EMBO/SourceData huggingface dataset:
Figure 7 A: Immunohistochemistry analysis recapitulates the biochemistry described in vitro for mithramycin. G401 tumor sections at 20X magnification stained with H&E, cleaved caspase 3 (CC3; apoptosis) or H3K27me3. A marked increase in CC3 that correlates with H3K27me3 staining is seen only in mice treated with the continuous infusion schedule but not vehicle. Scale bar (lower left): 50μm.  B: Prolonged durable response and cure of mice bearing G401 xenografts treated with 30mg/kg EC8042 administered continuously over 72-hours. Treatment duration indicated by gray shaded box. Asterisk indicate an animal sacrificed due to unknown causes not related to tumor progression or drug toxicity (see text).  C: Kaplan-Meier survival curves indicating extended survival for mice bearing established G401 xenografts treated with the 3-day continuous infusions of EC8042 in (B). The shaded box indicates the duration of treatment. Asterisk indicate an animal sacrificed due to unknown causes not related to tumor progression or drug toxicity (see text).  D: 20X image of section of G401 treated tumors stained for H3K27me3, cleaved caspase 3, and γH2AX. The sections compare vehicle to treatment started on day 1 with 30 mg/kg of EC8042 administered continuously for 72-hours (3-day pump). H3K27me3 increases and correlates with apoptosis (CC3), however induction of CC3 is modest. γH2AX staining does not increase with treatment indicating DNA damage is not responsible for these effects. Scale bar (lower left): 50μm.
Figure 8 Immunohistochemistry analysis of H&E stains from G401 xenograft tumors on 1, 3, and 7-days after treatment with vehicle or 3-day EC8042 infusion. EC8042 treated xenograft tumors exhibit evidence of mesenchymal differentiation compared to vehicle. microCT analysis of xenograft tumors on 7-days after treatment exhibit enhanced calcification compared to vehicle. IHC scale bar (lower left): 100μm.
Figure 1B. RNAseq data are plotted in log2 fold change (FC) levels of protrusions over cell bodies against adjusted -log10 false discovery rate (FDR) (n=2 replicates, average FC values are represented). The horizontal dashed line marks the FDR (q) = 0.05 threshold; vertical dashed lines mark the FC = 0.625 (left) or FC = 1.6 (right) thresholds.C. Heat map represents the k-means clustering of transcript log2 FC levels (protrusions over cell bodies) extracted from RNAseq datasets published elsewhere. The corresponding HUVEC FC levels are shown in parallel.D. smFISH co-detection of k 5 mRNAs and GAPDH in subconfluent motile HUVECs. arrows indicate orientation of RNA localisation; yellow dashed lines outline cell borders; red circles highlight smFISH spots; scale bars = 20 µmE. Polarisation Index (PI) of k 5 mRNAs and GAPDH in co-detected in HUVECs (n≥28 cells; **P<0.01, ***P<0.001, ****P<0.0001; Wilcoxon test).F. k 5 mRNAs PIs plotted against respective GAPDH PIs. The slope of the coloured lines represents the average k 5 mRNA/GAPDH PI ratio; the dashed grey line represents a 1:1 ratio (n≥28 cells).G. Top: distribution pattern of mRNAs clustered in k 2, k 5, k 7 and GAPDH. Bottom: smFISH co-detection of exemplar k7 / k 2 mRNAs and GAPDH in subconfluent motile HUVECs. arrows indicate orientation of RNA localisation; yellow dashed lines outline cell borders; red circles highlight smFISH spots; scale bars = 20 µmH. PIs of k 7 mRNAs and GAPDH co-detected in HUVECs (n≥25 cells; *P<0.05, **P<0.01, ***P<0.001; paired t test).I. PIs of k 2 mRNAs and GAPDH co-detected in HUVECs (n≥19 cells; *P<0.05; Wilcoxon test).
