EMBO/SourceData huggingface dataset:
Figure 2Western blots of BHLHE40 expression across tissues (A), BHLHE40 expression in MIN6 cells cultured under 20% or 5% O2 for the indicated time (B) and the quantification of B (n = 3 biological replicates) Data information: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or β-actin was used as a loading control. HPT, hypothalamusD-F. The effect of oxidative stress, endoplasmic reticulum stress, and energy stress on BHLHE40 expression. qRT-PCR analysis of Bhlhe40 in MIN6 cells incubated with 10μM H2O2 (n = 3 biological replicates; D), 2μM thapsigargin (Thap) or 5 μg/ml tunicamycin (Tun) (n = 7 biological replicates; E), or 2mM metformin (Met) for 24 hours (n = 3 biological replicates; F). Data information: Data are mean ± SEM; *p < 0.05, **p < 0.01, and ***p < 0.001 by unpaired two-tailed Student's t test.H-K. BHLHE40 expression in islets from diabetic mice. BHLHE40 expression was analyzed in ob/ob mouse islets by qRT-PCR (n = 4 biological replicates; H) and Western blotting (I) and in db/db mouse islets by Western blotting (J). Subcellular localization of BHLHE40 in ob/ob mice islets by immunohistochemical analysis (K). Data information: Data are mean ± SEM; *p < 0.05, **p < 0.01, and ***p < 0.001 by unpaired two-tailed Student's t test. β-actin was used as a loading control. Scale bar, 100 μm.
Figure 3A. Glucose-stimulated insulin secretion in MIN6 cells expressing short hairpin RNA against a non-targeting Ctrl or Bhlhe40 knockdown (B40 KD) were cultured under 20% or 5% O2 for 24 hours (n = 8 biological replicates). B. Glucose-stimulated insulin secretion in MIN6 cells infected with retroviruses generated with pMx-Ctrl (Ctrl) or pMx-Bhlhe40 (B40 OE; n = 4 biological replicates). Data information: Data are mean ± SEM; *p < 0.05, **p < 0.01, and ***p < 0.001 by unpaired two-tailed Student's t test. Ctrl, control; n.s., not significant.D. KCl-stimulated insulin secretion in Ctrl and B40 OE MIN6 cells (n = 3 biological replicates). Data information: Data are mean ± SEM; *p < 0.05, **p < 0.01, and ***p < 0.001 by unpaired two-tailed Student's t test. Ctrl, control; n.s., not significant.G. Cellular ATP content in Ctrl and B40 KD MIN6 cells cultured under 20% or 5% O2 for 24 hours (n = 3 biological replicates). Data information: Data are mean ± SEM; *p < 0.05, **p < 0.01, and ***p < 0.001 by unpaired two-tailed Student's t test. Ctrl, control; n.s., not significant.I-K. Mitochondrial DNA (mtDNA) content (n = 4 biological replicates; I), mitochondrial mass (n = 4 biological replicates; J), and mitochondrial membrane potential (n = 4 biological replicates; K) in Ctrl and B40 KD MIN6 cells cultured under 20% or 5% O2 for 24 hours. Data information: Data are mean ± SEM; *p < 0.05, **p < 0.01, and ***p < 0.001 by unpaired two-tailed Student's t test. Ctrl, control; n.s., not significant.
Figure 4B, Expression of DEGs shown in A was confirmed by qRT-PCR in Ctrl and B40 OE MIN6 cells (n = 3 biological replicates; B) Data information: Data are mean ± SEM; *p < 0.05, **p < 0.01 and ***p < 0.001 by unpaired two-tailed Student's t test.D. qRT-PCR of MAFA target genes in Ctrl and B40 KD MIN6 cells cultured under 20% or 5% O2 for 24 hours (n = 3 biological replicates). Data information: Data are mean ± SEM; *p < 0.05, **p < 0.01 and ***p < 0.001 by unpaired two-tailed Student's t test.G. Western blot of oxidative phosphorylation complex proteins and TOM20 protein in Ctrl and B40 KD MIN6 cells cultured under 20% or 5% O2 for 24 hours, and quantification of the band intensities (n = 3 biological replicates). Data information: Data are mean ± SEM; *p < 0.05, **p < 0.01 and ***p < 0.001 by unpaired two-tailed Student's t test. β-actin was used as a loading control. Ctrl, control.
