EMBO/soda-nel huggingface dataset:
Neural G0: a quiescent-like state found in neuroepithelial-derived cells and glioma (2021)
Figure 5

Figure 5: Identifying GBM specific Neural G0 marker genes and application to 641 human gliomas.

A, A GBM specific Neural G0 expression signature was developed using four GBM scRNA-seq datasets with a total of 47,169 cells (Darmanis et al., 2017; Neftel et al., 2019; Bhaduri et al., 2020; Wang et al., 2020). The ccAF classifier was applied to each dataset, marker genes were discovered for the Neural G0 phase in each dataset, and 22 common marker genes were identified between all four datasets.

B, A network showing the inter-relatedness of the 22 common GBM-specific Neural G0 marker genes. Genes with red circles were previously known to be associated with GBM in the DisGeNET database.

C, Violin plot showing PLP1 expression across all cell types from Darmanis et al., 2017. Data are log 10-transformed before plotting. Expression of PLP1 in single cells is much higher in oligodendrocytes (Oligo) than in Neural G0 or oligodendrocyte progenitor cells (OPC).

D, Violin plot showing GFAP expression across all cell types from Darmanis et al., 2017. Data are log 10-transformed before plotting. Expression of GFAP in single cells is more highly expressed in Neural G0 relative to all other cell types.

E, Violin plot showing AQP4 expression across all cell types from Darmanis et al., 2017. Data are log 10-transformed before plotting. Neural G0 expression of AQP4 in single cells is similar to astrocytes but has a lower median expression and lower upper limit.

F, Relative Neural G0 eigengene expression between grade II (n = 226), III (n = 244), and IV (n = 150) tumors (TCGA; LGG and GBM). An eigengene represents the common variation across each patient tumor for the Neural G0 genes, i.e., first principal component corrected for direction if necessary. All pairwise Student's t-tests comparisons had p-values <0.003.

G, Comparison of cell cycle (54 genes annotated to the Seurat G2M classifier gene list) and Neural G0 eigengene relative expression in each glioma. Each tumor is colored by its grade (green = II, red = III, and purple = IV).

H, Kaplan Meier survival plot of tumors with top 25% (n = 171) and bottom 25% (n = 171) of Neural G0 eigengene expression of Neural G0 genes. A Fleming-Harrington survival p-value was used to determine significance. Shaded region is the 95% confidence interval for the survival curve.

I, Distribution of tumor grade between tumors with top 25% and bottom 25% of Neural G0 eigengene expression.
Neural G0: a quiescent-like state found in neuroepithelial-derived cells and glioma (2021)
Figure 6

Figure 6: Reduction of G0/G1 transit time in NSCs after KO of CREBBP, NF2, PTPN14, TAOK1, or TP53.

A, Representative contour plot of flow cytometry for FUCCI (Sakaue-Sawano et al. 2008) in U5-NSCs after targeting of a non-growth limiting (NGL) control gene, GNAS1. Values are similar to wild-type and non-targeting control (NTC) U5-NSCs under similar culture conditions. The system relies on cell-cycle dependent degradation of fluorophores using the degrons from CDT1 (amino acids (aa) 30-120) (present in G0 and G1; mCherry) and geminin (aa1-110) (present in S, G2, and M; monomeric Azami-Green (mAG)).

B, Representative contour maps of flow cytometry for FUCCI following the loss of NF2, PTPN14, TAOK1, CREBBP, and TP53.

C, Ratio of G0/G1 (mCherry-CDT1+) to S/G2/M (mAG-Geminin+) from (A) and (B). Values are mean from 4 individually-tested LV guides per gene at 21 days post-selection.

D, G0/G1 and S/G2/M transit times using time-lapse microscopy and FUCCI. Differences in G0/G1 are statistically significant with p<0.001 for targeted U5-NSCs and p=0.0006 for GSC-131 compared to NTC.

Data information: The data are presented as the mean ± SD. Supporting information is provided in Appendix Figure S14. Significance was assessed using a two-tailed student's t-test (C) or Mann-Whitney test (D).
Glucocorticoids limit lipopolysaccharide-induced lethal inflammation by a double control system (2020)
Figure 1

Figure 1. Glucocorticoids protect against LPS-induced lethality, intestinal permeability and intestinal expression of ISGs.

