EMBO/soda-nel huggingface dataset:
Phytohormone cytokinin guides microtubule dynamics during cell progression from proliferative to differentiated stage (2020)
Figure 5

Fig. 5 | Cytokinin and auxin affect distinct root growth zones.

A. Time lapse imaging of Arabidopsis root expressing the plasma membrane marker (PM) pUB10::EYFP-NPSN12 (wave line W131Y)(Geldner et al, 2009). Root growth recorded for 2 min and overlay of root tip images at time point 0 (magenta) and 2 min (cyan) used for the calculation of the root growth rate (RGR) and the cell elongation rate (CER). Scale bar 25 µm.

B. RGR of wild type (grey bars) and cre1-12 (blue bars) treated for 1 and 4 hours with mock (DMSO), CK (10 µM BAP) or auxin (0.1 µM NAA). Mean ± s.d., (* P < 0.05and **** P < 0.0001 by the Student's t-test, n= 5-8 roots per condition in 3 independent replicates).

C-E. Analysis of CK and auxin effects on CER of epidermal cells. Epidermal cells of the TZ and the EZ in the wild type and cre1-12 with PMs visualized with the YFP-NPSN12 marker. Overlay of images of epidermal cells at time 0 (magenta) and 2 min (cyan) is presented. Scale bar 10 µm (C). CER (µm/min) of root epidermal cells of the TZ (D) and the EZ (E) of the wild type (grey bars) and cre1-12 (blue bars) mutant. Hormone concentrations and conditions of treatment (D, E) were as described for (C). Mean ± s.d., (* P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 by Student's t-test, n=15-20 cells per root growth zone in 3-6 roots per condition, in 3 independent).
Phytohormone cytokinin guides microtubule dynamics during cell progression from proliferative to differentiated stage (2020)
Figure 6

Fig. 6 | Cytokinin modulates MT dynamics in animal cells.

A. MT plus-end growth rates (µm/min) in leukocytes expressing the EB3-mCherry plus-end marker before (dark grey) and after (light grey) treatment with mock (DMSO), CK (10 µM BAP or 10 µM trans-zeatin) and auxin (0.1 µM NAA). MT plus-end growth rates were monitored for 10 min before and for 10 min after each treatment. The number of MT plus-end events (EB3-mCherry) tracked per cell was 100-200/min. Mean ± s.d., (*** P < 0.001 by Student's t-test, n = 1-3 cells per condition in 4 independent replicate).

B. Kymograph representation of individual EB3-mCherry comets tracked for 30 sec in leukocytes cells, before and after treatment with mock (DMSO) or CK (10 µM BAP). Scale bar 2 µm.

C-E. Immunostaining of α-tubulin (green) in leukocytes mounted in media containing DAPI (blue, nuclear marker) and quantification of immunolabeled MTs. Cells were not or treated with 300 nM nocodazole (N) for 10 min (C). Leukocytes pretreated for 30 min with mock (DMSO), CK (10 µM BAP or 10 µM trans-zeatin), adenine (10 µM) or auxin (10 µM NAA) prior to a 10-min treatment with 300 nM nocodazol (D). Scale bar 50 µm (C, D). E. Relative fluorescence intensity of MTs in leukocytes treated with nocodazole, or nocodazole and hormones as described for (D) compared to untreated (black bar) cells. Mean ± s.d., (P < 0.05 by two-way Anova, n = 18-33 cells per condition in 3 independent replicates).
COCO/DAND5 inhibits developmental and pathological ocular angiogenesis (2020)
Figure 1

Figure 1. COCO inhibits sprouting angiogenesis. (A,B) Representative images of HUVECs (A; n=6) and HRMECs (B; n=4) sprouting in a fibrin gel with VEGF (25ng/ml) in the presence or absence of COCO. Scale bar, 75 μm. (C) Representative images of choroidal explants cultured for 5 days in the presence or absence of COCO. Scale bar, 500 μm. (D,E) Quantification of tube surface area of micrographs shown in (A,B). Results are presented as mean ± sem, statistical analyses were performed using Mann-Whitney test. (D: *P=0.0119 (- vs COCO 30ng/ml); *P=0.0108 (- vs COCO 60ng/ml); (E: **P=0.0013 (- vs COCO 30ng/ml); ***P=0.00026 (- vs COCO 60ng/ml)). (F) Quantification of sprouting surface area of micrographs shown in (C; n=6). Results are presented as mean ± sem, statistical analyses were performed using Mann-Whitney test. *P=0.0107. (G) COCO decreases scratch wound migration after 18h. Scale bar, 150 μm. (H) Quantification of wound closure. Results are presented as mean ± sem, statistical analyses were performed using Mann-Whitney test. **P=0.0010; (n=6). (I) COCO decreases EdU incorporation in cultured HUVECs. (J) Quantification of EdU incorporation. Results are presented as mean ± sem, statistical analyses were performed using Mann-Whitney test. *P=0.0317; (n=5). (K) COCO does not induced apoptosis in HUVECs. (L) Quantification of apoptotic (Annexin V-positive) and viable (Annexin V-negative) cells (n= 4).
COCO/DAND5 inhibits developmental and pathological ocular angiogenesis (2020)
Figure 2

Figure 2. COCO inhibits developmental retinal angiogenesis. (A) Schematic of the experimental strategy to assess early formation of the retinal vasculature (P1-P5). (B) Retinal flat mounts of P5 mice injected with PBS, COCO or Flt1Fc are stained with IB4 (negative images of the fluorescent signal). Scale bar, 100 μm. (C) Quantification of vascular length and number of branchpoints (p-values are versus PBS treatment). Vessel length: **P=0.0059 (PBS vs COCO); *P=0.0169 (PBS vs Flt1Fc). Vessel junctions: **P=0.0063 (PBS vs COCO); *P=0.0159 (PBS vs Flt1Fc); (n=4 mice/group). (D) COCO does not increase empty collagen IV sleeves. Scale bar, 50 μm. (E) Quantification of % of IsoB4+ vessels to Coll IV+ vessels; (n=9). (F) Visualization of pericyte coverage (NG2:green; IsoB4:red) in PBS- or COCO-injected retinas. Scale bar, 50 μm. (G) Quantification of percentage of IsoB4 vascular staining covered by NG2 staining (% overlay) (n=8). (H) COCO injections (P1) do not result in apoptosis in P5 retinas. Scale bar, 40 μm. (I) Quantification of apoptotic cells in control and COCO-injected mice (n=3). Results are presented as mean ± sem and statistical significance was analyzed by Mann-Whitney test. *P<0.05, **P<0.01.
COCO/DAND5 inhibits developmental and pathological ocular angiogenesis (2020)
Figure 3

Figure 3. Chronic injections of COCO decrease blood vessel development but do not result in retinal apoptosis. (A) Schematic of the experimental strategy to assess early formation of the retinal vasculature (P1-P28). (B) Retinal flat mounts of P28 mice injected with PBS, COCO or Flt1Fc are stained with IB4 (negative images of the fluorescent signal). Scale bar, 100 μm. (C) Quantification of vascular length and number of branchpoints (p-values are versus PBS treatment). Vessel length: **P=0.0019 (PBS vs COCO); *P=0.0128 (PBS vs Flt1Fc). Vessel junctions: **P=0.0073 (PBS vs COCO); *P=0.0302 (PBS vs Flt1Fc); (n=4). (D) COCO injections (P1, P7 and P14) do not result in ONL thinning in P28 retinas. Scale bar, 40 μm. (E) Quantification of apoptotic cells (cleaved caspase-3; CC3) in control and COCO-injected mice (p-values are versus PBS treatment). (n=4). Results are presented as mean ± sem and statistical significance was analyzed by Mann-Whitney test. *P<0.05, **P<0.01, ***P<0.001.
COCO/DAND5 inhibits developmental and pathological ocular angiogenesis (2020)
Figure 4

Figure 4. COCO injections do not induce vascular regression in adult mice. (A) Schematic of the experimental strategy to assess injections of COCO in the adult retinal vasculature. (B) Retinal flat mounts of adult mice injected with PBS or COCO for 5 days are stained with IB4 (negative images of the fluorescent signal). Scale bar, 100 μm. (C) Quantification of vascular length and number of branchpoints; (n=4). (D) Schematic of the experimental strategy to assess repeated injections of COCO in the adult retinal vasculature. (E) Retinal flat mounts of adult mice injected with PBS or COCO for 28 days are stained with IB4. Scale bar, 90 μm. (F) Quantification of vascular length and number of branchpoints; (n=4). Results are presented as mean ± sem and statistical significance was analyzed by Mann-Whitney test.
COCO/DAND5 inhibits developmental and pathological ocular angiogenesis (2020)
Figure 5

Figure 5. COCO inhibits retinal and choroidal pathological angiogenesis. (A) Schematic of the experimental strategy to assess the effects of COCO in the Oxygen-induced Retinopathy model. (B) Retinal flat mounts of P17 OIR mice injected with PBS, COCO or Flt1Fc are stained with IB4. Red areas highlight vascular tufts. Scale bar, 500 μm. (C) Quantification of neovascular tuft area in P17 OIR. **P=0.0021 (PBS vs COCO); *P=0.0122 (PBS vs Flt1Fc); (n=6). (D) Quantification of central avascular area in P17 OIR; (n=6). (E) Schematic of the experimental strategy to assess the effects of COCO in the Laser-induced Choroidal Neovascularization (CNV) model. (F) Choroidal flat mounts of adult mice subjected two weeks prior to Laser-CNV injected with PBS, COCO or Flt1Fc stained with IB4 (green) and phalloidin (red). Scale bar, 100 μm. (G) Quantification of CNV surface area. *P=0.0210 (PBS vs COCO); *P=0.0113(PBS vs Flt1Fc); (n=12-14 burns from 5 animals/group). Results are presented as mean ± sem and statistical significance was analyzed by Mann-Whitney test. *P<0.05, **P<0.01.
COCO/DAND5 inhibits developmental and pathological ocular angiogenesis (2020)
Figure 6

Figure 6. COCO does not impair VEGF signaling in cultured endothelial cells. (A) Western blot analysis of HUVECs treated 24 hours prior with COCO and stimulated with VEGF for up to 60 min. Immunoblots are representative of 4 independent experiments. (B) Densitometric analysis of western blot experiments. * P=0.0457; (n=4). (C) COCO treatment does not alter VEGF receptor expression in HUVECs. Immunoblots are representative of 3 independent experiments. (D) Densitometric analysis of western blot experiments; (n=3). Results are presented as mean ± sem and statistical significance was analyzed by Two-Way Anova. *P<0.05.
COCO/DAND5 inhibits developmental and pathological ocular angiogenesis (2020)
Figure 7

Figure 7. COCO is expressed in human and mouse retina and localizes to mitochondria in COCO-exposed HUVECs. (A) Western blot of whole adult human retina extracts incubated with an anti-human COCO antibody and revealing a unique band at ~36 kDa (arrow). Recombinant human COCO was used as positive control. (B) Western blot of photoreceptors produced from human embryonic stem cells incubated with an anti-mouse COCO antibody and revealing a unique band at ~36-38 kDa (arrow). Western blots are representative of 3 independent experiments. (C) Immunofluoresence analysis of adult human retina sections with an anti-mouse COCO antibody. Specific immuno-reactivity was observed in multiple areas (arrows) when compared with sections only exposed to the secondary antibody. Scale bar, 40 μm. (D) RNAscope in situ hybridization (Dand5, top; Negative probe (dapB); down) and hematoxylin staining of P5 mouse retinas. Scale bar, 40 μm. (E) Dual RNAscope in situ hybridization and visual-arrestin immunohistochemistry of P5 mouse retinas. Scale bar, 40 μm. Images are representative of 4 animals. (F) Immunofluoresence analysis of HUVECs exposed to COCO for 5 hrs prior to fixation. Exogenously added COCO was detected using an anti-human COCO antibody, showing co-localization with human mitochondria (MTCO1 antibody). Scale bar 25 μm.
COCO/DAND5 inhibits developmental and pathological ocular angiogenesis (2020)
Figure 8

Figure 8. Transcriptional changes associated with COCO stimulation in ECs. (A) Scheme of the protocol to determine differential gene expression after treatment of HUVECs 24h with COCO. (B) Differential gene expression heatmap generated by DESeq2 of top-altered genes of HUVECs treated with COCO compared to PBS-treated control cells (n = 4 independent samples/group). (C) GSEA pathway analysis of control vs COCO-treated HUVECs for TGFβ, BMP or ROS pathways.
COCO/DAND5 inhibits developmental and pathological ocular angiogenesis (2020)
Figure 9

Figure 9. COCO alters the energy metabolism of endothelial cells. (A) Seahorse analysis of mitochondrial respiration (OCR) and glycolysis (ECAR) in HUVECs stimulated with COCO for 1, 2 and 24 hours. **P=0.0032 for ECAR, 1hour; **P=0.0079 for ECAR, 2hours; *P=0.0255 for OCR, 2hours; (n=5). (B) Media glucose levels (BioNova analysis) in HUVECs cultured with COCO for 1 or 6 hours, expressed as % of initial levels *P=0.0459; (n=4). (C) Evaluation of ATP content in HUVECs stimulated with COCO for 1, 6 or 24 hours. *P=0.0120 (- vs 1hour); *P=0.0109 (- vs 6hours); *P=0.0219 (- vs 2-DG); (n=4). (D) Determination of NAD+/NADH ratio in HUVECs for 6 or 24 hours with COCO. **P=0.0028 (6hours); *P=0.0207 (24hours); (n=3). (E) Evaluation of pyruvate content in HUVECs stimulated with COCO for 1, 3, 6, 12 or 24 hours. *P=0.0244; (n=4). (F) Determination of cellular ROS using DCFDA fluorescence in HUVECs treated with COCO and/or N-acetylcystein. *P=0.0343 (- vs 48hours-NAC); *P=0.0114 (- vs H2O2-NAC); *P=0.0252 (48hours-NAC vs 48hours+NAC); **P=0.0067 (H2O2-NAC vs H2O2+NAC); (n=4). (G) Determination of mitochondrial superoxide using MitoSox fluorescence in HUVECs treated with COCO. *P=0.0372 (- vs 24hours); *P=0.0137 (- vs 3-NP); (n=3). (H) Representative images of choroidal explants cultured for 4 days in the presence or absence of COCO and/or N-acetylcystein. Scale bar, 500 μm. (I) Quantification of sprouting surface area of micrographs shown in (H). *P=0.0126 (- vs COCO-NAC); *P=0.0200 (COCO-NAC vs COCO+NAC); (n=6). Results are presented as mean ± sem and statistical significance was analyzed by Mann-Whitney test. *P<0.05, **P<0.01.
A universal SARS-CoV DNA vaccine inducing highly crossraective neutralizing antibodies and T cells (2022)
Figure 1

Figure 1. Immunogenicity of the universal SARS-CoV-2 vaccine

A) Schematic representation of the universal SARS-CoV-2 DNA vaccine OC-2.4 gene design has been given.

B-E) Ability of the SARS-CoV-2 DNA vaccine OC-2.4 to induce antibodies against S protein of the huCoV-19/WH01 (B) and Beta variants (D), and the ability to neutralize these viruses in vitro (C and E).

F,G) Also shown is the ability of the universal SARS-CoV-2 DNA vaccine OC-2.4 to prime and boost antibodies to S protein (huCoV-19/WH01) following priming with a recombinant S protein in adjuvant (F) and the ability of these antibodies to neutralize the Delta and Omicron variants in vitro (G).

H,I) Finally, three doses of the universal SARS-CoV-2 DNA vaccine OC-2.4 induce antibodies that cross react with S proteins from the huCoV-19/WH01, Beta, and Delta variants in mice (H), and four 840 µg doses of the same vaccine induces antibodies in female Zeeland White rabbits that neutralize both the Delta and Omicron variants in vitro (I).

Data information: All samples were run individually in serial six-fold dilutions dilutions from 1:60 to 1:466560 with groups of five to 10 mice (B, E, F, and H). In the NT assay all serum samples from a group were pooled and serial two-fold dilutions from 1:16 to to 4096 were run in quadruplicate (C, E and G). In I) individual rabbit sera were run as serial two-fold dilutions from 1:16 to to 4096 in quadruplicate.
A universal SARS-CoV DNA vaccine inducing highly crossraective neutralizing antibodies and T cells (2022)
Figure 2

Figure 2. The universal SARS-CoV-2 vaccine induces broadly crossreactive T cell responses

A-D) T cell responses as determined by ELISpot in Balb/c mice immunized three times with either spike protein in QS21 adjuvant (rS/QS21; A), 50µg universal SARS-CoV-2 vaccine OC-2.4 (B), primed with rS/QS21 and boosted with two doses of OC-2.4 (C), or empty pVAX plasmid (D), using in vivo electroporation. Spleens were harvested and individual mice were analyzed for the presence of IFNγ producing T cells using the indicated antigens. Data has been given as the number of IFNγ producing (spot forming) cells (SFCs) per million splenocytes. Each color indicate an individual mice.

E-H) Also shown is analysis of T cell responses in New Zeeland White rabbits, which were immunized four times with either vehicle only (E), or 84µg (F) or 840 µg (G) universal SARS-CoV-2 DNA vaccine OC-2.4 using in vivo electroporation. Peripheral blood mononuclear cells (PBMCs) were harvested and analysed for presence of IFNγ producing T cells using the indicated antigens. Data has been given as the number of IFNγ SFCs per million PBMCs. The dotted line indicates the 50 SFC cut off. Also shown (H) is the cumulated IFNγ SFCs per million PBMCs to the RBD, M and N peptide pools added together for each group of immunized rabbits (84µg DNA, 840µg DNA, and vehicle only) at each time point (day). The numbers on the x-axis of graphs A) to D) indicates the number of the respective peptide pool (WH1 variant) covering a part of the indicated protein.

Data information: All samples from mice and rabbits were run individually in triplicate and the mean number of SFCs/million have been given (A to G). In H) the cumulated SFCs from all positive peptide pools from immunized rabbits have been given. The dotted line indicated the 50 SFCs/million cut off.
Transcriptionally active enhancers in human cancer cells (2020)
Figure 3

Figure 3: Transcription at functionally verified enhancers.

(A) UCSC genome browser view of normalized TT-seq coverage on the plus and minus strand at the TAL1 locus (hg38; chr1:47,194,268-47,242,880; (Kent et al., 2002)) in the leukemia cell lines Jurkat and K562. For better visualization, TT-seq coverage is cut at 200 (purple lines). eRNAs are shown between the plus and minus strand. Red line indicates the short 12 bp insertion in Jurkat cells, introducing novel binding sites for the transcription factor MYB (Mansour et al., 2014).

(B) UCSC genome browser view of normalized TT-seq coverage on the plus and minus strand at the 2.8 Mbp gene desert surrounding MYC (hg38; chr8:127,042,406-129,779,109; (Kent et al., 2002)) in fourteen cancer cell lines. eRNAs represent the union from all cell lines. Genome-wide association studies (GWAS) risk loci contain GWAS single nucleotide polymorphisms (SNPs) for prostate cancer (PrCa), acute lymphocytic leukemia (ALL), breast cancer (BrCa), colorectal cancer (CRC), and glioblastoma (GBM) including SNPs in linkage disequilibrium (r2 ≥ 0.8). TT-seq coverage is displayed between 8-250 to allow for better visualization of the eRNA signal surrounding MYC. Dashed rectangle is shown at full scale on the right. Note: TT-seq reads were mapped against the hg38 reference genome and we cannot rule out the existence of genomic rearrangements (e.g. translocations) affecting the shown region in the used cancer cell line clones.
Transcriptionally active enhancers in human cancer cells (2020)
Figure 4

Figure 4: eRNA synthesis at super-enhancers and in large regulatory regions.

(A) Med1-defined enhancers ranked by Med1 ChIP-seq signal (Yan et al., 2013) in LoVo colorectal cancer cells. Super-enhancers (black rectangles) were determined as enhancer regions lying above the inflection point of the curve (Materials and Methods).

(B) Increased eRNA synthesis from super-enhancers. Boxplots comparing eRNA synthesis (TT-seq signal) at Med1-defined typical enhancers (n=2,111) and super-enhancers (n=130) in LoVo cells (analysis was restricted to intergenic enhancers; Materials and Methods). Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Notches represent 95% confidence intervals for the median values. Outliers not shown. P-value by two-sided Mann-Whitney U-test.

(C) Super-enhancers are associated with the production of long eRNAs. Boxplots showing length of TT-seq-defined eRNAs originating from Med1-defined super-enhancers (n=552) or other enhancer regions (n=6,934). Median, hinges, whiskers and notches are shown as in (B). P-value by two-sided Mann-Whitney U-test.

(D) Barplot showing the number of TT-seq enhancer regions per Med1-defined super-enhancer region in LoVo cells. Super-enhancers spanning a protein-coding TSS (n=32) are excluded from this comparison as regions TSS ± 1 kbp were excluded from TT-seq enhancer calling.

(E) UCSC genome browser view of normalized TT-seq coverage on the plus and minus strand at the PHLDA1 locus (hg38; chr12:75,612,350-76,042,552; (Kent et al., 2002)) in the colorectal cancer cell line HCT 116. TT-seq coverage is cut at 250 (purple lines) to allow for better visualization. eRNAs are shown between the plus and minus strand. Aggregated transcription factor ChIP-seq peak regions (TF ChIP-seq) comprise ChIP-seq binding profiles of 19 TFs in HCT 116 cells from CISTROME (Zheng et al., 2019). The height of the signal indicates the number of different TFs binding at a particular region. Binding regions for selected TFs (TCF7l2, YY1, SP1) are shown below together with p300 and RNA polymerase II (Pol II). TT-seq enhancer regions are shown at the bottom together with a H3K27ac-defined super-enhancer annotation, which was downloaded from SEdb (Jiang et al., 2019). At date of publication no Med1 ChIP-seq data was available for HCT 116.
Transcriptionally active enhancers in human cancer cells (2020)
Figure 7

Figure 7: Putative enhancers regulating the oncogene BMP4.

UCSC genome browser view of normalized TT-seq coverage on the plus and minus strand at the BMP4 locus (hg38; chr14:53,935,697-54,264,430; (Kent et al., 2002)) in LoVo colorectal cancer cells. TT-seq coverage is cut at 500 (purple lines) to allow for better visualization. eRNAs and enhancers are shown above and below the minus strand, respectively. Aggregated transcription factor ChIP-seq peak regions (TF ChIP-seq) include ChIP-seq binding profiles of 326 TFs in LoVo cells (Yan et al., 2013). The height of the signal indicates the number of different TFs binding at a particular region. Significant promoter capture Hi-C (CHi-C) interactions are shown in purple (Orlando et al., 2018). See Source Data for Fig 7 for original view of CHi-C interactions from WashU epigenome browser (Li et al., 2019).

EXPANDED VIEW FIGURE LEGENDS
H3K9 methylation extends across natural boundaries of heterochromatin in the absence of an HP1 protein. (2015)
Figure 1

Swi6 is highly mobile

Schematic of single‐plane acquisition FRAP on endogenously tagged Swi6‐EGFP. Representative still images of a FRAP experiment are shown. See also Movie EV1. Green sphere: yeast nucleus containing Swi6‐EGFP; green dots: heterochromatic Swi6‐EGFP; red circle: bleached area.

Normalized average intensities (black dots) of FRAP on Swi6‐EGFP heterochromatic loci (n = 31) fitted to a one‐component model (blue line). The dashed lines indicate the final relative intensity that is set to 1 and the fluorescence half‐recovery time (t
1/2).

Fluorescence t
1/2 values of FRAP on Swi6‐EGFP heterochromatic loci. The box bounds the interquartile range (IQR) divided by the median, and whiskers extend to a maximum of 1.5 × IQR beyond the box.

Schematic indicating the volumes measured to calculate the normalized fluorescence recovery of a locus relative to the entire nucleus.

Normalized fluorescence recovery values of FRAP on Swi6‐EGFP heterochromatic loci. The box bounds the interquartile range (IQR) divided by the median, and whiskers extend to a maximum of 1.5 × IQR beyond the box.

FLIP experiment showing a bleached (FRAP, red circle) and unbleached, adjacent locus (FLIP, blue circle) in the same nucleus. The graph displays average relative fluorescence of FLIP (blue), FRAP (red), and control loci (no bleach, green) in other non‐bleached cells.

Schematic of a line‐scan FRAP experiment. Fluorescence intensities are measured along a line of a non‐bleached nucleus (Ctrl nucleus) and a bleached nucleus (FRAP nucleus) in Swi6‐EGFP
clr4∆ cells. Red box: bleached region. Right: raw data example.

Average relative intensities of a line‐scan FRAP experiment on Swi6‐EGFP in heterochromatin (blue line), euchromatin (green line), and clr4∆ (red line). The curves have a gliding average of 40.