EMBO/soda-nel huggingface dataset:
Bhlhe40 and Bhlhe41 transcription factors regulate alveolar macrophage self-renewal
and identity (2019)
Figure 4

Figure 4. Accumulation of lipids in AMs of DKO mice.

(A) Staining of bronchoalveolar lavage cytospins from WT and DKO mice with the lipophilic dye Oil Red O. Representative results of two independent experiments.

(B) Quantification of Oil Red O-positive cells in WT and DKO mice for individual animals. Horizontal lines indicate the mean and error bars (s.d.). * P < 0.05 (Student's t-test). Results pooled from two independent experiments; 4 WT and 5 DKO mice were analyzed in total.

(C) Flow cytometric analysis of AMs from WT and DKO mice stained with the lipophilic dye BODIPY 493/503. Representative results of two independent experiments with three mice per group.

(D) Analysis as in C performed with WT:DKO mixed BM chimeras. Gating on CD45.1 and CD45.2 was applied to identify cells of WT and DKO donor origin. Representative results of two independent experiments with at least three chimeras.

(E) RNA-seq analysis of Acaa1b and Hsd17b4 expression in WT and DKO AMs, which were isolated by flow cytometry from mice in steady state (top) or from mixed BM chimeras (bottom). Error bars represent SEM; two biological replicates per genotype.
Bhlhe40 and Bhlhe41 transcription factors regulate alveolar macrophage self-renewal
and identity (2019)
Figure 5

Figure 5. Genome-wide analysis of Bhlhe40 and Bhlhe41 function in AMs.

(A) Comparison of changes in gene expression induced by Bhlhe40/Bhlhe41-deficiency in steady-state knockout mice (horizontal axis) and in mixed BM chimeras (vertical axis). Log2 transformed DKO/WT fold changes are plotted. Genes with expression of > 5 TPM in at least one of the samples are displayed. Genes highlighted in magenta indicate known and putative regulators of AMs. Closed symbols - Bhlhe40-bound genes (Bhlhe40 ChIP-seq peak assigned to the nearest gene within a 50 kb window). Open symbols - genes without Bhlhe40 binding.

(B) Presence of RNA transcripts (RNA-seq; top) at the Gas7 and Acaa1b genes in WT and DKO AMs; the ChIP-seq analysis of H3K27 acetylation in WT and DKO AMs (middle) and Bhlhe40 binding in WT AMs (bottom). All analyses performed on ex vivo AMs.

(C) Consensus Bhlhe40-binding motif identified by de novo motif-discovery (MEME-ChIP suite; E-value, 8.6 × 10−121).

(D) Distribution of Bhlhe40 peaks at intergenic regions, the gene body or the promoter in AMs.

(E) Gene set enrichment analysis (GSEA) on dataset ranked by DKO/WT fold change in steady-state AM RNA-seq using set of genes assigned to top 1,000 Bhlhe40 peaks (ranked by P value) (left) or a random set of 1,000 genes (right). Similar results were obtained with all three random sets tested. NES - normalized enrichment score. FDR False Discovery Rate.

(F) Pairwise comparison of H3K27 acetylation in WT and DKO AMs performed on 2-kb regions around the Bhlhe40 peak summits (right - for all Bhlhe40 peaks called with P value of < 10−10; center - for top 3,000 Bhlhe40 peaks (ranked by P value)) and around WT AM ATAC-seq peaks (Immgen) that did not have a Bhlhe40 peak in their close proximity (filtered as described in Materials and Methods) (left). Volcano plots show adjusted P values (vertical axes) and log2 fold change (horizontal axes) for H3K27ac pairwise comparison in WT vs DKO AMs; genes assigned to some of the Bhlhe40-binding regions are labeled on the left plot.
Bhlhe40 and Bhlhe41 transcription factors regulate alveolar macrophage self-renewal
and identity (2019)
Figure 6

Figure 6. Repression of AM lineage-inappropriate genes by Bhlhe40 and Bhlhe41.