(A-C) WT C57BL/6J mice were injected with 12.5 mg/kg LPS, with or without pre-treatment with 10 mg/kg DEX 30 minutes before LPS. (A) Lethality was followed-up. LPS-treated mice (n=5) are depicted as black circles and DEX/LPS-treated mice (n=5) are depicted as white circles. P-values for survival curves were analyzed with a Fisher's exact test. (B) Blood samples were taken 2 hours after LPS and TNF levels were measured in serum (n=4-5). (C) Three hours after LPS challenge, dextran labeled with FITC was administered by oral gavage. Five hours after gavaging, blood plasma was collected and fluorescence was measured as a degree of intestinal permeability (λexc/λem = 488/520 nm) (PBS: n=3, LPS and DEX/LPS: n=5).

(D-E) RNA sequencing (RNAseq) results of IECs of WT mice (n=3) treated with PBS or 12.5 mg/kg LPS for 6 hours. (D) Heatmap with selection of LPS-induced genes. Color key shows the log2 of normalized counts. (E) HOMER motif analysis of LPS-induced genes. Motifs with the highest rank and their p-value and q-value are displayed.

(F-G) WT mice (n=5) were treated with PBS or 12.5 mg/kg LPS with or without 10 mg/kg DEX pre-treatment. Ileum was isolated and gene expression was measured via qPCR.

Data information: P-values were calculated using 1-way ANOVA, unless otherwise stated. All bars represent mean ± SEM. ****P< 0.0001; *** P<0.001; ** P≤ 0.01; * P≤ 0.05.
Glucocorticoids limit lipopolysaccharide-induced lethal inflammation by a double control system (2020)
Figure 2

Figure 2. LPS leads to induction of necroptosis-stimulating ISG genes in IECs.

(A-D) WT mice (n=3-5) were treated with PBS or 12.5 mg/kg LPS with or without 10 mg/kg DEX pre-treatment. Ileum was isolated 6 hours after LPS challenge. (A) p-MLKL and MLKL protein levels and (B) RIPK3 protein levels were analyzed via western blot using ACTIN as a loading control. Expression levels were quantified using FIJI and normalized to ACTIN levels. P-values were calculated using 1-way ANOVA. (C) Gene expression (Defa6, lysozyme) was measured via qPCR. P-values were calculated using 1-way ANOVA. All bars represent mean ± SEM. ****P< 0.0001; *** P<0.001; ** P≤ 0.01; * P≤ 0.05.

(D) H&E staining was performed on ileum samples. Scale bar: 50µm.

(E) Lysozyme expression (in red) was detected via IHC, with Hoechst counterstaining. Scale bars: 50µm.

(F) Quantification of lysozyme expression. For every picture (n=3-4 per treatment), a selection of 10 crypts was made. Lysozyme positive signal was quantified in this region of interest (ROI) using an automated script in QuPath. Values are the positive area relative to total area of ROI. P-values were calculated using 1-way ANOVA. Data is shown as mean ± SEM. ****P< 0.0001; *** P<0.001; ** P≤ 0.01; * P≤ 0.05.
Glucocorticoids limit lipopolysaccharide-induced lethal inflammation by a double control system (2020)
Figure 3

Figure 3. TNF and IEC TNFR1 play critical roles in LPS-induced lethality and ISG induction in IECs.

(A) TNFR1+/+ (n=9) and TNFR1-/- mice (n=11) were treated with 12.5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.

(B) TLR4fl/fl mice (n=7) and TLR4VillinKO (n=9) mice were treated with 12.5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.

(C) TNFR1fl/fl mice (n=13) and TNFR1VillinKO (n=10) mice were treated with 12.5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.

(D) Venn diagram of LPS- and TNF-upregulated genes in IEC, as identified by RNA-seq (n=3). HOMER motif analysis was performed on the 692 overlapping genes. Motifs with the highest rank and their p-value and q-value are displayed.

