EMBO/soda-nel huggingface dataset:
RNA m6A methylation regulates sorafenib-resistance in liver cancer through FOXO3-mediated autophagy (2020)
Figure 4

Figure 4. RNA m6A-Seq identified FOXO3 as a downstream target of METTL3-mediated m6A modification under hypoxia.

A Gene Set Enrichment Analysis (GSEA) output images of cellular response to stress pathways displaying a correlation of differentially regulated genes in METTL3-knockdown HepG2, SMMC-7721 and Bel-7402 cells with the C5. biological process symbol set. (p < 0.001).

B The KEGG analysis shows that FOXO3 involved in hypoxia and autophagy.

C Protein level of FOXO3 in METTL3-knockdown HCCs under hypoxia for 48h.

D Half-life of FOXO3 mRNA in Bel-7402 cells and correspondent METTL3-knockdown Bel-7402 cells treated with actinomycin D.

E Half-life of FOXO3 protein in Bel-7402 cells and correspondent METTL3-knockdown Bel-7402 cells treated with cycloheximide. Quantification of the protein optical density by Image J in E-1.

F Analysis of FOXO3 mRNA in non-ribosome portion (<40S), 40S, 60S, 80S and polysome for the METTL3-knockdown Bel-7402 cells compare to control cells under hypoxia for 48h.

G Knockdown of YTHDF1 and YTHDF2 in Bel-7402 cells by lentiviral shRNAs.

H Knockdown of YTHDF1 but not YTHDF2 abolished the increase of FOXO3 protein level in METTL3-overexpressing Bel-7402 cells. Quantification of the protein optical density by Image J in H-1 and H-2.

I RNA level of FOXO3 in YTHDF1-knockdown Bel-7402 cells by RT-PCR.

J The m6A-IP sequencing under hypoxia proved the m6A modification participated in regulation of FOXO3. The YTHDF1 binding site locates at the 3'UTR of FOXO3.

K 800 bp of FOXO3 3'UTR containing the m6A modification site was cloned into luciferase reporter vector. Mutation of m6A consensus sequence was generated by replacing adenosine with thymine.

L Relative luciferase activity of the wild-type and mutant form of FOXO3 3'UTR reporter vectors in Bel-7402 cells with various levels of METTL3 and YTHDF1.

M Knockdown of METTL3 reduced the m6A methylation in FOXO3 mRNA by the m6A MeRIP analysis.

N Knockdown of METTL3 reduced the m6A methylation in FOXO3 mRNA by the YTHDF1-RIP analysis.

O Correlation analysis of the RNA expression levels of METTL3 and FOXO3 in liver tumor cohort (Kim-90) by Pearson.

P Correlation analysis of the RNA expression levels of YTHDF1 and FOXO3 in liver tumor cohort (Kim-90) by Pearson.

Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments.
RNA m6A methylation regulates sorafenib-resistance in liver cancer through FOXO3-mediated autophagy (2020)
Figure 5

Figure 5. Overexpression of FOXO3 rescued the m6A-dependent sorafenib-sensitivity under hypoxia by inhibiting autophagy.

A Validation of biomarker genes related to autophagy in FOXO3-knockdown Bel-7402 cells by RT-PCR.

B Protein level of FOXO3 in naïve HepG-2 and sorafenib-resistant HepG-2 cells under hypoxia for 48h.

C Protein levels of LC3 I/II in FOXO3-knockdown SMMC-7721 cells.

D Overexpression of FOXO3 in sorafenib-resistant HepG-2 cells by Western blot.

E Protein levels of LC3 I/II in METTL3-knockdown & FOXO3-overexpression SMMC-7721 cells under hypoxia for 48 h.

F Electron micrographs of METTL3-knockdown SMMC7721 cells and METTL3-knockdown & FOXO3-overexpression SMMC7721 cells under hypoxia for 48 h.

G Representative Immunostaining images of LC3 in METTL3-knockdown SMMC7721 cells and METTL3-knockdown & FOXO3-overexpression SMMC7721 cells under hypoxia for 48 h. Scale bar, 200μm. The quantification of the numbers of GFP-LC3 puncta/cell by Imaris in G-1.

