EMBO/soda-nel huggingface dataset:
p53 transcriptionally regulates SQLE to repress cholesterol synthesis and tumor growth (2021)
Figure 6

Fig. 6 p53 suppresses cell growth through SQLE and cholesterol

A Cell proliferation of p53+/+ and p53-/- HepG2 cells treated with DMSO or GGTI-2133 (1μm).

B Proliferation of p53+/+ and p53-/- HepG2 cells treated with control or SQLE siRNA. Protein expression was examined after transfection 48 hours.

C Growth of p53+/+ and p53-/- HepG2 cells transfected with and without exogenous SQLE (HA-SQLE) in LPDS medium. Protein expression is assayed after transfection 24 hours.

D Colonies number of p53+/+ and p53-/- HepG2 cells expressing control or SQLE siRNA. Number of colonies with a diameter greater than 10μm were quantified.

E and F Cholesterol concentration, protein expression (E) and cell proliferation (F) of p53+/+ and p53-/- HepG2 cells in LPDS medium containing with or without 5μg cholesterol.

G and H Cholesterol levels, protein expression (G) and cell growth (H) of SK-HEP-1 cells stably expressing control shRNA or p53 shRNA in LPDS medium in presence or absence of 5μg cholesterol.

I-K p53+/+ and p53-/- HCT116 cells transfected with control or SQLE siRNA for 48 hours were injected into nude mice separately. (I) Tumor volume were recorded after inoculation (top). Western blot was performed using lysates of xenograft tumors (bottom). (J) Average tumor weights (top) and images (bottom) of xenograft tumors (3weeks, means ± SD, n=7) are shown. (K) Cholesterol concentrations of xenograft tumors were examined.

L Percentage of SA-β-gal-positive cells of p53+/+ and p53-/- HepG2 cells expressing SQLE siRNA or control siRNA (upper panel). Protein expression (middle panel) and representative images (lower panel) are shown . Sacle bar, 100 μm.

Data information: A-H, I, J, K, L, Bars represent mean ± s.d., *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; A-H, K, L, n=3 biological replicates; J, n=7 biological replicates; Statistical significance was determined by two-tailed unpaired t-test.
p53 transcriptionally regulates SQLE to repress cholesterol synthesis and tumor growth (2021)
Figure 7

Fig. 7 Terbinafine inhibits HFD-induced lipid accumulation and liver cancer growth in p53 knock out mice

A-H. (A) Experimental design for the mouse model in Fig. 7A-H. p53+/+ and p53-/- C57BL/6N male mice (p53+/+ mice n=8;p53-/- mice n=6) were fed with HFD diet for 10 weeks and were administered PBS or terbinafine (80mg/kg) every day for 8 weeks. (B) Mice body weight of p53 +/+ (left) and p53-/- (right) mice. (C) Representative liver photos of mice (left), changes in liver weight (right), H＆E staining (D) and Oil-Red staining (E) of liver tissues are shown respectively. Total liver cholesterol concentrations (F) and triglyceride levels (G) of each group were examined. (H) SQLE mRNA expression of mice liver tissues by qRT-PCR.

I-O. (I) Experimental design for the mouse model in Fig. 7I-O. p53-/- C57BL/6N male mice (n=7) were injected with a single dose of diethylnitrosamine (DEN) (25mg/kg) at two weeks and then fed with HFD diet at 4 weeks. Oral administration of terbinafine were given at 13 weeks old as indicated. Body weight (J) and liver/body weight ratio (K) of mice are shown. Shown are liver tumor incidence (L) and tumor numbers (M). (N) H&E (left), Ki-67 (middle) and Oil Red O (right) staining of livers of mice. (O) Total liver cholesterol concentrations of mice were examined.

