24h. n = 4 in each group.

G, Murine pancreatic tumour cells R211 were treated with the vehicle, α, β, β/δ, γ-specific or pan-PI3K inhibitors and living cells were quantified after 3 days with a MTT colorimetric assay. Metabolically active cells are considered as living cells. n ≥ 3 in each group.

H, Murine pancreatic tumour cells R211 were treated with the vehicle, α or pan-PI3K inhibitors and apoptotic (IncuCyte Annexin V) cells were quantified after 2 days. n = 3 in each group.

I, Relative p110α, β, γ or δ mRNA expression (compared to scramble siRNA) after inhibition of expression by siRNA targeting p110α or a combination of siRNA targeting each class I PI3K isoform. n ≥ 3 in each group.

J, R211 and PDAC8661 cells were treated with siRNA scramble, siRNA targeting p110α or a combination of siRNA targeting each class I PI3K isoform (pools) and subjected to a Boyden chamber migration assay. Migrating cells were quantified after 24h. n = 3 in each group.

K, Relative p110α or β mRNA expression (compared to scramble shRNA stably transduced-cells) in human pancreatic tumour cells PANC-1 stably transduced with shRNA targeting p110α or β. n = 4 in each group.

L, PANC-1-transduced cells were subjected to a Boyden chamber migration assay. Migrating cells were quantified after 24h. n = 5 in each group.

Data information: Mean +/- SEM (* p<0.05, ** p<0.01, *** p<0.001, n ≥ 3 independent experiments except for WB experiment, Student's t-test. When not precised, comparisons are performed with vehicle.
Pancreatic Cancer Intrinsic PI3Ka Activity accelerates Metastasis and rewires Macrophage Component (2021)
Figure 3

Figure 3: PI3Kα inhibition regulates selective PI-3,4,5-P3 species.

A-B A, Murine pancreatic tumour cells R211 were treated for 15 min with the vehicle, α-specific A66 or pan-PI3K inhibitor BKM120 at 0.01, 0.05, 0.1, 0.5, 1 or 5 µM in the presence of 10% FBS and protein levels of P-Akt (Ser473), total Akt and β Actin were observed by western blot. B, P-Akt on Ser473 was quantified and normalised with β Actin. n = 3 in each group.

C-D R211 cells were treated with the vehicle, α-specific inhibitor A66 or pan-PI3K inhibitor BKM120 at 1 µM and C, living cells were quantified after 3 days with a MTT colorimetric assay or D, migrating cells were quantified after 24h in a Boyden chamber assay and compared to vehicle (DMSO). n = 3 in each group.

E, Murine pancreatic tumour cells R211 were treated for 15 min with the vehicle (0.01% DMSO) or α-specific A66 or pan-PI3K inhibitor BKM120 at 1µM. Phospholipids were extracted, total PIP, PIP2 and PIP3 were quantified and compared to the vehicle (DMSO). n = 3 in each group.

F, the proportion of each PIP3 sub-type was represented. Sub-types whose proportion was modified compared to DMSO were underlined. n = 3 in each group.

Data information: Mean +/- SEM (* p<0.05, ** p<0.01, *** p<0.001, n ≥ 3 independent experiments, Student's t-test).
Pancreatic Cancer Intrinsic PI3Ka Activity accelerates Metastasis and rewires Macrophage Component (2021)
Figure 4

Figure 4: PI3Kα inhibition is effective on pro-tumoural features regardless of the genetic landscape of pancreatic adenocarcinoma.

A-B A, Migration inhibition capacities of PI3K inhibitors at 1 µM were tested on pancreatic tumour cells (R211, PDAC8661, PANC-1, 10593, 10158, R6344, R6430, R6065, R6141). The mean of these values was plotted against the in vitro IC50 for each class I PI3K and each inhibitor. IC50 is determined on recombinant proteins. Pearson correlation tests were performed and its p-values were presented separately for each class I PI3K isoform. B, individual p-values of these correlation tests were represented for each of the 9 pancreatic (R211, PDAC8661, PANC-1, 10593, 10158, R6344, R6430, R6065, R6141,) breast (MDA-MB-231, MDA-MB-468) and prostate (PC3) cancer cell lines; Pearson correlation p values for each PI3K isoform are plotted separately. n = 3 in each group.

C, Cytotoxic capacities of PI3K inhibitors at 10 µM were tested on the 10 pancreatic (R211, PDAC8661, DT4994, PANC-1, 10593, 10158, R6344, R6430, R6065, R6141), breast (MDA-MB-231, MDA-MB-468), prostate (PC3) and acute myeloid cells (NOMO-1, HL-60) and the correlation with the in vitro IC50 for each class I PI3K of each inhibitor was determined. Pearson correlation tests were performed, and p-values represented for each isoform and cell line. n ≥ 3 in each group.

Data information: The dotted red line corresponds to a threshold p-value of 0.05 obtained with a Pearson correlation test. Mean +/- SEM; n≥3 independent experiments.
Pancreatic Cancer Intrinsic PI3Ka Activity accelerates Metastasis and rewires Macrophage Component (2021)
Figure 5

Figure 5: cfDNA-positive PDAC present increased lethality and metastatic dissemination.

A, Representative images of PI3K activation (as shown by positive pAkt substrate staining) on the primary tumours of PDAC patients, with values of quantification of cfDNA and mutated KRAS allele frequency.

