EMBO/soda-nel huggingface dataset:
Targeting the SHP2 phosphatase promotes vascular damage and inhibition of tumor growth (2021)
Figure 2

Figure 2. EphrinB2, TIE2 and SHP2 phosphorylation in human melanoma and colon carcinoma. SHP2 inhibition in endothelial cells.

(A) Representative tissue immunostaining of human melanoma, colon carcinoma and colon adenoma; confocal images from serial sections (scale bars: 50μm tissues). Arrowheads point to CD31+/p-EphrinB+; CD31+/p-SHP2+; CD31+/p-Tie2+ and CD31+/Ang2+ endothelial cells.

(B) Gene expression in tumor (black bar) and control (white bar); top panel: melanoma (GSE7553, primary and metastatic; tumor n=54; control skin n=5); bottom panel: colon cancer (TCGA, primary and metastatic; tumor n=269; control n=41. **P<0.01, ****P<0.0001; P values from Mann Whitney U-test.

(C) Impact of high and low (75% quartile) mRNA levels on the survival probability of patients with primary melanoma; Kaplan-Meier analysis using TCGA primary melanoma datasets (n=103); P values from Mann Whitney U-test.

(D) Impact of high and low (median) mRNA levels on the survival probability of patients with colon cancer; Kaplan-Meier analysis using TCGA colon cancer dataset (n=262).

(E) Proliferation of endothelial (HUVEC, BMEC and HDMED) and tumor (B16F10, LLC1 and 4T1) cells in three-day cultures supplemented with SHP099. Representative experiments of 3-8; error bars: ± S.D.; 5-10 replicate cultures/experiment.

(F) SHP2 depletion in BMEC and B16F10 by 6 distinct shRNAs (#1-#6); pLKO: vector; none: no infection. Immunoblotting with SHP2 antibody followed by reblotting for β-actin.

(G) SHP2 shRNAs #1, #2, #3 reduce BMEC, not B16F10 cell proliferation; representative of 3 experiments; error bars: ± S.D.; 5 replicate cultures/experiment.

(H) SHP099 (5μM HUVEC and HDMEC; 20μM BMEC) minimally alters endothelial cell cycle distribution measured by flow cytometry; 3-day culture; 3 independent experiments; error bars: ± S.D.

(I) Non-viable endothelial cells identified by flow cytometry (DRAQ5+/DAPI+) after 3-day culture with or without SHP099 (5μM HUVEC and HDMEC; 20μM BMEC); quantification of 3 independent experiments; error bars: ± S.D. P values from two-tailed Student's t-test; ***P<0.001;
Targeting the SHP2 phosphatase promotes vascular damage and inhibition of tumor growth (2021)
Figure 3

Figure 3. SHP099 reduces ERK1/2 and increases STAT3 phosphorylation in endothelial cells.

(A) Kinetic SHP099 modulation of p-STAT3 (Tyr705) and p-ERK1/2 (Thr202/Tyr204) in endothelial and B16F10 cells; SHP099: 5μM HUVEC; 20μM BMEC and B16F10; representative immunoblotting of 3-5 experiments; h: hours.

(B) Quantification of band intensities in panel (a); p-STAT3/STAT3 and p-ERK1/2/ERK1/2.

(C-F) (C,D) The JAK inhibitor Tofacitinib (Tofa, 50nM) enhances the proliferation of HUVEC and BMEC compared to control (C) and reduces endogenous p-STAT3 levels in HUVEC and BMEC compared to control (D) after 72-hour culture. SHP099 (5μM HUVEC; 20μM BMEC) reduces cell proliferation and increases p-STAT3 in HUVEC and BMEC compared to control; error bars: ± S.D.; (E,F) The MAP kinases inhibitor PD098059 (PD0980, 10μM) and SHP099 (5μM HUVEC; 20μM BMEC) inhibit HUVEC and BMEC proliferation compared to control (E) and reduce p-ERK1/2 levels by immunoblotting after 72-hour culture (F). Results of proliferation in panels C and E (% of control) from triplicate cultures are representative of 3-4 experiments; *P<0.05, **P<0.01 ***P<0.001; two-tailed Student's t-test; error bars: ± S.D. Results of immunoblotting in panels D and F are representative of 3 independent blots.

