EMBO/soda-nel huggingface dataset:
The onset of PI3K-related vascular malformations occurs during angiogenesis (2022)
Figure 4

Figure 4. Pik3ca-vascular malformations in murine retinas reproduce hallmarks of human disease.

(A) Representative images of EC-Pik3caH1047R/WT P6 retinas isolated from mice treated with 0.125 mg/kg of 4-OHT on P1. Retinas were immunostained for blood vessels (IB4). Red asterisks show arteries and arterioles and yellow arrowheads veins. Scale bars: 150 μm (left panel) and 30 μm (right, high magnification panels).

(B) Representative images of P6 retinas from control and EC-Pik3caH1047R/WT mice treated with 0.125 mg/kg 4-OHT on P1, following immunostaining for p-S6 (S235/236) and blood vessels (IB4). Scale bars: 150 μm (left panels) and 30 μm (right panels, high magnification).

(C) Representative control and EC-Pik3caH1047R/WT P6 retinas immunostained for blood vessels (IB4), EC nuclei (Erg) and EdU. Scale bars: 150 μm (left panels) and 30 μm (right panels, high magnification).

(D-G) Quantification of (D) p-S6 (S235/236) intensity (presented as a fold change of vehicle-treated control), (E) EC proliferation by EdU staining, (F) EC number by Erg positive cells and (G) retinal vascularity by IB4-positive area in control and EC-Pik3caH1047R/WT retinas. Error bars are s.e.m. n > 5 retinas per genotype. Statistical analysis was performed by nonparametric Mann-Whitney test. **p < 0.01 was considered statistically significant.
The onset of PI3K-related vascular malformations occurs during angiogenesis (2022)
Figure 5

Figure 5. Miransertib prevents the formation of Pik3ca-vascular malformations in mice.

(A) 4-OHT and miransertib dosing scheme used for a prevention therapeutic experimental setup.

(B, C) Representative images of P6 retinas isolated from control and EC-Pik3caH1047R/WT mouse littermates. Blood vessels were stained with IB4. Lower panels showing high magnification images of the representative areas showing (B) blood vessels (IB4), EC nuclei (Erg), EdU incorporation and (C) pS6 (S235/236). Scale bars: 150 μm (upper panel) and 30 μm (lower panels).

(D-G) Quantification of (D) retinal vascularity by IB4 staining, (E) EC number by Erg immunostaining, (F) EC proliferation by EdU staining, and (G) pS6 (S235/236) intensity (presented as a fold change of vehicle-treated control). Bars represent the mean ± s.e.m. n ≥ 4 retinas per genotype. Statistical analysis was performed by nonparametric Mann-Whitney test. *p < 0.05 and **p < 0.01 were considered statistically significant.
The onset of PI3K-related vascular malformations occurs during angiogenesis (2022)
Figure 6

Figure 6. Low dose of miransertib prevents the growth of Pik3ca-vascular malformations. (A) 4-OHT and miransertib dosing scheme used for a prevention experimental setup.

(B, C) Representative confocal images of P6 retinas isolated from control and EC-Pik3caH1047R/WT mouse littermates. Blood vessels were stained with IB4. Lower panels showing high magnification images of the representative areas showing (B) blood vessels (IB4), EC nuclei (Erg), EdU incorporation and (C) pS6 (S235/236). Scale bars: 150 μm (upper panels) and 30 μm (lower panels).

(D-G) Quantification of (D) retinal vascularity by IB4 staining, (E) EC number by Erg immunostaining, (F) EC proliferation by EdU staining, and (G) pS6 (S235/236) intensity (presented as a fold change of vehicle-treated control). Bars represent the mean ± s.e.m. n ≥ 3 retinas per genotype. Statistical analysis was performed by nonparametric Mann-Whitney test. *p < 0.05 and **p < 0.01 were considered statistically significant.
The onset of PI3K-related vascular malformations occurs during angiogenesis (2022)
Figure 7

Figure 7. Low dose miransertib induces regression of Pik3ca-vascular malformations in vivo.

(A) 4-OHT and miransertib dosing scheme used for a curative experimental setup.

