EMBO/soda-nel huggingface dataset:
RASSF1C oncogene elicits amoeboid invasion, cancer stemness and extracellular vesicle release via a SRC/Rho axis (2021)
Figure 5

Figure 5 RASSF1C oncogene induces invasion and metastatic dissemination via EV transfer in vivo

A. Cartoon of in vivo experimental approach. MDA-MB-231CFP;Cre and reported T47DDsRed cells were co‑injected in one mammary gland of 10 mice per experiment.

B. Left: representative confocal images of mammary tumor formed by the co-injection of T47DDsRed reporter cells with either MDA-MB-231CFP;Cre;Control or MDA-MB-231CFP;Cre;HA-RASSF1C. Scale 200 μm. Right: averaged values for all mice for the percent of the eGFP+ or CFP+ cells as part of the whole tumor.

C. Representative 3 hour migration paths of either MDA-MB-231CFP;Cre;Control (Control, left) or MDA‑MB‑231CFP;Cre;HA-RASSF1C (RASSF1C, right) within a single intravital imaging field of tumors co‑injected with T47DDsRed reporter cells.

D. In vivo migration speed of MDA‑MB‑231CFP;Cre;Control or MDA‑MB‑231CFP;Cre;HA-RASSF1C in a mixed tumor with T47DDsRed cells. Values are an average of the totals from 5 mice each, measured from intravital time-lapse images.

E. The normalized in vivo motility of T47DeGFP and T47DDsRed reporter cells in mixed tumors. A Non‑parametric Mann-Whitney U test was used to derive statistical significance.

F. Cartoon representing the Cre-LoxP model exploited to follow up amoeboid motility in recombined reporter (T47DeGFP) cells.

G. Quantification of distinct morphology of T47DeGFP in tumors from observed from a total of 30 positions in n=5 mice/group (Control or RASSF1C). Mesenchymal, Amoeboid or Hybrid motility were scored manually as described in Fig. 1K.

H. Quantification of the number of T47DeGFP metastatic events found in the lungs of mice with mammary gland tumors. Whole lung tile images were used for quantification, 6 of 10 μm sections each, 100 μm apart.

Data information: All data are from n=3 independent experiments. Data are analyzed by Student T-test and represented as mean ± SEM.* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.
RASSF1C oncogene elicits amoeboid invasion, cancer stemness and extracellular vesicle release via a SRC/Rho axis (2021)
Figure 6

Figure 6 RASSF1 gene methylation as a prognostic biomarker in breast cancer patients

A. Schematic representation of the RASSF1 gene showing the domains in RASSF1A and C. Bars shaded dark and light green denote the CpG islands associated with RASSF1A and RASSF1C respectively.

B. Kaplan-Meier plots of overall survival of 496 stage I and II breast cancer patients with different levels of promoter/CpG island methylation of RASSF1A, previously reported to be associated with outcome in breast cancer patients. P values were obtained from a log-rank test.

C. Left: heatmap depicting methylation patterns across the RASSF1 gene. In light green, RASSF1-2γ CpG island is depicted; in dark green, RASSF1-1α CpG island is depicted. Right: Kaplan‑Meyer survival curves depicting interaction of RASSF1A and RASSF1C methylation to predict the outcome of early stage breast cancer patients. P values were calculated from a log-rank test. Color legend: yellow, low RASSF1A and low RASSF1C; green, low RASSF1A and high RASSF1C; red, high RASSF1A and low RASSF1C; grey, high RASSF1A and high RASSF1C.

D. Model representing how RASSF1C expression in RASSF1A‑silenced breast cancer cells (MDA-MB-231) activates a signaling cascade involving SRC kinase, RhoGDI and culminates in RhoA activation and generation of amoeboid contractility and cancer stemness features. RASSF1C-driven amoeboid cancer stem cells contribute to alteration of the tumor microenvironment by rendering recipient cells (MCF7, T47D) more invasive and able to upregulate stem cell markers, due to EV-mediated communication.

