EMBO/soda-nel huggingface dataset:
TGIF1 functions as a tumor suppressor in pancreatic ductal adenocarcinoma (2019)
Figure 7

Figure 7: TGIF1 phosphorylation in PDAC disrupts its anti-tumor activity

A Representative microscope pictures (20x) of MIAPaCa-2, Suit-2, Capan-2 and Panc-1 cells. Scale bars, 200 μM.

B Expression of TGIF1 and Twist1 in MIAPaCa-2, Suit-2, Capan-2 and Panc-1 cells was analyzed by immunoblotting.

C,D MIAPaCa-2, Suit-2, Capan-2 and Panc-1 cells were transfected with empty vector (EV), wild-type (TGIF1) or phosphorylation mimic mutant (TGIF1.2TD) of TGIF1. Cell extracts were analyzed 48 hr following transfection by immunoblotting using anti-Flag antibody (C). Cells were selected with neomycin for 2-3 weeks, and counted using an automatic cell counter (D) (n=6). Data are expressed as mean ± SEM. **p< 0.01; ***p< 0.001; based on a two-tailed Student's test.

E Model of suppression of TGIF function by KrasG12D/MPK-mediated phosphorylation. In this model, acquisition of KrasG12D leads to constitutive activation of MAPK/ERK, which in turn phosphorylates TGIF1. Phosphorylation of TGIF1 disrupts its ability to inhibit Twist1 expression, thereby leading to suppression of CDH1 and p16INK4A expression and attendant PDAC formation and progression.
Autophagy mediates temporary reprogramming and dedifferentiation in plant somatic cells (2019)
Figure 1

Fig 1. Autophagy is rapidly engaged upon perception of diverse stimuli. GFP-ATG8a expressing seedlings in Murashige & Skoog (MS) growth medium or 30 min after treatment with MS containing ACC, ABA, ATP, BL, 6-BA, Flg22, NAA or PEP1. (A) Representative maximum intensity projection images of 10 Z-stacks per image. Scale bar: 10 µm. (B) Quantification of GFP foci per 0.0025 mm2. Values are presented as mean ± standard deviation of the mean and were calculated from at least three independent experiments with 3 individuals per replicate. Bars marked with an asterisk (*) are statistically significant (P<0.05) according to the T -test. (C) GFP-ATG8a cleavage immunoblot for plants exposed to the same treatments as in (A). Numbers below the blots represent ratio for given sample normalized to input and relative to non-treated control. Experiments were repeated minimum 3 times with similar results. (D) NBR1 immunoblot for atg2-2 samples for given treatments. Numbers below the blots represent ratio for given sample normalized to input and relative to non-treated control. Experiments were repeated minimum 3 times with similar results.
Autophagy mediates temporary reprogramming and dedifferentiation in plant somatic cells (2019)
Figure 2

Fig 2. Autophagy is reactivated upon contrasting stimuli perception. (A) Seedlings were acclimated for 16 h in MS containing ABA and then imaged 30 min after being swapped to MS containing ABA/NAA (control), MS or MS containing flg22. Images are representative maximum intensity projection of 10 Z-stacks per condition. Experiments were repeated 3 times independently with similar results. Scale bar: 10 µm.

(B) Quantification of GFP foci per 0.0025 mm2, for samples treated as described in A. Values are presented as mean ± standard deviation of the mean and are based on 3 independent experiments, with 3 individuals per condition. Bars marked with an asterisk (*) are statistically significant (P<0.05) according to the T -test.

(C) GFP-ATG8a cleavage immunoblot for given treatments. The ratio of free GFP to loading control, normalized to the 16h pre-treated sample (set to 1) is provided below each band.

(D) Seedlings were acclimated for 16 h in MS containing NAA and then imaged 30 min after being swapped to MS containing ABA/NAA (control), MS or MS containing 6-BA. Images are representative maximum intensity projection of 10 Z-stacks per condition. Experiments were repeated 3 times independently with similar results. Scale bar: 10 µm.

(E) Quantification of GFP foci per 0.0025 mm2, for samples treated as described in (D). Values are presented as mean ± standard deviation of the mean and are based on 3 independent experiments, with 3 individuals per condition. Bars marked with an asterisk (*) are statistically significant (P<0.05) according to the T -test.