(B, C) Representative confocal images of P6 retinas isolated from control and EC-Pik3caH1047R/WT mouse littermates. Blood vessels were stained with IB4. Lower panels showing high magnification images of the representative areas showing (B) blood vessels (IB4), EC nuclei (Erg), EdU incorporation and (C) pS6 (S235/236). Scale bars: 150 μm (upper panels) and 30 μm (lower panels).

(D-G) Quantification of (D) retinal vascularity by IB4 staining, (E) EC number by Erg immunostaining, (F) EC proliferation by EdU staining, and (G) pS6 (S235/236) intensity (presented as a fold change of vehicle-treated control). Bars represent the mean ± s.e.m. n ≥ 4 retinas per genotype. Statistical analysis was performed by nonparametric Mann-Whitney test. *p < 0.05, **p < 0.01 and ***p < 0.001 were considered statistically significant.
The onset of PI3K-related vascular malformations occurs during angiogenesis (2022)
Figure 8

Figure 8. Miransertib impairs cell viability of PIK3CA- and TEK-mutant patient-derived ECs. (A) Illustration showing the strategy of EC isolation from patient-derived biopsies.

(B) Representative sequencing chromatograms of PIK3CA and TEK mutant VM-derived ECs. Arrows show the detected point mutations.

(C) Representative confocal images of PIK3CA and TEK patient-derived ECs immunostained for VE-cadherin (EC-specific junctional protein) and ERG (EC-specific transcription factor). Cell nuclei were visualised with DAPI. Scale bars: 30 μm.

(D) Immunoblot showing the activation of PI3K/AKT/mTORC1 pathway (by assessing the levels of p-AKT and p-S6) among different PIK3CA and TEK patient-derived ECs. Primary HUVEC were used as wild-type control.

(E-F) Immunoblot showing the impact of miransertib at increasing doses on PI3K/AKT/mTORC1 pathway (by assessing p-AKT and p-S6 levels) in (E) PIK3CA and (F) TEK mutant patient-derived ECs.

(G) Wild-type (HUVECs), PIK3CA and TEK mutant EC viability upon the treatment with miransertib for 72h at different doses assessed by MTS assay. Fitting curves and 95% CI IC50 values for wild-type (HUVECs), PIK3CA and TEK ECs are shown. For PIK3CA and TEK-mutant ECs n = 3 biological replicates (patients); for wild-type ECs (HUVECs) n = 4 technical replicates PIK3CA-mutant vs. TEK-mutant p = 0.501; PIK3CA-mutant vs. HUVECs p = 0.0815; TEK-mutant vs. HUVECs: p = 0.4085. Statistical analysis was performed by comparison of best-fit values using the extra sum-of-squares F test.

Expanded View Figure Legends
Convergence of cMyc and β-catenin on Tcf7l1 enables endoderm specification (2015)
Figure 1

Figure 1. Suppression of GSK3 activity promotes endoderm production from naïve ES cells

(A) Quantification of CXCR4+ cells by flow cytometry and Sox17+ cells by immunostaining at day 7 following differentiation in the presence or absence of 3 µM CH. Average and standard deviation (SD) of eight independent experiments.

(B) Gene expression profiling by RT-PCR at day 7 differentiation. Average and SD of three independent experiments.

(C) Immunostaining for Ecadherin (left) and FoxA2 (right) at day 7 of differentiation in presence of 3 µM CH.

(D) Quantification of CXCR4+ by flow cytometry over a seven-day differentiation time course in the presence of 3 µM CH. Average and SD of three independent experiments.

(E) Immunostaining for Sox17 at day 4 to day 7 of differentiation in the continuous presence or absence of 3 µM CH. Scale bars = 200 µM
Convergence of cMyc and β-catenin on Tcf7l1 enables endoderm specification (2015)
Figure 2

Figure 2. GSK3 inhibition reduces activity of the transcriptional repressor Tcf7l1

(A) Assay of Tcf7l1 mRNA by RT-PCR for the first 4 days of differentiation in the presence or absence of 3 µM CH. Average and SD of three independent experiments. Day2 *p= 0.048, Day3 *p=0.036.

(B) Immunoblot for Tcf7l1 at day 3 of differentiation in the presence (+) or absence (-) of 3 µM CH.