Expanded View Figure Legends
Myc suppresses male-male courtship in Drosophila (2022)
Figure 3

Fig. 3. Loss of Myc enhances Ddc transcription and Ddc-mediated dopamine level.

(A-C) DA level (pictograms per head) in males of the indicated genotypes. n = 3, 3, 3 (A); n = 4, 5, 3, 3 (B); n = 3, 3, 3 (C) times (100 heads per genotype).

(D) Posterior confocal sections of UAS-mCD8-GFP; Ddc-Gal4 adult male brain in combination with UAS-LacZ or UAS-Myc.RNAi-1 stained with antibody to nc82 (pan-neuropil marker; magenta). Scale bars represent 50 μm.

(E) Quantification of GFP and nc82 fluorescence intensity in GFP positive somata area (n = 10 heads per genotype). Statistical significance was compared with relative control.

(F, G) Western blot of proteins extracted from adult heads of indicated genotypes. Statistical analysis showing the protein level of GFP (F) or Ddc (G) to that of β-actin in Western blot.

Data information: Data are presented as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Asterisks indicate significance by unpaired t test. Ordinary one-way ANOVA and the Dunn's multiple comparison test was used to assess significance for multiple samples.
Myc suppresses male-male courtship in Drosophila (2022)
Figure 4

Fig. 4. Myc binds to its target site in Ddc promoter and inhibits Ddc transcription.

(A) Structure of Drosophila Ddc gene. The open reading frame (ORF) is represented as gray boxes separated by introns in lines, 5' and 3' UTR are shown as open box. Two putative Myc target sites (E1 and E2) are indicated.

(B) Drosophila S2 cells transfected with the pUAST-HA empty vector or the pUAST-HA-Myc plasmid were co-transfected with each of the firefly luciferase reporter vector bearing the fragment shown in (A). After 48 hours, the luciferase activity was detected in each cell lysate (n = 5 times).

(C) Drosophila S2 cells transfected with the pUAST-HA-Myc plasmid or the empty vector were used for quantification of ChIP-PCRs (n = 4 times).

(D) RNA from adult male heads (n = 50 heads per genotype) of indicated genotypes was subjected to real-time qPCR.

(E) DA level (pictograms per head) in the head of males of the indicated genotypes. n = 4 (100 heads per genotype).

(F) Courtship indices of wild type and DdcΔE1 males. Changes were determined by comparing the data with control. n = 10, 10 flies.

(G) Chaining indices of males of the indicated genotypes. Statistical significance was compared with relative control. n = 8, 6, 12 (3 days); 5, 5, 7, 7, 8, 8 (9 days) groups (10 flies per group).

(H) Courtship indices for one target (Canton-S) male and one tester male of the indicated genotypes. Changes were determined by comparing the data to relative control. n = 15, 15, 11 (3 days); 14, 13, 14, 15, 16, 18 (9 days) flies.

Data information: Data are presented as means ± SEM. **p < 0.01, ***p < 0.001, ****p < 0.0001. Asterisks indicate significance by unpaired t test in (B, C). Analyses of two samples employed unpaired t test in (D, E). Analyses of two samples employed Mann-Whitney test in (F). The Kruskal-Wallis test and the Dunn's multiple comparison test was used to assess significance for multiple samples (G, H).
Transcription factor EB (TFEB) is a new therapeutic target for Pompe disease. (2013)
Figure 1

TFEB stimulates clearance of enlarged lysosomes and reduces glycogen burden in PD myotubes.

A. Confocal microscopy image of PD myotubes (cl. 3LE8) infected for 72 h with adenovirus containing TFEB (PD + Ad-TFEB 72 h) shows a dramatic reduction in the number of large LAMP1-positive lysosomes (red) compared to that in untreated (PD) or adenovirus (PD + Ad-null)-treated PD myotubes. WT myotubes are shown on the left panel. Nuclei are stained with Hoechst (blue). TFEB was detected with anti-Flag antibody (green).