(A) Comparison of changes in gene expression induced by Bhlhe40/Bhlhe41-deficiency in steady-state knockout mice (horizontal axis) and in mixed BM chimeras (vertical axis) as shown in Fig. 5 A, but filtered for the indicated signatures (no TPM filter applied). Closed symbols - genes with associated Bhlhe40 binding (as in Fig. 5 A) revealed by ChIP-seq. Open symbols - genes without Bhlhe40 binding. Inserts show Bhlhe40 ChIP-seq track for selected dysregulated genes.

(B) Flow cytometric comparison of expression of the indicated cell-surface markers (identified in panel A) on WT and DKO AMs. A narrow forward/side scatter gate was applied to correct for possible differences in autofluorescence. Representative results of two independent experiments with three and four mice per group.
Targeting netrin-3 in Small Cell Lung Cancer and Neuroblastoma. (2021)
Figure 1

Figure 1: Netrin-3 expression in cancer cell lines

Exploring the expression by RNA-sequencing of netrin-1 and -3 among 675 cancer cell lines spanning 16 cancer types.

A. Expression heat map of netrin-3 and netrin-1. Receptors of netrin-1 and putative receptors of netrin-3: DCC, UNC5-A, -B, -C, -D, Neogenin, APP, CD146 are presented. The color legend (right-hand side) describes all tumor types included in the heat map. Asterisks indicate the two main pathologies in which netrin-3 expression is detectable.

B. Analysis of netrin-1 and -3 expression in tumor cell lines and ranked according to netrin-1 expression

C. Percentage of cell lines positive for netrin-1 and/or -3 expression. Co-occurrence of expression was calculated using Fisher's exact test. P-value is indicated in the table.
Targeting netrin-3 in Small Cell Lung Cancer and Neuroblastoma. (2021)
Figure 2

Figure 2: Analysis of netrin-3 expression on Neuroblastoma (NB) prognosis.

A. Quantification of netrin-3 gene expression by q-RT-PCR in a panel of 181 human NB stages 1, 2, 3, 4 and 4S. Number of cases is indicated on the graph. Error bars indicate s.e.m. Statistical treatment of the data was performed using a two-sided Student t-test.

B. Quantification of netrin-3 gene expression by q-RT-PCR in a panel of 181 human neuroblastoma patients, defined as low- and high-risk NB. The number of cases is indicated on the graph. Error bars indicate s.e.m. Statistical treatment of the data was performed using a two-sided Student t-test.

C. Netrin-3 high expression is a marker of poor prognosis in NB. 280 months overall Kaplan-Meier survival curves in a panel of 181 patients of all NB stages. The cohort was dichotomized base on netrin-3 median expression (in green and red). Statistical treatment of the data: Mantel-Cox; p value is indicated below the graph.

D. Netrin-3 high expression is a marker of poor prognosis in aggressive NB. 280 months overall Kaplan-Meier survival curves in a panel of 54 stage 4 patients. The cohort was dichotomized base on netrin-3 expression, as presented in panel C. Statistical treatment of the data: Mantel-Cox; p-value is indicated below the graph.

E. Confirmation in 498 RNA-seq NB patients that netrin-3 high expression is a marker of poor prognosis in NB. 6,000 days overall Kaplan-Meier survival curves. The cohort was dichotomized based on netrin-3 median expression (in green and red). Statistical treatment of the data: Mantel-Cox; p-value is indicated below the graph.
Targeting netrin-3 in Small Cell Lung Cancer and Neuroblastoma. (2021)
Figure 3

Figure 3: Netrin-3 is a target gene of MYCN in NB.

A. Gene Set Enrichment Analysis (GSEA) in high netrin-3 NB patients. The cohort was dichotomized with the n=50 lowest vs 50 highest: amplification the MYCN pathway (p=0.039; fdqr=0.025).

B. Netrin-3 expression is higher in MYCN amplified NB patients. The cohort was dichotomized based on mycn amplification and sorted for netrin-3 expression. Statistical treatment of the data: Welsh test; p-value is indicated on the graph. Error bars indicate s.e.m (boxes= 25th to 75th percentile; central band= mean; whiskers = min to max).

