EMBO/soda-nel huggingface dataset:
Regulation of TORC1 by Rag GTPases in nutrient response (2008)
figf4

(a) dRagA positively regulates wing compartment size. Wild-type or mutant dRagA transgenes were expressed in posterior compartments with the en–GAL4 driver. The ratios of representative posterior to anterior compartment areas are shown. Posterior compartment area was significantly increased in response to dRagAQ61L expression and decreased in response to dRagAT61N expression. Data are mean ± s.d., *P = 7.32 × 10−4 (n = 7), **P = 6.18 × 10−4 (n = 12), Student's two-tailed t-test, (n is the number of adult wings analysed). (b) dRagA positively regulates wing cell size. The average area of posterior compartment cells from en–GAL4 UAS–dRagA adult wings is shown. Cells expressing dRagAQ61L are significantly larger and dRagAT61N-expressing cells are smaller than controls. Data are mean ± s.d., *P = 1.15 × 10−3 (n = 7), **P = 0.025 (n = 12), Student's 2-tailed t-test (n represents number of adult wings analysed). (c, d) dRag GTPases positively regulate larval fat body cell size. (c) Cell area of clonally-induced dRagA-expressing cells or dRagC homozygous mutant cells relative to neighbouring wild-type control cells is shown. Cell area was determined from phalloidin-stained fixed fat body samples from fed or 48 h starved larvae. Expression of dRagAWT or dRagAQ61L significantly increased relative cell area under starvation but not fed conditions. Cells expressing dRagAT61N and dRagC loss-of-function cells were significantly smaller than control cells only under nutrient replete conditions. Data are means ± s.d., *P = 2.04 × 10−3 (n = 5), **P = 2.94 × 10−6 (n = 14), ***P = 3.79 × 10−7 (n = 14), ****P = 1.36 × 10−5 (n = 30), Student's two-tailed t-test. (d) Representative examples of fat body cells with altered dRagA activity. Cells expressing dRagA transgenes are marked by the expression of GFP in the left and middle panels, and dRagC homozygous mutant cells are marked by absence of GFP in the right panel. Scale bar represents 50 μm.
Regulation of TORC1 by Rag GTPases in nutrient response (2008)
figf5

(a) Rag acts through TORC1 to regulate S6K phosphorylation. HEK293 cells were transfected with constructs as indicated. Co-expression of of mTORKD construct (600 ng) or rapamycin treatment (rapa, 20 nM, 30 min) abolished the effect of RagAQ66L and RagCS75N on S6K phosphorylation. The protein level of mTORKD was determined by immunoblotting with anti-mTOR antibody. (b) RagA/RagC and TSC1/TSC2 independently regulate S6K phosphorylation. HEK293 cells were transfected with 200 ng of each Rag and/or ′ constructs as indicated. Amino acid starvation for 1 h (–AA) is indicated. Phosphorylation and protein levels of the transfected proteins were determined by immunoblotting with appropriate antibodies, as indicated. (c) TSC2 and RagA/B independently affect S6K phosphorylation. HA–S6K (20 ng) was transfected into HeLa cells with or without RNAi against human TSC2, RagA and RagB as indicated. (d) RagAT21N and RagCQ120L do not block Rheb-induced S6K phosphorylation. RagAT21N and RagCQ120L (200 ng each) were transfected into HEK293 cells with or without Rheb construct (20 ng). S6K was included in the co-transfection. Phosphorylation and protein levels of the transfected proteins were determined by immunoblotting with appropriate antibodies, as indicated. Full scans of blots are provided in Supplementary Information, Fig. S6.
Regulation of TORC1 by Rag GTPases in nutrient response (2008)
figf6