Figure 2E. Left: representative subconfluent motile cells co-transfected with plasmids expressing Lyn-mCherry, MCP-GFPnls and 24xMS2-RAB13 3'UTR or 24xMS2. Right: percentage of cells with MCP-GFP localised to protrusions when co-transfected with full length or deletion versions of RAB13 3'UTR (n≥3 experiments). white arrowheads indicate non-nuclear localisation of MCP-GFPnls; arrows indicate the orientation of RNA localisation; yellow dashed lines outline cell borders; scale bars = 20 µm For each k 5 mRNA 3'UTR, a diagram of the full length 3'UTR and the positions of the RNA motif is shown together with the respective RNAseq mapped reads from HUVEC protrusions; black arrowheads indicate predicted polyadenylation sitesF. Left: representative cells co-transfected with plasmids expressing Lyn-mCherry, MCP-GFPnls and 24xMS2-k 5 3'UTRs. Right: percentage of cells with MCP-GFP localised to protrusions when co-transfected with full length or deletion versions of k 5 3'UTR (n≥3 experiments). white arrowheads indicate non-nuclear localisation of MCP-GFPnls; arrows indicate the orientation of RNA localisation; yellow dashed lines outline cell borders; scale bars = 20 µm For each k 5 mRNA 3'UTR, a diagram of the full length 3'UTR and the positions of the RNA motif is shown together with the respective RNAseq mapped reads from HUVEC protrusions; black arrowheads indicate predicted polyadenylation sites
Figure 3B. Representative genotyping PCR demonstrates the band size shift in ∆LE HUVECs.D. Wt and ∆LE HUVEC RNAseq mapped reads depicting RAB13 exon usage.E. Quantification of RAB13 mRNA smFISH spot number in Wt and ∆LE HUVECs (n=3 experiments; ns: not significant; unpaired t test).F. Number of RAB13 mRNA smFISH spots plotted against the respective Polarisation Index (PI) (n=29 cells; ns: not significant; linear regression).G. Left: representative Western blotting (WB) of Wt and ∆LE HUVECs. Right: densitometry analysis of WB data (n=3 samples; ns: not significant; unpaired t test).H. smFISH co-detection of RAB13 and control GAPDH in Wt and ∆LE motile HUVECs cultured under subconfluent conditions. Arrows indicate orientation of RNA localisation; yellow dashed lines outline cell borders; red circles highlight smFISH spots; scale bars = 20 µm (H). Bar charts are presented as means±s.d.I. PI of RAB13 and GAPDH co-detected in Wt and ∆LE HUVECs (n=29 cells; P***<0.001, ns: not significant; Mann-Whitney test).J. RAB13 PI plotted against respective GAPDH PI. The slope of the coloured lines represents the average RAB13/GAPDH PI ratio; the dashed grey line represents a 1:1 ratio (n=29 cells).
Figure 4A. Representative time-lapse microscopy of a bEnd.3 cell co-transfected with plasmids expressing Lyn-mCherry, MCP-GFPnls and 24xMS2-RAB13 3'UTR. Arrowheads indicate filopodiaB. Frequency of newly formed filopodia formed within 5 µm intervals relative to the nearest MCP-GFPnls particle or a randomised (ctrl) position (n=99 filopodia; **P<0.01, ns: not significant; Pearson r correlation).C. Distance of newly-formed filopodia to MCP-GFPnls or a ctrl position plotted against filopodia duration (n=99 filopodia; *P<0.05, ns: not significant; Spearman r correlation).D. Wt and ∆LE HUVECs co-cultured on fibroblast monolayers. Endothelial cells were identified either with an antibody against the endothelial cell marker PECAM-1 (left) or through expression of a nucleofected plasmid encoding the cytoskeletal marker Lifeact-GFP (right). Arrowheads indicate filopodiaE. Number of filopodia detected in co-cultured HUVECs (n=30 cells; **P<0.01; unpaired t test)F. Number of filopodia detected in co-cultured HUVECs within 12 µm intervals relative to cell distal tip (n=30 cells; **P<0.01, P***<0.001, ns: not significant; one-way ANOVA with Bonferroni's correction)G. Number of filopodia detected in individual clones of co-cultured HUVECs within 12 µm intervals relative to cell distal tip.
Figure 5B. Representative Puro-PLA experiments detecting newly synthesised RAB13 in HUVEC protrusions present in the lower side of Transwell membranes. Puro: puromycin; Aniso: anisomycin; 1ary Abs: primary antibodies. white arrowheads indicate Puro-PLA punctae; yellow dashed lines outline protrusion borders (B);C. Quantification of RAB13 Puro-PLA punctae normalised to protrusion area (n≥40 protrusions; *P<0.05, ****P<0.0001; Kruskal-Wallis test with Dunn's correction).D. Representative RAB13 IF assay on migrating HUVECs.E. Left: representative Western blotting (WB) of siRNA transfected HUVECs. Right: densitometry analysis of WB data (n=3 samples; *P<0.05; paired t test).F. Control (ctrl) and RAB13 siRNA-treated HUVECs co-cultured on fibroblast monolayers. Endothelial cells were identified with an antibody against the endothelial cell marker PECAM-1. black arrowheads indicate filopodia (F)G. Number of filopodia detected in co-cultured HUVECs (n≥35 cells; **P<0.01; unpaired t test).H. Number of filopodia detected in co-cultured HUVECs within 12 µm intervals relative to cell distal tip (n≥35 cells; *P<0.05, ns: not significant; Kruskal-Wallis test with Dunn's correction).