B. Distribution of lysosomal size differs significantly in Ad-null and Ad-TFEB PD myotubes (p = 6.32 × 10−8; Kolmogorov–Smirnov test). Lysosomal size is expressed as number of pixels representing lysosomal area (LAMP1-positive structures). The median lysosomal size of Ad-TFEB infected myotubes (m = 367.13 pixels, n = 703, range 208–2659) was significantly lower than that of Ad-null infected myotubes (m = 491.16 pixels, n = 1395, range 200–2857; p = 6.5 × 10−12; Wilcoxon rank sum test).

C. Confocal microscopy image of PD myotubes infected for 24 h with Ad-TFEB shows relocation of lysosomes to the plasma membrane (top). Images showing LAMP1 staining (red) on plasma membrane in a PD myotube infected with Ad-TFEB (bottom; arrows) but not in a non-infected cell (middle). Non-permeabilized cells were incubated with anti-LAMP1 antibody at 4°C for 40 min, followed by fixation and staining with secondary antibody.

D. Confocal microscopy images of live non-infected PD myotubes (left) or PD myotubes infected for 72 h with Ad-TFEB (right) show a dramatic reduction in the amount of accumulated glycogen in TFEB-treated cells. The cells were incubated with the fluorescent glucose (2-NBDG; green), extensively washed, and analysed by confocal microscopy. Bar: 10 µm for all panels.
Transcription factor EB (TFEB) is a new therapeutic target for Pompe disease. (2013)
Figure 2

Therapeutic enzyme is trapped in the area of autophagic build-up in PD fibres.

Confocal microscopy of live unstained fibres from a GFP-LC3:WT mouse (WT) and from untreated (GAA−/−) or ERT-treated (GAA−/−; +ERT) GFP-LC3:GAA−/− mice. Images show typical centrally located autophagic build-up with multiple clusters of LC3-positive autophagosomes. Structures like these are never seen in WT muscle. Autophagic build-up is present virtually in all fibres derived from EDL or gastrocnemius muscles (not shown), but only in 60–70% of FDB fibres. Labelled recombinant human GAA (red) was administered i.v. into 3–4 month-old GFP-LC3:GAA−/− mice (n = 5) at a dose of 100 mg/kg twice with a 24 h interval. Mice were sacrificed the next day, and live fibres (shown for EDL muscle) were analysed by confocal microscopy. The labelled recombinant GAA was detected almost exclusively in the autophagic vesicles. At least 500 fibres were analysed for each condition. Bar: 10 µm.
Transcription factor EB (TFEB) is a new therapeutic target for Pompe disease. (2013)
Figure 3

TFEBmt reduces lysosomal size, but induces apoptosis in a subset of PD myotubes.

A. Immunostaining of non-infected cells (day 13 in differentiation medium) and cells infected with a mutant form of TFEB (TFEBmt) with LAMP1 (red) and Flag (green). Ad-TFEBmt was added to the myotubes for 72 h on day 10 in differentiation medium. Ad-TFEBmt-infected cells show massive accumulation of TFEB in the nuclei and significant reduction in lysosomal size, similar to that seen with TFEB.

B. Western blot of cell lysates confirms the presence of TFEB in the nuclear fraction; the different intensities of the two bands corresponding to LAMP1 protein in untreated and TFEBmt-treated samples may reflect the differences in the glycosylation pattern.

C. Western blot of protein lysates from untreated, Ad-null, TFEB, and TFEBmt-treated myotubes with anti-caspase-3 antibody. No activated (cleaved) products are detected in any condition. α-Tubulin was used as a loading control.

D. TUNEL assay shows the presence of some apoptotic cells in Ad-TFEB-infected cultures, but not in control cultures infected with adenovirus alone (n = 3). Bar: 10 µm for all panels.
Transcription factor EB (TFEB) is a new therapeutic target for Pompe disease. (2013)
Figure 4

TFEB promotes clearance of enlarged lysosomes and increases lysosomal motility in PD fibres.