(E) TNFR1+/+ and TNFR1-/- mice (n=4-6) were treated with 12.5 mg/kg LPS. Ileum was isolated and gene expression was measured via qPCR. Fold changes are shown on the graph.

(F) TNFR1fl/fl and TNFR1VillinKO mice (n=4-6) were treated with 12.5 mg/kg LPS. Ileum was isolated and gene expression was measured via qPCR. Fold changes are shown on the graph.

Data information: P-values were calculated using 2-way ANOVA, unless otherwise stated. All bars represent mean ± SEM. ****P< 0.0001; *** P<0.001; ** P≤ 0.01; * P≤ 0.05.
Glucocorticoids limit lipopolysaccharide-induced lethal inflammation by a double control system (2020)
Figure 4

Figure 4. First GR control point: to suppress TNF production in macrophages.

(A) GRfl/fl (n=8,) and GRLysMKO mice (n=4) were treated with 2.5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.

(B) GRLysMKO mice were treated with PBS (n=12) or 10 mg/kg DEX (n=13) before a lethal dose of LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.

(C) TNF measurement in the serum of GRfl/fl (n=4) and GRLysMKO mice (n=3). Mice were treated with 2.5 mg/kg LPS and blood was drawn 2 hours later. TNF concentration was measured with ELISA.

(D) TNF measurement in the serum of GRfl/fl and GRLysMKO (n=4). Mice were treated with PBS or a lethal dose of LPS (12.5 and 2.5 mg/kg for GRfl/fl and GRLysMKO mice respectively) , with or without pre-treatment with 10 mg/kg DEX and blood was drawn 2 hours later. TNF concentration was measured with ELISA.

(E-F) GRfl/fl (n=4) and GRLysMKO mice (n=3-4) were treated with 2.5 mg/kg LPS. Ileum was isolated after 6 hours and gene expression was measured via qPCR. Fold changes are depicted on the graphs.

(G) GRfl/fl and GRLysMKO mice (n=4) were treated with a PBS or LPS (12.5 and 2.5 mg/kg for GRfl/fl and GRLysMKO mice respectively) with or without 10 mg/kg DEX pre-treatment of 30 minutes. Ileum was isolated after 6 hours and gene expression was measured via qPCR. Fold changes are depicted on the graphs.

Data information: P-values were calculated using 2-way ANOVA, unless otherwise stated. All bars represent mean ± SEM. ****P< 0.0001; *** P<0.001; ** P≤ 0.01; * P≤ 0.05.
Glucocorticoids limit lipopolysaccharide-induced lethal inflammation by a double control system (2020)
Figure 5

Figure 5. Second GR control point: to suppress TNF-induced ISG expression in intestinal epithelium.

(A) GRfl/fl (n=8) and GRVillinKO mice (n=10) were treated with 5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.

(B) GRVillinKO mice were treated with PBS (n=9) or 10 mg/kg DEX (n=10) before 5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test. (C) TNF measurement in the serum of GRfl/fl (n=4) and GRVillinKO mice (n= 4). Mice were treated with 5 mg/kg LPS and blood was drawn 2 hours later.

(D) TNF measurement in the serum of GRfl/fl (n=4) and GRVillinKO mice (n=4). Mice were treated with PBS or LPS (12.5 and 5 mg/kg for GRfl/fl and GRVillinKO mice respectively) with or without pre-treatment with 10 mg/kg DEX for 30 minutes. Blood was taken 2 hours after LPS challenge and TNF concentrations were measured with ELISA.

(E) GRVillinKO mice were treated with PBS (n=8) or a neutralizing P55 (TNFR1) antibody (n=9) 2 hours before and 8 hours after 5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.

(F) Ileum was isolated from non-stimulated GRfl/fl (n=4) and GRVillinKO mice (n=4) and gene expression was measured via qPCR. Expression levels are shown as fold induction with GRfl/fl expression values set at 1. P-values were calculated using a student's t-test.

(G-H) GRfl/fl (n=4) and GRVillinKO mice (n=4) were treated with 5 mg/kg LPS. Ileum was isolated and gene expression was measured via qPCR.