Figure 6A. Left: Tg(fli1ep:MCP-GFPnls) zebrafish embryo at 26 hours post fertilisation (hpf) displaying vascular-specific expression of MCP-GFPnls. Inset shows the nuclear expression of MCP-GFPnls in the intersomitic vessels (ISVs) sprouting from the dorsal aorta (DA). Middle: scheme depicts the in vivo MS2 system strategy with fli1 enhancer/promoter (fli1ep)-driven expression of reporter constructs, simultaneous translation of Lyn-mCherry reporter and binding of MCP-GFPnls to 24xMS2-rab13 3'UTR. Right: scheme illustrates ISV cells expressing Lyn-mCherry imaged in panels B-D. TC: tip cell; SC: stalk cell. Arrowheads indicate non-nuclear localisation of MCP-GFPnls; arrows indicate direction of ISV sprouting; yellow dashed lines outline ISV (A)(B-D) Time-lapse microscopy of Tg(fli1ep:MCP-GFPnls) tip (C) and stalk (B,D) cells displaying mosaic expression of Lyn-mCherry-24xMS2-rab13 3'UTR in ISV cells. Arrowheads indicate non-nuclear localisation of MCP-GFPnls; arrows indicate direction of ISV sprouting; yellow dashed lines outline ISV cell (B-D) borders
Figure 7B. Representative genotyping PCR demonstrates the band size shift in zebrafish harbouring a ∆482 rab13 3'UTR. Asterisk marks a heteroduplex formed between Wt and ∆482 rab13 3'UTR PCR amplicons.D. RNAseq mapped reads depicting rab13 exon usage in Tg(kdrl:EGFP) rab13+/+ and rab13∆3'UTR /∆3'UTR zebrafish embryos. Coloured lines indicate SNPs.E. qPCR analysis of rab13 mRNA levels in individual 26-28 hpf clutch-matched sibling embryos (n≥9 embryos; ns: not significant; Kruskal-Wallis test with Dunn's correction). : +/+, +/∆ and ∆/∆ represent Tg(kdrl:EGFP) rab13+/+, rab13+/∆3'UTR and rab13∆3'UTR /∆3'UTR embryos, respectivelyF. smFISH detection of rab13 and kdr mRNA in cultured GFP-expressing endothelial cells extracted from 48 hpf Tg(kdrl:EGFP) rab13+/+ and rab13∆3'UTR /∆3'UTR zebrafish embryos. Arrows indicate orientation of RNA localisation; yellow dashed lines outline cell borders; red circles highlight smFISH spots; scale bars = 10 µm (F).G. Polarisation Index (PI) of rab13 and kdr detected by smFISH in individual zebrafish cells (n≥8 cells; **P<0.01, ns: not significant; one-way ANOVA with Bonferroni's correction). : +/+, +/∆ and ∆/∆ represent Tg(kdrl:EGFP) rab13+/+, rab13+/∆3'UTR and rab13∆3'UTR /∆3'UTR embryos, respectivelyH. rab13 PI plotted against respective kdr PI. The slope of the coloured lines represents the average rab13/kdr PI ratio; the dashed grey line represents a 1:1 ratio (n≥8 cells). : +/+, +/∆ and ∆/∆ represent Tg(kdrl:EGFP) rab13+/+, rab13+/∆3'UTR and rab13∆3'UTR /∆3'UTR embryos, respectively
Figure 8A. Time-lapse confocal microscopy of representative Tg(kdrl:EGFP) rab13+/+ and rab13∆3'UTR /∆3'UTR embryos. DLAV: dorsal longitudinal anastomotic vessel; HM: horizontal myoseptum. T0= 25 hpf; arrowheads indicate extra branches emerging from the main ISVs at the HM position; scale bars = 50 µm (A).B. Frequency of ISV ectopic branching occurring at the HM (n=4 experiments; *P<0.05; one-way ANOVA with Bonferroni's correction). +/+, +/∆ and ∆/∆ represent Tg(kdrl:EGFP) rab13+/+, rab13+/∆3'UTR and rab13∆3'UTR /∆3'UTR embryos, respectively (B).