Data information: B, C, F, G, H, K, L, M, O, Bars represent mean ± s.d., ns, not significant; *P<0.05; **P<0.01; B, C, p53+/+ mice (n=8), p53-/- mice (n-6) biological replicates; F, G, H, O, n=3 biologically independent samples; J, K, L, M, n=7 biologically independent samples; Statistical significance was determined by two-tailed unpaired t-test.
p53 transcriptionally regulates SQLE to repress cholesterol synthesis and tumor growth (2021)
Figure 8

Fig. 8 SQLE knockdown restricts liver tumor initiation by p53 loss

A Experimental design for the mouse model in Fig. 8. Control mice (p53flx/flx) and liver specific Alb-Cre p53 knockout mice (Alb-cre p53flx/flx) (n=5) were injected with a single dose of DEN at 13 days and then fed with HFD diet at 4 weeks. A tail vein injection of an associate adenovirus serotype 8(AAV8) expressing either control or SQLE shRNA at 13 weeks as indicated. p53flx/flx and Alb-cre mice were examined by genotyping (right panel). p53 flx/flx product size: 370bp. p53+/+ product size: 288bp. Alb-cre product size: 385bp.

B-D Liver weight (B), representative liver photos (C, left), liver tumor numbers (C, right), representative images (D, left) and quantification (D, right) of Ki-67 staining are shown. Red arrows indicate the tumors in the liver (C).

E H&E (left), Ki-67 (middle) and Oil Red O (right) staining of livers of mice

F Total liver triglyceride concentrations of mice were examined.

G Total liver cholesterol concentrations of mice were examined. Protein levels are shown (bottom).

Data information: B, C, D, F, G, Bars represent mean ± s.d., *P<0.05; **P<0.01; ***P<0.001; B, C, n=5 biologically independent samples; D, F, G, n=3 biologically independent samples; Statistical significance was determined by two-tailed unpaired t-test.

Figure legends for Expanded View
Nix is a selective autophagy receptor for mitochondrial clearance (2009)
Figure 1

Nix interacts with Atg8/LC3/GABARAP proteins. (A) Yeast clones harbouring empty bait vector (pYTH9, mock) or those encoding ScAtg8, LC3B, Atg12 and SUMO1 were transformed with empty prey vector (pACT2, mock) or those encoding fragments of ATG4A, ATG4B, NBR1, p62 and full‐length Nix. Interaction was assessed by yeast growth on the SD‐W/‐L/‐H medium with a β‐gal assay. (B) GST pulldown assays using cell extracts of Flag‐Nix‐expressing COS7 cells and immobilized GST or the indicated GST fusions. Coprecipitated Nix was detected with Flag antibodies. (C) Purified GST‐tagged NixΔTM was cleaved from the GST moiety by thrombin, purified and used for precipitation by GST fusion proteins. Precipitated proteins were analysed by western blotting (WB) with Nix antibodies. Ponceau S staining was used to visualize GST‐fusion proteins. (D) HeLa cells were treated as indicated and endogenous proteins were immunoprecipitated (IP) from cell extracts using Nix antibody and analysed by using Nix and LC3 antibodies. β‐gal, β‐galactosidase; BafA1, bafilomycin‐A1; CCCP, carbonyl cyanide m‐chlorophenyl hidrazone; GST, glutathione‐S‐transferase; IgG, immunoglobulin G; NixΔTM, recombinant Nix lacking a TM; SUMO1, small ubiquitin‐like modifier 1; TCL, total cell lysate; TM, transmembrane domain.
Nix is a selective autophagy receptor for mitochondrial clearance (2009)
Figure 2

Identification of Nix‐LIRs. (A) Amino‐acid sequences of Nix and related BNIP3 protein from the indicated species were aligned using the ClustalW2 program (EMBL‐EBI, Cambridge, UK). The published LIR sequences were aligned manually alongside Nix/BNIP3 for comparison. The shaded regions indicate highly conserved residues in the two putative LIRs. Consensus symbols * (identical residues), : (conserved substitution) and . (semi‐conserved substitution) are valid for the Nix/BNIP3 alignment only. (B) A schematic of Nix showing its domain organization and the point mutations used in this study to identify LIRs. (C) Lysates of COS7 cells transfected with Flag‐wt‐Nix or the indicated mutants were incubated with immobilized GST‐LC3A. The coprecipitated proteins were detected by western blotting (WB) with Flag antibodies. BNIP3, Bcl2/E1B 19kDa‐interacting protein 3‐like protein; Dr, Danio reio; GST, glutathione‐S‐transferase; Hs, Homo sapiens; LIR, LC3‐interacting region; Mm, Mus musculus; wt, wild type; Xl, Xenopus laevis.
Nix is a selective autophagy receptor for mitochondrial clearance (2009)
Figure 3

