EMBO/soda-nel huggingface dataset:
KRAS signaling in malignant pleural mesothelioma (2021)
Figure 7

Fig 7 - Transplantable KRAS/TP53-mutant murine mesothelioma (KPM) cell lines.

KRASG12D;Trp53f/f pleural mesothelioma (KPM), pleural mesothelial (PMC), and asbestos-induced AE17 mesothelioma cells (all from C57BL/6 mice) were analyzed.

A KPM cell culture showing anoikis (white arrows) and spindle-shaped morphology (black arrows).

B Representative colonies of KPM1 cells (7.5 x 103 cells/vessel) seeded on a soft agar-containing 60 mm Petri dish and stained with crystal violet after a month (n = 3/group).

C Data summaries from in vitro MTT reduction (top; 2 x 104 cells/well; n = 6 independent experiments) and in vivo subcutaneous tumor growth after injection of 106 cells per C57BL/6 mouse (bottom; n = 5/group).

D KRAS/Kras mRNA Sanger sequencing shows wild-type Kras (KrasWT) of PMC and mutant murine Kras/human KRAS alleles (KRASG12D and KrasG12C) of KPM and AE17 cells (arrows).

E, F RT-PCR (E) and qPCR (F) of KPM cells and parental tumors show Trp53f/f allele deletion (Δ) and Bap1 and Cdkn2a overexpression compared with PMC.

Data information: In (C), data are presented as mean (circles) and 95% confidence interval (bars). P, overall probability, two-way ANOVA. ****: P < 0.0001 for AE17 cells (top) or for KPM cells (bottom) compared with all other groups, Bonferroni post-tests. In (F), data are presented as raw data points (circles), rotated kernel density distribution (violins), and medians (lines). P, overall probability, two-way ANOVA. *, **, and ****: P < 0.05, P < 0.01, and P < 0.0001, respectively, for comparison with PMC, Bonferroni post-tests.
Phosphorylation-dependent Akt-Inversin interaction at the basal body of primary cilia (2016)
Figure 1

Figure 1. INVS specifically associates with Akt in mammalian cells.

Flag-INVS interacted with HA-Akt (lanes 1–3), but not with HA-PDK1 (lanes 4–6) or HA-PrKA (lanes 7–9) in HEK293T cells. Expression levels of HA-Akt, PDK1, PrKA, and Flag-INVS were similar, as determined by immunoblotting (HA: anti-HA antibody; F: anti-Flag antibody; C: control antibody). The results presented (A–E) are representative of at least two independent experiments.

B. Flag-tagged INVS interacted with HA-tagged Akt1 (lane 1–3), Akt2 (lane 4–6), and Akt3 (lane 7–9) in co-immunoprecipitation assays. Expression of each Akt isoform and INVS were similar.

C. INVS was co-immunoprecipitated with endogenous Akt from HEK293 cells, in which INVS is also endogenously expressed (lane 2).

D. To determine the Akt domain responsible for interacting with INVS, Akt subfragments were generated. The C-terminus of Akt is the primary domain for INVS interaction (lanes 7–9). Similar levels of each Akt fragment were immunoprecipitated using anti-HA antibody.

E. Schematic showing the structure and the functional domains of Flag-tagged full-length and fragmented INVS in mammalian expression vectors (N-Term: 1–421, Intermediate domain: 421–678, and C-Term: 678–1065) used in the current study. Intermediate (lanes 6–9) and C-terminal (lanes 10–12) fragments of INVS were important for interaction with Akt.
Phosphorylation-dependent Akt-Inversin interaction at the basal body of primary cilia (2016)
Figure 2

Fig. 2. INVS is a novel substrate of Akt.

A. The indicated recombinant INVS proteins (lanes 1–4) were used for IVK. Akt phosphorylates WT (lane5), 1–970 (lane 6), and 1–898 (lane 7) INVS, but failed to phosphorylate 1–670 (lane 8) INVS.