C. Q-RT-PCR analysis of netrin-3 gene expression in IMR32 (n=5) and IGRN91 (n=5) NB cell lines after MYCN silencing by siRNA (U-test). Error bars indicate s.e.m.

D. ChIP-seq analysis of MYCN binding, active enhancer epigenetic marks H3K27ac, H3K4me1, and active promoter epigenetic mark H3K4me3, on netrin-3 locus (red line). An enrichment of MYCN, associated with active enhancer marks, was detected in three different neuroblastoma cell lines NB-1643, COGN415 and LAN5.

E. Quantitative analysis showing the size of IGRN91 primary tumors implanted on CAM and silenced or not for netrin-3 (n=11 siScr, n=9 siNetrin-3; U-test). Error bars indicate s.e.m.

F. Quantitative analysis showing the size of IMR32 primary tumors silenced or not for netrin-3 (n=5 siScr, n=7 siNetrin-3; U-test). Error bars indicate s.e.m.
Targeting netrin-3 in Small Cell Lung Cancer and Neuroblastoma. (2021)
Figure 4

Figure 4: Characterization of Netrin-3 in SCLC.

A. Deep analysis of netrin-3 gene expression in lung cancer cell lines (n=76; RNA-Seq).

B. Q-RT-PCR analysis of netrin-1 and -3 expression, in lung cancer cell Lines (Error bars indicate SD, n=3).

C. Representative netrin-3 and -1 mRNA detection, using RNAscope on SCLC paraffin embedded tumor sections. Negative control DAPB, positive control PPiB. Each brown dot is a unique molecule of mRNA of each targeted gene (n=10, scale bars 20µm).

D. Quantification of netrin-3 and -1 expression in human SCLC paraffin embedded tumor sections (High≥ 50% marked cells; Low≤ 5%).
Targeting netrin-3 in Small Cell Lung Cancer and Neuroblastoma. (2021)
Figure 5

Figure 5: Netrin-3 is a target gene of NeuroD1 and ASCL1 in SCLC.

A. ChIP-seq analysis of NeuroD1 and ASCL-1 binding on the netrin-3 gene promoter. A major binding site was detected (red and blue peaks for NeuroD1 and ASCL1 respectively). Input controls are shown below (black).

B. ChIP-seq analysis of NeuroD1 and ASCL-1 chromatin enrichment, which was associated with broad H3K27ac-enriched regions at the netrin-3 locus (red line), indicating a transcriptionally active region in SCLC cell lines. (rep= experimental replicates).

C. Q-RT-PCR analysis of netrin-3 gene expression in NCI-H82 (n=6) and NCI-H69 (n=4) SCLC cell lines after Neurod-1 and ASCL-1 silencing by siRNAs (U-test). Error bars indicate s.e.m.
Targeting netrin-3 in Small Cell Lung Cancer and Neuroblastoma. (2021)
Figure 6

Figure 6: Tumor promoting role of netrin-3.

A. Bio-layer interferometry analysis of netrin-2L/receptor interactions. Other members of the netrin family were also tested: netrin-1, -4 and -G2. (red: netrin-2L; bleu: netrin-1; green netrin-4)

B. Kd calculation after bio-layer interferometry analysis, *= no interaction detected.

C. NCI-H82 CRISPR/Cas9 were subjected to immunoblots. sGx, designates the number of guide RNAs, control cells (sGc).

D. Netrin-3 silencing delays or inhibits SCLC engraftment in vivo. NMRI nude mice were subcutaneously engrafted with either NCI-H82-CRIPSR/cas9-NTN3 with three different guides RNAs (sG1 p=0.0022; sG2 p≤0.001; and sG3 p≤0.001) or control cells (sGc) n=6/group. Positive tumor engraftment was report when tumors reached 50 mm3 (Mantel Cox).

