EMBO/SourceData huggingface dataset:
Figure 1(a) Representative image of a Control (left) and Myo1cKO (right) embryo. Top: max projections of embryos stained with Phalloidin (green) and DAPI (gray). Bottom: magnifications at dashed rectangles of Phalloidin (grey) with white arrows pointing at fragments. Data information: Mann-Whitney test p values are indicated. Scale bars, 10 µm.(b-c) Number of fragments without DNA (b) per embryo and mean radius (c) in Control (grey) and Myo1cKO (salmon) embryos (left, Control n = 20, Myo1cKO n = 20). Boxplot shows median, upper and lower quartiles, min and max values. Whisker plot shows median, upper and lower quartiles. Data information: Mann-Whitney test p values are indicated. Scale bars, 10 µm.(d) Representative images of a time-lapse of Myo1cKO embryo undergoing fragmentation outside of division (top) and during division (bottom). Max projections of embryos expressing mTmG (grey) and mCherry-EB3 (grey) imaged with light sheet microscopy are shown. White arrows point at the forming fragments. (e) Ratio of fragmentation events during and outside of division (n = 13 cells from 7 embryos). Data information: Mann-Whitney test p values are indicated. Scale bars, 10 µm.
Figure 2(a) Representative images of Control (left) and Myo1cKO (right) embryos expressing mTmG and mCherry-EB3 (grey) shown as max projections. White arrows point at the mitotic spindle. (b-c) Spindle persistence in Control (n = 6) and Myo1cKO (n = 9) embryos (b) and in non-fragmenting and fragmenting cells of Myo1cKO embryos (c, n = 42 and 12 cells from 7 Myo1cKO embryos). Spindle persistence measures the time from appearance to disassembly of the spindle. Data information: Mann-Whitney test p values are indicated. Boxplots show median, upper and lower quartiles, min and max values. Scale bars, 10 µm.(f) Representative images of embryos treated with DMSO (left) and 37.5 µM Ciliobrevin D (right), stained with Phalloidin. Top: Maximum projection. Bottom: magnifications of a single plane at dashed rectangles of Phalloidin with white arrows pointing at fragments. Data information: Mann-Whitney test p values are indicated. Boxplots show median, upper and lower quartiles, min and max values. Scale bars, 10 µm.
Figure 3(d) Top: Representative images of a time-lapse of a Myo1cKO embryo expressing Myh9-GFP (green) and mTmG (magenta) while labelled with SiRDNA (grey) undergoing fragmentation during mitosis shown as max projections. Bottom: magnifications of Myh9-GFP (grey) at dashed rectangles showing cortical accumulation (middle) followed by fragmentation (right). Data information Boxplots show median, upper and lower quartiles, min and max values. Scale bars, 10 µm.(e) Control (grey) and Myo1cKO (salmon) embryos Myh9-GFP Ic/Iref values at DNA-cortex distances < 20 µm and > 20 µm (Control embryos n = 10, cells n = 35; Myo1cKO embryos n = 9, cells n = 26) Data information: Kruskal-Wallis and pairwise Mann-Whitney tests (e) p values are indicated. Boxplots show median, upper and lower quartiles, min and max values. Scale bars, 10 µm.(f) Comparison of the increase in Myh9-GFP intensity in Control and Myo1cKO embryos when DNA is in close proximity to the cortex. The ratio Ic0-20/Ic>20 is calculated from Myh9-GFP intensities at the contact region when DNA-cortex distances are < 20 µm (Ic0-20) divided by intensities when distances are > 20 µm (Ic>20) (Control embryos n = 10, cells n = 33; Myo1cKO n = 9, cells n = 26). Data information: Mann-Whitney test p values are indicated. Boxplots show median, upper and lower quartiles, min and max values. Scale bars, 10 µm.
Figure 4(a) Representative images of Control (left) and Myo1cKO (right) embryos labelled with SiRDNA (grey) and expressing a reporter of Cdc42 activity (yellow) in one blastomere, which is undergoing mitosis, shown as max projections. The second blastomere is outlined with a dashed line. White arrows point at the accumulation of active Cdc42 at the cortex in close proximity to DNA. (b) The ratio of active Cdc42 Ic/Iref was compared between < 20 µm and > 20 µm DNA-cortex distances for Control (dark yellow) and Myo1cKO (yellow) embryos (Control embryos n = 10; Myo1cKO embryos n = 10). Data information: Kruskal-Wallis and pairwise Mann-Whitney tests (b Boxplots show median, upper and lower quartiles, min and max values. Scale bars, 10 µm.(c) Representative images of Myo1cKO embryos expressing LifeAct-GFP (green) alone (left) or together with dominant negative Cdc42 (DNCdc42, right) labelled with SiRDNA (grey) shown as max projections. White arrows point at the cortex in close proximity to DNA. (d) LifeAct-GFP Ic/Iref values at DNA-cortex distances < 20 µm and > 20 µm for Myo1cKO (salmon) and Myo1cKO + DNCdc42 (yellow) embryos (Myo1cKO embryos n = 8, cells n = 19; Myo1cKO + DNCdc42 embryos n = 12, cells n = 30). Data information: Kruskal-Wallis and pairwise Mann-Whitney tests d) Boxplots show median, upper and lower quartiles, min and max values. Scale bars, 10 µm.(e) Representative images of a GFP (left) and Ect2-GFP (right) expressing embryo stained with Phalloidin (green) and DAPI (grey) shown as max projections. White arrows indicate fragments. (f) Number of fragments without DNA per embryo in GFP (grey) and Ect2-GFP (blue) embryos (GFP n = 14; Ect2-GFP n = 18). Data information Mann-Whitney test (f) p values are indicated. Boxplots show median, upper and lower quartiles, min and max values. Scale bars, 10 µm.