(a, b) dRagC is not required for Rheb-induced cell growth. (a) The area of Rheb-overexpressing cells in control or dRagC mutant (dRagC−/−) backgrounds under fed conditions, relative to that of neighbouring control cells which were assigned a value of 1. Overexpression of Rheb led to a significant increase in cell area in both control and dRagC mutant backgrounds. Data are mean ± s.d., *P = 0.034 (n = 5), **P = 6.1 × 10−3 (n = 5), Student's two-tailed t-test (n represents number of experimental samples). (b) A representative example of a clone of Rheb-overexpressing cells in a dRagC−/− animal. Rheb transgene-expressing cells are marked by co-expression of GFP. Cell boundaries are labelled by phalloidin staining in red; nuclei are labelled by DAPI in blue. Scale bar represents 50 μm. (c, d) Expression of dRagAQ61L fails to rescue the growth impairment of Rheb mutant cells. (c) Relative area of clonally-induced Rheb26.2 homozygous mutant cells in a control background and in animals expressing dRagAQ61L throughout the fat body. Clonally induced Rheb26.2 homozygous mutant cells were significantly smaller than neighbouring control cells both in wild-type and in dRagAQ61L expressing backgrounds. Data are mean ±s.d., *P = 2.91 × 10−4 (n = 7), **P = 2.59 × 10−8 (n = 5), Student's two-tailed t-test (fed conditions where n represents number of experimental samples). (d) A representative example of Rheb homozygous mutant cells (marked by lack of GFP, arrows) in fat body ubiquitously expressing UAS–dRagAQ61L. GFP-positive control cells in this experiment are a mixture of Rheb+/− and Rheb+/+. Scale bar represents 50 μm.
Regulation of TORC1 by Rag GTPases in nutrient response (2008)
figf7

(a–d) dRagAQ61L suppresses autophagy. (a) Drosophila fat body cells clonally expressing dRagAQ61L (marked in green by GFP expression) failed to accumulate autolysosomes (shown in surrounding control cells by punctate Lysotracker Red staining) in response to starvation for 4 h. Nuclei are marked in blue by DAPI. (b–d) Induction of autophagosomes in response to 4-h starvation is shown by the punctate pattern of GFP–Atg8a expression in control fat body cells (b), but not in cells expressing dRagAQ61L (c). The average number of GFP–Atg8a-marked autophagosomes per cell in control and dRagAQ61L-expressing clones is shown (d). Data are mean ± s.d., *P = 2.91 × 10−6, Students two-tailed t-test (n = 33 fat body samples imaged per genotype). Scale bars represent 25 μm in each panel. (e) RagA regulates LC3 conversion in mammalian cells. Myc–LC3 was co-transfected with RagAQL and RagCSN or RagATN and RagCQL into HEK293 cells as indicated. One day after transfection, cells were cultured in amino-acid-sufficienct medium (+AA) or amino-acid-depleted medium (−AA) for 4 h before collection. Western blotting for Myc–LC3 and HA–Rag were performed. Autophagic conversion of LC3I into the lipidated LC3II form was blocked by active RagA and stimulated by dominant-negative RagA. Full scans of blots are provided in Supplementary Information, Fig. S6.
Regulation of TORC1 by Rag GTPases in nutrient response (2008)
figf8

(a, b) dRagA activation increases sensitivity to starvation. Expression of dRagAQ61L using the fat-body-specific Cg–GAL4 driver significantly decreased survival of adult female flies under starvation (b) but not fed (a) conditions, relative to controls (Cg–GAL4 alone). Asterisks indicate significant difference, compared with controls. Data are mean ± s.d., *P 0.05, Students two-tailed t-test (n = 150 flies/genotype/treatment). (c) A proposed model of Rag GTPase in regulation of TORC1 activity. Rag GTPases act independently of and in parallel to TSC–Rheb to activate TOR signalling, possibly by transducing a nutrient-dependent signal. The mechanism of TOR regulation by Rag GTPases is indirect, and probably involves additional unidentified factors.
Modulation of phagosome phosphoinositide dynamics by a Legionella
phosphoinositide 3-kinase (2020)
Figure 1

Figure 1. The kinase motif embedded in MavQ is critical for its activity to inhibit yeast growth.