(C) Assay of FoxA2 mRNA by RT-PCR for the first 3 days of differentiation in the presence or absence of 3 µM CH. Average and SD of three independent experiments. **p=5x10-7.

(D) ChiP-seq data from a compendium of transcription factor ChIP-seq analyses (Sánchez-Castillo et al, 2015). Gene tracks represent binding of Tcf7l1 at the FoxA2, Nodal and Eomesodermin (Eomes) gene locus. Red lines indicate the regions analyzed by ChIP PCR for Tcf7l1.

(E) ChIP for Tcf7l1 followed by qPCR for the regions indicated in Fig. 2D. Analysis was performed in wild-type ES cells in 2i culture conditions. Average and SD of three independent experiments. Klf2 *p=0.025, FoxA2 *p=0.050, Eomes *p=0.023.

(F) ChIP for Tcf7l1 followed by qPCR. Analysis was performed in wild-type ES cells on day 3 of differentiation in the presence or absence of 3 µM CH. Average and SD of three independent experiments. FoxA2 *p = 0.049.
Convergence of cMyc and β-catenin on Tcf7l1 enables endoderm specification (2015)
Figure 3

Figure 3. Tcf7l1 null ES cells do not require CH to initiate endoderm differentiation programme

(A) Assay of FoxA2 mRNA by RT-PCR in Tcf7l1 null cells on day 1 to 4 of differentiation without CH. Average and SD of three independent experiments.

(B) Gene expression analysis of endodermal associated genes by RT-PCR in control wild-type and Tcf7l1 null cells on day 4 of differentiation in the presence and absence of 3 µM CH. Average and SD of three independent experiments.

(C) FoxA2 (green) and Oct4 (red) immunostaining of Tcf7l1 null ES cells on day 4 of differentiation in the presence or absence of 3 µM CH. DAPI staining in blue.

(D) Assay of CXCR4, Hex and Sox17 mRNA by RT-PCR in control wild-type and Tcf7l1 null cells on day 6 of differentiation in the presence and absence of 3 µM CH. Average and SD of three independent experiments.
Convergence of cMyc and β-catenin on Tcf7l1 enables endoderm specification (2015)
Figure 4

Figure 4. Induction of FoxA2 alleviates the requirement for CH

(A) Assay of FoxA2 mRNA by RT-PCR in FoxA2-Tet ES cell lines 48 hr after treatment with the indicated dose of DOX. Average and SD of two independent experiments from 2 independent lines.

(B) FoxA2 immunostaining of FoxA2-Tet ES cells 48 hr after DOX treatment. Left, no DOX control. Middle and right, 10ng/ml DOX. DAPI staining shown in blue. Right panel shows middle image without DAPI signal.

(C) Assay of FoxA2 and CXCR4 mRNA by RT-PCR on day 3 of differentiation with DOX at the indicated dose. Average and SD of two experiments from two independent cell lines.

(D) Assay of Sox17 mRNA by RT-PCR on day 7 of differentiation with addition of 3 µM CH and/or 10 ng/ml DOX at the indicated time points (in days). Average and SD of two independent experiments from two independent cell lines.

(E) Sox17 immunostaining of FoxA2-Tet ES cells at day 7 of differentiation in the presence or absence of 10 ng/ml DOX and the presence and absence of 3 µM CH as indicated. Far right panel shows image to its left without DAPI.
Convergence of cMyc and β-catenin on Tcf7l1 enables endoderm specification (2015)
Figure 5

Figure 5. Suppression of GSK3 activity reduces Tcf7l1 and induces FoxA2 through both β-catenin dependent and independent pathways

(A) TOPFlash reporter activity following differentiation for 3 days in 3 µM CH or 100 ng/ml (W100) or 200 ng/ml (W200) recombinant Wnt3a plus 500 ng/ml recombinant RSpondin1. Relative luciferase units are normalised to FOPflash. Average and SD of three independent experiments.

(B) Assay of Tcf7l1 and FoxA2 mRNA by RT-PCR at day 2 of differentiation in the presence or absence of 3 µM CH or recombinant Wnt3a (200 ng/ml plus 500 ng/ml RSpondin1(WR)). Average and SD of three independent experiments. ** p<0.01.