A. Confocal microscopy images of live fibres derived from 3 to 4 month-old GFP-LC3:WT (top left), untreated GFP-LC3:GAA−/− (bottom left) or TFEB-treated GFP-LC3:GAA−/− (right) mice. All fibres were transfected with mCherry-LAMP1 to visualize lysosomes (red). The effects of TFEB are clearly visible – overall reduction in lysosomal size, appearance of normal size lysosomes (similar to those in the WT), and lysosomal docking to the plasma membrane (inset). Bar: 10 µm.

B. FDB muscle of a GAA−/− mouse was transfected with both GFP-TFEB and mCherry-LAMP1 (GAA−/− + GFP-TFEB). Images were taken before (left) and after 4 h (right) of time-lapse microscopy. Lysosomal clearance is visible in the TFEB-transfected fibre at both time points. Lysosomes appear to “exit” the fibre (inset and arrow) at the 4 h time point when TFEB is activated as evidenced by its nuclear translocation (green nuclei). Bar: 10 µm.

C. The mean maximum velocity of lysosomes (top) and the number of large (>3.5 µm) lysosomes (bottom) in untreated and TFEB-treated PD fibres (note: all data for Flag- and GFP-TFEB-treated fibres are pooled). Lysosomal velocities were calculated from time-lapse images using ImageJ software with the manual tracking plug-in. For each condition the trajectories of multiple lysosomes were followed (n = 26 untreated; n = 43 TFEB-treated) and the three highest velocity measurements per lysosome were recorded. In TFEB-treated fibres, the maximum velocity of lysosomes was significantly increased (p = 2.07 × 10−17) and the number of large lysosomes was significantly decreased (p = 1.0 × 10−3). Ten untreated and 24 TFEB-treated fibres were analysed for the size calculations. * indicates statistically significant differences (p ≤ 0.001; Student's t-test). Error bars represent 95% confidence intervals.
Transcription factor EB (TFEB) is a new therapeutic target for Pompe disease. (2013)
Figure 5

TFEB stimulates lysosomal–autophagosomal fusion and clearance.

Confocal microscopy images of live fibres from GFP-LC3:GAA−/− mice.

Muscle was transfected with mCherry-LAMP1 only. The image (a single frame from the time-lapse series presented in Movie 5) shows lysosomes (red), LC3-positive autophagosomes (green) and a number of autolysosomes (yellow).

Muscle was transfected with Flag-TFEB and mCherry-LAMP1. Massive formation of autolysosomes is indicated by yellow structures; the three lower panels provide a snapshot of the process of exocytosis. The structures at the plasma membrane are labelled with both LC3 and LAMP1, indicating that they represent amphisomes (a product of fusion between autophagic vesicles and late endosomes) or autolysosomes (a product of fusion between autophagic vesicles and lysosomes). Bar: 10 µm for all panels.
Transcription factor EB (TFEB) is a new therapeutic target for Pompe disease. (2013)
Figure 6

TFEB promotes redistribution and docking of lysosomes to the plasma membrane in autophagy-deficient PD fibres.

Confocal microscopy images of live fibres from muscle-specific autophagy-deficient GAA−/− mice (Atg7:GAA DKO).

Muscle was transfected with mCherry-LAMP1 only.

Muscle was transfected with GFP-TFEB and mCherry-LAMP1 (Atg7:GAA DKO +GFP-TFEB). The TFEB-transfected fibres show realignment of the lysosomes and membrane detachment (most striking in top and bottom panels) similar to those in TFEB-transfected fibres from PD mice (see Fig 4B inset). Lysosomes can be seen in the space between the fibre and plasma membrane (arrows). Bar: 10 µm for all panels.

The mean maximum velocity of lysosomes (top) and the number of large (>3.5 µm) lysosomes (bottom) in untreated and TFEB-treated autophagy-deficient Atg7:GAA DKO fibres. The increase in maximum velocity (41%) is significant (p = 2.729 × 10−18; n = 57 lysosomes for untreated; n = 52 lysosomes for TFEB-treated), but there is only a slight trend toward smaller lysosomal size (p = 7.0 × 10−2; n = 10 fibres for untreated; n = 21 fibres for TFEB-treated). The corresponding values from GAA−/− mice are presented for comparison (for TFEB-treated condition: n = 19 lysosomes for velocity measurements, and n = 10 fibres for the lysosomal size measurements). * indicates statistically significant differences (p ≤ 1.0 × 10−5 for the top panel and p ≤ 0.01 for the lower panel; Student's t-test).
Transcription factor EB (TFEB) is a new therapeutic target for Pompe disease. (2013)
Figure 7

Intramuscular injection of AAV2.1-TFEB in GAA −/− mice promotes glycogen clearance and attenuates PD pathology.