Data information: P-values were calculated using 2-way ANOVA, unless otherwise stated. All bars represent mean ± SEM. ****P< 0.0001; *** P<0.001; ** P≤ 0.01; * P≤ 0.05.
Glucocorticoids limit lipopolysaccharide-induced lethal inflammation by a double control system (2020)
Figure 6

Figure 6 GR dimers play an essential role in protection against LPS-induced lethality and suppression of ISG expression in IECs.

(A) Left: LPS dose-response curves of GRwt/wt (n=3-7 per LPS dose) and GRdim/dim mice (n=3-7 per LPS dose) treated with 1.5 mg/kg LPS. Right: LD50 values of LPS are depicted on top of each bar for each group.

(B) GRdim/dim mice were treated with PBS (n=5) or 10 mg/kg DEX (n=5) before 1.5 mg/kg LPS and survival was monitored. P-values were analyzed with a Fisher's exact test.

(C) TNF measurement in the serum of GRwt/wt (n=5) and GRdim/dim mice (n= 5). Mice were treated with 1.5 mg/kg LPS and blood was drawn 2 hours later. TNF concentration was measured with ELISA.

(D) TNF measurement in the serum of GRwt/wt and GRdim/dim mice (n=4) treated with PBS or LPS (12.5 and 1.5 mg/kg in GRwt/wt and GRdim/dim mice respectively) with or without pretreatment with 10 mg/kg DEX. Blood was taken 2 hours after LPS challenge and TNF concentrations were measured with ELISA.

(E) GRdim/dim mice were treated with PBS (n=6) or a neutralizing P55 (TNFR1) antibody (n=8) 2 hours before and 8 hours after 1.5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.

(F) Venn diagram of LPS- and DIM-upregulated genes (in basal condition) in ileum, as identified by RNA-seq (n=3).

(G) Tofacitinib (n=6) or vehicle (n=7) was given orally to GRdim/dim mice. After 1.5 mg/kg LPS injection survival was monitored and p-values were analyzed with a Fisher's exact test.

(H) GRwt/wt (n=4) and GRdim/dim (n=4) mice were treated with 1.5 mg/kg LPS. Ileum was isolated and gene expression was measured via qPCR. Fold changes are depicted on the graphs.

(I) GRdim/dim mice (n=4) were treated with PBS or 1.5 mg/kg LPS with or without 10 mg/kg DEX pretreatment. Ileum was isolated and gene expression was measured via qPCR. Fold changes are depicted on the graphs.

Data information: P-values were calculated using 2-way ANOVA, unless otherwise stated. All bars represent mean ± SEM. ****P< 0.0001; *** P<0.001; ** P≤ 0.01; * P≤ 0.05.
mTOR inhibits autophagy by controlling ULK1 ubiquitylation, self-association and function through AMBRA1 and TRAF6 (2013)
figf1