A. Mutations in the kinase motif abolished the yeast toxicity of MavQ. S. cerevisiae was transformed with plasmids expressing Flag-tagged wild-type MavQ or the indicated MavQ mutants under the galactose-inducible promoter. Yeast cells were spotted on synthetic medium supplemented with glucose or galactose for 3 days before image acquisition.

B. The expression of MavQ in the yeast was detected by Flag antibody. The PGK (3-phosphoglyceric phosphokinase) was used as a loading control.

Data information: Data shown are from at least three independent experiments (biological replicates, n≥3).
Modulation of phagosome phosphoinositide dynamics by a Legionella
phosphoinositide 3-kinase (2020)
Figure 2

Figure 2. MavQ is a phosphoinositide 3 Kinase that converts PtdIns into PtdIns3P.

A. Biochemical assays for the kinase activity of MavQ. Purified MavQ was incubated with or without specific PI substrates, the conversion of ATP to ADP was measured by the ADP-Glo assay. RLU, relative luminescence units.

B. H149, N152 and D160 in MavQ are critical for its kinase activity. Purified wild-type and mutant MavQ were reacted with PtdIns and ATP. The kinase activity was determined by the ADP-Glo assay.

C. The PI product produced by MavQ determined by TLC assays. di-C8-Bodipy-FL-PtdIns was used to react with MavQ (lane 4) and samples were further incubated with the 3-phosphatase MTM which hydrolyzes both PtdIns3P and PtdIns(3,5)P2 (lane 5) or with the PI4K LepB_NTD which phosphorylates PtdIns3P into PtdIns (3,4)P2 (lane 6). A schematic illustration of the enzymatic reactions is shown in the lower panel.

D. Mutations in H149, N152 and D160 abolish the ability of MavQ to convert PtdIns into PtdIns3P. Experiments were performed with the same procedure as described in panel C.

Data information: In A and B, data are shown as mean ± SD of triplicates (technical replicates, n=3) and are representative of three independent experiments (biological replicates, n=3). In C and D, data are representative of at least three independent experiments (biological replicates, n≥3). Unpaired two tailed Student t test, p<0.05 indicates significant difference.
Modulation of phagosome phosphoinositide dynamics by a Legionella
phosphoinositide 3-kinase (2020)
Figure 3

Figure 3. The PI3K activity of MavQ is insensitive to wortmannin.

A. Representative fluorescence images of HeLa cells expressing the PtdIns3P probe GFP-2xFYVEHrs and MavQ with or without wortmannin treatment. Scale bars, 5 μm.

B. Percentage of HeLa cells transfected with the indicated plasmids in which GFP-2xFYVEHrs localizes to vesicle-like structures. At least 100 cells (n≥100) were counted for each sample. Wort, wortmannin.

C. Induction of the ATP hydrolysis activity of MavQ by PtdIns is not affected by wortmannin. Recombinant MavQ was incubated with increasing amounts of wortmannin for 30 min at room temperature prior to the addition of PtdIns and ATP. The wortamannin-sensitive PI3K p110α was used as a control. The ADP-Glo assay was used to measure the kinase activity.

D. Wortmannin does not affect the production of PtdIns3P by MavQ. Purified MavQ was pretreated with 100 μM of wortmannin for 30 min at room temperature. di-C8-Bodipy-FL-PtdIns and ATP were then added to the reaction mixtures. The TLC assay was employed to detect the generation of PtdIns3P by MavQ. The wortamannin-sensitive PI3K p110α was included as a control.

Data information: In panel B, data show the mean ± SD of three independent experiments (biological replicates, n=3). In panel C, data are shown as mean ± SD of triplicates (technical replicates, n=3) and are representative of three independent experiments (biological replicates, n=3). In panel D, data are representative of at least three independent experiments (biological replicates, n≥3). Unpaired two tailed Student t test, p<0.05 indicates significant difference.
Modulation of phagosome phosphoinositide dynamics by a Legionella
phosphoinositide 3-kinase (2020)
Figure 4

Figure 4. The association of MavQ with the LCV and its role in intracellular growth of L. pneumophila in macrophages.