A. Glycogen assay in TFEB-injected gastrocnemii and in the contralateral muscles. In TFEB-injected muscles glycogen levels were significantly decreased compared to those in untreated muscles. * indicates statistically significant differences (p = 1.0 × 10−4; n = 6; Student's t-test).

B. PAS staining of TFEB-treated muscle shows a reduction of lysosomal glycogen stores (puncta) compared to those in untreated muscle. Original magnification: 20×.

C. LAMP1 staining of TFEB-injected gastrocnemii and of the contralateral untreated muscles. In TFEB-treated muscles, the size of LAMP1-positive vesicles was reduced. Bar: 2 µm.
Transcription factor EB (TFEB) is a new therapeutic target for Pompe disease. (2013)
Figure 8

Impact of TFEB on muscle fibre ultrastructure in GAA−/− mice.

A. EM images of muscle injected with either AAV-GFP (untreated) or AAV-TFEB (TFEB-treated). Asterisks indicate glycogen-containing lysosomes. Bar: 1.5 µm (upper panels) and 0.45 µm (lower panels). Higher magnification images (lower panels) show that glycogen particles are less densely packed in TFEB-treated muscle. Black arrows indicate autophagosome profiles; the white empty arrow shows remnants of mitochondria engulfed by the lysosome.

B. Graphical presentations of lysosomal length (average ± SE; n = 100 lysosomes; p = 6.31 × 10−5), the number of lysosomes per 5 µm2 area of muscle fibre section (average ± SE; n = 50 fields; p = 4.80 × 10−3), and the number of autophagosomes flanking glycogen-containing lysosomes (average ± SE; n = 100 lysosomes; p = 4.39 × 10−5 < 0.001). Student's t-test was used for each comparison.

C. Graphical presentations of mitochondrial size (average ± SE; n = 100) and the number of mitochondria per 5 µm2 area of muscle fibre section (average ± SE; n = 50 fields). The differences were not significant by Student's t-test (p = 3.65 × 10−1 and 4.27 × 10−1, respectively).
mTOR-Controlled Autophagy Requires Intracellular Ca(2+) Signaling. (2013)
Figure 1

Time- and concentration-dependent stimulation of autophagy by rapamycin.

A–B) Western-blot analysis for GAPDH and LC3 of protein lysates obtained from HeLa cells treated with DMSO or 1 µM rapamycin (Rapa) for the indicated time periods (A) (n = 7) or for 5 h with the indicated concentrations (B) (n = 6). One hour before harvesting, 100 nM bafilomycin A1 was added. Upper panels: representative Western blots; lower panels: quantification of the LC3-II/GAPDH ratio. C) GFP-LC3-punctae quantification in HeLa cells treated for 5 h with different concentrations of rapamycin. Left: representative pictures. The scale bar represents 10 µm. Concentrations are mentioned in the right lower corner. Right: Quantification of the number of punctae per cell (n = 3). * p<0.05, repeated measurements ANOVA.
mTOR-Controlled Autophagy Requires Intracellular Ca(2+) Signaling. (2013)
Figure 3

Rapamycin reduces the ER Ca<sup>2+</sup>-leak rate.