(F) GFP-ATG8a cleavage immunoblot for given treatments. The ratio of free GFP to loading control, normalized to the 16h pre-treated sample (set to 1) is provided below each band.
Autophagy mediates temporary reprogramming and dedifferentiation in plant somatic cells (2019)
Figure 3

Fig. 3. Autophagy facilitates temporary reprogramming during perception of contrasting stimuli by removing old components and modulating the intensity of new responses. (A) Schematic representation of the strategy used for consecutive stress treatment. (B) Pattern correlation used to find proteins that accumulate upon ABA/NAA treatment and are removed in WT but not in atg2 after swapping to flg22/6-BA. (C-F) Protein clusters obtained after quantitative proteomics of WT (green) and atg2-2 (magenta) samples treated as described in (A). (C) Protein cluster fitting the pattern displayed in (B). (D) Protein cluster for proteins that accumulate to higher levels in atg2 than WT upon treatment with flg22. (E) Protein cluster of proteins which accumulate upon NAA treatment and are removed in WT but not in atg2 after swapping to 6-BA (F) Protein cluster for proteins that accumulate to higher levels in atg2 than WT upon treatment with 6-BA.
Autophagy mediates temporary reprogramming and dedifferentiation in plant somatic cells (2019)
Figure 4

Fig 4. Autophagy deficiency leads to reduced phenotipic plasticity and increased heterogeneity (A) Weather pattern recorded for the Swedish spring of 2013.

(B) Dry weight of WT, atg5-1 or atg2-2 plants grown under stable (21/16 ºC 16/8-h photoperiod) or following the Swedish Spring of 2013. Box plots: centerlines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend to the minimum and maximum. Significance calculated by Kruskal-Wallis or Feltz and Miller test for the equality of coefficients of variation as explained in materials and methods.

Asterisks represent significant pairwise differences ( ∗∗∗∗P< 0.0001)

(C) Box plots displaying the heterogeneity of samples in (B). Box plots: centerlines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend to the minimum and maximum. Significance calculated by Kruskal-Wallis or Feltz and Miller test for the equality of coefficients of variation as explained in materials and methods. Asterisks represent significant pairwise differences (∗∗P < 0.01, ∗∗∗∗P< 0.0001)
Autophagy mediates temporary reprogramming and dedifferentiation in plant somatic cells (2019)
Figure 5

Fig 5. Dedifferentiation is severely impaired in autophagy deficient cells.

(A) Representative images of root explants from WT, atg2-2 and atg5-1 in CIM (6 days) or CIM + SIM (6 + 21 days). Scale bar: 2 cm

(B) Quantification (%) of explants presenting shoots after 21 days after incubation on SIM. Results were obtained from 3 independent experiments with at least 20 calli per condition. Bars marked with an asterisk (*) are statistically significant (P<0.05) according to the T -test.
Autophagy mediates temporary reprogramming and dedifferentiation in plant somatic cells (2019)
Figure 6

Fig. 6. Autophagy deficient cells lose control over de novo organogenesis and senesce earlier upon prolonged cultivation in pluripotent cell inducing media (A) Representative images of root explants from WT, atg2-2 and atg5-1 in CIM (21 days) or CIM + SIM (21+ 21 days or 21+ 35 days). Scale bar: 2 cm (B and C) Fresh weights of calli after incubation on CIM (B) or CIM + SIM (21+ 21) (C). Box plots: centerlines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend to the minimum and maximum. Results were obtained from 3 independent experiments with at least 20 calli per condition, asteriscs mark statistical significance to WT according to the T -test (** P<0.001; *** P<0.0001).

Figure Expanded View legends
CANCER-ASSOCIATED MUTATIONS IN VAV1 TRIGGER VARIEGATED SIGNALING OUTPUTS AND T CELL LYMPHOMAGENESIS (2021)
Figure 3

FIGURE 3. New regulatory layers unveiled by the VAV1 mutations

(A) 3D structure of the Vav1 CSH3 domain. The previously described intramolecular inhibitory sites 1 and 2 of the Vav1 CSH3 domain are depicted in orange and yellow, respectively (Barreira et al., 2014). The regulatory Y836 phosphosite is highlighted in blue (Barreira et al., 2014). The residues present in the new inhibitory interface of the CSH3 are shown in raspberry color.

(B) 3D structure of the VAV1 SH2 (red) bound to a Syk tyrosine-phosphorylated peptide (yellow). The mutated residues are shown in purple. p-Y, tyrosine phosphorylated.