(G,H) The ERK1/2 inhibitors AZD6244 and Tamnitinib dose-dependently reduce B16F10 cell proliferation (G); representative experiment of 3 independent experiments; the results reflect the means of 5 replicate/condition; error bars: ± S.D.; and levels of endogenous p-STAT3 and p-ERK in B16F10 cells (representative of 3 blots) (H).
Targeting the SHP2 phosphatase promotes vascular damage and inhibition of tumor growth (2021)
Figure 4

Figure 4. Vascular effects of SHP099 and inhibition of B16F10 tumor growth.

(A-C) SHP099 (A, 75mg/kg; B, C: 100mg/kg) reduces B16F10 tumor growth in syngeneic mice. Panels depict tumor growth from initiation of treatment to endpoint (left) and tumor weight (right) at endpoint; no. mice/group: 6-11 control; 7-9 SHP099.

(D) Extravasation of FITC-dextran (green, pointed by arrowheads) in tumors treated with SHP099. Vessels identified by CD31 (red); DAPI (blue) identifies cell nuclei. Confocal images (scale bar: 200μm). Representative images from 3 tumors.

(E) "Vascular sleeves" (Collagen IV+ CD31-) identified in a representative SHP099-treated tumor. Representative confocal images from 6 tumors (scale bar: 50μm).

(F) Reduced CD31+ vascular area in SHP099-treated tumors compared to controls (n=6/group); quantification by ImageJ.

(G) The ratio of Collagen IV+/CD31+ area is increased in SHP099-treated tumors compared to controls (n=6/group); quantification by ImageJ; Coll: collagen.

(H) Cleaved caspase-3 detection in a CD31+ vessel (representative of 6 SHP099-treated tumors); arrowheads point to cleaved caspase-3+CD31+ cells. Cleaved caspase-3+ tumor cell is marked by *. Confocal images (scale bar: 10μm).

(I) Increased cleaved caspase-3 fluorescence intensity in CD31+ cells from SHP099-treated tumors compared to controls (n=3/group); at least 100 cells counted/sample.

(J) Ki67+ proliferating cells in representative of 3 SHP099-treated and 3 control tumors; CD31 identifies the vessels; DAPI identifies cell nuclei. Confocal images (scale bar: 50μm).

(K) Quantification of proliferating cells within SHP099 and control (n=4/group) tumors by ImageJ.

(L) Hypoxic tumor identified Hypoxyprobe (green) representative of 3 SHP099-treated tumors; confocal images (scale bar: 100μm), left; quantification of hypoxic areas in control (n=3) and SHP099-treated (n=3) tumors by ImageJ.

(M) Representative control (of 5) and SHP099-treated (of 5) B16F10 tumor sections through the maximum diameter; H&E staining (scale bar: 10mm).

(N) Cleaved caspase-3+ fluorescence intensity in SHP099 and control tumors (n=3/group).

Data information: Error bars: ± S.D.; P values from two-tailed Student's t-test; *P<0.05, **P<0.01 and ***P<0.001.
Targeting the SHP2 phosphatase promotes vascular damage and inhibition of tumor growth (2021)
Figure 5

Figure 5. Features of SHP099 tumor growth inhibition.

(A) Anti-tumor activity of SHP099 against B16F10 tumors established s.c. in NOD scid (NSG) mice (n=5/group); P values from two-tailed Student's t-test; *P<0.05, **P<0.01. Error bars: ± S.D.

(B) Extravasation of FITC-dextran (green) in tumors established in NSG mice treated with SHP099; scale bar (50μm). Representative images from 3 mice/group.

(C) Reduced CD31+ vascular area in SHP099-treated tumors compared to controls (n=5/group); ImageJ quantification. P values from two-tailed Student's t-test; *P<0.05. Error bars: ± S.D.