(a) FLAG–AMBRA1 was transfected into HEK293 cells together with ULK1WT or ULK1K46I Myc-tagged proteins. Protein extracts were immunoprecipitated (IP) using anti-FLAG antibody. (b) HEK293 cells were grown either in normal or in EBS medium for 1 h; protein extracts were immunoprecipitated using anti-ULK1 antibody or rabbit immunoglobulin as a negative control (IgG). Autophagy induction is shown by p62 degradation. (c) HEK293 cells were treated as in b, in the presence of leupeptin and stained by anti-AMBRA1 (red) and anti-ULK1 (green) antibodies. The merge of the two signals and DAPI (blue) for nuclear staining is shown in the right panels. Scale bar, 6 μm. The outlined areas are shown at a higher magnification in the lower panels. White arrows point to the co-localization puncta. Measurements by Metamorph indicate a co-localization ratio of 46% between ULK1 and AMBRA1 on autophagy induction. (d,e) ULK1 and AMBRA1 constructs are illustrated. Asterisks mark nonspecific bands corresponding to immunoglobulins. (d) HEK293 cells were co-transfected with vectors encoding AMBRA1 together with ULK1–Myc FL (full length) or its deletion mutants encoding the kinase and the proline-rich domains (Δ829–1,051), and the ULK1 carboxy-terminal domain (Δ1-828). Protein extracts were immunoprecipitated using anti-Myc antibody. (e) HEK293 cells were co-transfected with vectors encoding ULK1–HA together with β-galactosidase–Myc (βgal), AMBRA1 full-length (FL), and F1, F2 or F3 Myc-tagged proteins. Protein extracts were immunoprecipitated using anti-HA antibody. The relative amounts of co-immunoprecipitated ULK1 and F1 or F3, respectively, are the same (see upper and lower left panel). (f) HEK293 cells were co-transfected with vectors encoding AMBRA1, ULK1–Myc and ATG13–FLAG. Protein extracts were immunoprecipitated using anti-FLAG antibody. (g) HEK293 cells were transfected with scramble siRNA or ATG13 siRNA. Protein extracts were immunoprecipitated using ULK1 antibody or IgG. The band density ratio of immunoprecipitated AMBRA1 relative to immunoprecipitated ULK1 is analysed, with the control ratio arbitrarily defined as 1.00.
n = 3 extracts prepared from independent experiments; data are presented as means±s.d. and significance is
P = 0.04. Uncropped images of blots/gels are shown in Supplementary Fig. S6. Source data of statistical analysis are shown in Supplementary Table S1.
mTOR inhibits autophagy by controlling ULK1 ubiquitylation, self-association and function through AMBRA1 and TRAF6 (2013)
figf2

(a) HEK293 cells were transfected with AMBRA1 or the empty vector, as a control. ULK1, AMBRA1 and actin were analysed by western blotting. (b) Protein extracts from wild-type (Ambra1+/+) and homozygous (Ambra1gt/gt) embryos for the Ambra1 gene-trap mutation at developmental day 14.5 were analysed by western blotting using anti-Ulk1 or anti-Hsp90 (loading control) antibodies. Quantification is shown on the right.
n = 6 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is
P = 0.037. (c) AMBRA1, ULK1 and actin (as a protein loading control) were analysed by western blotting in HeLa cells transiently transfected with scramble siRNA or AMBRA1 siRNA. Quantification is shown on the right.
n = 5 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is
P = 0.007. (d) HeLa cells stably interfered for AMBRA1 were transiently transfected with empty vector or AMBRA1 vector; AMBRA1, ULK1, ATG13, p-ATG13 and actin levels were analysed by western blotting. Quantification of ULK1 and pATG13 bands is shown on the right.
n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is
P = 0.006 for ULK1 and
P = 0.044 and
P = 0.011 for pATG13. (e) HeLa cells were transfected or not with vector encoding AMBRA1 and treated with 35 μM cycloheximide (CHX) for 6 and 8 h before extraction. ULK1, actin and AMBRA1 were analysed by western blotting. Quantification is shown below.
n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is
P = 0.049 and
P = 0.02. (f) HEK293 cells were transfected as in a; ATG13, pATG13, ULK1, AMBRA1 and actin were analysed by western blotting. (g) Endogenous ULK1 is immunoprecipitated from HeLa cells transfected with β-galactosidase or AMBRA1 plasmid respectively using an anti-ULK1 antibody. As a control, rabbit IgG was used. The resulting precipitates were subjected to ULK1 kinase assay using MBP as the substrate. Purified complexes were analysed by autoradiography and by western blotting using an anti-ULK1 antibody. Total extracts were analysed using an anti-AMBRA1 antibody. Uncropped images of blots/gels are shown in Supplementary Fig. S6. Source data of statistical analysis are shown in Supplementary Table S1.
mTOR inhibits autophagy by controlling ULK1 ubiquitylation, self-association and function through AMBRA1 and TRAF6 (2013)
figf3