(C) Levels of phosphorylation of the Y174 phosphosite of the indicated Vav1 mutant proteins immunoprecipitated from Jurkat cells (upper panel). The total amount of Vav1 immunoprecipitated in each sample is shown in the bottom panel (n = 3 independent experiments).
CANCER-ASSOCIATED MUTATIONS IN VAV1 TRIGGER VARIEGATED SIGNALING OUTPUTS AND T CELL LYMPHOMAGENESIS (2021)
Figure 6

FIGURE 6. Vav1∆C-mediated proliferation requires engagement of several downstream pathways

(A) Schematic representation of the experiments used in this figure. See details in main text and Methods.

(B) Depiction of the Vav1 mutants used in these experiments. The E201A mutation is shown as a gray circle. The activity of each mutant is represented on the right. Green box, WT activity; red box, gain-of-function; blue box, loss-of-function.

(C) Representative immunoblot showing the abundance of the ectopic Vav1 proteins, EGFP and the endogenous tubulin α (loading control) in CD4+ T-cells transduced with retrovirus particles used in these experiments (top).

(D) Representative FACS plots showing EGFP epifluorescence in CD4+ T-cells transduced with retroviruses encoding the indicated proteins (top). Numbers indicate the relative percentage (%) of the EGFP+ cell population in each case. Grey shaded histograms represent the fluorescence obtained from CD4+ T-cells nontransduced with retrovirus particles. Similar results were obtained in CD4+ T cells transduced with virus encoding EGFP (n = 6), Vav1WT (n = 6), Vav1∆C (n = 6), Vav1∆C+E201A (n = 6) and Vav1∆N (n = 3).

(E, F) Representative FACS plots (E) and quantification (F) of the proliferation of EGFP+ CD4+ T cells expressing the indicated proteins using the Cell Trace Violet method. In E, the grey shaded histograms represent the fluorescence obtained from nonstimulated CD4+ T cells before stimulation and retroviral transduction. In F, each point represents the values obtained with a single experimental mouse. n = 6 per each experimental condition, except in the case of Vav1∆N (n = 3).

(G-J) Flow cytometry determination of Tox (G), ICOS (H), CD25 (I) and ICN1 (J) expression in EGFP+ CD4+ T cells expressing the indicated Vav1 proteins. NSt, Non stimulated cells (CD4+ T cells before stimulation and retroviral transduction). In all panels, each point represents the values obtained for a single experimental condition. f.i., mean fluorescence intensity relative to the isotype-matched control antibody. n = 6 per each experimental condition, except for the Vav1∆N and NSt conditions (n = 3).

(K-N) Example of a flow cytometry analysis (K and M) and final quantification of p-Akt (L) and p-Erk1/2 (N) levels in EGFP+ CD4+ T cells expressing the indicated Vav1 proteins in primed (blue bars) and restimulated (red bars) cells. In K and M, the grey shaded histograms represent the fluorescence obtained with the isotype-matched control antibody. In L and N, NSt, nonstimulated, St, stimulated. n = 3 per each experimental condition.

Data information: In panels F, G, H, I, J, L and N, values are shown as means ± SEM from three independent experiments. In panels L and N, P values are given relative to nonstimulated (blue asterisks) and stimulated (red asterisks) cells expressing Vav1WT. We also included P values for the values exhibited by each Vav1 mutant protein relative to those obtained in nonstimulated condition (black asterisks). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 (Student's t test).
CANCER-ASSOCIATED MUTATIONS IN VAV1 TRIGGER VARIEGATED SIGNALING OUTPUTS AND T CELL LYMPHOMAGENESIS (2021)
Figure 7

FIGURE 7. Vav1∆C-driven CD4+ T cell proliferation requires ICN1 and proper calcineurin signaling

(A, B) Representative FACS plots (A) and quantification (B) of the EGFP+ CD4+ T cell proliferation in the different experimental groups (right) and indicated experimental conditions (top) using the Cell Trace Violet detection method. In A, grey shaded histograms represent the fluorescence obtained from nonstimulated CD4+ T cells before stimulation and retroviral transduction. n = 3 independent experiments.

(C-E) Flow cytometry determination of intracellular Tox (C), intracellular ICN1 (D) and surface ICOS (E) levels in EGFP+ CD4+ T cells expressing the indicated Vav1 proteins. f.i., mean fluorescence intensity relative to the isotype-matched control antibody. n = 3 independent experiments.

(F) Summary of the Vav1-regulated signaling pathways that contribute to promote the proliferation of primary CD4+ T cells that have been unveiled in this work. The other downstream effectors and pathways of Vav1 are indicated. The main primary effectors as shown in red.

Data information: In panels B, C, D and E, values are shown as means ± SEM from three independent experiments. P values are given relative to nontreated (light blue asterisks) and treated (dark blue asterisks) EGFP+ cells. We also include P values for the values exhibited by each experimental group relative to those obtained in nontreated condition (black asterisks). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 (Mann-Whitney U test).