The indicated recombinant INVS proteins (lanes 1–10) were used for IVK. Akt phosphorylates WT (lane 6) and 1–898 (lane7) INVS, but failed to phosphorylate other subfragments of INVS (1–822, 1–746, and 1–670, in lane 8, 9, and 10, respectively), as detected using a phospho-Akt substrate antibody.

.

C. Using the indicated subfragments of INVS for IVK, we confirmed that Akt phosphorylates 675–1065 (lane 4), but not 675–822 (lane 5), or 675–746 (lane 6) INVS.

D. Amino acid alignment of 824–898. Motif scan analysis using the Scansite3 database identified three amino acids, T864, S865, and T866, as putative Akt phosphorylation targets.

E. Purified recombinant WT or 3A INVS were generated (top panel, lanes 1–4). In IVK, Akt phosphorylated the WT fragment (824–898, middle panel, lane 3), but failed to phosphorylate 3A INVS (middle panel, lane 4), as detected by using a phospho-Akt substrate antibody.

.

F. Akt phosphorylated WT (lane 10), 1–898 (lane 12) and 675–1065 (lane 15) INVS, but failed to phosphorylate both full-length and 675–1065, ∆727–896, 1–857, or 1–822, 3A INVS (lanes 16, 11, 14, and 15, respectively), all of which lacked the T864/S865/T866 target amino acid.

.
Phosphorylation-dependent Akt-Inversin interaction at the basal body of primary cilia (2016)
Figure 3

Figure 3. Both INVS and phosphorylated Akt are located at the basal region of primary cilia.

A. Confocal microscopy shows that active Akt (phospho-S473Akt) is localized in the basal body of primary cilia (right side panels show higher magnification).

B. INVS is localized in the proximal region of the INVS compartment in primary cilia (INVS compartment, right side panels show higher magnification).

C. INVS co-localizes with γ-tubulin, a centrosome marker.

D. Anti-phospho-Akt substrates stain positively at the basal region of the primary cilia (arrows), as determined by anti-acetylated tubulin (right side panels show higher magnification).

E. Silver-intensified immunogold electron microscopy shows that both phospho-Akt (left side panels) and INVS (right side panels) were localized to the ciliary pocket of primary cilia in the presence of PDGF-AA (white arrow).

F. Schematic representation of the view from an electron microscope.
Phosphorylation-dependent Akt-Inversin interaction at the basal body of primary cilia (2016)
Figure 4

Figure 4. Co-localization of INVS and phosphorylated-Akt increases with PDGF-AA treatment.

PDGF-AA stimulation enhances localization of active Akt (phospho-S473Akt) at the basal body of primary cilia (right side panels show higher magnification).

Quantification of co-localization of phosphorylated Akt (green) and INVS (red) at the indicated time points after PDGF-AA stimulation revealed a time-dependent increase. Colocalization was measured by counting yellow pixels (colocalized area) using Imaris software (Bitplane AG). Results presented are means ±SE (n=31 at time 0, n=35 at 1 min., and n=33 at 3 min, respectively). Three independent experiments were analyzed with similar results. Statistical significance was determined by student’s t test.

Co-immunoprecipitation assays using HEK293T cells show that INVS preferentially binds to WT-Akt (second panel, lane1) or phospho-mimetic Akt (T308D/S473D, lane2), but fails to interact with phosphorylation-defective forms of Akt (T308A/S473A, lane 3).

PDGF-AA stimulation of HEK293T cells results in augmented interaction of endogenous INVS with Akt (top panel, comparison between lane 1 and lane 2, untreated and PDGF-AA-stimulated conditions, respectively).