H Representative Immunostaining images of LC3 in naïve HepG-2 cells and sorafenib-resistant HepG-2 cells under hypoxia for 48 h. Scale bar, 200μm. The quantification of the numbers of GFP-LC3 puncta/cell by Imaris in H-1.

I The IC50 of METTL3-knockdown SMMC7721 cells with overexpressing FOXO3 after treated with sorafenib for 24h under hypoxia condition.

J The IC50 of naïve HepG-2 cells and sorafenib-resistant HepG-2 cells with overexpressing FOXO3 after treated with sorafenib for 24h under hypoxia condition.

K Cell survival assay of METTL3-knockdown SMMC7721 cells with overexpressing FOXO3 after treated with sorafenib for 24h under hypoxia condition.

L Cell survival assay of naïve HepG-2 cells and sorafenib-resistant HepG-2 cells with overexpressing FOXO3 after treated with sorafenib for 24h under hypoxia condition.

Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD and are representative of 3 independent experiments.
RNA m6A methylation regulates sorafenib-resistance in liver cancer through FOXO3-mediated autophagy (2020)
Figure 6

Figure 6. METTL3-depletion significantly enhanced sorafenib-resistance via the METTL3/FOXO3 axis in vivo.

A Depletion of METTL3 in Hepa1-6 cells by CRISPR-Cas9 knockout enhanced sorafenib-resistance in vivo.

B-C Growth curve (B) and tumor weight (C) of Hepa1-6 cells derived xenografts in the subcutaneous implantation mice model in six treatment groups for 12 days (n=5).

D Knockdown of METTL3 slightly increased liver tumor growth treated with sorafenib in orthotopic xenograft mouse model. Stable METTL3-knockdown Bel-7402 cells and control cells were injected into the liver of each NOD/SCID mouse. 3 weeks after injection, livers were separated for pathological analysis. Red circle indicates tumors in the livers.

E Liver tumors in orthotopic xenograft mouse model were confirmed by hematoxylin and eosin staining.

F Overexpression of FOXO3 rescued sorafenib-resistance mediated by METTL3-knockdown in xenograft liver tumors. Representative xenograft tumors at endpoint, containing 6 groups.

G-H Growth curve (G) and tumor weight (H) of Hepa1-6 cells derived xenografts in the subcutaneous implantation mice model in 6 treatment groups for 10 days (n=5).

I H&E and immunohistochemical images of Ki67, LC3-B, VEGF-A in Hepa1-6 cells derived xenografts in 6 treatment groups.

J-L Quantification of Ki67 (K), LC3-B (K), and VEGF-A (L) expression in immunohistochemical images by Image Pro Plus (IPP) analysis.

Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD.
RNA m6A methylation regulates sorafenib-resistance in liver cancer through FOXO3-mediated autophagy (2020)
Figure 7

Figure 7. METTL3-depletion significantly enhanced sorafenib-resistance in HCC patient-derived xenografts.

A Graphic illustration of two human liver cancer PDXs mouse model based sorafenib (SOR) treatment regimen.

B-D In vivo analyses of tumor (B), growth (C), and weight (D) in mice that were subcutaneously implanted with tumor tissues from two human liver cancer patients and divided into four groups.

E H&E and immunohistochemical images of Ki67, LC3-B, VEGF-A in randomly selected PDX#1 and PDX#2 tumors.

F-H Quantification of Ki67 (F), LC3-B (G), and VEGF-A (H) expression in immunohistochemical images from PDX tumors by Image Pro Plus (IPP) analysis.

I Knockdown of METTL3 by siRNA in PDX#1 and PDX#2 tissues were confirmed by Western blot.

J Working model. The molecular mechanisms of m6A-dependent sorafenib-resistance in liver cancer. The expression of METTL3 was down-regulated in sorafenib-resistant liver cancer, and caused decrease of FOXO3 by the m6A modification in an YTHDF1-dependent manner. Degradation of FOXO3 thus promoted autophagy-associated sorafenib-resistance in liver cancer.