figf1(a-d) Rotenone (Rot) increased the number of GFP-LC3 puncta and the level of co-localization with mitochondria (arrows) in SH-SY5Y cells (a-c; 1 μM) and primary cortical neurons (d; 250 nM), quantified in Fig. 3f and Supplementary Fig. S1b,c. Veh, vehicle.(e,f) Rotenone increased delivery of MitoTracker Green-stained mitochondria (mt) to LysoTracker Red (LTR)-stained lysosomes, inhibited by siRNA knockdown of ATG7 or LC3 in SH-SY5Y cells. Ctrl, control. Right: RNAi knockdown.(g) Rotenone decreased IMM (COXIV), OMM (TOM40) and matrix (MnSOD) protein expression levels in primary neurons, reversed by bafilomycin (Baf) inhibition of autolysosomal degradation, quantified in Supplementary Fig. S1e. Mean ± s.d. of n = 7 independent experiments for b,c, and n = 3 independent experiments for f,g (see Supplementary Table S4 for statistics source data); *P0.05 versus vehicle control; †P0.05 versus Rot/control siRNA. Scale bars, 10 μm. See Supplementary Fig. S7 for uncropped blots.
figf2(a,b) IMM and OMM fractions isolated from primary neurons following the indicated treatments were lipid extracted for LC-MS analysis. (a) MS spectra demonstrated increased cardiolipin (CL) content of the OMM after rotenone (Rot) treatment, with diversification of the cluster distribution to the 7 clusters exhibited by the IMM. Veh, vehicle. (b) Pie charts showing the CL distribution between IMM and OMM fractions from toxin-treated neurons, normalized to mitochondrial lipid phosphorus.(c,d) Treatment with rotenone caused significant increases in PLA2-hydrolysable (surface accessible) CL assessed by LC-MS in primary cortical neurons (c; 250 nM) and SH-SY5Y cells (d; 1 μM).(e,f) Treatment with 6-OHDA and CCCP increased surface exposure of CL probed with annexin V in SH-SY5Y (e, 120 μM) and parkin-expressing HeLa cells (f, 20 μM), respectively. Mean ± s.d. of n = 3 independent experiments for c-f (see Supplementary Table S4 for statistics source data); *P0.05 versus vehicle control.
figf3(a-e) SH-SY5Y cells expressing control (Ctrl) or scramblase-3 siRNA #508 (PLS3 siRNA) for 72 h were stained with MitoTracker Green FM, a transmembrane potential-independent dye, and treated with rotenone (Rot; 1 μM). Veh, vehicle. (a) Anionic phospholipids exposed to the outer surface of isolated mitochondria were quantified by flow cytometry for Alexa 647-annexin V binding.(a-e) SH-SY5Y cells expressing control (Ctrl) or scramblase-3 siRNA #508 (PLS3 siRNA) for 72 h were stained with MitoTracker Green FM, a transmembrane potential-independent dye, and treated with rotenone (Rot; 1 μM). Veh, vehicle. (b-d) PLS3 knockdown had no effect on rotenone-induced autophagy (b), but decreased mitophagy in SH-SY5Y cells treated with rotenone (c; 1 μM) or 6-OHDA (d; 120 μM).(a-e) SH-SY5Y cells expressing control (Ctrl) or scramblase-3 siRNA #508 (PLS3 siRNA) for 72 h were stained with MitoTracker Green FM, a transmembrane potential-independent dye, and treated with rotenone (Rot; 1 μM). Veh, vehicle. (d,e) The effects of PLS3 siRNA were recapitulated using a second siRNA #433 (d, Supplementary Fig. S3b,g), and reversed by transfection with an RNAi-resistant mouse PLS3 vector (e). Inset: PLS3 overexpression.(f-i) Primary neurons transfected for 72 h with CLS siRNA or scrambled control siRNA were treated with rotenone (250 nM) and analysed for autophagy (Supplementary Fig. S1b) or mitophagy by either co-localization analysis (f)(f-i) Primary neurons transfected for 72 h with CLS siRNA or scrambled control siRNA were treated with rotenone (250 nM) and analysed for autophagy (Supplementary Fig. S1b) or mitophagy by either co-localization analysis (f), or with mitochondrial protein immunoblotting (g) and densitometry (h,i and Supplementary Fig. S4c). Knockdown efficiencies are shown in Supplementary Figs S3 and S4. Mean ± s.d. of n = 4 independent experiments for b,c, and n = 3 independent experiments for a,d-f,h-i (see Supplementary Table S4 for statistics source data); *P0.05 versus vehicle; †P0.05 versus toxin-treated control siRNA; **P0.05 versus Rot/PLS3 siRNA. See Supplementary Fig. S7 for uncropped blots.