Nix recruits EGFP‐GABARAP‐L1 to stressed mitochondria in reconstituted Nix−/− MEFs. Nix−/− MEFs cotransfected with EGFP‐GABARAP‐L1 and wt‐Nix (A), or Nix‐W35A (B), were incubated with and without mitochondrial poison CCCP for 3 h. Cells were then fixed and analysed for colocalization between EGFP‐GABARAP‐L1 and Nix (stained with Flag antibodies). The final panel in each row shows colocalization between Flag‐Nix‐Cy3 and GABARAP‐L1 using the ‘colocalization’ highlighter plug‐in for ImageJ software (NIH, Bethesda, MA, USA). (C) Quantification was achieved by counting the number of complete colocalizations, EGFP/Cy3 per cell for approximately 30 cells per condition. Results are expressed as a percentage of colocalization (EGFP:Cy3) in control and CCCP‐treated cells. Error bars in (C) show the standard deviation (s.d.) of three independent experiments. Statistical analysis was performed using the Mann–Whitney t‐test. CCCP, carbonyl cyanide m‐chlorophenyl hidrazone; DMSO, dimethyl sulfoxide; EGFP, enhanced green fluorescent protein; MEF, mouse embryonic fibroblast; wt, wild type.
Nix is a selective autophagy receptor for mitochondrial clearance (2009)
Figure 4

Mitochondrial clearance is impaired in Nix−/− reticulocytes transduced with Nix‐W35A. (A) Flow cytometry of reticulocyte‐enriched blood, cultured in vitro for 3 days, stained with MTR. The blood was taken from mice transplanted with Nix−/− bone marrow and reconstituted with wt‐Nix and Nix‐W35A viruses. Reticulocytes were induced by phenylhydrazine treatment. The viruses also express GFP. (B) Mitochondrial clearance in the GFP‐positive fraction of the reticulocyte‐enriched blood, as described in panel (A). Mice were reconstituted with wt‐Nix, LIR‐W35A or empty vector viruses. Data from Nix−/− and Nix+/+ mice are shown for comparison. (C) Anti‐Flag, anti‐GFP and anti‐β‐actin western blots of fetal liver cells transduced with Flag‐Nix and Flag‐W35A‐Nix viruses, normalized to total protein. GFP, green fluorescent protein; LIR, LC3‐interacting region; MTR, MitoTracker Red; wt, wild type.
Foxm1 regulates neural progenitor fate during spinal cord regeneration (2021)
Figure 1

Figure 1 Analysis of differentially expressed genes during spinal cord regeneration

(A) Twenty spinal cords of NF50 tadpoles were isolated at 0, 1 and 3 day post amputation (dpa) and pooled for RNA sequencing.

(B) Genes with a |log2(FC)|>1 and p-adj<0.01 were used for hierarchical clustering. For each cluster, the gene list was uploaded on Fidea (http://circe.med.uniroma1.it/fidea/) (D'Andrea et al, 2013). The five most significant enrichment of GO (biological processes) terms are shown, and the -log10(p_value) with Bonferroni correction is shown.

(C) The dataset was uploaded on the Ingenuity Pathway Analysis software (Qiagen). Genes with a |log2(FC)|>1 and p_adj<0.01 were considered. The software identified upstream regulators based on the changes in expression levels of known dowstream targets. Each upstream regulator is attributed a z-score, corresponding to the negative log of the p-value derived from the Fisher's Exact test.

(D) Changes in the expression of foxm1 and known downstream targets in the whole tail comparing day 0 and day 3 (WT_d0d3) and in the spinal cord comparing day 0 and day 1(SC_d0d1) and day 0 and day 3 (SC_d0d3). The whole tail dataset was obtained from (Chang et al, 2017).

(E) Tadpoles at NF50 were amputated, fixed at the indicated time and then processed for whole mount in situ hybridisation using a probe specific for foxm1. The two last panels show transverse section in the non-regenerating spinal cord (nr) and the regenerate at 3dpa. The red circle highlights the spinal cord and the asterisk the notochord.