LEGENDS TO EXPANDED VIEW FIGURES
Desensitisation of Notch signalling through dynamic adaptation in the nucleus (2021)
Figure 1

Figure 1. Optogenetic induction of NICD nuclear translocation and target gene expression with minute-scale precision in live Drosophila embryos.

(A-B) Illustration depicting the optogenetic system employed to control NICD nuclear translocation (opto-Notch). NICD (purple) with its endogenous NLS deleted was tagged with the AsLOV2 domain (turquoise) from Avena Sativa is such a way that its Jα helix masks an engineered NLS in the dark (A). To ensure maximal nuclear exclusion, Zdark (blue dashed line on the mitochondrion), a protein domain which binds specifically to the dark conformation of AsLOV2, was localised to mitochondria through a TOM20 targeting motif (not depicted). Blue light illumination triggers a conformational change in the LOV2 domain, which results in the simultaneous unbinding from mitochondria and exposure of the engineered NLS, thus allowing NICD nuclear translocation and target gene (sim) expression, appearing as a green dot in the nucleus (B).

(C-D) Snapshots from confocal live imaging movies of a representative embryo (N=7) during cellularisation co-expressing opto-Notch, Mito-Zdk, MCP::GFP and sim-MS2. Continuous photo-activation from the onset of cellularisation with a stack size of z= 25μm (λ=488 nm) was alternated with mCherry excitation (λ=561 nm) at 1 min intervals. Single confocal z-slices of the mCherry channel are shown to record opto-Notch localization before, and 10 minutes after sustained photo-activation. Magnified insets show opto-Notch bound to the mitochondrial anchor in the dark (C) and in the nucleus upon photo-activation (D). Scale bar, 10 μm.

(E) Wild-type sim expression profile in a Drosophila embryo at the onset of gastrulation as revealed by in situ hybridization. Scale bar, 50 μm

(F-H) Maximum intensity z-projections (z = 25 μm) of the same embryo as in (C,D) simultaneously photoactivated and imaged in the GFP channel (λ=488 nm) to record sim expression at a time resolution of 30 s. Shown are the first cycle of photo-activation, which approximates the dark state (F), 15 min (G), and 35 min (H) after continuous photo-activation. Magnified insets show that sim is not expressed in the dark at this stage (F), but it is expressed in lateral ectodermal cells after 15 min of photo-activation (G), and appeared to be shut off in most nuclei after 35 mins (H). Scale bar, 10 μm.
Desensitisation of Notch signalling through dynamic adaptation in the nucleus (2021)
Figure 2

Figure 2. Transcriptional activation in response to NICD nuclear translocation undergoes near-perfect adaptation.

(A-C) The number of sim-expressing nuclei as counted by automated spot detection is shown over time for different optogenetic activation regimes (top blue bars). (A) Continuous opto-Notch activation initially induces sim expression across a large number of nuclei. Over time, the majority of nuclei adapt to the input and terminate expression. (B) Inactivation is not due to developmental timing, as nuclei remain responsive when activation is delayed to a later time point. (C) Sustained nuclear localization of NICD is required for adaptation to take place, as pulsatile activation can overcome adaptation. Note that sim expression could not be measured during dark phases of pulsatile photo-activation as its visualization requires 488 λ nm excitation which causes photo-activation; dashed lines are a visual aid connecting data from the same embryo.

(D-E) Quantification of nuclear localisation of mCherry::NICD during opto-Notch photo-activation. Raw data was bleaching-corrected with an exponential fit and shown is the nuclear/total ratio, which further normalises bleaching and embryo-to-embryo variability. NICD displays persistent nuclear enrichment during continuous activation (D) and shuttles back and forth between nucleus and cytoplasm under the pulsatile protocol (E).
Desensitisation of Notch signalling through dynamic adaptation in the nucleus (2021)
Figure 4

Figure 4. Molecular perturbations matched to model predictions reveal that adaptation is best explained by state-dependent inactivation.

(A-C) Simulated predictions of sim expression dynamics during continuous opto-Notch activation under different molecular perturbations. Row 1 shows the wild-type model behaviours. Rows 2-3 show the response to a knock-out of a negative regulator of sim expression, which could be either the internal inhibitor (B) of the adaptation motifs (row 2) or an attenuator that in wild-type dampens the activation of (A) (row 3). Row 4 simulates the stabilization of (A) in its active state by reduction of either the inhibitory action of (B) or the auto-inactivation of (A). Note that these perturbations generally lead to a loss of adaptive behaviour, with the exception of the attenuator knock-out on state-dependent inactivation (C; row 3).