E. NCI-H69-CRISPR/Cas9 were subjected to immunoblots. sGx, designates the number of guide RNAs, control cells (sGc)

F. NCI-H69-CRISPR/Cas9 cells presented in E. were subcutaneously engrafted with either NCI-H69- CRIPSR/cas9-NTN3 with three different guide RNAs (sG1 p=0.017; sG2 p=0.028; and sG3 p=0.001) or control cells (sGc) n=6/group. Positive tumor engraftment was report when tumors reached 20 mm3 (Mantel Cox).
Targeting netrin-3 in Small Cell Lung Cancer and Neuroblastoma. (2021)
Figure 7

Figure 7: Netrin-3 is a therapeutic target in SCLC.

A. Analysis of the NP137 antibody (anti-netrin-1) epitope across netrin family members.

B. Analysis of NP137 binding on netrin-2L, netrin-1; netrin-4 or netrin-G2 by bio-layer interferometry assays. Kd rhNTN1: 0.96 ± 0.30nM; Kd for rchNTN2: 1.53 ± 0.96nM

C. NP137 inhibits NTN1/UNC5B or NTN-2L/UNC5B interaction, detected by Elisa assays. Here the recombinant proteins are the murine forms (Control IgG= mouse IgG2A, mNP137= murine Fc, UNCB=rat UN5H2) bars indicate SD, n=3.

D. NMRI nude mice were engrafted with NCI-H82 cells by subcutaneous injection of 2 million cells. When the mean tumor volume reached approximately 80 mm3, animals were treated 3 x time/weekly by intra-abdominal injection of PBS; NP001 (Human IgG1, isotype); NP137 (anti-netrin1/3) during 20 days. n=10 animals/group (*p < 0.05, two-way Anova, Error bars indicate s.e.m.).

E. NP137 enhances survival of mice engrafted with NCI-H82 cell line (see D). Analysis of Kaplan-Meier survival curves of mice treated or not with NP137. Mantel Cox test; n=10 animals/group.

F. Quantification of cell death (cleaved PARP) in H82 treated tumors in D. Tumors were analyzed 7 days after the first treatment (ie: 400mm3; n=3/group. U-test, bars indicate SD).

FIGURE EV LEGENDS
HSD17B7 gene in self-renewal and oncogenicity of keratinocytes from Black versus White populations (2021)
Figure 5

Fig 5 HSD17B7 is a positive determinant of HKC and SCC cells proliferative and oncogenic potential.

(A) Colony forming assays of multiple HKC strains from White and Black donors (passages 2,3) infected with a HSD17B7 expressing lentivirus (HSD-oe) versus empty vector control (ctrl), plated in triplicate dishes at limited density (1000 cells per 60mm dish) at 5 days after infection. A week later cells were fixed and stained by crystal violet and number of colonies (>0.149mm2) was determined. Shown are numbers of colonies +/- SD. n(dishes per condition) =3. *p<0.05. 2-tailed unpaired t-test.

(B) Colony forming assays of the indicated SCC cell lines stably infected with a HSD17B7 expressing lentivirus (HSD-oe) versus empty vector control (ctrl), utilizing the same conditions as in the previous panel. Shown are numbers of colonies +/- SD. n(dishes per condition) =3. *p<0.05; ****p<0.0001. 2-tailed unpaired t-test.

(C) Proliferation live-cell imaging assays of SCC cell lines plus/minus HSD17B7 overexpression as in the previous panel. Cells were plated in triplicate wells in 96-well plates followed by cell density measurements (IncuCyte), taking four images per well every 2h for 150-160h. n (wells per condition)=3. ****p<0.0001. Pearson r correlation.

(D-F) Intradermal tumorigenicity assays with HKCs (269W) stably infected with an HSD17B7 overexpressing lentivirus (HSD17B7-oe) versus control (Ctrl). Experimental conditions were the same as in Fig 2. Mice were sacrificed 15 days after injection. Shown are representative images and quantification of tumor cells density assessed by immunohistochemical staining with anti-pan keratin antibodies (D,) as well as % of Ki67 positive cells (E) and K1 positivity (F) of pan-keratin positive areas. For (D), n(tumors per condition) = 9; for (E and F) n(3 fields per tumor, 5 tumors per condition) = 15. *p<0.05, ****p<0.0001. Horizontal line: median. 2-tail unpaired t-test. Scale bar: 500μm (D), 100μm (F).