Both WT and 3A-INVS, which are localized at the INVS compartment of primary cilia under serum-starved condition (left side panels, lower panels show higher magnification), translocate to the basal body of the cilium after PDGF-AA stimulation (right side panels, lower panels show higher magnification). Results presented are means ± SE of yellow pixels demonstrating colocalization of EGFP-INVS (green) with γ-tubulin (red) in hTERT-RPE1 cells (n=28). Three independent experiments showed similar results. Statistical significance was determined by student’s t test. Fluorescence intensities of INVS (green), acetylated-α-tubulin (blue), and γ-tubulin (red) along the line (a-b) are plotted underneath.
Phosphorylation-dependent Akt-Inversin interaction at the basal body of primary cilia (2016)
Figure 5

Figure 5. Decrease in ciliary development by phosphorylation-defective INVS at T864/S865/T866.

A. PDGF-AA stimulation resulted in the phosphorylation of WT INVS (top panel, lane 2), but not 3A INVS (top panel, lane 6). PDGF-AA-stimulated INVS phosphorylation is inhibited by Akt inhibitors (top panel, LY294002, MK2206, and GSK690693, lanes 3, 4, and 5, respectively).

B. Myr-Akt, a constitutively active form of Akt, was expressed together with WT or 3A INVS in HEK293 cells and INVS phosphorylation was quantified. WT, but not 3A INVS, can be phosphorylated in the presence of Myr-Akt.

C. 3A INVS exhibited a weaker interaction with Akt as compared to WT INVS (top panel, compare lanes 1 and 2, WT and phospho-defective mutant, respectively). Expression of Akt and INVS are shown underneath.

D. 3A INVS exhibited weaker dimerization as compared to WT INVS (top panel, compare lanes 1 and 2, WT and 3A, respectively). Expression of Akt and INVS are shown below each.

E. Length of primary cilia in confluent hTERT-RPE cells was analyzed after siRNA-mediated knock down of Akt1/2. Ciliary length decreased in cells expressing siRNA. Results presented are means ±SE of the longitudinal length of acetylated-tubulin (red), a marker of primary cilia, in siRNA-transfected (green) hTERT-RPE1 cells (n=175 for control siRNA and Akt siRNA, and n=195 for Akt siRNA plus siRNA resistant Akt, respectively). Three independent experiments showed similar results. Statistical significance was analyzed by student’s t test. Note that re-introduction of siRNA-resistant Akt (Matsuda-Lennikov et al., 2014) in Akt knockdown cells rescued the effect on ciliary length.

F. Expression of 3A INVS (bottom panels) but not WT INVS (middle panels) inhibited the development of primary cilia as compared to control cells (top panels).

Quantification of ciliary length. Results are mean ± SE of longitudinal length of acetylated-tubulin (blue), a marker of primary cilia, in EGFP-INVS (green) transfected in hTERT-RPE1 cells (n=58 for EGFP vector and for EGFP-INVS WT, and n=52 for EGFP-INVS 3A mutant, respectively). Three independent experiments showed similar results. Statistical significance was determined by student’s t test.
Phosphorylation-dependent Akt-Inversin interaction at the basal body of primary cilia (2016)
Figure 6

Fig. 6. INVS controls spindle axis alignment during cell division.

Schematic describing the measurement of spindle angle (θ) (Kikuchi et al., 2010). Z-stack images were obtained from 0.45 mm-thick sections of metaphase cells and the angle between the axis of metaphase spindle and that of the substrate surface, which is termed the spindle angle, was calculated. Results presented are scatter plots of the angle (θ) of the mitotic spindle (n=28 for all samples of vector, HA-WT, -3D, -3A, -R899X, -Q671X, and -R603X INVS stably transfected MDCK cells). Three independent experiments showed similar results. Statistical significance was determined by Mann-Whitney’s non-parametric median test.

Spindle angles were measured in MDCK cells transfected with the indicated INVS plasmids (+). The spindle axis was misaligned in cells expressing mutant forms of INVS as compared to cells expressing WT INVS or sham-transduced cells. Expression of 3A INVS and of clinically relevant truncated forms of INVS but not WT INVS, resulted in disorientation of the spindle axis during metaphase. Similar expression of all INVS constructs was verified by immunoblotting (Appendix figure S4A).