(D-E) Results of continuous activation experiments in a Hairless heterozygous hypomorphic mutant (Hairless H2) and under overexpression of Twist (Twist OE), which could act as a stabiliser of (A). The observed outcomes are most consistent with the predictions from the state-dependent inactivation model.

(F) Proposed model of Notch nuclear adaptation: NICD induces target gene expression by triggering an active state of the Promoter-Enhancer Complex (EPC). Continuous activation leads to a state-dependent transition of the EPC into an inactivated refractory state. Hairless reduces the effective activation rate whereas Twist reduces the effective inactivation rate.

Expanded View Figure Legends
Hfq-dependent mRNA unfolding promotes sRNA-based inhibition of translation (2019)
Figure 4

Figure 4. Footprinting of the OmrA/ OmrB/ Hfq binding sites on dgcM mRNA by chemical and enzymatic probing

A - 5' end-labeled dgcM mRNA (10 nM) was subjected to probing as described in Materials and Methods. All components added to the labeled RNA are indicated above the autoradiogram. When added, Hfq hexamers were at 50 nM, the sRNAs OmrA and OmrB at 250 nM. The accessibility of each nucleotide was probed with water (lanes 1-4, negative control for RNA integrity), RNase T1, or lead(II) acetate. Lane T1: RNase T1 under denaturing conditions. Lane OH: alkaline ladders. Binding sites of Hfq (green arrows and boxes), and site 1 (red arrows and boxes) and 2 (blue arrows and boxes) of the sRNAs, are highlighted. Note that site 2 is bound differently by OmrA and OmrB (blue boxes on gel). Black boxes indicate Hfq-dependent T1 cleavage enhancements.

B - Secondary structure of dgcM mRNA ± Hfq, based on results in Figure 4A. Nucleotides are color-coded as in 4A. Unchanged T1 cleavages are indicated by filled arrows, enhanced T1 cleavages by arrow and star, protection from T1 cleavages by dashed arrows. Lead(II) acetate-protected sequences are indicated by full (Hfq+OmrA/B), dashed (Hfq + OmrA), or dotted (Hfq alone) frames. In the lower panel Q = Hfq, A = OmrA, and B = OmrB.
mTORC2 sustains thermogenesis via Aktinduced glucose uptake and glycolysis in BAT (2015)
Figure 5

Figure 5. mTORC2 in adipose tissue is required for cold-induced glucose uptake and glycolysis (A) 2-Deoxyglucose-6-phosphate (2DG6P) accumulation in BAT of AdRiKO and control mice housed at 22°C or at 4°C for 4h (n=6/group). (B) Extracellular acidification rate (ECAR) of BAT explants from AdRiKO and control mice housed at 22°C or at 4°C for 4h (n=7/group). (C) Immunoblot analysis of BAT from AdRiKO and control mice housed at 22°C or at 4°C for 8h for the indicated proteins (n=6/group, each lane represents a mix of 3 mice). (D) Immunoblot analysis of isolated plasma membranes from BAT of AdRiKO and control mice housed at 22°C or at 4°C for 8h for the indicated proteins (n=6/group, each lane represents a mix of 3 mice). (E) Immunoblot analysis of mitochondrial and cytosolic fractions from BAT of AdRiKO and control mice housed at 22°C or at 4°C for 4h for the indicated proteins (n=6/group, each lane represents a mix of 3 mice). (F) Cytosolic hexokinase activity in BAT of AdRiKO and control mice housed at 22°C or at 4°C for 4h (n=5 (control 22°C), n=5 (AdRiKO 22°C), n=7 (control 4°C), n=7 AdRiKO 4°C)). (G) Mitochondrial hexokinase activity in BAT of AdRiKO and control mice housed at 22°C or at 4°C for 4h (n=5 (control 22°C), n=5 (AdRiKO 22°C), n=7 (control 4°C), n=7 AdRiKO 4°C)). Data represent mean ± SEM. Statistically significant differences between AdRiKO and control mice were determined with unpaired Student’s t-test and are indicated with asterisks (*=p<0.05; **=p<0.01, ***=p<0.001). Statistically significant differences between temperatures were determined with unpaired Student’s t-test and are indicated with a number sign (#=p<0.05; ##=p<0.01; ###=p<0.001). The exact p-value for each significant difference can be found in Appendix Table S2.