(F) Total RNA was isolated from regenerating tails at indicated time points post amputation, reversed transcribed into cDNA and analysed for foxm1 expression by qPCR, using ef1α as a reference gene. The graph represents the mean +/- SD of three independent experiments. One-way ANOVA with Dunnett's multiple comparisons test was used. * p=0.0149
Foxm1 regulates neural progenitor fate during spinal cord regeneration (2021)
Figure 2

Figure 2 Foxm1 is required for spinal cord regeneration but does not regulate the length of the cell cycle

(A) NF40 tadpoles with the following genotypes foxm1-/- (mut), foxm1+/- (het) and foxm1+/+ (wt) were amputated and left to regenerate for 7 days. The images show representative tails at 3 and 7 dpa. The white arrowheads indicate the amputation site.

(B) Quantification of the rate of regeneration. The ratio of the length of the regenerate to the length that has originally be amputated was compared for the whole tail at 3 and 7 dpa. The graph represents the mean+/-SD of 5 independent experiments from 3 different clutches with at least 5 tadpoles in each experiment.

(C) Experimental set up for EdU labelling, foxm1 knockout and wt tadpoles were amputated and left to regenerate for 3 days. Tadpoles were then injected with EdU and 2 days later the tails were fixed, sectioned and stained for EdU and DAPI.

(D) Representative images of EdU (red) and DAPI (blue) staining at 5 dpa.

(E) The graph represents the mean +/- SD of EdU+ cells over the total number of cells in the spinal cord of 5 to 12 tadpoles. Each data point represents a tadpole, with an average of 9 sections analysed per animal.

(F) Experimental set up for Dual-Pulse S-phase Labelling: NF50 tadpoles at 3 dpa were injected with EdU and three hours later, the same tadpoles were injected with BrdU. Six hours after the first injection, the tails were fixed, sectioned and labelled for Sox3, Edu, BrdU and DAPI.

(G) Representative images of EdU (green), BrdU (magenta), Sox3 (white) and DAPI (blue) staining at 3 dpa.

(H) Quantification of images in (G). The graph represents the mean +/- SD of 6 tadpoles with an average of 13 sections per tadpole analysed.

Data information: two-way ANOVA with Tukey's post-hoc tests were used for B and E, and an unpaired t-test for H. ns: non-significant, **p<0.01, **** p<0.001. Scale bar is 500µm in A and 25µm in D and G.
Foxm1 regulates neural progenitor fate during spinal cord regeneration (2021)
Figure 4

Figure 4 Changes in cell cycle dynamics during regeneration

(A) UMAP projection with inferred cell cycle phase for each cell

(B) Bar plot showing the proportion of cells in G1, G2/M and S phase of clusters of progenitor cells (prog., expressing sox2 and sox3) and differentiating progenitors (diff. prog., expressing neurod1, neurog1) and neurons (neur., expressing snap25). The total does not always amount to 100% as some clusters have cells from both 0 and 3 dpa. The bar boxed in red represent the foxm1 positive cluster and the cluster numbering refers to the cluster identity defined in Fig 3G.

(C) Representative sections labelled with an anti-PCNA antibody (red) and DAPI (blue) at the indicated day after amputation (dpa). The white arrowheads point to cells in S phase in the spinal cord and the yellow arrowhead at cells in G1, G2 and M phases. The right panels correspond to the inset indicated as a white box in the middle panels.

(D) The ratio of PCNA+ per total number of cells (DAPI) in the spinal cord was determined at the indicated times after amputation (n=3, with a mean of x sections per data point).

(E) The PCNA+ cells were then distributed in G1/G2 (diffuse signal), S (punctated signal) or M phase (condensed chromatin) at the indicated stage of regeneration.

Data information: D-E data are the mean+/-SD of three independent experiments and one-way ANOVA with Tukey's post-hoc tests was used ns: non significant, **p<0.01, ****p<0.0001. In C, scale bar represents 25µm for the left and middle panels, and 5µm for the right panels
Foxm1 regulates neural progenitor fate during spinal cord regeneration (2021)
Figure 5

Figure 5 Foxm1 promotes the differentiation of neural progenitors in the regenerating spinal cord

(A) Representative sections of spinal cords from tadpoles injected with EdU at 3 dpa and fixed at 5 dpa. After sectioning, the samples were labelled with antibodies against Sox3 (green), EdU (magenta) and DAPI (blue). White arrowheads show Sox3 positive extensions.