WT INVS robustly inhibited the transactivation of INVS in a luciferase reporter assay, which could be reverted by C-terminal truncated INVS (R899X, Q671X, or Q603X), but not by a phospho-mimetic mutant or 3A INVS. Results presented are mean ± SE of the ratio of triplicates between sequentially measured firefly TCF/LEF signal and Renilla luciferase signal in HA-INVS transfected 293T cells. Dual-Luciferase reporter® Assay System (Promega) was used and experiments were performed in triplicates. Two independent experiments showed similar results. Statistical significance was determined by student’s t test.

The expression of HA-Dvl (upper panels) and the indicated forms of Flag-INVS (lower panels) are shown by immunoblots; α-tubulin was used as an internal control.
Phosphorylation-dependent Akt-Inversin interaction at the basal body of primary cilia (2016)
Figure 7

Fig. 7. The C-terminus and phosphorylation sites of INVS are required for proper tubular differentiation.

WT INVS- transduced MDCK cells in 3D cultures form uniform round tubules. However, expression of 3A or NPHP2-related truncated INVS mutants, which lack Akt phosphorylation site (s), disrupts this uniformity. Results presented are representative of three independent experiments and show mean ± SE of percent normal acini in vector- or WT INVS-transduced MDCK cells (n=422 for Vector, n=398 for HA-WT, n=401 for -3D, n=365 for -3A, n= 344 for -R899X, n=331 for -Q671X, and n=325 for -R603X INVS stably transfected MDCK cells, respectively). Three independent experiments showed similar results. Statistical significance was determined by student’s t test.

WT transduced MDCK cells form a relatively smaller lumen; however, expression of 3A INVS or the indicated NPHP2-related truncation mutants result in relatively larger acini. Both truncation mutants (Q671X or R603X) lack the Akt phosphorylation site and exhibit a larger lumen size. Three independent experiments showed similar results.

C. Quantification of normal lumen in cells transduced with WT or mutant INVS. Results presented are mean ±SE of percent normal acini in vector- or WT INVS-transduced MDCK cells (n=166 for Vector, n=204 for HA-WT, n=233 for -3D, n=150 for -3A, n=164 for -R899X, n=128 for -Q671X, and n=137 for -R603X INVS stably transfected MDCK cells, respectively). Three independent experiments showed similar results. Statistical significance was determined by student’s t test.

D. Size of lumen in cells transduced with WT or mutant INVS is shown. Results presented are scatter plots of acini size (n=205 for Vector, n=143 for HA-WT, n=131 for -3D, n=167 for -3A, n=144 for -R899X, n=128 for -Q671X, and n=144 for -R603X INVS stably transfected MDCK cells, respectively). Two independent experiments showed similar results. Statistical significance was determined by Mann-Whitney’s non-parametric median test.
ULK1 induces autophagy by phosphorylating Beclin-1 and activating VPS34 lipid kinase (2013)
figf1