(C) Assay of Tcf7l1 and FoxA2 mRNA by RT-PCR in Ctnnb1 null cells at day 1 and 3 of differentiation in the absence or presence of 3 µM CH. Average and SD of three independent experiments.

(D) Immunoblot for Tcf7l1 in Ctnnb1 null cells during the first 3 days of differentiation in the absence (-) or presence (+) of 3 µM CH. Results from two independent, clonally-derived Ctnnb1 null ES cell lines are shown. . Tubulin was used as a loading control.
Convergence of cMyc and β-catenin on Tcf7l1 enables endoderm specification (2015)
Figure 6

Figure 6. cMyc protein is stabilized by GSK3 inhibition and reduces Tcf7l1 expression independently of β-catenin.

(A) Assay of cMyc mRNA by RT-PCR in wild-type ES cells at day 1 to 3 of differentiation. Average and SD of three independent experiments.

(B) Immunoblot for total cMyc and phospho Ser62 (pSer62) cMyc during the first 3 days of differentiation in the absence (-) or presence (+) of 3 µM CH.

(C,D) Immunoblot for the destabilized form of cMyc protein (pThr58) during the first 3 days of differentiation in the absence (-) or presence (+) of 3 µM CH in wild-type and Ctnnb1 null cells respectively.

(E) Assay of Tcf7l1 mRNA by RT-PCR in wild-type cells at day 2 of differentiation in the presence or absence of a small molecule inhibitor of cMyc (cMyc Inhibitor II; Myci). Treatment with Myc inhibitor 10058-F4 (Sigma) generated similar results (data not shown). Average and SD of three independent experiments. **p=0.0002.

(F) Assay of Tcf7l1 and FoxA2 mRNA by RT-PCR in cMyc null/nMyc heterozygous cells (C-/-;N+/-) and floxed parental cells (cMycfl/fl;nMycfl/fl (control)) at day 3 of differentiation in the presence or absence of 3 µM CH. Average and SD of four or six independent experiments. Tcf7l1 *p=0.01, FoxA2 *p=0.034.

(G) FoxA2 immunostaining of control and cMyc null/nMyc heterozygous (cMyc-/-;nMyc+/-) cells at day 3 of differentiation in the presence of 3 µM CH.

(H) Assay of cMyc and Tcf7l1 mRNA by RT-PCR in cMycT58A inducible cell lines (icMycT58A) 24 hr after DOX treatment. Average of 2 independent experiments.

(I) Assay of Tcf7l1 mRNA by RT-PCR in icMycT58A clone 1 cell line at day 3 of differentiation in the absence of 3 µM CH following cMyc induction with 10 ng/ml DOX. Average and SD of 3 independent experiments. **p=0.007.

(J) Immunoblot for cMyc and Tcf7l1 in icMycT58A clonal cell line 1 48 hr after DOX induction at 10ng/ml.
Convergence of cMyc and β-catenin on Tcf7l1 enables endoderm specification (2015)
Figure 7

Figure 7. cMyc binds to the Tcf7l1 genomic locus in response to CH.

(A) Schematic representation of the genomic region upstream of the Tcf7l1 transcriptional start site (TSS).

(B) ChIP for cMyc performed in wild-type ES cells differentiated for 2 days in the absence or presence of 3 µM CH. qPCR was carried out for the regions indicated in Fig. 7a. Average and SD for five independent experiments, *p=0.028.

(C) ChIP for cMyc performed in wild-type ES cells differentiated for 2 days in the presence of 3 µM CH plus or minus Myc inhibitor 10058-F4. Average and SD for three independent experiments, *p=0.026.

(D) Assay for Miz1 mRNA by RT-PCR for the first three days of differentiation in the presence or absence of 3 µM CH. Average and SD of three independent experiments.

(E) ChIP for Miz1 or IgG control performed in E14 ES cells differentiated for 2 days in presence of 3 µM CH. qPCR was carried out for the regions indicated in Fig. 7a. Average and SD for five independent experiments, **p=0.0015.
PAQR3 controls autophagy by integrating AMPK signaling to enhance ATG14L-associated PI3K activity (2016)
Figure 1

Figure 1. PAQR3 regulates autophagosome formation without altering the activities of AMPK and mTOR.