(G, H) Intradermal tumorigenicity assays with SCC13 cells overexpressing HSD17B7 (HSD17B7-oe) versus control (Ctrl). Mice were sacrificed 15 days after injection. Shown are representative images and quantification of tumor cells density, assessed by immunohistochemical staining with anti-pan keratin antibodies (G) as well as % of Ki67 positive cells in pan-keratin positive areas (H). For (G), n(tumors per condition) = 4; for (H), n(3-5 fields per tumor, 4 tumors per condition) 15, 19. *p<0.05; Horizontal line: median. One-tailed paired t-test. Scale bar in (G, H): 100μm.
HSD17B7 gene in self-renewal and oncogenicity of keratinocytes from Black versus White populations (2021)
Figure 6

Fig 6 HSD17B7 plays an essential function in HKC and SCC cells proliferation.

(A) Colony forming assays of multiple HKC strains from donors of White (213W) and Black (246B, 261B, 285B) origin, which were infected with two HSD17B7 silencing lentiviruses (shHSD2, shHSD4) versus vector control (shCtrl). Gene silencing efficiency is shown in Appendix Fig S6. Cells were plated in triplicate dishes at limited density (1000 cells per 60mm dish) at 5 days after infection and fixed a week later for determination of colony formation as in Fig. 5A. Shown are representative dishes together with data quantification, expressed as numbers of colonies +/- SD. n(dishes per condition)= 3 **p<0.01. 2-tailed unpaired t-test.

(B) Sphere forming assays of multiple HKC strains plus/minus HSD17B7 silencing as in the previous panel. Cells were cultured in Matrigel in triplicate dishes, utilizing the same conditions as for the experiments of Fig 2F. Data are expressed as ratio of large spheres (> 2000 pixels>=0.0095mm2) versus total number of spheres (>100 pixels>=0.00047mm2). +/- SD. n(dishes per condition)= 3 **p<0.01; ****p<0.0001, 2-tailed unpaired t-test.

(C) EdU labeling assays of multiple HKC strains plus/minus HSD17B7 silencing as in the previous panels. Cells were plated in triplicate dishes 5 days after lentiviral vector infection and selection and pulse labeled with EdU for 3 hrs. Shown are % of EdU positive cells +/- SD. n(dishes per condition)= 3. *p<0.05, **p<0.01; ***p<0.0005; p****<0.0001, 2-tailed unpaired t-test.

(D) Colony forming assays of the indicated SCC cell lines plus/minus HSD17B7 silencing as in the previous panels. Cells plated in triplicate dishes 5 days after lentiviral vector infection and selection were tested for clonogenic capability as in (A). Data are represented as number of colonies +/- SD. n(dishes per condition)= 3 *p<0.05, **p<0.01; p****<0.0001, 2-tailed unpaired t-test.

(E) EdU labeling assays of the indicated SCC cell lines plus/minus HSD17B7 silencing . Experimental conditions were as in (C). n(dishes per condition)= 3. ****p<0.0001, 2-tailed unpaired t-test. Data are represented as % of EdU positive cells +/- SD.

(F) Colony forming assays of SCC13 cells plus/minus lentivirally-mediated HSD17B7 overexpression and subsequent infection with two shRNA silencing vectors as indicated. Experimental conditions were as in (D). n(dishes per condition)= 3 *p<0.05, **p<0.01; ***p<0.0005, 2-tailed unpaired t-test. Data are represented as number of colonies +/- SD.
HSD17B7 gene in self-renewal and oncogenicity of keratinocytes from Black versus White populations (2021)
Figure 8

Fig 8 Differential mitochondrial OXPHOS activity in Black versus White HKCs and as a function of HSD17B7 expression.

(A) Purified mitochondrial preparation from multiple HKC strains from White and Black donors (12 per ancestry; passage 2,3; triplicate cultures) were analyzed for levels of Electron Transport Chain activity (ETC, nmol of reduced cytochrome c/min/mg protein), ATP (nmol/mg protein) and ROS (MtROS (nmol/mg mitochondrial protein). In parallel, Acid Soluble Metabolites (ASM, pmol/h/mg protein) levels were measured as a readout of β-oxidation. Similar measurements of HKCs strains at a later passage are shown in Appendix Fig S6. Data are displayed as average values for all tested HKC strains (3 cultures per strain for ETC, ATP and FAO, 1 culture per strain for mtROS, white and black dots, depending on ancestry) together with mean +/- SD. n(HKC strains per ancestry)= 12 ***p<0.0005; ****p<0.0001, 2-tailed unpaired t-test. Individual experimental values are provided in Dataset EV3.

(B) Multiple HKC strains (249W, 260W, 275W) stably transduced with an HSD17B7-expressing lentivirus (HSD-oe) versus empty vector control (Ctrl) were analyzed as in (A). Data are shown as individual values for 3 parallel cultures per strain together with mean +/- SD. *p<0.05; **p<0.01; ***p<0.0005; 2-tailed unpaired t-test. Individual experimental values are provided in Dataset EV3.

(C, D) SCC13 cells stably infected with an HSD17B7-expressing lentivirus (HSD-oe) versus empty vector control (Ctrl) (C) or two HSD17B7-silencing lentivirus (shHSD2, shHSD4) versus vector control (shCtrl) (D) were analyzed as in the previous panels. Shown are individual values for 3 parallel cultures per condition together with mean +/- SD. **p<0.005; ***p<0.0005; ****p<0.0001. 2-tailed unpaired t-test. Additional metabolic measurement experiments of SCC cells plus/minus HSD17B7 overexpression and silencing, in the context of additional manipulations are shown in Fig 9.
HSD17B7 gene in self-renewal and oncogenicity of keratinocytes from Black versus White populations (2021)
Figure 9

Fig 9 HSD17B7 functions are dependent on catalytic activity and involve zymosterol production.

(A) Upper panel: schematic representation of the 17-beta-dehydrogenase domain shared between the HSD17B1 and HSD17B7 enzymes with positions of the NADP and steroid binding sites and the active site (Breton et al., 1996). Two conserved charged residues in these domains, Tyrosine (Y) 193 and Lysine (K) 197, were changed into Glycine (G). Lower panel: closeup views of active site of HSD17B7 showing the NADP and steroid binding pocket and position of the Y193G and K197G amino acid substitutions.

(B) Clonogenicity assays with two different SCC cell lines stably infected with lentiviruses overexpressing HSD17B7 in wild type (HSD-wt) or Y193G and K197G mutated (HSD-mut) forms versus empty vector control (Ctrl). SCC13 cells were cultured in either DMEM + 10% FBS (*) or HKC growth media. HSD17B7 overexpression was verified by RT-qPCR and immunoblot analysis shown in Appendix Fig S6. Experimental conditions were as in Fig 5A. Data are represented as number of colonies (>0.149mm2) +/- SD. n(dishes per condition)= 3 *p<0.05, **p<0.005; ***p<0.0005, ****p<0.0001. 2-tailed unpaired t-test.

(C) Purified mitochondrial preparation from SCC13 cells infected with lentiviruses expressing wild type (HSD-wt) and Y193G and K197G mutated (HSD-mut) forms versus empty vector control (Ctrl) as in (B) were assayed for levels of Electron Transport Chain activity, ATP and mtROS production as in Fig 8A. Data are displayed as average values of triplicate measurements together with mean +/- SD. n(dishes per condition)= 3. **p<0.005; ***p<0.0005. 2-tailed unpaired t-test. Individual experimental values are provided in Dataset EV3.

(D) SCC13 cells were infected with two HSD17B7 silencing lentiviruses (shHSD2, shHSD4) versus vector control (shCtrl) followed, 3 days after infection and antibiotic selection, by treatment with Zymosterol (2.5uM) or ethanol vehicle alone for 48hours. Purified mitochondrial preparation were assayed for levels of Electron Transport Chain activity, ATP and mtROS production as in Fig 8A. Data are displayed as average values of triplicate measurements together with mean +/- SD. n(dishes per condition)= 3. ****p<0.0001, 2-tailed unpaired t-test. Individual experimental values are provided in Dataset EV3.