(D, E) SHP099 reduces growth (D) and weight (E) of control (p-LKO vector) and SHP2-depleted (shSHP2) B16F10 tumors compared to untreated controls: p-LKO untreated (n=5), p-LKO treated with SHP099 (n=9), shSHP2 untreated (n=5), and shSHP2 treated with SHP099 (n=13). P values by analysis of variance with Dunnett's multiple comparison test; ***P<0.001.

(F) Persistent depletion of SHP2 in tumors induced by shSHP2-B16F10 cells (n=5) compared to control tumors induced by p-LKO-B16F10 (n=5); tumors removed from mice 18 days after cell inoculation.

(G) SHP099 induces extravasation of FITC-dextran (green) in tumors from inoculation of shSHP2-B16F10 (representative image from 3 tumors); scale bar: 50μm.

(H) Reduced CD31+ vascular area in SHP099-treated tumors from s.c. inoculation of shSHP2-B16F10 cells compared to controls from p-LKO-B16F10 cells (n=5/group); ImageJ quantification. Error bars: ± S.D.; P values from two-tailed Student's t-test; *P<0.05.

(I) Schematic of experiment.

(J,K) After footpad inoculation of B16F10 cells, mice were treated orally with SHP099 (n=20) or vehicle only (n=20). Tumor size (J) was estimated from foot weight (g); size of popliteal lymph nodes (K) was measured (mm2) by ImageJ software; Student's t-test; **P<0.01, ***P<0.001.

(L,M) Proportion of mice in the SHP099 and control groups with macroscopic (L) and microscopic (M) evidence of metastases; Fisher's exact test; ***P<0.001.

(N) Representative popliteal lymph nodes from control and SHP099-treated mice with microscopic evidence of metastases; H&E stain; scale bars 500 μm (low magnification) 20 μm (high magnification).
Targeting the SHP2 phosphatase promotes vascular damage and inhibition of tumor growth (2021)
Figure 6

Figure 6. SHP099 and AMG386 cooperatively reduce B16F10 tumor growth.

(A,B) AMG386 (5.6mg/kg s.c.; twice/week) and SHP099 (a: 200 mg/kg; b: 100mg/kg orally/daily) were administered individually or together to B16F10 tumor-bearing mice (A: control n=7; SHP099: n=8; AMG386: n=8; SHP099+AMG386 n=11; B: control, SHP099 and AMG386: n=4; SHP099+AMG386: n=8). Panels depict tumor growth from initiation of treatment to endpoint (left) and tumor weight (right) at endpoint; error bars ± s.d.; P values by analysis of variance with Dunnett's multiple comparison test; **P<0.01, ***P<0.001.

(C,D) p-TIE2 in tumor vessels from control and AMG386-treated mice; representative confocal images; arrowheads point to CD31+/p-TIE2- endothelial cells (scale bars: 50μm) (C), and quantification (D) of p-TIE-2/CD31+ in control (n=3) and AMG386 treated (n=3) tumors (1,200 CD31+ cells/group). P values from two-tailed Student's t-test; ***P<0.001.

(E,F) p-EphrinB (E) in representative tumor vessels from control (n=3) and AMG386-treated (n=3) tumors; confocal images (E), (scale bar: 50μm) and quantification of p-EphrinB/CD31+ (F); P values from two-tailed Student's t-test; ***P<0.001.

(G) Vessel perfusion visualized by FITC-dextran (green); CD31 (red) identifies the endothelium; DAPI (blue) identifies the nuclei; representative tumor confocal images (scale bar:50μm) of 3 mice/group; arrow heads point to FITC-dextran extravasation; asterisks to extravasated erythrocytes.

(H) Quantification of cleaved caspase-3 in the tumor vasculature (CD31+ cells); n=3 tumors/group; number of CD31+ cells counted: control (n=311); SHP099 (n=425); AMG386 (n=451); combined SHP099+AMG386 (n=484). Error bars: ± S.D.; P values by analysis of variance with Dunnett's multiple comparison test; **P<0.01, ***P<0.001.