(B) Sections of spinal cords at 5 dpa in the stump (non-regen.) or in the regenerate (regen) labelled with anti-Sox3 (green), anti-Acetylated Tubulin (AcTub, magenta) and DAPI (blue). White arrowheads show Sox3 positive extensions.

(C) Quantification of the images shown in (A). The ratio of Sox3+ per total number of cells (DAPI) in the spinal cord was determined in the stump (non-regen.) and the regenerating spinal cord (regen.) in wt and foxm1-/- tadpoles (n=3 to 8, with an average of 15 sections per data point).

(D) Quantification of the proportion of cycling progenitors (Sox3+EdU+/DAPI, S3+/E+/DAPI), the proportion of progenitors having divided (Sox3+EdU+/Sox3+, S3+E+/S3+) and the proportion of progenitors self-renewal (Sox3+EdU+/EdU+, S3+E+/E+) in the regenerate. The graph represents the mean +/- SD from 9 tadpoles (wt) and 8 tadpoles (foxm1-/-) with an average of 18 sections per data point.

(E) Representative sections of spinal cords from tadpoles injected with EdU at 3 dpa and fixed at 5 dpa. After sectioning, the samples were labelled with antibodies against Myt1 (green), EdU (magenta) and DAPI (blue).

(F) Quantification of images shown in (E). The graph represents the mean +/- SD from 3 tadpoles with an average of 12 sections each.

Data information: For C, D and F, a two-way-ANOVA with Tukey's post-hoc tests was used and the graphs represent the mean +/- SD. ns=non-significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. For A, B and E, the scale bar represents 25µm
A Crohn’s disease variant in Atg16l1 enhances its degradation by caspase 3 (2014)
f1

a, Multiple sequence alignment of ATG16L1 (NP_110430.5). Protein accession numbers shown in Supplementary Table 3. b, Carboxy-terminal V5 immunoblot of full-length (68 kDa) and cleaved (36 kDa) ATG16L1&bgr;. Silver stain (bottom gels) depicts total protein. Data represent 3 independent experiments. IB, immunoblot. c, TNF-&agr;-mediated processing of human ATG16L1 in monocyte-derived macrophages. Scatter plot of immunoblot densitometry depicts a ratio of cleaved:full-length ATG16L1 (Extended Data Fig. 2). n = 7 donors. d, TNF-&agr;-mediated processing of murine ATG16L1 in macrophages. Immunoblot represents 5 independent experiments. Scatterplot represents data pooled from 5 independent experiments. WT, wild type.
A Crohn’s disease variant in Atg16l1 enhances its degradation by caspase 3 (2014)
f2

a, HeLa cells transfected with control siRNA (siCTL) or siCaspase 8 were stimulated with 2.0 &mgr;M staurosporine or 20 ng ml−1 TNF + 10 &mgr;g ml−1 CHX. b, HeLa cells transfected with control siRNA or siCaspase 9 were stimulated as in a. c, HeLa cells transfected with control, caspase-3- or caspase-7-specific siRNAs were stimulated with 20 ng ml−1 TNF + 10 &mgr;g ml−1 CHX. d, Wild-type or caspase-3-knockout macrophages were stimulated with 2.0 &mgr;M staurosporine or 20 ng ml−1 TNF + 10 &mgr;g ml−1 CHX. Data in a–c represent 2 independent experiments; data in d represent 4 independent experiments.
A Crohn’s disease variant in Atg16l1 enhances its degradation by caspase 3 (2014)
f3

a, Intracellular staining for LC3 and ATG16L1 following glucose starvation in the presence of bafilomycin A1. White mask in LC3 channel illustrates punctate LC3 staining, representing LC3-II. Histogram depicts mean fluorescence intensity of 10,000 cells per sample. b, Quantification of mean ATG16L1 fluorescence intensity in a, n = 4 mice. Glu., glucose. c, Immunoblot of phosphorylated (p-) AMPK&agr; (Thr 172) and lipidated LC3 (LC3-II). d, Quantification of mean LC3-II area in murine macrophages, n = 4 mice. e, Caspase 3 activity in murine macrophages following glucose starvation, n = 3 mice. RLU, relative light units. f, Quantification of mean LC3-II area in wild-type (Casp3+/+) or Casp3-knockout (Casp3−/−) macrophages, n = 3 mice. Data in a–f represent 2 independent experiments.
A Crohn’s disease variant in Atg16l1 enhances its degradation by caspase 3 (2014)
f4