Data information: In all relevant panels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Two-tailed t-test. Data are presented as mean ± SD.
The TFEB orthologue HLH-30 regulates autophagy and modulates longevity in Caenorhabditis elegans (2013)
f1

(a) Nuclear localization of HLH-30 was visualized by fluorescence microscopy in day 1 adult wild-type (WT) (upper panel) and glp-1(e2141) (lower panel) animals expressing HLH-30::GFP raised at the non-permissive temperature (25 °C). Inserts show enlarged intestinal cells. Graph shows percentage of animals with HLH-30 in the nuclei of intestinal cells (four biological replicates, ~50 animals each, mean±s.d., **P0.01, Student’s t-test). Magnification × 100; scale bar, 100 μm. (b) Expression of hlh-30 and putative autophagy-related and lysosomal target genes was measured by quantitative PCR (qPCR) in day 1 adult WT and glp-1(e2141) animals raised at 25 °C (mean±s.d. of three biological replicates, *P0.05, **P0.01, Student’s t-test). (c) Expression of hlh-30 and putative autophagy-related and lysosomal target genes was measured by qPCR in day 1 adult glp-1(e2141) and glp-1(e2141); hlh-30(tm1978) animals raised at 25 °C (mean±s.d. of three biological replicates, *P0.05, **P0.01, Student’s t-test). ctsa* is a cathepsin A orthologue (cosmid C08H9.1 (ref. 30)). See Supplementary Fig. 1 for qPCR analyses of hlh-30(tm1978) (control for c) and WT and glp-1(e2141) animals fed bacteria expressing hlh-30 dsRNA. (d,e) GFP::LGG-1 punctae were quantified in (d) hypodermal seam cells or (e) proximal intestinal cells of L3 larvae of WT and glp-1(e2141) animals. Animals were fed bacteria expressing control or hlh-30 dsRNA for two generations at 20 °C. Eggs were then transferred to plates seeded with the appropriate dsRNA-expressing bacteria at 25 °C and analysed at the L3 larval stage (mean±s.e.m. of ~300 seam cells and ~25 intestines, **P0.01, Student’s t-test). (f,g) glp-1(e2141) animals expressing (f) LMP-1::GFP or (g) SQST-1::GFP were raised at the non-permissive temperature (25 °C) and fed bacteria expressing control or hlh-30 dsRNA from hatching. Micrographs of the posterior intestine were taken on day 1 of adulthood, and LMP-1::GFP fluorescence (mean±s.d. of ~10 animals, **P0.01, Student’s t-test) and SQST-1::GFP foci (mean±s.d. of ~30 animals, **P0.01, Student’s t-test) were quantified. Experiments were performed at least three times with similar results. See Supplementary Fig. S2a,c for images of whole animals and Supplementary Fig. S2b,d for replicates. Magnification, × 200; scale bar, 100 μm.
The TFEB orthologue HLH-30 regulates autophagy and modulates longevity in Caenorhabditis elegans (2013)
f2

(a) Nuclear localization of HLH-30 was quantified in day 1 adult animals expressing HLH-30::GFP. Animals were fed bacteria expressing control or tor dsRNA from hatching and raised at 20 °C (three biological replicates, ~50 animals each, mean±s.d., **P0.01, Student’s t-test). (b,c) Expression of putative autophagy-related and lysosomal target genes was measured by quantitative PCR in day 1 (b) WT (N2) and (c) hlh-30(tm1978) animals fed bacteria expressing control or tor dsRNA from hatching (20 °C). Data are mean±s.d. of biological triplicates. *P0.05, **P0.01; Student’s t-test. csta* is a cathepsin A orthologue (cosmid C08H9.1 (ref. 30)).(d) Lifespan analysis of WT animals and hlh-30(tm1978) mutants fed bacteria expressing control or tor dsRNA from day 1 of adulthood was carried out at 20 °C. See Supplementary Table S3 for details of lifespan analyses and replicate experiments.
The TFEB orthologue HLH-30 regulates autophagy and modulates longevity in Caenorhabditis elegans (2013)
f3