(A) GFP-PAQR3 transfected HeLa cells were fixed for immunofluorescence staining with indicated antibodies before or after glucose starvation (GS) for 4 h. The nuclei were stained with Hoechst 33342. Scale bar: 10 μm.

(B) Immunoblotting (IB) analysis of wild type (WT) and PAQR3-deficient MEFs after GS for different time as indicated.

(C) WT and PAQR3-deficient MEFs were treated with 80 mM chloroquine (CQ) for 2 h to induce LC3-II accumulation. Whole-cell lysates were harvested for IB analysis of LC3-II.

(D) Autophagosome-like structures in WT and PAQR3-deleted MEFs were detected after GS for 4 h by transmission electron microscopy. * indicates autophagosome-like structures. N, nucleus.

(E) Quantitative analysis of autophagic vacuoles in transmission electron microscopy images. Thirty cells were quantified from each independent experiment，which was repeated for three times with similar results. Values are presented as mean ± SD, ***p < 0.001.

(F) Immunofluorescence staining of LC3 in WT or PAQR3-deficient MEFs after GS for 4 h. The arrowhead indicates representative LC3-positive puncta. Scale bar: 10 μm.

(G, H) HeLa cells were infected by PAQR3 knockdown or overexpression lentivirus together with their respective control virus. After GS for 1 h, the cell lysates were harvested for IB.
PAQR3 controls autophagy by integrating AMPK signaling to enhance ATG14L-associated PI3K activity (2016)
Figure 2

Figure 2. PAQR3 modulates ATG14L-linked VPS34 lipid kinase activity.

(A~C) HeLa cells stably expressing GFP-DFCP1 were transfected with or without PAQR3-specific shRNA. GFP signals were observed in these cells before or after glucose starvation (GS, shown in A). The knockdown efficiency of PAQR3 is shown in B and the statistics of GFP-positive puncta per cell is illustrated in C. Values are presented as mean ± SD, *** P < 0.001. Scale bar: 10 μm.

(D) GFP-WIPI1 was transfected into WT or PAQR3-deficient MEFs. 24 h later, the cells were incubated under GS for 4 h, followed by fluorescence observation. Scale bar: 10 μm.

(E) Different VPS34 complexes of WT or PAQR3 knockout HeLa cells were immunoprecipitated by ATG14L and UVRAG antibodies respectively in normal medium (NM) or GS. Then PI(3)P level was determined by a quantitative ELISA assay. The PI(3)P level was normalized to the amount of ATG14L or UVRAG used in the assay (n = 5; ***p < 0.001; ns, not significant).

(F) HeLa cells infected with control or PAQR3-overexpressed lentivirus were treated with GS for 4 h. PI(3)P levels were determined as in Figure 2E(n = 5; **p < 0.01; ns, not significant).

(G, H) PAQR3 knockout HeLa cells were transfected with plasmids of multiple PAQR3 deletion mutants. At 24 h after the transfection, the cell lysates were used to immunoprecipitate VPS34 complex by ATG14L antibody, followed by immunoblotting. The activity of ATG14L-linked VPS34 activity was detected by a quantitative ELISA assay. The PI(3)P level was normalized to the amount of ATG14L (n = 5; **p < 0.01).
PAQR3 controls autophagy by integrating AMPK signaling to enhance ATG14L-associated PI3K activity (2016)
Figure 3

Figure 3. PAQR3 is a component of ATG14L-Beclin1-VPS34 complex, mutually exclusive with UVRAG.

(A) Interaction between endogenous PAQR3 and the components of VPS34 complex in HEK293T cells. The cell lysates were used for immunoprecipitation (IP) and immunoblotting (IB) with the antibodies as indicated.

(B) Cytosol and Golgi fractionations were isolated from the livers of PAQR3-deleted mice and their littermate controls. Equal protein amounts of cytosol and Golgi fractions were subjected to IB.

(C) HEK293T cells were transiently transfected with Myc-tagged PAQR3. At 24 h after transfection, the cells were treated with normal medium (NM)，amino acid starvation (AS) or glucose starvation (GS), HBSS solution, or rapamycin (Rapa, 50 nM) for 4 h respectively. The cell lysates were then used in IP and IB.