Figure 5C. Luciferase reporter assay was performed with MIN6 cells transfected with BHLHE40 expression plasmids, pRL-SV40 plasmid, and pGL3-Mafa plasmids (wildtype [black] and E-box mutated [red] sites; n = 4 biological replicates). D. Luciferase reporter activity in MIN6 cells cultured under 20% or 5% O2 for 24 hours (n = 3 biological replicates). Data information: Data are mean ± SEM; *p < 0.05 **p < 0.01, and ***p < 0.001 by unpaired two-tailed Student's t test. Ctrl, control; n.s., not significant.H-J. Proteins sampled from Ctrl and B40 OE MIN6 cells with FLAG-Pdx1 expression (H) or from MIN6 cells with FLAG-Pdx1 expression pre-cultured under 20% or 5% O2 for 24 hours (I) were immuno-precipitated by IgG or anti-FLAG antibody, after which qRT-PCR was performed for the indicated regions (n = 9 biological replicates for H and n = 6 biological replicates for I). FLAG (PDX1) expression in I was confirmed by Western blotting (J). Data information: Data are mean ± SEM; *p < 0.05 **p < 0.01, and ***p < 0.001 by unpaired two-tailed Student's t test. Ctrl, control; n.s., not significant.
Figure 6C, D. Glucose tolerance test of Ctrl:ob/ob and βB40KO:ob/ob mice (n = 9 mice and n = 8 mice, respectively; 6 weeks old) (C) and AUC (D). Data information: Data are mean ± SEM; *p < 0.05 **p < 0.01, and ***p < 0.001 by unpaired two-tailed Student's t test.E. Glucose-stimulated insulin secretion in Ctrl:ob/ob and βB40KO:ob/ob mice (n = 9 mice and n = 6 mice, respectively; 8 weeks old). F. Glucose-stimulated insulin secretion in isolated islets from Ctrl and βB40KO mice after culture under 5% O2 for 24 hours (n = 8 biological replicates). Data information: Data are mean ± SEM; *p < 0.05 **p < 0.01, and ***p < 0.001 by unpaired two-tailed Student's t test.I, J. Representative images of pancreatic islets stained for insulin and glucagon in Ctrl:ob/ob and βB40KO:ob/ob mice (12 weeks old) (I). The ratios of total islet area to whole pancreas area (%) are shown (n = 3 biological replicates; J). Data information: Data are mean ± SEM; *p < 0.05 **p < 0.01, and ***p < 0.001 by unpaired two-tailed Student's t test. Scale bar, 10 μm.K, L. Representative images of pancreatic islets stained for insulin, MAFA, and BHLHE40 in Ctrl:ob/ob and βB40KO:ob/ob mice (12 weeks old) (K). Fluorescence intensities of nuclear and cytosolic MAFA in K were quantified (n = 30 cells; L). Data information: Data are mean ± SEM; *p < 0.05 **p < 0.01, and ***p < 0.001 by unpaired two-tailed Student's t test. Scale bar, 10 μm.M. qRT-PCR of Mafa and its target genes in Ctrl:ob/ob and βB40KO:ob/ob mice (n = 4 mice). Data information: Data are mean ± SEM; *p < 0.05 **p < 0.01, and ***p < 0.001 by unpaired two-tailed Student's t test.
Figure 1A-C Volcano plots depicting significantly up (blue) and down (pink) regulated phosphorylation events for DMXAA vs DMSO groups for 5 min (A), 10 min (B) and 30 min (C) timepoints. -Log10(p-value) is plotted on the y-axis vs Log2(Fold change [FC]) on the x-axis. Significantly regulated events display p-value>0.01 and Log2(FC) >1 or<-1.
Figure 2A Sting-/- iBMDMs expressing mRuby3-STING (magenta) and eGFP-Golgin84 (green) were imaged live on the 3i marianas spinning disk microscope. Z stack images were acquired either before (i.e., untreated) or after 50 μg/mL DMXAA treatment (i.e., image captured at 12 min post DMXAA treatment). Data is shown as a maximum intensity projection (MIP) of Z stack images.D Sting-/- iBMDMs expressing mRuby3-STING were imaged live on the spinning disk microscope. Representative images of timelapse showing STING vesicles exiting the Golgi for a recording starting 27 min after 50 μg/mL DMXAA treatment. Data is shown as a MIP of Z stack images. Square region of interest (ROI) indicates zoomed insert. Scale bar = 5 μm.F Sting-/- iBMDMs expressing eGFP-STING were imaged live on the spinning disk microscope. A representative image of timelapse showing transport of STING vesicles for a recording starting 1 h 18 min after 50 μg/mL DMXAA treatment. Data is shown as a MIP of Z stack images. Square ROI indicates zoomed insert. Scale bar = 5 μm.
Figure 3A Primary BMDMs were left untreated or treated with 25 μg/mL DMXAA for 2, 4 or 6 h. Cells were lysed for immunoblot with the indicated antibodies. Data shown is representative of 3 independent experiments. B Primary BMDMs were left untreated or treated with 10 μg/mL 2'3'-cGAM(PS)2 for 2, 4 or 6 h. Cells were lysed for immunoblot with the indicated antibodies. Data shown is representative of 3 independent experiments.D Sting-/-, WT or TBK1/IKKε dKO iBMDMs were left untreated or treated with 10 μg/mL 2'3'-cGAM(PS)2 for 1, 2, or 3 h. Cells were lysed for immunoblot with the indicated antibodies. Data shown representative of 3 independent experiments.
Figure 4B Primary BMDMs were treated with 2 μM TAK243 or DMSO vehicle control for 30 min before being either left untreated or treated with 25 μg/mL DMXAA for 1, 2 or 4 h. Cells were lysed for immunoblot with the indicated antibodies. Data shown is representative of 3 independent experiments. C Primary BMDMs were treated with 2 μM TAK243 or DMSO vehicle control for 30 min before being either left untreated or treated with 10 μg/mL 2'3'-cGAM(PS)2 for 4 h. Cells were lysed for immunoblot with the indicated antibodies. Data shown is representative of 3 independent experiments.D Primary BMDMs were treated with 2 μM TAK243 or DMSO vehicle control for 30 min before being either left untreated (UT) or treated with 25 μg/mL DMXAA for 1, 2 or 4 h. Cell were fixed and stained for p-STING (white) and the nucleus (DAPI; blue), before Z stack images were acquired on the LSM980 confocal microscope. Images are displayed as a maximum intensity projection (MIP) of Z stack images. Scale bar = 5 μm. Data shown is representative of 3 independent experiments.G-I Primary BMDMs were treated with 2 μM TAK243 or DMSO vehicle control for 30 min before being either left untreated (UT) or treated with 25 μg/mL DMXAA or 10 μg/mL 2'3'-cGAM(PS)2 for 4 h. Cells were lysed for RNA purification and the expression of Ifnb1 (G) , Isg15 (H) and Il6 (I) was analysed by qPCR. Data is shown as mean ± SEM combined from N=3 independent experiments. Statistical analysis was performed using two-way ANOVA using Bonferroni's multiple comparisons test, where * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.
Figure 5B Sting-/- iBMDMs expressing HA-STING were left UT or treated with 50 μg/mL DMXAA for 1 or 3 h. Cells were lysed and a portion of lysate underwent immunoblot with the indicated antibodies (i.e., lysate). The remaining lysate underwent immunoprecipitation with an anti-HA antibody (i.e., HA pull down). Samples then underwent immunoblot with the indicated antibodies. Data representative of 3 independent experiments.D Square ROI indicates zoomed insert from C, displayed as both merged and single colour images. Scale bar = 5 μm.
Figure 6A dCas9-KRAB expressing iBMDMs, without (i.e., dCas9 alone) or with HRS-targeting sgRNAs (i.e., sg1, sg2 or sg3) were treated for 48 h with 1 μg/mL doxycycline (Dox) to induce sgRNA expression. Cells were then left untreated (UT), or treated with 20 μg/mL DMXAA or 10 μg/mL 2'3'-cGAM(PS)2 for 6 h. Cells were lysed for immunoblot with the indicated antibodies. Data shown is representative of 3 independent experiments.B-C dCas9-KRAB expressing iBMDMs, without (i.e., dCas9 alone) or with HRS-targeting sgRNAs (i.e., sg1, sg2 or sg3) were treated for 48 h with 1 μg/mL Dox to induce sgRNA expression. Cells were then left UT, or treated with 20 μg/mL DMXAA or 5 μg/mL 2'3'-cGAM(PS)2 for 6 h. Cell supernatant was collected and assayed for secreted IFNβ (B) and IL6 (C) by ELISA. Data is shown as mean ± SEM combined from N=3 independent experiments. Statistical analysis was performed using two-way ANOVA using Bonferroni's test, where * p<0.05, *** p<0.001, **** p<0.0001. n.d. = not detectedD-F dCas9-KRAB expressing iBMDMs, without (i.e., dCas9 alone) or with HRS-targeting sgRNAs (i.e., sg1, sg2 or sg3) were treated for 24 h with 1 μg/mL Dox to induce sgRNA expression. iBMDMs were further treated with 1 μg/mL (i.e. 1000 ng/mL) or 100 ng/mL H-151 as indicated, and incubated for a further 24 h. Cells were lysed for RNA purification and the expression of Ifnb1 (D), Isg15 (E) and Irf7 (F) was analysed by qPCR. Data is shown as mean ± SEM combined from N=3 independent experiments. Statistical analysis was performed using two-way ANOVA using Tukey's multiple comparisons test, where * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Bars display significant differences between dCas9 and sgRNA knockdowns in the absence of H-151. Additional asterisks represent significant differences between the absence or presence of H-151 across the same cell line.
Figure 7A-B VPS4aWT or VPS4aE228Q dominant negative BMDMs were left untreated (UT) or treated with 1 μg/mL doxycycline (Dox) for 4 h. BMDMs were then further left UT or treated with 25 μg/mL DMXAA (A) or 10 μg/mL 2'3'-cGAM(PS)2 (B) for 4 h. Cells were lysed for immunoblot with the indicated antibodies. Data shown is representative of 2 independent experiments for A and 3 independent experiments for B.C-D VPS4aWT or VPS4aE228Q dominant negative BMDMs were left UT or treated with 1 μg/mL Dox for 4 h. BMDMs were then further left UT or treated with 25 μg/mL DMXAA for 4 h. Cell supernatant was collected and secreted IFNβ (C) or IL-6 (D) was measured by ELISA. Data is shown as mean ± SEM combined from N=3 independent experiments. Statistical analysis was performed using one-way ANOVA. using Bonferroni's test, where * p<0.01. n.d. = not detected
Figure 1C PICT assay for the interactions between indicated pairs of proteins tagged to FRB and GFP. Representative images before and after adding rapamycin (RAP) of RFP-tagged anchor and GFP-tagged prey are shown in the upper and middle row, respectively. Bottom row shows the merged images. White zoom in boxes, 0.9µm. Scale bar, 5µm.
Figure 2A Ape1 processing was analyzed by western blot in the indicated strains and temperatures and normalized to the processing achieved in wild-type cells ± SD, n = 3.D Representative EM images of pep4Δ and pep4Δdrs2Δ strains. Black squares show a zoom-in in the vacuole. White arrowheads point to Cvt bodies.E Representative CLEM images of drs2Δ cells. Ape1-GFP (top) correlates with a membrane-free ribosome exclusion area (middle). Black square (bottom) shows a zoom-in at the position correlating with Ape1-GFP (white arrowhead).
Figure 3Ape1 processing was analyzed by western blot in the indicated strains, and normalized to the processing achieved in cells expressing wild-type Drs2 ± SD, n ≥ 3 biological replicates.
Figure 4D Ape1 processing was analyzed by western blot in the indicated strains, and normalized to the processing achieved in cells expressing wild-type Drs2 ± SD, n = 3 biological replicates.
Figure 5A Ape1 processing was analyzed by western blot in cells overexpressing N-terminal Drs2 (1-212 aa) and normalized to the processing achieved in cells with an empty vector ± SD, n = 3.C Left, PICT assay for the interaction of Trs85-FRB (TRAPPIII) and Drs2-GFP or drs2-5A-GFP after adding rapamycin (RAP). The Interaction score was normalized to the measurement of Drs2-GFP. Error bars: mean ± SD, n = 3. Asterisk indicates significant difference as determined by a two-tailed Student's t-test (**P<0.01). Right, representative images of the PICT assay. RFP-tagged anchor and GFP-tagged prey are shown in the upper and middle row, respectively. Bottom row shows the merged images. White zoom in boxes, 0.9µm. Scale bar, 5µm.
Figure 6A Histograms representing the mean speed of Atg9 puncta observed in wild-type, drs2Δ and drs2-5A cells. The histograms were fitted to a mixture of Gaussian distributions with two components; the percentage of each population and the means of the fitting curves are indicated on top of the plot.B Representative images (left) and quantification (right) of the co-localization between Atg9-GFP and Ape1-mCherry (upper and middle row, respectively) in cells expressing wild-type Drs2 or drs2-5A. White arrowheads point to Ape1-mCherry clusters co-localizing with Atg9-GFP. C Values were normalized to the measurements in cells expressing wild-type Drs2. Error bars: mean ± SD, n = 3 biological replicates. Asterisks indicate significant difference as determined by a two-tailed Student's t-test (**P<0.01). Scale bar, 5µm.C Representative images (left) and quantification (right) of the PICT assay for the interaction of Trs85-FRB (TRAPPIII) and Atg9-GFP in cells expressing wild-type Drs2 or drs2-5A after adding rapamycin (RAP). RFP-tagged anchor and GFP-tagged prey are shown in the upper and middle row, respectively. Bottom row shows the merged images. White zoom in boxes, 0.9µm. , C Values were normalized to the measurements in cells expressing wild-type Drs2. Error bars: mean ± SD, n = 3 biological replicates. Asterisks indicate significant difference as determined by a two-tailed Student's t-test (**P<0.01). Scale bar, 5µm.
Figure 1B) Western blot detection of DOT1L and TUBULIN (TUB) in whole testicular protein extracts from CTL and Dot1l-KO (KO) adult mice.D) Western blot detection of DOT1L and H3K79me2 in Dot1l-KO (KO) and CTL germ cells. Normalization was performed by detecting the membrane with anti-TUBULIN (TUB). SC = primary spermatocytes, RS = round spermatids.
Figure 2B) Histology of Dot1l-KO and CTL testes (periodic acid-Schiff staining). Scale bars indicate 200µm.D) Results from the tests of fertility (natural mating) of Dot1l-KO and CTL males (7 males in each group mated for ~3 months with wild-type females). Schematic diagrams representing the number of litters and the litter size from progeny of Dot1l-KO or CTL males (mean ± SEM).The p-value obtained with a Student t-test is indicated on each graph.
Figure 3C) Scatter plots (mean values following angular transformation ± SEM) of the percentage of motile and progressively motile spermatozoa in Dot1l-KO and CTL adult mice (N= 12 biological replicates for CTL and 10 biological replicates for Dot1l-KO) obtained following CASA (computer-assisted sperm analysis). ***p-value <0.0005 (Mann Whitney test performed after angular transformation of the percentages).F) Western blot detection of transition protein 2 (TNP2) in spermatozoa from Dot1l-KO and CTL males. The same quantity of material was loaded in each well (i.e. extracts from 2 million spermatozoa).G) Coomassie-stained protamine extracts from CTL and Dot1l-KO spermatozoa following acid urea gel electrophoresis (same gel, two different intensities). The same quantity of material has been loaded in each well (i.e. extracts from 1.4 million spermatozoa). Protamine 1 and 2 bands (PRM1 and PRM2, respectively) are detected at the bottom of the gel. The rectangle highlights the bands that are likely immature forms of Protamine 2 (i.e. non-cleaved precursor forms). The right panel shows a western blot detection using anti-PRM2 antibody which confirms that one of the high molecular weight band only observed in Dot1l-KO spermatozoa is an immature form of Protamine 2 (Pre-PRM2). The corresponding Coomassie-stained gel is also shown.H) Quantification of histones TH2B and H3 (N= 11 and 10 biological replicates for CTL and KO, respectively), of TNP2 (N= 8 biological replicates for CTL and for KO) and of PRM1/PRM2 ratio (N= 3 biological replicates for CTL and for KO) in Dot1l-KO and CTL spermatozoa, related to Fig 3E-G. The data shown are normalized to control mean values (± SEM). *p-value <0.05, ***p-value <0.0005 (unpaired t-tests). A.U. = arbitrary units.
Figure 4(A, B) Bar plots showing the quantification of post-translational modifications (PTMs) in histones H3 and H4 in ES (elongating/condensing spermatids) by LC-MS/MS. After normalization to be at constant amount of histone H3 or H4 in each analyzed sample mass spectrometry signals were divided by the average signal in both conditions (CTL and KO), so as to be able to represent all peptides in the same figure, whatever their MS intensity. Quantitative data obtained on biological replicates (N= 3 for CTL and 3 for KO) were plotted as dots, and the height of the bars indicates the average value (mean ± standard deviation, SD). T-tests were performed to estimate the possible significant difference of modified peptide abundances between the two genotypes. *p-value <0.05, ** p-value<0.005 and ***p-value <0.0005. When interpreting our mass spectrometry data against the database MS_histoneDB, we were able to identify various H3 variants, namely canonical H3 and variants H3.3, testis-specific H3t and H3mm13. "H3" indicates sequences shared among several variants; "H3.3", "H3t" or "H3mm13", each variant. A.U. = arbitrary units.
Figure 5D) H3K79me2 enrichment profile at selected deregulated genes, in two biological replicates of ChIP-Seq performed in wild type RS (Rep#1 and 2). The input value is shown in grey.
Figure 1A Super-resolution fluorescence microscopy images showing relative distribution between MATR3 and DAPI. Scale bars, 5μm (Upper) or 0.5μm (Lower). Arrows point to the representative MATR3/DAPI co-staining regions. B Coefficient of correlation between MATR3 and histone modification H3K9me3 (n=105), H3K9me2 (n=98), H3K27me3 (n=107), H3K27ac (n=97) and H3K4me3 (n=99) in AML12 cells. Quantifications were performed on randomly selected ROIs in cell nuclei. Each point represents one cell.C (Upper) Schematic diagram of dox-inducible shRNA system for MATR3 knockdown and MATR3 rescue in AML12 cells. (Lower) Western blotting detected the expression level of MATR3 after 3 days of Dox treatment (+Dox) and followed by 3 days of Dox removal (±Dox) in AML12 cells. Rep, replicate.D (Upper) Representative cross-section images showing distribution of histone modifications upon Ctrl and MATR3 knock down (+Dox). (Lower) Quantify the distribution pattern of histone modifications by Standard Deviation of Pixel Intensity in cell nuclei. For H3K9me3, n=102 (Ctrl) or 84 (+Dox); for H3K9me2, n=100 (Ctrl) or 101 (+Dox); for H3K27me3, n=98 (Ctrl) or 98 (+Dox); for H3K27ac, n=117 (Ctrl) or 124 (+Dox); for H3K27me3, n=98 (Ctrl) or 98 (+Dox); for H3K4me3, n=107 (Ctrl) or 97 (+Dox). Each point represents one cell. The P values were calculated using unpaired two-tailed Student's t test; ns, not significant, *p<0.05, ****p<0.0001. Error bars indicate mean ± s.e.m.K Representative cross-section images showing nuclear localization of H3K27me3 in ES cells after 6h addiction of 500μM IAA (+IAA 6h) or equal-volume of alcohol (-IAA). Middle: The zoom-in view of H3K27me3 in one ES cell. The outer dotted line colocalizes with nuclear membrane; the inner dotted line has the 80% diameter of the outer dotted line. The average pixel intensity of regions within outer dotted line (ROI.o) and regions within inner dotted line (ROI.i) were measured separately for each cell. Scale bars, 5μm.L Statistics for relative intensity (ROI.o/ ROI.i) after 6h addiction of 500μM IAA (+IAA 6h) (n=46) or equal-volume of alcohol (-IAA) (n=38). Each point represents one cell. The P values were calculated using unpaired two-tailed Student's t test; ****p<0.0001. Error bars indicate mean ± s.e.m.
Figure 2A Western blotting showing the distribution of MATR3 proteins in chromatin-non-associated and chromatin-associated extracts before and after DRB (75μM for 12h) or RNase A (pre-treat with 0.05% Triton x-100 for 30s, followed by 10μg/ml RNase A for 1h) treatment in AML12 cells. Representative of two independent replicates with similar results.B (Left) The representative cross-section image showing nuclear distribution of DAPI, MATR3 and H3K27me3 before and after 24h treating of 75μM DRB in AML12 cells. (Right) Line charts showing pixel intensity of each channel on the ROIs. r, coefficient of correlation. Scale bars, 5μm.G (Left) Representative cross-section images showing relative distribution between AS L1 RNA with MATR3 and with histone modification marks (H3K27me3, H3K9me3 and H3K27ac) in AML12 cells. Probes for RNA FISH were designed towards the consensus sequence of antisense L1_Mus1 RNAs. (Right) Line charts showing pixel intensity of each channel on the ROIs. Scale bars, 5μm. r, coefficient of correlation.H Normalized average density of the marks (top) and heatmaps(bottom) for the two groups of L1 loci. The two groups are antisense L1 RNAs or sense L1 RNAs that interacted with MATR3. L1 loci with RIP (MATR3 -IgG) count number >= 10 of antisense RNA were identified as MATR3-associated antisense L1, and the same cutoff for MATR3-associated sense L1.