A. Representative immunofluorescence images of the association of MavQ with the LCV. BMDM cells infected with wild-type or Dot/Icm-deficient (dotA-) L. pneumophila were sequentially stained with antibodies against L. pneumophila and MavQ. Images were taken under a fluorescence microscope. Insets represent 3x magnification of regions defined by dash lines. Scale bar, 5 μm.

B. The rates of of LCVs positively stained for MavQ 2 h post-infection. At least 100 LCVs (n≥100) were scored for each sample.

C. Dynamics of MavQ association with LCV. BMDMs were infected with the wild-type L. pneumophila strain at an MOI of 2 for the indicated experimental durations. At least 100 phagosomes (n≥100) were counted for each sample to obtain the percentage of MavQ-positive LCVs.

D. Intracellular growth of L. pneumophila in macrophages. BMDMs were infected with indicated L. pneumophila strains at an MOI of 0.05. At the indicated time points, CFUs were determined by plating the saponin-solubilized lysates of infected cells on CYE plates.

E. Expression of mavQ in L. pneumophila strains used for infection. Total proteins of bacteria cultured to the post-exponential phase separated by SDS-PAGE were probed with MavQ-specific antibodies. ICDH was detected as a loading control.

Data information: In panels B and C, data are shown as mean ± SD of three independent experiments (biological replicates, n=3). In panel D, results shown are from one representative experiment done in triplicate (technical replicates, n=3) and are representative of three independent experiments (biological replicates, n=3). In panel E, results show one representative of three independent experiments (biological replicates, n=3). Unpaired two tailed Student t test, p<0.05 indicates significant difference.
Modulation of phagosome phosphoinositide dynamics by a Legionella
phosphoinositide 3-kinase (2020)
Figure 5

Figure 5. MavQ functions together with LepB and SidF to produce PtdIns4P on the LCV.

A. Biochemical synthesis of PtdIns4P from PtdIns by sequential reactions catalyzed by MavQ, LepB_NTD and SidF. MavQ was reacted with di-C8-Bodipy-FL-PtdIns, resulting in the production of PtdIns3P (lane 6). The PtdIns3P-specific PI4K LepB-NTD was then added into the reaction, yielding PtdIns(3,4)P2 (lane 7). The product of the sample receiving LepB_NTD was further incubated with the 3-phosphatase SidF to yield PtdIns4P (lane 8). The lipid products were detected and visualized by TLC. A schematic illustration of the enzymatic reactions is shown in the lower panel.

B. Representative images of SidC anchoring on LCVs. BMDMs infected with the indicated L. pneumophila strains at an MOI of 2 for 2 h were sequentially stained with antibodies against the bacteria and SidC. Images were acquired using a fluorescence microscope. Insets represent 3x magnification of regions defined by dash lines. Scale bar, 5 μm.

C. Quantitation of SidC-positive LCVs. At least 100 phagosomes (n≥100) were scored for each sample.

D. Expression and translocation of SidC. The lysates of L. pneumophila strains used for infection were probed for protein expression, ICDH was detected as a loading control (top panel). SidC translocation was detected by probing the saponin-soluble fractions of infected U937 cells with SidC-specific antibodies. Tubulin was probed as a loading control (bottom panel).

Data information: In panels A and D, data show one representative of three independent experiments (biological replicates, n=3). In panel C, results are shown as mean ± SD from three independent experiments (biological replicates, n=3). Unpaired two tailed Student t test, p<0.05 indicates significant difference.
Modulation of phagosome phosphoinositide dynamics by a Legionella
phosphoinositide 3-kinase (2020)
Figure 6

Figure 6. SidP does not affect the enzymatic activity of MavQ.