(I) Quantification of tumor vascular area; n=5/tumors group. Error bars: ± S.D.; P values by analysis of variance with Dunnett's multiple comparison test; **P<0.01, ***P<0.001.

(J) Quantification of "vascular sleeves" (CD31-Collagen IV+) n=5/tumors group. Error bars: ± S.D.; P values by analysis of variance with Dunnett's multiple comparison test; **P<0.01, ***P<0.001.

(K, L) (K) % necrotic tumor area (n=4/tumors group) and (L) % proliferating tumor cells (n=3/tumors group) in control and treated groups. Error bars: ± S.D.; P values by analysis of variance with Dunnett's multiple comparison test; *P<0.05, **P<0.01, ***P<0.001.

(M) Representative control and treated (n=5/group) B16F10 tumor sections through the maximum diameter; H&E staining; (scale bars: 10mm).

(N) Proliferating Ki67+ cells (green) in representative control and treated tumors (n=3/group); CD31 identifies the vessels; DAPI identifies cell nuclei. Confocal images (scale bar: 50μm).
Targeting the SHP2 phosphatase promotes vascular damage and inhibition of tumor growth (2021)
Figure 7

Figure 7. SHP099 and AMG386 modulate STAT3, ERK and AKT signaling in the B16F10 tumor vasculature.

(A-D) Nuclear p-STAT3 (A,B) and p-ERK1/2 (C,D) in tumor CD31+ endothelial cells of mice treated for 14 days with SHP099 (200 mg/kg), AMG386 (5.6mg/kg) or the combination of SHP099 (200 mg/kg) + AMG386 (5.6mg/kg). Representative (3 tumors/group) immunostaining (A,C) and quantification of staining intensity/CD31+ cell (B, D); horizontal line: mean intensity/group; n=3 tumors/group; number of CD31+ cells counted in B: control (n=180), SHP099 (n=308), AMG386 (n=300), combined SHP099+AMG386 (n=322); number of CD31+ cells counted in D: control (n=1,733), SHP099 (n=821), AMG386 (n=1,399), combined SHP099+AMG386 (n=783). Scale bars: A, 50μm; C, 10μm.

(E,F) FOXO1 detection in tumor CD31+ endothelial cells after treatment with SHP099 (200 mg/kg), AMG386 (5.6mg/kg) or the combination of SHP099 (200 mg/kg) + AMG386 (5.6mg/kg). Representative (3 tumors/group) confocal images (E) and quantification (n=3 tumors/group) (F) of FOXO1 fluorescence intensity/CD31+ cell. Number of CD31+ cells counted: control (n=2,285), SHP099 (n=2,262), AMG386 (n=2,300), combined SHP099+AMG386 (n=2,361).

Data information: P values by analysis of variance with Dunnett's multiple comparison test; **P<0.01, ***P<0.001.
Targeting the SHP2 phosphatase promotes vascular damage and inhibition of tumor growth (2021)
Figure 8

Figure 8. SHP099 and AMG386 reduce MC38 tumor growth.

(A,B) AMG386 (5.6mg/kg s.c.; twice/week) and SHP099 (100mg/kg orally/daily) were administered individually or together to MC38 tumor-bearing mice (control n=10; SHP099: n=15; AMG386: n=10; SHP099+AMG386 n=15). Tumor growth from initiation of treatment to endpoint (left) and tumor weight (right) at endpoint; error bars ± S.D.; P values by analysis of variance with Dunnett's multiple comparison test; *P<0.05, **P<0.01, ***P<0.001 (A). Representative control and treated (n=4/group) MC38 tumor sections through the maximum diameter (B); H&E staining; scale bar: 10mm.

(C) Vessel perfusion visualized by FITC-dextran (green); CD31 identifies endothelial cells (red); DAPI (blue) identifies the nuclei; representative (of 3 mice/group) confocal images (scale bar: 50μm); arrow heads point to FITC-dextran extravasation.