(D) Four VPS34 complexes of MEFs were immunopurified using the indicated antibodies under NM, AS or GS conditions. The relative abundance of VPS34-binding partners was determined by IB. Each IP was normalized to the amount of VPS34.

(E) HEK293T cells were transfected with the plasmids as indicated and the cell lysates were used in IP and IB.

(F) The lysates of WT or PAQR3 knockout MEFs were used in IP and IB.

(G) MEFs were infected with control or PAQR3-expressing lentivirus. Then a quantitative immunodepletion assay was performed with increasing amounts of the indicated antibodies. Supernatants of MEF lysates after immunodepletion (Post-IP) were examined to determine the level of VPS34 complex proteins.
PAQR3 controls autophagy by integrating AMPK signaling to enhance ATG14L-associated PI3K activity (2016)
Figure 4

Figure 4. PAQR3 is a scaffold of ATG14L-Beclin1-VPS34 complex.

(A) A two-step co-IP assay in HEK293T cells to determine the ternary complex containing PAQR3, Beclin1 and ATG14L. The procedures of the two-step co-IP are outlined in the top. The samples were subjected to immunoprecipitation (IP) and immunoblotting (IB) with the antibodies as indicated.

(B) HeLa cells were transiently transfected with the plasmids as indicated. 24 h after transfection, the cells were treated with or without glucose starvation (GS) for 4 h. The cell lysates were used in IP and IB.

(C, D) Different truncations of ATG14L (C) or Beclin1 (D) were co-transfected with Myc-tagged PAQR3 into HEK293T cells as indicated, followed by IP and IB.

(E) Purified GST or GST-fused PAQR3 (1-71) (GST fused NH2-terminal 71aa of PAQR3) on glutathione agarose beads was mixed with purified His tagged proteins as indicated. After incubation at 4 °C for 3 h, the samples were washed extensively and subjected to IB.

(F, G) Different PAQR3 deletion constructs were co-transfected with Flag-tagged Beclin1 (F) or GFP-fused ATG14L (G) into HEK293T cells. The cells were then subjected to IP and IB.

(H) A schematic diagram depicts critical domains of PAQR3 involved in the interaction with ATG14L and Beclin1 respectively.
PAQR3 controls autophagy by integrating AMPK signaling to enhance ATG14L-associated PI3K activity (2016)
Figure 5

Figure 5. PAQR3 is phosphorylated by AMPK during autophagy, dependent on ATG14L.

(A) HeLa cells were cultured in normal medium (NM) or glucose starvation (GS) for 4 h. The starved lysates were treated with calf intestinal phosphatase (CIP) to remove phosphate groups and analyzed by immunoblotting (IB) with Phos-tag gel or regular SDS-PAGE.

(B) The cell lysates from HeLa cells were collected and analyzed after GS for indicated time (lane 1~5), or GS for 2 h followed by 2 h chase in NM (lane 6). The cell lysates were subjected to Phos-tag gel and regular SDS PAGE respectively.

(C) WT or AMPKα1/2 double knockout MEFs were incubated under NM or GS for 4 h, followed by IB with Phos-tag gel or regular SDS-PAGE.

(D) HeLa cells were incubated with compound C (20 mM, 30 min) with or without GS, followed by IB with Phos-tag gel or regular SDS-PAGE.

(E) Bacterial purified His-tagged NH2-terminal 71 amino acid of PAQR3 was incubated with or without purified Flag-tagged ATG14L. Then the complexes were treated with WT AMPK or DN-AMPK (dominant negative AMPK) for the indicated time in vitro. PAQR3 phosphorylation was examined by Phos-tag gel.

(F) HeLa cells infected with control or ATG14L-expressing lentivirus were incubated under GS for different time as indicated. Whole cell lysates were analyzed by both Phos-tag gel and regular SDS-PAGE.

(G) HeLa cells were infected by lentivirus expressing control shRNA or ATG14L-specific shRNA. Then the ATG14L knockdown cells were stably re-expressed with shRNA-resistant ATG14L (rATG14L) or Beclin1 and incubated under NM or GS for 4 h. Whole cell lysates were analyzed by both Phos-tag gel and regular SDS-PAGE.