Unless otherwise stated, experiments were repeated three times; data shown are representative. (a) Different VPS34 complexes were immunoprecipitated (IP) from MEFs in the presence (N) or absence (−A) of amino acids using the indicated antibodies and assayed for kinase activity (left, top panel). Inputs were immunoblotted using the antibodies indicated (left, lower panels). Quantification of VPS34 activity is from 3 biological repeats (right panel; data shown are mean+s.d.). AR, autoradiography. (b) Immunoprecipitation of three VPS34 complexes, normalized for VPS34, was performed using the indicated antibodies under nutrient-rich (N) or starvation (-A) conditions. VPS34-binding partners were analysed by western blotting. (c) ATG14L-containing VPS34 complexes were immunopurified from WT, ULK1−/− 2KD (ULK def.) and FIP200−/− MEFs, and measured for lipid kinase activity as in a. (d) VPS34 was immunopurified from WT, ULK-deficient and FIP200−/− MEFs. A kinase assay was performed as in a. (e) Immunoprecipitation of Beclin-1-containing VPS34 complexes from WT, ULK-deficient and FIP200−/− MEFs. A kinase assay was performed as in a. (f) LC3B puncta and PtdIns(3)P levels were analysed with anti-LC3B and the biotin–2XFYVE domain probe. Representative immunofluorescence images of LC3B and 2XFYVE domain binding are shown (scale bars, 10 μm). (g) Quantification of LC3B puncta from f. Details of quantification are provided in the Methods; Data shown in g–i are mean+s.d. from a minimum of 6 unique fields of view from a representative experiment (see statistical source data in Supplementary Table S1). (h) Total PtdIns(3)P was quantified from the experiment in f. Error bars were calculated as in g. (i) Quantification of PtdIns(3)P that co-localizes with LC3B on amino-acid withdrawal from the experiment described in f. (j) VPS34 activity with or without ULK1 overexpression was assayed. A representative experiment of four repeats is shown.
*P0.05(a,g,h,i) as determined by Student’s
t-test (see statistical source data in Supplementary Table S1 and Methods). Quantification of PtdIns(3)P/VPS34 provided under the PtdIns(3)P panel in c–e,j nutrient-rich conditions was normalized to 1. Uncropped images of blots are shown in Supplementary Fig. S4.
ULK1 induces autophagy by phosphorylating Beclin-1 and activating VPS34 lipid kinase (2013)
figf2

Unless otherwise stated, all experiments were repeated three times and the data shown are representative. (a) HEK293 cells were transfected with ATG14L, VPS34 and Beclin-1. ATG14L-containing VPS34 complexes were immunopurified and subjected to an in vitro ULK1 kinase assay in the presence of γ-32P[ATP]. Bound ATG14L complexes and soluble ULK1 were separated and phosphorylation was detected by autoradiography (AR, left panels). Western blotting was performed (right panels). Results are representative of two unique experiments. (b) Full-length murine GST–Beclin-1 and various truncations (as labelled) were subjected to an in vitro HA–ULK1 kinase assay. GST–Beclin-1 6(S/T)A has serine/threonine residues 4, 7, 10, 14, 29 and 42 mutated to alanine. ULK1 inputs were determined by western blotting, Beclin-1 inputs by Coomassie (Coom) and target phosphorylation by autoradiography. (c) GST–Beclin-1 (1–85) was subjected to an in vitro ULK1 kinase reaction and analysed by mass spectrometry. Ser 14, Ser 15 in humans (outlined), is the main phosphorylation site identified (for mass spectrometry data, see Supplementary Fig. S2b,c). Mass spectrometry was performed on a single experiment. (d) Beclin-1 Ser 14 is the main in vitro ULK1 phosphorylation site. Beclin-1 WT, and S4A and S14A mutants were subjected to an in vitro ULK1 kinase assay. The reaction was developed by autoradiography and stained for Beclin-1 input levels by Coomassie blue. Results are representative of two unique experiments. (e) HEK293 cells were transfected with the indicated plasmids under nutrient-rich conditions. Beclin-1 was immunoprecipitated and immunoblotted with pBeclin-1(Ser 14), or anti-Beclin-1 as a loading control. ULK1 inputs are included below immunoprecipitated samples. (f) Purified GST–Beclin-1 (1–85) was subjected to in vitro phosphorylation by GST–ULK1 (top panel) and GST–ULK2 (bottom panel). Reactions were immunoblotted with the indicated antibodies. (g) HEK293 cells were transfected with ATG14L, VPS34 and Beclin-1 and grown under nutrient-rich conditions. ATG14L-containing VPS34 complexes were immunoprecipitated and lipid kinase activity was assayed as described in Fig. 1j. Inputs were immunoblotted with the indicated antibodies. Representative of four unique experiments. (h) Stable lines containing Beclin-1(WT or S14A) were used for Beclin-1 immunoprecipitation. Binding partners were determined by SDS–PAGE analysis and western blot using the indicated antibodies. Uncropped images of blots are shown in Supplementary Fig. S4.
ULK1 induces autophagy by phosphorylating Beclin-1 and activating VPS34 lipid kinase (2013)
figf3