(D-G) Quantification of "vascular sleeves" (CD31-Collagen IV+); n=4 tumors/group (D); cleaved caspase-3+/CD31+ cells; n=4 tumors/group; at least 1000 CD31+ cells counted/sample (E); tumor vascular area; n=7 tumors/group (F); Ki67+ tumor cells; n=4 tumors/group; at least 1000 CD31+ cells counted/sample (G); error bars: ± S.D. P values by analysis of variance with Dunnett's multiple comparison test; *P<0.05, **P<0.01, ***P<0.001.

(H) Representative images depicting Ki67+ cells in control and treated MC38 tumors; representative images (scale bar: 50μm) of 4 tumors/group.

(I) Quantification of necrotic tumor area in control and treated mice; 4 tumors/group; error bars: ± S.D. P values by analysis of variance with Dunnett's multiple comparison test; *P<0.05, ***P<0.001.

(J-L) Quantification of p-STAT3, p-ERK and FOXO1 in tumor CD31+ endothelial cells in control and treated mice; (n=3 tumors/group); CD31+ cells counted in J: control (n=3,842), SHP099 (n=21,212), AMG386 (n=12,786), combined SHP099+AMG386 (n=11,746). CD31+ cells counted in K: control (n=12,125), SHP099 (n=8,033), AMG386 (n=3,916), combined SHP099+AMG386 (n=4,230). CD31+ cells counted in L: control (n=16,076), SHP099 (n=6,555), AMG386 (n=13,715), combined SHP099+AMG386 (n=7,260). P values by analysis of variance with Dunnett's multiple comparison test; *P<0.05, **P<0.01, ***P<0.001.

(M) Schematic representation of the effects of AMG386 and SHP099 on signaling pathways active in the tumor vasculature.

Legends for Expanded View (EV) Figures
Host autophagy mediates organ wasting and nutrient mobilization for tumor growth (2021)
Figure 1

Figure 1. Tumor-induced organ wasting.

(a) Representative 2D μ-CT scans of RasV12; ctrl at day 6, RasV12; scrib-/- larvae at day 6 and day 10. Muscle (green), fat body (blue) and eye-antennal discs/tumor (red) are outlined.Scale bar: 1mm. Anterior (A), Right (R).

(b) Representative 3D rendering of genotypes indicated in (a). Anterior (A), Dorsal (D), Left (L).

(c) Representative 3D rendering of RasV12; scrib-/- tumors (red) and central nervous system (yellow), over time.
Host autophagy mediates organ wasting and nutrient mobilization for tumor growth (2021)
Figure 2

Figure 2. Tumor-induced organ wasting.

(a) Quantification of tumor growth of RasV12; ctrl day 6 (n=15) RasV12; scrib-/- tumors, day 6 (n=15), day 7 (n=5), day 8 (n=4), day 9 (n=5) and day 10 (n=5).

(b) Quantifications of muscle volume, of larvae carrying tumors of RasV12; ctrl (n=15), and RasV12; scrib-/- at day 6(n=15), day 7 (n=5), day 8 (n=5), day 9 (n=5) and dat 10 (n=5).

(c) Quantification of width of Dorsal Oblique 3 (DO3) muscle in larvae carrying tumors of RasV12; ctrl at day 6 (n=29) and RasV12; scrib-/-at days 6 (n=29) and day 10 (n=31).

(d) Quantification of adipose tissue volume in larvae carrying tumors of RasV12; ctrl at day 6 and RasV12; scrib-/-at days 6 and day 10.

(e) Representative confocal images of adipose tissue in RasV12; ctrl and RasV12; scrib-/- tumors bearing animals at indicated ages. Lipid droplets are highlighted with Lipid Tox staining. Scale bar: 50μm

(f) Quantification of dry weight of RasV12; ctrl (n=9), RasV12; scrib-/- day 6 (n=15) and day 10 (n=18) tumor bearing larvae excluding the tumor weight.