(a) Protein extracts from HeLa cells were immunoprecipitated using anti-ULK1 antibody or with IgG as a negative control and analysed by western blotting using anti-Lys63 specific ubiquitin and anti-ULK1 antibodies. (b) HeLa cells were co-transfected with ULK1–Myc alone or in combination with wild-type ubiquitin or its mutant (K48R or K63R) HA-tagged constructs. Protein extracts were immunoprecipitated using an anti-Myc antibody. Purified complexes and total extracts were analysed by western blotting using anti-Myc and anti-HA antibodies. (c) In HeLa cells, AMBRA1 was downregulated by RNAi. Protein extracts were immunoprecipitated using anti-ULK1 antibody or with IgG as a negative control. Purified complexes and total extracts were analysed by western blotting using anti-ULK1, anti-Lys-63 ubiquitin and anti-AMBRA1 antibodies. Of note, ULK1 levels in the co-immunoprecipitation experiment do not change in this case in the AMBRA1 siRNA lane, owing to saturation of the anti-ULK1 antibody (see also Fig. 4h). (d) HeLa cells were co-transfected with vectors encoding for ubiquitin Lys-63–HA alone or in combination with AMBRA1. Protein extracts were immunoprecipitated using an anti-ULK1 antibody or with IgG as a negative control and Ub-Lys-63–HA, ULK1 and AMBRA1 were analysed by western blotting. (e,f) HeLa cells were cultured in complete or starvation medium for 30 min or in the presence of 100 nM of rapamycin for 3 h and then protein extracts were immunoprecipitated using anti-ULK1 antibody or with IgG as a negative control. Purified complexes and total extracts were analysed by western blotting as in a. Quantifications are shown on the right. In both panels,
n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is
P = 0.003 and
P = 0.0015, respectively. Uncropped images of blots/gels are shown in Supplementary Fig. S6. Source data of statistical analysis are shown in Supplementary Table S1.
mTOR inhibits autophagy by controlling ULK1 ubiquitylation, self-association and function through AMBRA1 and TRAF6 (2013)
figf4

(a) HeLa cells were transfected with FLAG–TRAF6 or grown in starvation medium for 1 h. Some of them were treated with chloroquine. LC3, p62 and actin were analysed by western blotting. Quantification of LC3II and p62 bands is shown on the right.
n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is
P = 0.017 for LC3 and
P = 0.01 and
P = 0.04 for p62. (b) HeLa cells were transduced with AMBRA1 lentiviral shRNA and transfected or not with FLAG–TRAF6. Quantification of LC3II and p62 bands is shown on the right.
n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is
P = 0.001 and
P = 0.0018 for LC3 and
P = 0.004 for p62. (c) Count of LC3 puncta in HeLa cells transduced as in b. Five different confocal microscopy images for each experiment were randomly chosen and GFP-positive dots were examined on the images with identical brightness and contrast settings. The results are represented as the average number of LC3 dots with 100 cells analysed per sample. Confocal images are shown in Supplementary Fig. S3b. (d) HEK293 cells were transfected with AMBRA1 and FLAG–TRAF6 as indicated. Protein extracts were immunoprecipitated using an anti-FLAG antibody. (e) HeLa cells were transfected as in b and FLAG–TRAF6 was purified as in d. Quantification of immunoprecipitated TRAF6 is shown on the right.
n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is
P = 0.0003. (f) In HeLa cells, TRAF6 was downregulated by RNAi. AMBRA1, ULK1, TRAF6, ATG13 and actin were analysed by western blotting. (g) HeLa cells were co-transfected with vectors encoding ULK1–Myc, FLAG–TRAF6 and Ub-Lys-63–HA, as indicated. Protein extracts were immunoprecipitated using an anti-Myc antibody. (h) HeLa cells stably interfered for AMBRA1 were transfected with FLAG–TRAF6 and Ub-Lys-63–HA as indicated. Protein extracts were immunoprecipitated using anti-ULK1 antibody. (i) Endogenous ULK1 is immunoprecipitated from HeLa cells transfected as in b. The resulting precipitates were subjected to a ULK1 kinase assay using MBP as the substrate. Purified complexes were analysed by autoradiography and by western blotting using an anti-ULK1 antibody. Uncropped images of blots/gels are shown in Supplementary Fig. S6. Source data of statistical analysis are shown in Supplementary Table S1.