A–B) Western-blot analysis for luminal Ca2+-binding proteins in HeLa cells treated with the indicated concentrations of rapamycin (Rapa) for 5 h: calreticulin (CRT) (A) and BiP/Grp78 (BiP) (B). Upper panels: representative Western blots; lower panels: quantification of the protein/GAPDH ratio (n = 4). C) Western-blot analysis for SERCA2 in HeLa cells treated with the indicated concentrations of rapamycin for 5 h. Upper panels: representative Western blots; lower panel: quantification of the SERCA2/GAPDH ratio (n = 4). D) Representative plot showing the decrease in ER 45Ca2+ content (logarithmic scale) in a Ca2+-free efflux medium without ATP as a function of time in permeabilized HeLa cells pretreated for 5 h with 1 µM rapamycin or with DMSO. The passively bound Ca2+ was determined by loading the cells with 45Ca2+ in the presence of 10 µM of the Ca2+ ionophore A23187 and then subtracted from the stored 45Ca2+. The ER Ca2+-leak rate can be estimated as the rate of decline of the ER 45Ca2+-store content as a function of time. E) Quantification of the mean 45Ca2+-store content at the beginning of the measurement (t0) (n = 5). F) Quantification of the mean slope of the curve in D after transformation to a linear scale, which is a measure of the 45Ca2+-leak rate (n = 5). * p<0.05; ** p<0.01, paired Student's t-test.
mTOR-Controlled Autophagy Requires Intracellular Ca(2+) Signaling. (2013)
Figure 4

Changes in Ca<sup>2+</sup> signaling are independent of autophagy stimulation and occur upstream of the Atg12-Atg5 complex.

A) Representative Western-blot analysis for Atg12 (showing the autophagic Atg12-Atg5 complex), GAPDH and LC3 of protein lysates obtained from MEF cells pretreated with (+Dox) or without (-Dox) doxycycline and treated with DMSO or 0.1, 1 or 5 µM rapamycin (Rapa) for 5 h (n = 3). B–C) Representative measurements of cytosolic Ca2+ signals, displayed as Fura2 ratio (F340/F380), showing the effect of 1 mM ATP (B) or 10 µM ionomycin (Iono) in intact MEF cells pretreated with or without doxycycline and treated with different concentrations of rapamycin for 5 h. Prior to the addition of ATP or Iono, EGTA (3 mM) was added to chelate the extracellular Ca2+ as indicated. D) Quantification of the average amplitude of the response (F−F0) (n = 3, 4, 5 and 6 for ATP-Dox, ATP+Dox, Iono-Dox and Iono+Dox, resp.) * p<0.05; ** p<0.01; *** p<0.001, repeated measurements ANOVA.
mTOR-Controlled Autophagy Requires Intracellular Ca(2+) Signaling. (2013)
Figure 5

Rapamycin-induced autophagy is Ca<sup>2+</sup>-dependent.

Western-blot analysis for GAPDH and LC3 of protein lysates obtained from HeLa cells treated for 5 h with DMSO, 1 µM rapamycin (Rapa), 10 µM BAPTA-AM or both. One hour before harvesting, 100 nM bafilomycin A1 was added. Left: representative Western blots; right: quantification of the LC3-II/GAPDH ratio (n = 6). * p<0.05, repeated measurements ANOVA.
ROCK2 inhibition triggers the collective invasion of colorectal adenocarcinoma (2019)
Figure 1

Figure 1. Colorectal adenocarcinomas organize as cohesive and polarized epithelial glands

A) Representative specimen of colorectal (CRC) primary tumour stained with haematoxylin/eosin/saffron (HES), or antibodies against E-cadherin or Vimentin. i) The blue, orange and pink dotted lines highlight the normal mucosa, the submucosa and the muscularis propria respectively. Red dotted line highlights the neoplastic tissue. Black arrowheads indicate the direction of invasion. Boxed regions ii and iii show high magnification of normal colonic glands (ii) and the CRC invasive front (iii). Scale bar: 2 mm and 500 μm.

B) Representative images of histological sections of normal colon and primary CRC stained for EpCam and Villin. Boxed regions i, ii and iii are high magnifications of the luminal cavity of normal colonic gland (i) and colorectal carcinoma glands (ii and iii). Aarrowheads point to the apical pole enriched in villin. Scale bars: 50 μm.

(C) Graph presenting the percentage of the tumour area displaying a glandular architecture from a cohort of 16 patients (see Fig EV1).