figf4(a) Western blot analysis showed beclin 1 cleavage products (Mr49 K) were not observed in primary neurons exposed to mitophagy-inducing sublethal concentrations of rotenone (250 nM×2 h: Rot low) or STS (100 nM×2 h). However, beclin 1 was cleaved on exposure to a lethal dose of rotenone (1 mM×24 h: Rot high). Veh, vehicle; CTD, C-terminal domain.(b,c) Likewise, SH-SY5Y cells did not show beclin 1 cleavage on exposure to rotenone (b) or 6-OHDA (c; duplicate lanes). Data are representative of 2-3 independent experiments per toxin.(d) Cell lysates from stable PINK1-Flag-expressing SH-SY5Y cells were treated with 6-OHDA (120 μM), rotenone (1 μM) or FCCP (2 μM), and analysed by Flag immunoblot for PINK1-Flag levels.(e) SH-SY5Y cells transfected with HA-parkin were treated with the indicated toxins, immunolabelled for HA (green) and mitochondrial p60 antigen (red) and analysed for HA-parkin redistribution to mitochondria. FCCP elicited translocation of parkin to mitochondria (arrows), which was not seen with the other mitophagy stimuli.(f) Cytosolic and mitochondrial pellets were prepared from SH-SY5Y cells treated with vehicle (V), 6-OHDA (O, 120 μM) or rotenone (R, 1 μM). Gels were loaded with cytosolic or mitochondrial proteins, and immunoblotted for p62/SQSMT1, and the indicated fractionation markers.(g,h) Confocal analysis of p62/SQSMT1 distribution in SH-SY5Y cells (g) and in primary cortical neurons (h) that were co-transfected with mCherry-tagged p62/SQSMT1 and mitochondrially targeted GFP. Note recruitment of p62 to large mitochondrial aggregates (g, yellow, arrows) in FCCP-treated cells, which was not observed in control or rotenone-treated cells. Scale bars, 10 μm. See Supplementary Fig. S7 for uncropped blots.
figf5(a) Recombinant LC3 incubated with tetraoleoyl-CL (TOCL) or dioleoylphosphatidylcholine (DOPC) liposomes at the indicated molar ratios was analysed by blue native PAGE; liposome binding impaired the gel entry of proteins.(b) Titration of phospholipids (for example, TOCL) to LC3 was performed to evaluate the ratio that prevents 50% of the LC3 from entering the gels (IC50) as an index of relative affinity. Bottom left inset: representative gel for TOCL/LC3 = 12; top right inset: comparison of IC50 values for TOCL versus dioleoyl-phosphatidic acid (DOPA) and tetralinoleoyl-CL (TLCL) versus monolyso-trilinoleoyl-CL (lyso-CL). The IC50 for DOPG was >15. *P0.05 versus TOCL; †P0.05 versus TLCL.(f-j) The arrowhead in e shows the position of the N-terminal truncation used to create GFP-LC3 deletion mutants, which were analysed for GFP-LC3 puncta formation (f,g,i) and participation in mitophagy (h,j) in response to the indicated stimuli. Rot, rotenone; Veh, vehicle.(e,k) Dual point mutations were prepared on the basis of predicted CL contact residues (e), and analysed for participation in mitophagy elicited by Rot or 6-OHDA (k). Inset: expression of GFP-LC3 plasmids (uncropped blots in Supplementary Fig. S7). UT, untransfected. Mean ± s.d. of n = 3 independent experiments for f-h, and n = 4 independent experiments for i,j (see Supplementary Table S4 for statistics source data); *P0.05 versus respective vehicle. †P0.05 versus toxin-treated wild type (WT).
Figure 1 D. Time course of pyrene actin polymerization showing the influence of different SPIN90 constructs in reactions containing 50 nM Bos taurus Arp2/3 complex and 3 μM 15% pyrene labeled actin. E. Plot of maximum polymerization rate versus concentration of SPIN90 for reactions shown in (D)  F. Plot of maximum polymerization rate versus concentration of SPIN90 for pyrene actin polymerization assays containing 2.5 μM SPIN90, 500 nM Arp2/3 complex, 3 μM actin and the indicated concentration of the SPIN90 ARM domain construct  G. Time courses of pyrene actin polymerization as described in panel (D), except the influence of the SPIN90 middle segment is tested on its own or in the presence of the 351-722 construct
Figure 4 B. Time courses of pyrene actin polymerization showing the influence of mutations on the ability of SPIN90(269-722) to activate Arp2/3 complex. Reactions contained 3 μM 15% pyrene labeled actin, 50 nM Bos taurus Arp2/3 complex, and 20 μM wild type or mutant SPIN90(269-722). C. Plot of maximum polymerization rate versus concentration of SPIN90(269-722) for reactions as shown in panel (B)
Figure 5 A. Plot of maximum polymerization rate versus concentration of the small molecule Arp2/3 complex inhibitor CK-666. Reactions contained 3 μM 15% pyrene labeled actin, 50 nM Bos taurus Arp2/3 complex and the indicated concentrations of CK-666
Figure 6B. SDS-PAGE gel showing results of actin filament copelleting assay. Actin filaments at 3.5 μM were used to co-pellet 0.6 μM Bos taurus Arp2/3 complex in the presence or absence of the indicated concentrations of SPIN90(269-722) (S: Soluble fraction, P: Pellet) C. Plot of co-pelleting data as shown in (B), run in quadruplicate with error bars showing the standard deviation
Figure 1A) Principal component analysis (PCA) of RNA-seq data derived from 54 single embryos in 8 developmental stages. X axis shows the first (PC1) and y axis the second (PC2) principal component. The variance explained by each component is indicated in parenthesis. Morphological stages are differentially coloredB) Ratio of X-chromosomal over autosomal median transcript abundances (X/A) of preblastoderm (PB), female and male embryos in 7 developmental stages. Each data point is a single embryo. A ratio of 1 represents balanced transcript levels between the X chromosome and autosomes (dotted horizontal line).C) Log2 Fold Change (log2FC) of RNA-seq between female (F) and male (M) embryos of expressed genes on X chromosome (X, left, total=1515) and on autosomes (A, right, total=7451) in 7 developmental stages.D) Log2 Fold Change (log2FC) of RNA-seq between female (F) and male (M) embryos of X- chromosomal genes which are constitutively (left, total=303) or developmentally expressed (right, total=303) in 7 developmental stages. P-values (pval) are calculated by one-sample Wilcoxon signed rank test to assess the median log2FC values against zero (balanced expression); ns: non-significant (p-value > 0.001).
Figure 2A) Example genomic region of MSL2 MNase-ChIP-seq profiles in early (stage 5-8, orange) and late (stage 13-15, purple) mixed-sex embryos. DNA binding is defined as sub-nucleosomal fragments (10 - 130 bp), while chromatin binding as mono-nucleosomal fragments (130 - 220 bp). Called peaks/regions are represented as boxes above the tracks and HAS below. Late-appearing regions are marked by a plus (+) sign. Graph represents pooled IP/Input signal of 3 biological replicates.B) Average composite plots of sub-nucleosomal MSL2 MNase-ChIP-seq (DNA) centered at PionX (left) or HAS (right) in early and late mixed-sex embryos. Total number of PionX = 56 and HAS = 247. Graph represents average IP/Input signal of 3 biological replicates. Shaded area indicates the 95% confidence interval across sites.C) Average composite plots of mono-nucleosomal MSL2 ChIP-seq (Chromatin) centered at PionX (left) or HAS (right) in early and late mixed-sex embryos.
Figure 3B) Average composite plots of MSL2 [Chromatin] profiles (stage 5-8, orange and stage 13-15, purple) centered at MSL2 DNA peaks which do not overlap (DNA) or overlap MSL2 chromatin regions (DNA + Chromatin), and MSL2 chromatin regions which do not overlap MSL2 DNA peaks (Chromatin). Graphs represent average IP/Input signal of 3 biological replicates. Shaded area indicates the 95% confidence interval across sites.C) Average composite plots of MSL2 [Chromatin] profiles centered at MSL2 chromatin regions called in both early and late (left) or only in late stages (right). Graphs represent average IP/Input signal of 3 biological replicates. Shaded area indicates the 95% confidence interval across regions. Total number of early = 212 and late = 87.D) Average composite plots of MOF [Chromatin] profiles centered at MSL2 chromatin regions called in both early and late (left) or only in late stages (right).