a, Quantification of ATG16L1 mean fluorescence intensity by image-based flow cytometry of 10,000 cells, n = 4 (wild-type, WT), 5 (T300A). Data represent 3 independent experiments. b, Quantification of caspase 3 activity upon glucose starvation of 2 × 104 cells, n = 5. Data represent 2 independent experiments. c, Quantification of mean LC3-II area of samples in a. Data represent 3 independent experiments. d, Visualization of early autophagosomes by transmission electron micrographs (TEM). Red arrows, early autophagosomes, characterized by vesicular structures with a double-membrane. Blue arrows, multivesicular lysosomal structures, identified by degraded intravesicular cargo. Micrographs representative of 10–12 images per donor, n = 4 (WT), n = 3 (T300A). A total of 29 (WT) and 26 (T300A) images containing autophagosomes was analysed. Histogram quantifies autophagic vesicles as a ratio of autophagosome:cytosolic area.
A Crohn’s disease variant in Atg16l1 enhances its degradation by caspase 3 (2014)
f5

a–c, Y. enterocolitica colony forming units (c.f.u.) in human and murine macrophages infected for 6 h (20 m.o.i.). Data in a are pooled from 4 independent cohorts of 3 or 4 donors per genotype, total n = 16 (non-risk, WT), 15 (T300A, risk). Data in b represent 3 independent experiments, n = 4 mice. Data in c represent 3 independent experiments, n = 6. d, TNF-&agr;, IL-1&bgr; and IL-6 levels in culture media of WT and T316A macrophages infected with 20 m.o.i. Y. enterocolitica. Data represent 2 independent experiments, n = 4 mice. e, Ileum and colon histology (haematoxylin and eosin staining) of WT mice. Images are representative of 3 (PBS) and 8 (Y. enterocolitica) treated male mice. f, Measurement of cytokine mRNA transcripts in mesenteric lymph nodes. Data are pooled from 2 independent experiments of 3 mice (PBS) and 7 mice (Y. enterocolitica) per experiment. Transcripts are normalized to Gapdh (2−Dgr;Ct). g, Y. enterocolitica c.f.u. in mesenteric lymph nodes of WT and T316A knock-in mice 48 h following oral gavage. Data represent 3 independent experiments, n = 9 male mice.
Antisense transcription represses Arabidopsis seed dormancy QTL DOG1 to regulate drought tolerance (2017)
Figure 1

Figure 1. DOG1 antisense expression is selectively regulated by ABA and drought in Arabidopsis leaves. Application of the plant hormone ABA but not GA affects luciferase activity driven by the antisense promoter.

A Representative picture of plants carrying pASDOG1::LUC sprayed with mock solution, ABA or GA and imaged 24 h later. The full pictures are shown in Fig EV2 and Appendix Fig S1. Scale bar, 1 cm.

B Quantification of emitted light intensity per plant after treatment with ABA and GA, compared with mock treatment.

C Antisense expression is reduced in response to drought conditions. The full pictures are shown in Fig EV3. Scale bar, 1 cm.

D Quantification of emitted light intensity per plant for control and drought-treated plants.

E Strand-specific RT-qPCR asDOG1 expression analysis of 3-day Col-0 (WT) drought-treated vs. control plants.

F The asDOG1 expression level (black line) is reduced while DOG1 sense expression (grey line) is increased after application of ABA. Leaves of mature Col-0 (WT) 40-day-old plants were sprayed with ABA and collected 0, 6 and 10 h later for RNA extraction and RT-qPCR. Signals were normalized against the level of the UBC transcript. The data points are the averages for at least three biological replicates and are normalized against the mock-treated value. Error bars represent standard deviation.* and *** represent t-test P-values of < 0.05 and < 0.001, respectively.