Lifespan analyses of (a) WT (N2) and (b) germline-less glp-1(e2141) animals raised at the non-permissive temperature (25 °C) and fed bacteria expressing control or hlh-30 dsRNA from day 1 of adulthood were carried out at 20 °C. Lifespan analyses of (c) dietary-restricted eat-2(ad1116) mutants, (d) insulin/IGF-1 receptor daf-2(e1370) mutants, (e) mitochondrial respiration clk-1(e2519) mutants and (f) mRNA translation rsks-1(sv31) mutants, fed bacteria expressing control or hlh-30 dsRNA from day 1 of adulthood (c,d,f) or larval L4 stage (e), were carried out at 20 °C. See Supplementary Table S2 for details of lifespan analyses including at least three independent experiments.
The TFEB orthologue HLH-30 regulates autophagy and modulates longevity in Caenorhabditis elegans (2013)
f4

(a) Expression of hlh-30 was measured by quantitative PCR (qPCR) in day 1 adult WT (N2), eat-2(ad1116) (dietary restriction, DR), daf-2(e1370) (insulin/IGF-1 signaling), clk-1(e2519) (mitochondrial respiration) and rsks-1(sv31) (mRNA translation) animals (mean±s.d. of three biological replicates, *P0.05, Student’s t-test). (b) Nuclear localization of HLH-30 was quantified in day 1 adult WT, eat-2, daf-2, clk-1 and rsks-1 mutants (mean±s.d. of four biological replicates, ~50 animals each, **P0.01, analysis of variance). (c) Lifespan analysis of WT and transgenic animals overexpressing HLH-30::GFP, and raised and maintained on OP50 was carried out at 20 °C. See Supplementary Table S7 for details of lifespan analyses and replicate experiments. (d) GFP::LGG-1 punctae were quantified in hypodermal seam cells of WT animals or animals overexpressing HLH-30 (n>300, ±s.e.m., **P0.01, Student’s t-test). The increase in GFP::LGG-1 punctae observed in animals overexpressing HLH-30 could be reversed by atg-18 RNAi treatment (data not shown). (e) TFEB expression was measured by qPCR in the livers of 4.5-month-old female (F) and male (M) mice fed AL or subjected to DR for 5.5 weeks starting at 3 months of age (mean±s.e.m. of ~20 mice per group, *P0.05, Student’s t-test). (f) TFEB protein was detected by western blotting of nuclear fractions from the livers of five female and five male mice fed AL or subjected to DR. Actin was included as a loading control, see Supplementary Fig. S8 for additional controls. (g) TFEB protein levels in Fig. 4f were quantified by densitometry and normalized to actin (mean±s.d., *P0.05, Student’s t-test).
Polarized sorting of Patched enables cytoneme-mediated Hedgehog reception in the Drosophila wing disc (2020)
Figure 1

Figure 1. The SNARE proteins Synaptobrevin (Syb) and Synaptotagmin (Syt1) are located at receiving cytonemes.

A) GFP tagged Syb protein expressed in Hh-receiving cells co-localizes with endogenous immuno-labelled Ptc along the apico-basal axis (3D reconstruction). Vesicles containing Syb and Ptc are visualized at lateral and basal planes (arrows), and can be detected along cytonemes marked with Syb-GFP (arrowhead).

B) Expression of the GFP tagged Syt1 also co-localizes with inmmuno-labelled endogenous Ptc at vesicles (lateral plane, arrows point at vesicles with co-localization) and decorates the cytoneme membranes (basal plane, arrowheads indicate cytonemes).

C) GRASP experiment shows exocytic events in Hh-receiving cells at basal membrane contacts. Hh-receiving cells express the GFP barrel 1-10 tagged to Syb and Hh-producing cells express the complementary GFP barrel 11 tagged to the membrane protein CD4. Note the fluorescent discrete punctae (arrows) along Hh producing cells cytonemes (stabilized with Ihog-RFP) crossing the reception area.

D) Expression in receiving cells of the Syb functional partner Syx1A tagged to GFP. Note a strong localization in clusters close to the basal membrane in the 3D reconstruction (left panel) and in confocal lateral and basal planes, including cytonemes (right panels, arrows and arrowhead indicate vesicle-like structures along cytoenemes).