A. Direct interactions between SidP and MavQ in the GST pull-down assay. GST beads coated with GST-SidP or GST were incubated with His6-MavQ. After washing the beads with GST binding buffer, the proteins resolved by SDS-PAGE were detected by Coomassie brilliant blue staining.

B, C. SidP does not impair the kinase activity of MavQ in vitro. MavQ was preincubated with increasing amounts of SidP, and the kinase activity of MavQ was measured by the ADP-Glo assay (B) or the TLC assay (C).

D. Co-expression of SidP with MavQ in HeLa cells does not influence the production of PtdIns3P caused by MavQ. The association of the fluorescence probe GFP-2xFYVEHrs with vesicle-like structures was used to indicate the distribution of PtdIns3P in cells. At least 100 cells were scored in each sample (n≥100) (left panel). Wort, wortmannin. Expression of MavQ and SidP in the samples were shown in the right panel. EV represents empty vector.

E. Deletion of sidP does not impact the association of SidC with the LCV. BMDMs infected with the indicated L. pneumophila stains were stained as described in Fig. 5 and the association of SidC with bacterial phagosomes was similarly determined (n≥100) (left panel). Representative images of infected cells harboring bacterial phagosomes were also shown (right panel). Insets represent 3x magnification of regions defined by dash lines. Scale bar, 5 μm.

Data information: In panels A and C, data show one representative of three independent experiments (biological replicates, n=3). In panel B, data are shown as mean ± SD of triplicates (technical replicates, n=3) and are representative of three independent experiments (biological replicates, n=3). In panels D and E, data are shown as mean ± SD of three independent experiments (biological replicates, n=3). Unpaired two tailed Student t test, p<0.05 indicates significant difference.

Expanded View Figure legends
Nf1 Loss Promotes Kras-Driven Lung Adenocarcinoma and Results in Psat1-Mediated Glutamate Dependence (2019)
Figure 1

Figure 1. Nf1 Loss Activates Fak1 and Accelerates Murine LUAD Tumorigenesis

(A) Schematic of KrasLSL-G12D/+; p53fl/fl (KP) mice intratracheally infected with pSECC lentiviruses containing sgNf1 or control sgTom. Mouse tumor burden was tracked by micro-computed tomography (micro-CT) for five months post-infection, and lungs were finally harvested 21 weeks post-infection. Tumors were dissected out for immunohistochemical (IHC) staining and generation of the tumor-derived parental KP cell line.

(B) Quantification by micro-CT of mean tumor volumes in KP mice derived from 50 randomly-selected tumors at 8, 12, 16, and 20 weeks post-infection with control sgTom, sgNf1.1, sgNf1.2, or sgNf1.3 (n = 18 mice, 21 mice, 21 mice, and 20 mice, respectively).

(C) Quantification by micro-CT of total tumor volume per mouse in KP mice after infection with control sgTom, sgNf1.1, sgNf1.2, or sgNf1.3 at 8, 12, 16, and 20 weeks post-infection.

(D) Depictions and quantitation of total tumor burden (total tumor area/total lung area) in KP mice after infection with control sgTom, sgNf1.1, sgNf1.2, or sgNf1.3 at 21 weeks after infection.

(E) Distribution of histological tumor grades in KP mice after infection with control sgTom, sgNf1.1, sgNf1.2, or sgNf1.3 (n = 50 tumors each) at 21 weeks after infection.

(F) Assessment of the mitotic index of tumor cells by phosphorylated-histone H3 (pHH3)-positive nuclei density in KP mice LUAD tumors at 21 weeks after infection with control sgTom, sgNf1.1, sgNf1.2, or sgNf1.3 (n = 50 tumors each).

(G) Nf1 mRNA expression in LUAD tumor sections 21 weeks after infection with control sgTom, sgNf1.1, sgNf1.2, or sgNf1.3.