(g) Quantification of larval motility measure by crawling distance and crawling pattern in RasV12; ctrl (n=14), RasV12; scrib-/- day 6 (n=15), day 8 (n=16) and day 10 (n=14), each colored line represents a single larva.

(h) Coomassie feeding assay to asses food intake of RasV12; scrib-/- over time, three repeated measurements of an average food intake of 20 larvae.

Data information: Values depict mean±s.e.m. of minimum three independent pooled experiments. ns, not significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.0001 and ****P < 0.0001, from unpaired, two-tailed test.
Host autophagy mediates organ wasting and nutrient mobilization for tumor growth (2021)
Figure 3

Figure 3. Autophagy is required for systemic wasting.

(a-e) Cartoon (top) illustrates the genotypes of the larvae, eye-antennal disc (EAD, circle: tumor cells in green and microenvironment cell in black), systemic cells are illustrated as a square (wild type cells in light brown and atg13 mutant cells in light orange) at indicated ages. Cartoon (left) illustrates the structure of cephalic complex attached to mouth hook. Representative images of Larva (image of larva using the backlight of microscope), cephalic complex (green highlights the GFP-labelled tumor clones), muscle (Phalloidin in green stains actin and Hoechst in blue stains nucleus) and adipose tissue (Lipid Tox in red stains for lipid droplets, Hoechst stains nucleus) from top to bottom. (a) RasV12; ctrl tumor bearing animal at day 6. (b) RasV12; scrib-/- tumor bearing animal at day 6. (c) RasV12; scrib-/- tumor bearing animal at day 8. (d) RasV12, scrib-/-, atg13-/-//atg13-/- at day 8. (e) RasV12, scrib-/-, atg13-/-//atg13-/- animal complemented with transgenic atg13, rescuing the tumor growth, at day 8.

(f) Quantification of area of space occupied with lobes of fat body within larval segments 4 to 8 (shown in yellow dashed line in 2a), of animals bearing tumors at day 6 RasV12; ctrl (n=45), RasV12; scrib-/- (n=30), RasV12, scrib-/-, atg13-/-//atg13-/- (n=30) and ey3.5-atg13; RasV12, scrib-/-, atg13-/-//atg13-/- (n=29) and at day 10 RasV12; scrib-/- (n=40), RasV12, scrib-/-, atg13-/-//atg13-/- (n=25) and ey3.5-atg13; RasV12, scrib-/-, atg13-/-//atg13-/- (n=28).

(g) Quantification of tumor volumes day 6 RasV12; ctrl (n=7), RasV12; scrib-/- (n=11) and day 8 RasV12; scrib-/- (n=13), RasV12, scrib-/-, atg13-/-//atg13-/- (n=9) and ey3.5-atg13; RasV12, scrib-/-, atg13-/-//atg13-/- (n=9).

(h) Quantification of Ventral Longitudinal muscle 4 (VL4) (shown in yellow dashed line in 2a) of third segment of larvae carrying at day 6 RasV12; ctrl (n=15), RasV12; scrib-/- (n=14), and at day8 RasV12; scrib-/- (n=11), RasV12, scrib-/-, atg13-/-//atg13-/- (n=12) and ey3.5-atg13; RasV12; scrib-/-, atg13-/-//atg13-/- (n=14).

(i) Quantification of dry weight excluding tumor of RasV12; scrib-/-, atg13-/-//atg13-/-at day 6 (n=8) and day 10 (n=7).

(j) Quantification of larval motility measured by crawling distance and crawling pattern for RasV12; scrib-/-, atg13-/-//atg13-/- at day 6 (n=15) and at day 8 (n=15), each colored line represents a single larva.

Data information: Values depict mean±s.e.m. of minimum three independent pooled experiments. ns, not significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.0001 and ****P < 0.0001, from unpaired, two-tailed test. Scale bar: muscles 100 μm and adipose tissue 50 μm.
Host autophagy mediates organ wasting and nutrient mobilization for tumor growth (2021)
Figure 4

Figure 4. Autophagy-driven wasting releases metabolites into circulation.