Data information: Scale bars 10 μm.
Polarized sorting of Patched enables cytoneme-mediated Hedgehog reception in the Drosophila wing disc (2020)
Figure 2

Figure 2. Polarized Ptc vesicle fusion to basolateral membranes is SNARE complex dependent:

A) Ptc protein distribution in a wing disc (3D reconstitution) under down-regulation of the SNARE proteins Syb and Syt1 in the dorsal compartment (D), keeping the ventral compartment (V) as a WT internal control. Note the basal accumulation of endogenous Ptc (arrowheads) when knocking down Syb or Syt1.

B) Confocal images of immuno-labelled endogenous Ptc and cytonemes stabilized with Ihog-RFP protruding from Hh producing cells in wild type receiving cells (left panel), or B') when either blocking exocytosis by down-regulating Syb (middle panel) or B'') blocking endocytosis by expressing a dominant negative form of the Drosophila Dynamin, Shibire (right panel) in the Hh-receiving cells. Endogenous Ptc in wild type conditions cannot be visualized due to its rapid internalization and processing after Hh reception; while blocking exocytosis causes an accumulation of basal Ptc in intracellular punctae (arrowheads), and endocytosis inhibition leads to Ptc accumulation at the plasma membrane (arrows).

C) Confocal apical (left) and basal (right) images of a shits mutant wing disc expressing Syb RNAi dorsally (D) to also block exocytosis, keeping the ventral compartment (V) as an internal control where just endocytosis is inhibited. After dorsal Syb RNAi induction, endocytosis was inhibited in the whole shits disc by incubation at restrictive temperature. Note the dorsal reduction of Ptc at plasma membrane after blocking exocytosis.

D) Quantification of Ptc-GFP levels (Yac construct) along the wing disc apico-basal axis, integrating both fluorescence intensity and signal area (Integrated Density). The graph on the left shows values for wild type situations in purple (average in blue), while Syb RNAi treatment values are shown in green (average in orange). Note a clear shift of higher values towards the basal side of the discs after inhibition of Syb function. The graph to the right shows correlation coefficients between the maximum Ptc value and distance from the basal side (-1=basal, +1=apical) of discs after RNAi expression for different SNARE proteins and the wild type. Central horizontal lines show median values of N=7-16, box shows lower and upper quartiles and the whiskers show the maximum and minimum excluding outliers. A basal association is particularly noticeable for Syb, Syx1A and α-Snap down-regulation. Right panel is a scheme illustrating the wild type and RNAi-treated patterns of quantified Ptc-GFP levels distribution along the wing disc apico-basal axis.

Data information: Scale bars 10 μm.
Polarized sorting of Patched enables cytoneme-mediated Hedgehog reception in the Drosophila wing disc (2020)
Figure 3

Figure 3. Patched traffics from the apical to the basal side of the wing disc through Multi Vesicular Bodies:

A) Electron Microscopy imaging of wing discs expressing the UAS-Ptc-GFP construct for 24 hours in receiving cells using Ptc-Gal4; TubGal80ts. Left panel shows areas with anti-GFP immuno-labelling (marked in blue) throughout the apico/basal axis of the wing disc epithelium a) anti-GFP gold-labelling corresponding to Ptc is located on the apical membrane and early endosomes towards MVB formation. b) Ptc immuno-gold labelling is present in multivesicular bodies (MVBs) subapically (top right panels). b1) Note that subapical MVBs appear as less dense with greater Ptc labelling on the MVB outer membranes. c) Ptc label is also present in MVBs close to the basal membrane (lower right panels), c1) Note that basolateral Ptc MVBs are significantly denser and richer in Ptc positive intraluminal vesicles (ILV).

B) Endogenous Ptc subcellular localization after endocytosis inhibition by the dorsal expression of a dominant negative form of Rab5. The top panel is a schematic representation of the localized expression. The left panel is a digital 3D reconstruction and the right panel shows an apical and basal confocal sections. Note that accumulation of Ptc occurs at both the apical and basal sides of the dorsal part of the disc, compared to the wild type control on the ventral side.