Unless otherwise stated, all experiments were repeated three times and data shown are representative. (a) WT MEFs were cultured with or without amino acids. ATG14L-associated Beclin-1 was immunoprecipitated and treated with lambda phosphatase treatment (PPase) as indicated. Western blotting (immunoblotting, IB) was performed with the indicated antibodies. Beclin-1 Ser 14 phosphorylation was quantified (shown under top panel) and normalized to total Beclin-1. (b) WT MEFs were starved for the indicated times. Beclin-1 was immunopurified by ATG14L immunoprecipitation and immunoblotted as indicated (top panels). Whole-cell lysates were immunoblotted with pULK1 S757 (mTORC1-target site), pS6K and ULK1 antibodies (bottom panels). Two unique experiments were performed. (c) WT or FIP200−/− MEFs were incubated under nutrient-rich, amino-acid-deprived or Torin-1 (+T, an mTOR inhibitor) conditions. Beclin-1 was purified and immunoblotted as in Fig. 3b. (d) WT or ULK-deficient MEFs were incubated with or without amino acids. Beclin-1 was purified and immunoblotted as in a. Two unique experiments were performed. Uncropped images of blots are shown in Supplementary Fig. S4.
ULK1 induces autophagy by phosphorylating Beclin-1 and activating VPS34 lipid kinase (2013)
figf4

Unless otherwise stated all experiments were repeated three times and data shown are representative. (a) Beclin-1 alone (lanes 1–4) or Beclin-1 and ATG14L (lanes 5–8) were overexpressed in HEK293 cells. Beclin-1 was purified either by direct immunoprecipitation (lanes 1–4) or by ATG14L immunoprecipitation (lanes 5–8). Immunoprecipitated samples were subjected to an in vitro ULK1 kinase assay with increasing amounts of ULK1. Reactions were immunoblotted with the indicated antibodies. Black line denotes discontinuous lanes from the same gel. Two unique experiments were performed. (b) Beclin-1 alone or bound to ATG14L was purified as described in a. Equal amounts of ULK1 were added to each complex and reactions were quenched at the indicated time points. Western blotting was performed with the indicated antibodies. (c) An ATG14L–FLAG-6His-inducible U2OS cell line was induced for 16 h in the presence of amino acids. Endogenous Beclin-1 was immunoprecipitated and immunoblotted as in Fig. 3a. ATG14L input levels were detected by immunoblotting. Two unique experiments were performed. (d) HEK293 cells transfected with either ATG14L or Beclin-1, or both, in conjunction with ULK1, were immunoprecipitated as indicated and blotted with the indicated antibodies. (e) HEK293 cells were transfected with Beclin-1 and ULK1 in the presence of ATG14L WT or ATG14LΔCCD, which is defective in Beclin-1 binding, under nutrient-rich conditions. Lysates were resolved by SDS–PAGE and blotted with the indicated antibodies. (f) HEK293 cells were transfected with ULK1 and Beclin-1 in conjunction with either ATG14L WT, or one of two mutants (ΔBATS,ΔN) that are defective in phagophore localization. Samples were handled as in e. Uncropped images of blots are shown in Supplementary Fig. S4.
ULK1 induces autophagy by phosphorylating Beclin-1 and activating VPS34 lipid kinase (2013)
figf5

(a) HEK293 cells were transfected with Beclin-1, with or without UVRAG, in conjunction with ULK1 as indicated in the presence of amino acids. Lysates were immunoblotted with the indicated antibodies. A representative experiment of three repeats is shown. (b) UVRAG bridges the interaction between Beclin-1 and ULK1. HEK293 cells were transfected with Beclin-1, with or without UVRAG, in conjunction with ULK1 as indicated. Lysates were immunoprecipitated with anti-HA(Beclin-1) antibody and blotted with the indicated antibodies. A representative experiment of three repeats is shown. Uncropped images of blots are shown in Supplementary Fig. S4.