(H) Representative hematoxylin and eosin (H&E) and p-Fak1 immunohistochemical (IHC) staining of LUAD tumor sections 21 weeks after infection with control sgTom (grade 1 depicted), sgNf1.1 (grade 3 depicted), sgNf1.2 (grade 3 depicted), or sgNf1.3 (grade 3 depicted) (n = 50 tumors each). H&E scale bars (low-magnification top row = 100 μm, high-magnification bottom row = 250 μm); p-Fak1 IHC scale bars (low-magnification top row = 250 μm, high-magnification bottom row = 500 μm).

(I) Quantification of p-Fak1 IHC signals in LUAD tumor sections 21 weeks after infection with control sgTom, sgNf1.1, sgNf1.2, or sgNf1.3.

(J) Quantification of p-Fak1 IHC signals in sgNf1.3 LUAD tumor sections analyzed by tumor grade.

(K) Quantification of Psat1 mRNA expression in LUAD tumor sections 21 weeks after infection with control sgTom, sgNf1.1, sgNf1.2, or sgNf1.3.

(L) Quantification of Psat1 mRNA expression in sgNf1.3 LUAD tumor sections analyzed by tumor grade.

P-values are reported in Appendix Table S3. For bar charts, data presented as means with error bars representing standard deviations (SDs). For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.
Nf1 Loss Promotes Kras-Driven Lung Adenocarcinoma and Results in Psat1-Mediated Glutamate Dependence (2019)
Figure 2

Figure 2. Nf1 Loss Accelerates Tumor Growth in p53-Independent Manner

(A) Western blotting analysis of p53 expression in various p53/TP53-WT cell lines incubated with the DNA-intercalating agent doxorubicin (DOXO, 0.2 µg/ml for 6 hours) to induce p53 stabilization (n = 3 biological replicates). Full experimental data provided in Appendix Fig. S2A.

(B, C) Western blotting analysis confirming NF1 upregulation and p-FAK1 downregulation in (B) DOXO-treated PDKN1 cells and (C) DOXO-treated PDKN2 cells transduced with PGK-NF1 (n = 3 biological replicates). Full experimental data provided in Appendix Fig. S2B-C.

(D) Western blotting analysis confirming NF1 downregulation and p-FAK1 upregulation in DOXO-treated SW1573 cells transduced with sgNf1.3 (n = 3 biological replicates). Full experimental data provided in Appendix Fig. S2D.

(E, F) Western blotting analysis confirming Nf1 downregulation and p-Fak1 upregulation in (E) DOXO-treated LKR10 and (F) DOXO-treated LKR13 clones transduced with sgNf1.3 (n = 3 biological replicates). Full experimental data provided in Appendix Fig. S2E-F.

(G) FAK1 target gene expression in DOXO-treated PDKN1 cells transduced with PGK-control or PGK-NF1 (n = 3 biological replicates).

(H, I) Subcutaneous tumor volumes of DOXO-treated PDKN1 and PDKN2 cells transduced with PGK-control or PGK-NF1 (n=18 each).

(J-L) Subcutaneous tumor volumes of (J) SW1573, (K) LKR10, and (L) LKR13 cells transfected with sgTom or sgNf1.3 (n=18 each).

(M) Depictions and quantitation of KrasG12D/+; p53+/+ (K-only) autochthonous tumor burden (total tumor area/total lung area) in pSECC-sgTom (n = 18 mice) or pSECC-sgNf1.3 mice (n = 21 mice).

(N) Analysis of tumor grades in K-only autochthonous tumors derived from pSECC-sgTom (n = 18 mice) or pSECC-sgNf1.3 mice (n = 21 mice).

(O) Quantification of Ki-67-positive nuclei per mm2 in K-only autochthonous tumors derived from pSECC-sgTom (n = 18 mice) or pSECC-sgNf1.3 mice (n = 21 mice).

P-values are reported in Appendix Table S3. For bar charts, data presented as means with error bars representing standard deviations (SDs). For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean.