EMBO/SourceData huggingface dataset:
Figure 4(A KMTR2, KMTR2+Z-VAD treated MDA-MB-436 lysates for indicated early time points were analyzed for caspase-8, caspase-3, ROCK1 PARP as indicated.B) lexatumumab treated OVCAR3 lysates for indicated early time points were analyzed for caspase-3, ROCK1, pMLC, PARP, PD-L1 and CMTM6 etc. as indicated.(C) OVCAR3 cells were treated with pMLC activating ionophore A23187 (positive control) and lexatumumab ± ROCK1 inhibitors or ± rDR5 or ± rFOLR1. Lysates were later analyzed for ROCK1, pMLC, caspase-3 and PARP with GAPDH as loading control. Blue arrows indicate cleaved and activated ROCK1 and caspase-3.(D) MDA-MB-436 and OVCAR3 cells were treated with indicated ROCK1 inhibitors 2 hours prior to DR5 agonist KMTR2 treatment (20nM). After 6 hours flow cytometry was used to analyze surface PD-L1. (GSK269: GSK269962A, GSK429: GSK429286A)(E) MDA-MB-436 cell survival assay after treatment with DR5 agonist antibody KMTR2 ± ROCK1 inhibitors, error bars indicate SD (n=2). (GSK269: GSK269962A, GSK429: GSK429286A)Cultured MDA-MB-436 cells were treated with KMTR2 (IgG1-Fc) or IgG1 control ± ROCK1i (GSK269962) for 6 hrs. After 6 hrs, 500nM anti-PD-L1 avelumab (IgG4-Fc) was added to the media for additional 1 hr. Cellular lysates were pulled down with anti-hu-IgG4 specific beads. (G) Immunoprecipitated lysates and leftover supernatant after various treatments were run at the same time followed by blotting with ROCK1, PD-L1 and CMTM6. Total lysates as a control were also generated in exactly similar conditions and run next to supernatant and IP samples. Blue arrows indicate cleaved and activated ROCK1.(H) Immunoprecipitation assays with anti-PD-L1 (avelumab) using OVCAR-3 cell lines. Blue arrow indicates cleaved and activated ROCK1.(J) Relative luciferase activity ata is from 4 different DR5 agonist antibodies used in combination of two different ROCK1i inhibitors (n=3).(K) Tumor regression efficacy of MD5-1 (50μg), GSK269962A (<2mg/kg) and MD5-1+ GSK269962A (50μg + 2mg/kg) 4T1 tumors (n=4-7). 6-8 weeks old BALB/c mice bearing 4T1 tumors (~100mm3) were intraperitoneally (i.p.) injected with 50μg of indicated antibody every third day (n=4-6). GSK269962A (ROCK1i) was injected directly into the tumors. Tumor volumes were quantified at indicated days by caliper measurements.
Figure 5(B-D) FACs plots confirming expression of human DR5 (huDR5) and Chi-G4S-DR5 in mouse 4T1 cells. Chi-DR5 was not expressed on cell surface (C).(E) Cell viability analysis of huDR5 and Chi-G4S-DR5 stable 4T1 cells with indicated human DR5 agonists lexatumumab and KMTR2 (n=3).(F) Comparison of tumor growth of grafted huDR5 and Chi-G4S-DR5 stable 4T1 cells in Balb/c mice (n=3).(G) Chi-G4S-DR5 stable 4T1 tumors (after reaching ~150-200mm3) were treated with 4 doses (100μg) of IgG1, lexatumumab, lexatumumab +avelumab followed by tumor recovery and surface PD-L1 analysis using flow cytometry(H) Chi-G4S-DR5 stable 4T1 tumors (after reaching ~100mm3) were treated with 6 doses of indicated treatment. Isolated tumors were imaged together (n=4-5). ROCK1i indicates GSK269962A and was administered to animals via intra-tumor injections (2mg/kg).(H) Chi-G4S-DR5 stable 4T1 tumors (after reaching ~100mm3) were treated with 6 doses of indicated treatment. ROCK1i indicates GSK269962A and was administered to animals via intra-tumor injections (2mg/kg). average tumor weight is shown (n=4).(J-K) Confirmation of selective CD8+ T cell population depletion in mice spleen and grafted tumors after injecting animals with either anti-PD-L1 avelumab alone or avelumab+anti-CD8a antibody. CD4+ T-cells remained unchanged.(L) Average of tumor weights (harvested at same time) from mice treated (i.p.) with IgG1, lexatumumab, ROCK1i, lexatumumab + ROCK1i and anti-CD8+ lexatumumab + ROCK1i (n=4-6, 50μg Lexatumumab, 2mg/kg ROCK1i, 6 doses). ROCK1i indicates GSK269962A and was administered to animals via intra-tumor injections. Various treatments were started when tumors were ~100mm3 size.(M) Average of tumor weights (harvested at same time) from mice treated (i.p.) with IgG1, lexatumumab, avelumab, lexatumumab +avelumab and anti-CD8+ lexatumumab +avelumab (n=4-6).(N) 6-8 weeks old C57BL/6 bearing Chi-G4S-DR5 expressing MC38 tumors were intraperitoneally (i.p.) injected with 50μg of indicated antibody every third day (n=4-6). Various treatments were started when tumors were ~100mm3 size. Tumor volumes were quantified at indicated days by caliper measurements.Chi-G4S-DR5 stable 4T1 tumors (after reaching ~100mm3) were treated with 6 doses of indicated treatment. Isolated tumors were imaged together (n=4-5). ROCK1i indicates GSK269962A and was administered to animals via intra-tumor injections (2mg/kg). (O) KMTR2 (8 doses) antibody used as the DR5 agonist for the experiment. (P) Average of tumor weights of data shown in (O). (Q) Kaplan-Meier plot depicting the survival of syngeneic Chi-G4S-DR5 4T1 tumor bearing animals injected i.p. with 100μg of indicated antibodies such as IgG1, KMTR2 and avelumab. Animals were injected with GSK269962A 2mg/kg (in PBS) directly into tumors wherever ROCK1i is indicated.
Figure 6(A) Chi-G4S-DR5 stable 4T1 tumors harboring mice were treated lexatumumab, lexatumumab +ROCK1i, and avelumab + lexatumumab and other controls as indicated. Antibodies were treated i.p at 100μg dose (6 total), ROCK1i (in PBS) was injected directly into tumors at 2mg/kg dose (6 total). Various treatments were started when tumors were ~400mm3 size. Harvested tumors were grouped together and sized matched (3 independent sets: n=2-6 tumors in each set) followed by TIL isolation CD8/CD45 and CD4/CD45 expressing cells were measured by flow cytometry.(B) Plots showing % of total double positive TILs (CD8+CD45+ + CD4+CD45+) in right upper quadrant after combining 3 independent experiments. (C) Ratio of CD8+CD45+/CD4+CD45+ isolated TILs from tumors in each indicated treatment (n=3).Chi-G4S-DR5 stable MC38 tumors harboring mice were treated (6 total doses) lexatumumab, avelumab, ROCK1i, lexatumumab +ROCKi, and avelumab + lexatumumab and IgG1 control as indicated. Harvested tumors homogenized followed by quantitation. Protein lysates were run on SDS-Page followed by immunoblotting using indicated CD8, CD4, Foxp3, caspase-3 and granzyme-b antibody. GAPDH is loading control. Red arrows indicated cleaved caspase-3 p19 and p17 fragments.(H) Cell killing assay of 4T1 cells treated with murine DR5 agonist MD5-1 and bispecific avelu-MD5 antibody.(I) Balb/c mice harboring luciferase stable 4T1 tumors were i.p. injected with indicated antibodies. 50μg dose and mice were imaged after 5 doses.(J) 6-8 weeks old C57BL/6 mice bearing MC38 tumors were intraperitoneally (i.p.) injected with 50μg of indicated antibody every third day (n=4-6). Indicated treatments were started when tumors were ~100mm3 size. Tumor volumes were quantified at indicated days by caliper measurements.(K) ~150-200mm3 size MC38 tumor bearing C57LB/6 mice were treated with indicated MD5-1, avelumab and bispecific antibodies along with control IgG1, 6 total doses. Harvested tumors were homogenized followed by quantitation. Protein lysates were run on SDS-Page followed by immunoblotting using indicated CD8, CD4, caspase-3 and granzyme-b antibody. GAPDH is loading control. Red arrows indicated cleaved caspase-3 p19 and p17 fragments.(L) 6-8 weeks old C57BL/6 mice were injected with MC38 cells. When tumors reached ~150-200mm3, animals were intraperitoneally (i.p.) injected with 50μg of indicated antibody every third day. On day 18, tumors were harvested, sized matched and pooled by treatment group, exposed to collagenase/DNase and were single cell suspensions enriched for CD8+ cells. Enriched CD8+ T-cells from various treatments were restimulated with anti-CD3 (OKT3) antibody for 4 additional hours. CD8 gated cells were next analyzed for IFN-γ intracellular expression using flow cytometry.(M) Percentage of IFN-γ+CD8+ double positive cells from 3 independent experiments.
Figure 1Median CSF protein abundance distribution as calculated from MS-intensities of quantified peptides of each protein. The top ten most abundant proteins and hemoglobin proteins are highlighted.
Figure 2 A-C Protein AD/non-AD fold changes plotted vs statistical significance for Sweden (A), Magdeburg/Kiel (B), and Berlin (C) cohort. Proteins associated with the GO annotation neuron projection labelled in orange. Proteins above the dashed green line are statistically significant (p < 0.05) and those above the black curves have a q-value below 0.05
Figure 3Correlation of protein AD/non-AD fold changes in pairwise combinations of two cohorts each. Combinations are Sweden vs Magdeburg/Kiel Proteins included differ significantly (p < 0.05) and consistently in abundance by AD status in both cohorts each. Correlation of protein AD/non-AD fold changes in pairwise combinations of two cohorts each. Combinations are Sweden vs Berlin (D), and Magdeburg/Kiel vs Berlin (E). Proteins included differ significantly (p < 0.05) and consistently in abundance by AD status in both cohorts each. Proteins that differ significantly (p-value < 0.05 in E; q-value < 0.05 in F) in abundance by AD status across all three cohorts. Z-scored abundances of proteins in the AD and non-AD groups of all cohorts shown by the heat map Hierarchical clustering separates AD from non-AD groups. Pyruvat kinase PKM (PKM) was quantified in two isoforms and UniProt IDs are given in parenthesis. Black frames highlight proteins with consistent AD/non-AD fold changes across cohorts.Proteins that differ significantly (p-value < 0.05 in E; q-value < 0.05 in F) in abundance by AD status across all three cohorts. Z-scored abundances of proteins in the AD and non-AD groups of all cohorts shown by the heat map Hierarchical clustering separates AD from non-AD groups. Pyruvat kinase PKM (PKM) was quantified in two isoforms and UniProt IDs are given in parenthesis. Black frames highlight proteins with consistent AD/non-AD fold changes across cohorts.
Figure 4 A-C) Correlation of proteins with ELISA-measured t-tau concentration across samples within the Sweden (A), Magdeburg (B) and Berlin (C) cohorts. Proteins with a q-value below 0.05 are labeled in yellow. Proteins of the 40 protein signature are colored in red for those with higher abundance in AD CSF and in blue for those with higher abundance in non-AD CSF.  D) Three cohort summary of proteins significantly correlating with ELISA-measured t-tau. Protein names given for the 29 proteins out of the 40 protein signature with significant (p < 0.05) correlation in each of the three cohorts. Pyruvat kinase PKM (PKM) was quantified in two isoforms and UniProt IDs are given in parenthesis.  Protein abundance distribution of CSF showing the abundances of AD-modulated CSF proteins. Proteins of our 40 protein signature are highlighted in red (elevated abundance in AD) and blue (elevated abundance in non-AD). Proteins linked to glucose metabolism are highlighted in purple and labelled.  Protein abundance distribution of brain showing the abundances of AD-modulated CSF proteins. Proteins of our 40 protein signature are highlighted in red (elevated abundance in AD) and blue (elevated abundance in non-AD). Proteins linked to glucose metabolism are highlighted in purple and labelled.
Figure 2Circular representation of DNA methylation levels for mothers (outer circle) and children (inner circle). The height of each bar indicates the methylation change between the smoking and non‐smoking group (dark hue: hypermethylation, light hue: hypomethylation).
Figure 1(A) Endogenous FAM134B forms oligomers. FAM134B knockout (KO) or wild type (WT) 293T cells were treated with 1μM of Thapsigargin (Tg) for different time and the cell lysates were prepared and analyzed by Western-blot.(B) Analysis the oligomer size of recombinant FAM134B WT using Native PAGE.(C) In vitro liposome fragmentation assay. After the injection of recombinant proteins (100μg/100μl) into the chamber (500μl), the morphological changes of liposomes were monitored by live imaging for 20 min. The images at different time points as indicated are presented. Scale bars, 10 μm. "ns" means no significance, one-way ANOVA, error bars indicate SEM (n=3).(D) Quantification of the time between protein addition and liposome fragmentation. ***P<0.001, one-way ANOVA, error bars indicate SEM (n=3).(E, F) Measurement of the intracellular ER-scission activity. U2OS cells transiently expressing EGFP-FAM134B (WT), EGFP-FAM134B (84-233) or EGFP-FAM134B (Δ84-233) at same levels, lysosomal degradation of EGFP-FAM134B was blocked by Bafilomycin A1 (Baf A1) or DMSO. GFP-positive puncta were quantified for each cell in (F). For Each group, at least 30 cells were counted. Scale bars, 10 μm. ***P<0.001, "ns" means no significance, one-way ANOVA, error bars indicate SEM.(G, H) Measurement of the ER-phagy activity. U2OS cells transiently expressing mCherry-EGFP-FAM134B (WT), mCherry-EGFP-FAM134B (84-233) or mCherry-EGFP-FAM134B (Δ84-233) at same levels. ER-phagy is indicated by mCherry-positive but EGFP-negative puncta were quantified for each cell in (H). For mCherry-EGFP-FAM134B (WT), 50 cells were counted (n=50); for mCherry-EGFP-FAM134B (84-233), n=56; for mCherry-EGFP-FAM134B (Δ84-233), n=66. Scale bars, 10 μm. The scale bars in the magnification boxes are 2μm. ***P<0.001, one-way ANOVA, error bars indicate SEM.(I) Lysosomal cleavage of GFP was analyzed by Western blot for the cells co-expressing GFP-SEC61B and FAM134B WT-Flag, FAM134B (84-233)-Flag or FAM134B (Δ84-233)-Flag.
Figure 2(A) Mass spectrometry analysis uncovered that FAM134B was phosphorylated at serine 151.(B) Comparison of the self-interaction of FAM134B mono-phosphorylation mutants using co-IP.(C) Comparison of the self-interaction of FAM134 tri-phosphorylation mutants using co-IP.(D) Comparison of membrane scission activity using in vitro liposome fragmentation assay. Scale bars, 10 μm. ***P<0.001, one-way ANOVA, error bars indicate SEM (n≧3).(E) Lysosomal cleavage of GFP was analyzed by Western blot for the cells co-expressing GFP-SEC61B and FAM134B (WT)-Flag, FAM134B (SA)-Flag or FAM134B (SD)-Flag.(F, G) Measurement of the intracellular ER-scission activity. FAM134B knockout (KO) U2OS cells was engineered to express EGFP-FAM134B (WT), EGFP-FAM134B (SA) or EGFP-FAM134B (SD) at endogenous levels, lysosomal degradation of EGFP-FAM134B was blocked by Bafilomycin A1 (Baf A1). GFP-positive puncta were quantified for each cell in (G). For control, 25 cells were counted (n=25); For EGFP-FAM134B (WT), n=27; for EGFP-FAM134B (SA), n=29; for EGFP-FAM134B (SD), n=25. Scale bars, 10 μm. ***P<0.001, one-way ANOVA, error bars indicate SEM.(H, I) Measurement of the ER-phagy activity. FAM134B knockout (KO) U2OS cells was engineered to express mCherry-EGFP-FAM134B (WT), mCherry-EGFP-FAM134B (S151A) or mCherry-EGFP-FAM134B (S151D) at endogenous levels. Lysosomal mCherry-positive but GFP-negative puncta were quantified for each cell in (I). For mCherry-EGFP-FAM134B (WT), 25 cells were counted (n=25); for mCherry-EGFP-FAM134B (SA), n=23; for mCherry-EGFP-FAM134B (SD), n=21. Scale bars, 10 μm. The scale bars in the magnification boxes are 2 μm. ***P<0.001, one-way ANOVA, error bars indicate SEM.
Figure 3(A, B) Endogenous CAMK2B redistribution to ER membrane structures labeled by mCherry-FAM134B and BAP31 upon Tg treatment. Scale bars, 10 μm. The scale bars in the magnification boxes are 2 μm. The colocalization was analyzed by Pearson's correlation coefficient (PCC) in (B). For Ctrl, 16 cells were counted(n=16); for Tg treatment, 18 cells were counted (n=18). ***P<0.001, one-way ANOVA, error bars indicate SEM.(C) In vitro FAM134B S151 phosphorylation by CAMK2B was detected by Western blot. Recombinant proteins for FAM134BWT and FAM134BS151A were purified from E. coil were incubated with purified CAMK2B by IP in kinase buffer. FAM134B phosphorylation was analyzed by a specific antibody recognizing phos-Serine151 of human FAM134B.(D) FAM134B S151 phosphorylation in cells treated with CAMK2 activator (100nM EB1089 for 1h) or/and inhibitor (10μM KN93 for 2h). 293T or SKN-SH (a cell line derived from neuroblastoma) cells were treated with drugs as indicated, whole cell lysates were analyzed by phospho-FAM134B (S151) antibody.(E) FAM134B S151 phosphorylation in CAMK2B knockdown 293T cells.(F) In vitro reconstitution of CAMK2B-FAM134B-mediated membrane fragmentation using liposome assay. Purified recombinant FAM134B and CAMK2B were preincubated in kinase buffer with ADP or ATP at 30°C for 10min, the resultant protein mixtures were transferred to chamber coated with liposomes. Liposome fragmentation was monitored by live imaging. Scale bars, 10 μm. ***P<0.001, one-way ANOVA, error bars indicate SEM (n=3).(G, H) Measurement of the ER-phagy activity. U2OS cells transiently expressing mCherry-EGFP-FAM134B (WT) were treated with compounds as indicated. Lysosomal mCherry-positive but GFP-negative puncta were quantified for each cell in (H). For vehicle control, 53 cells were counted (n=53), for Ionomycin, n=56; for EB1089, n=54; for KN-93, n=58. Scale bars, 10 μm. The scale bars in the magnification boxes are 2 μm. ***P<0.001, one-way ANOVA, error bars indicate SEM.(I) FAM134B WT or knockout (KO) U2OS cells were treated with CAMK2 activator Ionomycin (Iono), EB1089 or inhibitor KN93 for 24 hours, Western-blot was performed to analyze the proteins as indicated.(J) CAMK2B WT or knockdown (KD) U2OS cells were treated with 1 μM Thapsigargin (Tg) to induce ER stress for different time as indicated. Cells were collected and analyzed for proteins as indicated by Western blot.(K) GFP-cleavage assay. CAMK2B WT or knockdown (KD) U2OS cells were co-transfected with FAM134B-Flag and GFP-SEC61B, 24 hours post transfection, cells were treated with drugs as indicated and analyzed for GFP-cleavage by Western blot. Note: the levels of Flag-FAM134B, CAMK2B, GFP-SEC61B and Tubulin were analyzed by Western blot using different membranes.
Figure 4(A) Comparison of the self-interaction of FAM134B WT with mutants as indicated using co-IP. SA-G216R was constructed by simultaneously mutating S149, S151 and S153 to alanine (SA) on the basis of FAM134B-G216R.(B) In vitro liposome fragmentation assay. Scale bars, 10 μm. ***P<0.001, one-way ANOVA, error bars indicate SEM (n≧3).(C, D) Measurement of the intracellular ER-scission activity of FAM134B WT, G216R, S151A-G216R and SA-G216R. GFP-tagged FAM134B proteins were expressed in U2OS cells at same levels. The autolyosomal degradation of ER fragments labelled by GFP-FAM134B were blocked by Baf A1 or DMSO. GFP-positive puncta were quantified for each cell in (D). For all of the groups, at least 30 cells were included for quantification; Scale bars, 10 μm. ***P<0.001, one-way ANOVA, error bars indicate SEM.(E, F) Measurement of the ER-phagy activity of FAM134BG216R. U2OS cells transiently expressing mCherry-EGFP-FAM134B (WT), mCherry-EGFP-FAM134B (G216R), mCherry-EGFP-FAM134B (S151A-G216R) or mCherry-EGFP-FAM134B (SA-G216R) at same levels. Lysosomal mCherry-positive but GFP-negative puncta were quantified for each cell in (F). For WT, 50 cells were counted (n=50); for G216R, n=55; for S151A-G216R, n=51; for SA-G216R, n=52. Scale bars, 10 μm. The scale bars in the magnification boxes are 2 μm. *P<0.05, ***P<0.001, one-way ANOVA, error bars indicate SEM.
Figure 5(A,A') Specific expression of FAM134B mRNA (green) in sensory neurons (red) of P4d vGlut2Cre;R26lsl-tdTommouse. Scale bars, 100μm. The white dotted line indicates the dorsal root ganglia (DRG) where sensory neurons are genetically labeled with Tomato. Insets of (a) and (a') are high magnifications of sensory neurons showing the co-localization of FAM134B mRNA and Tomato. Scale bars, 10 μm.Cultured sensory neurons from DRGs of E14.5 vGlut2Cre;R26lsl-tdTom embryos were infected with same titers of recombinant lentivirus expressing GFP-FAM134B WT (B), G216R(C), S151A-G216R(D), SA-G216R(E), G216R-ΔLIR (F) respectively. Infected sensory neurons (GFP+/Tom+) are indicated with arrowheads. The nuclei of all cells are shown with DAPI staining. Scale bars, 20 μm.Cultured sensory neurons from DRGs of E14.5 vGlut2Cre;R26lsl-tdTom embryos were infected with same titers of recombinant lentivirus expressing G216R supplemented with 5μM KN93 (G) Infected sensory neurons (GFP+/Tom+) are indicated with arrowheads. The nuclei of all cells are shown with DAPI staining. Scale bars, 20 μm.(H) Quantification of the viability of sensory neuron after 48h, 72h and 96h of infection. Sensory neuron viability was calculated as the percentage of transfected sensory neuron (GFP+/Tom+) in total sensory neurons (Tom+). For each time point, four biological replicates were included for statistical analysis. *P<0.05, **P<0.01, ***P<0.001, two-way ANOVA, error bars indicate SEM. "ns" is the abbreviation for "not significant". For all of the groups, at least 10 fields of the slides were included.
Figure 1.Entry of MLV pseudotyped with SARS-CoV-2 S, SARS-CoV-2 Sfur/mut and SARS-CoV S in VeroE6 cells.Entry of MLV pseudotyped with SARS-CoV-2 S or SARS-CoV-2 Sfur/mut in BHK cells transiently transfected with hACE2. The experiments were carried out in triplicate with two independent pseudovirus preparations and a representative experiment is shown.. Western blot analysis of SARS-CoV-2 S-MLV, SARS-CoV-2 Sfur/mut-MLV and SARS-CoV S-MLV pseudovirions using an anti-SARS-CoV S2 antibody.
Figure 2.Biolayer interferometry binding analysis of the hACE2 ectodomain to immobilized SARS-CoV-2 SB (B) or SARS-CoV SB
Figure 3.Two orthogonal views of the closed SARS-CoV-2 S trimer cryoEM map. C. Atomic model of the closed SARS-CoV-2 S trimer in the same orientation as in panel A.D-E. Two orthogonal views of the partially open SARS-CoV-2 S trimer cryoEM map (one SB domain is open). F. Atomic model of the closed SARS-CoV-2 S trimer in the same orientation as in panel D. The glycans were omitted for clarity.
Figure 5.Entry of SARS-CoV-2 and SARS-CoV S pseudotyped viruses is potently inhibited by four SARS-CoV S mouse polyclonal immune plasma.
Figure 1(A) Western blot showing the protein expression of ASCL1 and/or NEUROD1 in NCI-H69, NCI-H82, NCI-H146, NCI-H510A, NCI-H526, SHP-77 and DMS-53 SCLC cells (lanes 4-10); A549 and NCI-H460 NSCLC cells and a IMR-90 HFL cell lines (lanes 1-3). Tubulin (α-Tub) is shown as loading control. Each blot is representative from at least three independent experiments.
Figure 2(A-E) ChIP-seq experiments indicates the localization of (A) ASCL1, (B) NEUROD1 and (E) RNAPII. ATAC-seq (C) and (D) the presence of H3K27Ac mark indicate the chromatin accessibility around the TSS at t=0 (blue) and t=4h (green) after lurbinectedin treatment; -2.0 and +2.0 kb to TSS coordinates of all hg19 genes.
Figure 3(A-D) Chip-seq genome track of ASCL1/NEUROD1, RNAPII, and H3K27Ac on ASCL1, BCL2; INSM1 and MYB (hg19), in untreated (dark blue) and in lurbinectedin treated (green) conditions respectively; to be noticed the location of CGG rich motifs (lower part of each panel) which parallel the presence of lurbinectedin (red).