C) Digital 3D reconstruction (left) and apical and basal confocal sections (right) of endogenous Ptc localization after endocytosis inhibition by the dorsal expression of a dominant negative form of shibire. Note the same apical and basal accumulation as after expression of Rab5DN, and comparing to the ventral wild type half.

Data information: Scale bars (B, C), 10 μm.
Polarized sorting of Patched enables cytoneme-mediated Hedgehog reception in the Drosophila wing disc (2020)
Figure 4

Figure 4. Basal polarization of the Ptc receptor through MVB requires the ESCRT complex:

A) 3D reconstructions of immuno-labelled endogenous Ptc of wing discs in wild type condition and after RNAi expression for different ESCRT complex components. All RNAis' expression result in Ptc accumulation in large structures, located apically under Hrs (ESCRT-0 component) inhibition, and basally under inhibition of the ESCRT components I (Tsg101), II (Vps22) and III (Shrub). Graphs depicting signal values from apical to basal show a shift of higher intensity curves either towards acute apical or basal accumulation.

B) Detailed confocal imaging of immuno-labelled endogenous Ptc localization after clonal Tsg101 down-regulation. The image shows abnormal accumulation of Ptc clusters in basal cytonemes emanating from receiving cells (arrows), since in wild type conditions Ptc visualization at basal membranes is not possible due to its rapid internalization and processing after Hh reception (see Fig 2B).

C) Ptc accumulation in these punctated structures (arrows) is also visualized on receiving cytonemes stabilized with the co-receptor Ihog-RFP and after Tsg101 inhibition.

D) 3D reconstruction of Hh producing cytoneme network stabilized by Ihog-RFP shows that Ptc accumulation after Tsg101 treatment localizes above the signal sending cytoneme extensions (arrow).

Data Information: Scale bars 10 μm.
Polarized sorting of Patched enables cytoneme-mediated Hedgehog reception in the Drosophila wing disc (2020)
Figure 5

Figure 5. Ptc basal vesicles are enriched with extracellular vesicle markers:

A) Confocal image of wing disc expressing the human tetraspanin CD63-GFP expressed in the Hh receiving cells colocalizing with Ptc (Bac.Ptc-cherry) in vesicle-like structures (yellow arrows) along basal cytonemes.

B) In vivo confocal frames of abdominal histoblasts showing colocalization of CD63-Cherry and Ptc (Yac.Ptc-GFP) at punctae moving in anterograde direction along cytonemes (white arrowheads show trajectory in time-lapse frames).

C) Lateral confocal plane of wing disc showing endogenous Ptc colocalizing with the Drosophila tetraspanin Tsp96F-RFP (orthologous to the mammalian tetraspanin CD9/CD81) (yellow arrows indicate co-localization in punctate structures).

D) Basolateral in vivo imaging of abdominal histoblasts expressing Tsp96F-RFP in the Hh receiving cells shows colocalization with Ptc-GFP at mobile punctae (yellow arrows).

E) Tsp96F-RFP expressed in wing disc Hh-receiving cells also shows localization in basal cytonemes stabilized by Ihog-CFP expression (yellow arrows indicate Tsp96F-RFP-positive punctate structures).

F) Frames showing in vivo localization of Tsp96-RFP punctae to cytonemes stabilized with Ihog-CFP expressed in the abdominal histoblasts Hh-receiving cells, moving in an anterograde fashion towards the P compartment (white arrowheads show trajectory in time-lapse frames).

G) Confocal imaging of receiving cells expressing the HA tagged Tsp96F construct also shows co-localization with endogenous Ptc at cytonemes (yellow arrows indicate Tsp96F-HA and Ptc co-localizing punctate structures).

H) Western blot against HA-tag of Immuno-Precipitated sample (IP), Depleted Sample (Dep), High Speed Sample (HSS) Whole Sample and Control sample from a co-immunoprecipitation experiment isolating GFP of Tsp96F-HA and Ptc-GFP expressing larvae or GFP and Tsp96F-HA expressing larvae as negative control. Arrowhead indicates the positive band corresponding to Tsp96F-HA expected molecular weight (30-40 kDa), present in all samples including the immuno-precipitated one and not present in the negative control sample.

Data Information: Scale bars 10 μm.