(a) Changes in groups (carbohydrates, amino acids and fatty acids) of storage metabolites measured in circulating haemolymph with progressing wasting, shown as log2 changes measured by LC-MS and calculated per larvae relative to RasV12; ctrl at day 6.

(b) Volcano plot showing autophagy-dependent changes to amounts of circulating metabolites at day 6. X-axis shows the log2 of fold change of RasV12;atg13 -/-scrib-/- // atg13 -/- vs. RasV12; scrib-/- , y-axis shows -log10 p-value, calculated by t-test. Metabolite names are shown for metabolites with log2(FC) >± 1 and/or -log10(P) < 2. Green points indicate log2(FC) >± 1, blue indicate -log10(P) < 2 and red indicates for above both thresholds.

(c) Autophagy-driven wasting releases metabolites into circulation of 113 reliably detected metabolites, those showing significant differences in any of the three comparisons are shown. Color indicates the log2(fold change) difference and the numbers show the p-value of the comparison. The statistical test to define significance was FDR-adjusted t-test p-value < 0.05
Host autophagy mediates organ wasting and nutrient mobilization for tumor growth (2021)
Figure 5

Figure 5. Host-derived nutrients contribute to tumor biomass.

(a) The amount of glycogen in the whole larvae measured by biochemical assay (n=4), normalized to RasV12; ctrl at day 6, and per number of larvae.

(b-e) Representative confocal images of muscle and adipose tissue of larvae carrying RasV12; ctrl, RasV12; scrib-/- and RasV12; scrib-/-, atg13-/-//atg13-/- showing glycogen levels (white) at day 6 and day 8.

(f) Cartoon illustrating tumor growth (green) incorporation of molecules derived from food (in orange) or from host tissues (in blue).

(g) Sources of carbon incorporated into tumor biomass were differentiated by changing the isotopic carbon content of the food source 25 hours before measuring the total carbon content and isotopic ratio of the tumor. A similar, but independent experiment is reported in Holland P., et al., 2021 BMC Biol.

Data information: Values depict mean±s.e.m. of minimum three independent pooled experiments. ns, not significant, **P ≤ 0.01, from unpaired, two-tailed test. Scale bar: muscles 100 μm and adipose tissue 50 μm.
ESCRT machinery mediates micro-autophagy of the endoplasmic reticulum in yeast. (2019)
Figure 1

Figure 1 - Expression of ER-resident transmembrane proteins can induce autophagy of ER whorls.

A Freeze-fracture electron micrograph of wild-type yeast treated with DTT for 3 h. The left panel shows two spherical structures, one in a vacuole membrane invagination and one inside the vacuole. The middle panel shows the boxed area of the left panel at higher magnification. Arrows indicate membrane layers that are free of intramembrane particles. The right panel shows the protoplasmic fracture face (P face) of cortical ER with a high density of intramembrane particles. Cy, cytosol; N, nucleus; V, vacuole.

B Fluorescence images of cells expressing GPD promoter-driven GFP-Pho8 and the ER marker Sec63-mCherry and stained with CMAC to label the vacuole. The upper and lower panel show vacuole-associated and perinuclear GFP-Pho8 stretches and an intravacuolar GFP-Pho8 ring, respectively. Scale bars: 2 µm.

C Correlative light and electron microscopy of cells expressing GPD promoter-driven GFP-Pho8 and Sec63-mCherry. The lower panel shows the boxed area in the electron micrograph at higher magnification. The ER ring corresponds to a vacuolar whorl and the ER stretch to a perinuclear stack.

D Fluorescence images of cells expressing Hmg2-GFP and either additionally expressing Sec63-mCherry or stained with CMAC. The upper and lower panel show cytosolic and vacuolar Hmg2-GFP rings, respectively. Scale bars: 2 µm.