B, Quantification of pAkt substrate staining in T3 patients (total number of patients = 6, n = 2 in each group). Mean +/- SEM.

C, Survival curve of KPC mice presenting different levels of cfDNA. n in each group is indicated. Log rank test, *** p<0.001

D, Quantification of cfDNA in KPC mice at different stages of the disease. n in each group is indicated. PanIn= Pancreatic Intraepithelial Neoplasia. Non parametric Mann Whitney, * p<0.05, ** p<0.01, *** p<0.001.
Pancreatic Cancer Intrinsic PI3Ka Activity accelerates Metastasis and rewires Macrophage Component (2021)
Figure 6

Figure 6: Pharmacological PI3Kα inhibition prevents the rapid progression of cfDNA-positive PDAC. A, KPC mice diagnosed with an aggressive carcinoma were given daily oral doses of BYL-719 (50mg/kg) or of vehicule (0.5% methyl cellulose with 0.2% Tween-80). n = 6 in each group

B, The protein levels of pAkt (Ser473), total Akt, pERK (Thr202 and Tyr204) and total ERK were observed by western blot in spleen, lung and pancreas tissue lysates after treatment with oral doses of vehicle (0.5% methyl cellulose with 0.2% Tween-80) or of BYL-719 (50mg/kg).

C-E C, Quantification of tumour volume (expressed in fold change) and D,E, quantification of micro- and macro-metastatic foci and the relative metastasis area (comprising metastases in the spleen, liver and lung) of KPC mice treated with oral doses of vehicle (0.5% methyl cellulose with 0.2% Tween-80) or of BYL-719 (50mg/kg).

F, Development of ascites in KPC mice after treatment with oral doses of vehicle (0.5% methyl cellulose with 0.2% Tween-80) or of BYL-719 (50mg/kg).

G, Survival curve of KPC mice treated with the vehicle or BYL-719. Log rank test, * p<0.05.

H-J H, Representative images and i, quantification of Ki67 positive cells in pancreatic tumours and in J, metastasized liver sections after treatment with oral doses of vehicle (0.5% methyl cellulose with 0.2% Tween-80) or of BYL-719 (50mg/kg).

K-L K, Treatment regimen of the tail vein injection experiment in nude mice treated with the PI3Kα inhibitor or vehicle (n is indicated), quantification at two time points and L, representative images of luminescence via IVIS® Spectrum in vivo imaging system.

M, Quantification and representative CK19 IHC of R211-luc lung micro-metastasis from vehicle or BYL-719 treated mice. n=6 in vehicle; n=5 in BYL-719 group.

N, Tail vein injection experiment in C57/B6 mice of murine syngeneic pancreatic cancer cells (Kras mutated A338, Kras mutated and half PI3Kα inactive, A260; n>8 in each group), quantification of micro- and macro-metastasis at final time point.

Data information: Mean +/- SEM (* p<0.05, ** p<0.005) c-e, i, j, k, m, n: non parametric Mann Whitney; g: log rank test.
Pancreatic Cancer Intrinsic PI3Ka Activity accelerates Metastasis and rewires Macrophage Component (2021)
Figure 7

Figure 7: Tumour-intrinsic PI3Kα alters tumour cell chemokine secretion and promotes the acquisition of tumour-associated inflammatory macrophage characteristics in the peritumoural tissue.

A-D Blood count of A, white blood cells, B, monocytes, C, granulocytes and D, lymphocytes in KPC mice, with cohort size in each group.

E Basal level of IL-3 was quantified in KC;p110α+/+ (R211, A338 and A338L) and KC;p110α+/lox (A260 and A94L) cell lines isolated from mice primary tumours or lung metastases (L). Each point is a mean of n=3 independent values.

F, IL-3 relative level (compared to vehicle) was determined in 3 KC (R211, A338 and A338L) cell lines isolated from mice primary tumours under treatment with vehicle (DMSO) or 1µM of BYL-719. n=3 independent values.

G, Violin plot demonstrating the link between PI3Kα activation and activation of a selective population of γδLT in the PACA-AU cohort.

h-jH-J H, Representative images and quantification of I, F4/80 positive cells and J, CD206 positive cells after the pharmacological inhibition of PI3Kα. Black arrowheads show positive immune cells.

K,L Quantification of K, F4/80 and L, CD206 positive cells in KC and oncogenic PI3Kα mice.

M,N Quantification of CD206 positive cells in M, low-grade and N, high-grade PanIN lesions after the genetic inactivation of PI3Kα in epithelial pancreatic cells.

O, Quantification of CD206 positive in KPC mice at different stages of PDAC progression.

P, treatment of macrophage cell line with tumour-cell conditioned medium.

Q,R Quantification of secreted TNFα in indicated conditions. n=3 independent values for each cell line and treatment.

S, Murine pancreatic tumour cells R211 were treated with vehicle, TNF-α 20 ng/mL, BYL-719 1 µM or both treatments and subjected to a Boyden chamber migration assay. Migrating cells were quantified after 24h. n= 4 independent values.

Data information: n in each group is indicated in the figure. A-D, I-O, Mann-Whitney; F, Q-S, Student T-Test; G, ANOVA test corrected using the Benjamini & Hochberg method (BH) on SES (sample enrichment score). Mean +/- SEM (* p<0.05, ** p<0.005, *** p<0.0001).