Figure 1(B) Comparison of investigation duration and recognition index between the light (L, n = 12 animals) and dark control (D, n = 11 animals) groups. A significant reduction in the recognition index is observed in the L group compared to the D group (* p = 0.0156, Mann-Whitney test), while no significant difference is observed in investigation duration during the first trial. Data shown as mean ± SEM.(E) No statistically significant difference is observed in the investigation duration and recognition index between the L (n = 8 animals) and D (n = 7 animals) groups (p = 0.9551, Mann-Whitney test). Data shown as mean ± SEM.
Figure 2(C) OTA treatment (n = 6 animals) significantly reduces the recognition index in mice compared to the PBS injection control (n = 6 animals) mice (* p = 0.0260, Mann-Whitney test). Data shown as mean ± SEM.(F-G) Representative confocal images of c-fos staining showing oxytocin neurons in the supraoptic nucleus (SON) (E) and paraventricular nucleus (PVN) (F) in the L and D groups. Yellow arrows indicate oxytocin neurons co-labeled with c-fos. High magnification images are 10 μm Z-stack (63X, NA=1.4, pinhole = 1.26 AU), while low magnification images are 30 μm Z-stack (20X, NA=0.7, pinhole = 1.26 AU). Scale bar = 100 μm for low magnification images and 50 μm for high magnification images.
Figure 3(C) Average delta-F/F signal trace from SON oxytocin neurons with 10 seconds of dim light (5 lux) followed by 30 seconds of bright light (800 lux) in OPN4DTA/DTA; OxtCre/+ mice (red line, n = 4 animals) and OPN4+/+; OxtCre/+ mice (black line, n = 5 animals). The color shade indicates standard deviation.(G) The investigation duration and recognition index of the 595nm-control and the 470nm-activation group (n = 8 animals). There is a significant increase in the recognition index in the optogenetic activation group compared to the control (** p = 0.0078, Wilcoxon matched-pairs signed rank test). Data shown as mean ± SEM.
Figure 4(B) Light exposure significantly reduces the recognition index in control Opn4Cre/+ mice (n = 8 animals in both the L and D group), similar to WT mice (*p = 0.0499, Mann-Whitney test). Data shown as mean ± SEM. (C-E) There is no significant difference in recognition index between light exposure and dark control groups in OPN4DTA/DTA (C, n = 11 animals in both the L and D group, p = 0.7969), OPN4Cre/+; Brn3bz-DTA/+ (D, n = 9 animals in both the L and D group, p > 0.9999), and OPN4Cre/Cre mice (E, L group n = 9 animals, D group n = 8 animals, p = 0.9551) using the Mann-Whitney test. Data shown as mean ± SEM.(H) During the second trial, the investigation duration of the 470nm-activation group (red) was lower than the 595nm-control (black) (n = 7 animals, * p = 0.011, Sidak's multiple comparison test). Optogenetic activation of ipRGC terminals at SON with 470 nm light significantly enhances the recognition index compared to the 595 nm control (* p = 0.0156, Wilcoxon matched-pairs signed rank test). Data shown as mean ± SEM.
Figure 5(B) Representative confocal image of the pSON region stained with synaptophysin (blue), CTB-Alexa 568 (red), and GFP from GABAergic neurons (green). Arrowheads indicates colocalization. Scale bar = 10 μm.(I) Average delta-F/F signal trace from pSON GABAergic neurons during the transition from baseline to dim light (5 lux, black line, n = 7 trials) and from baseline to bright light (800 lux, red line, n = 12 trials) in vGATCre/+ mice (n = 5 animals). The color shade indicates standard deviation.
Figure 6(D) Minute-wise investigation duration of the 595nm-control (black) and 470nm-activation (red) groups (n = 8 animals). Data shown as mean ± SEM for each minute.(G) The investigation duration and recognition index of the GFP-control (n = 4 animals) and the TeLC-silencing (n = 6 animals) group. During the second trial, the investigation duration of the TeLC-silencing group was lower than the GFP-control group (* p = 0.0280, Sidak's multiple comparison test). The TeLC-silencing group had a higher recognition index than GFP-control group (* p = 0.0317, Mann-Whitney test). Data shown as mean ± SEM.
Figure 1(A, B) Transmission electron micrographs of negatively stained 2F (A) and 3F (B) fibrils before sonication and injection into mice. The two fibril polymorphs were grown in vitro from synthetic Aβ40 and show the morphological differences originally reported Fibrils are negatively stained with uranyl acetate. Bars represent 100 nm.(C) PK resistance profile of 2F and 3F fibrils. Values are expressed as the mean densitometric value at each PK concentration (technical triplicates) ± standard error. Statistical analyses were performed by student's t-test (*p<0.05).D) Seeding assays using 100 pM of 2F (blue) and 3F (green) fibrils as seeds as described in Materials and Methods. Curves were generated by measuring ThT emission values at different time points. Reaction kinetics were statistically different as assessed by using the multiple comparisons method to compare pairs of curves (***p<0.0001). Data in graphs is represented as averages depicting standard errors of five technical replicates.F-I) 2F and 3F fibrils were tested for their reactivity against a variety of luminescent conjugated thiophenes able to discriminate among conformational variants of misfolded proteins. Stock solutions of LCOs (1.5 mM) were diluted in distilled water to 15 μM and added to the wells to a final concentration of 0.3 μM. All wells contained 10 μM of either 2F or 3F fibrils. The samples were incubated at 37°C and the emission spectrum of each probe was collected after 30 minutes by exciting the samples at 440 nm (HS-68) or 535 nm (HS-194, HS-208 and HS-212).
Figure 2(A-H) Representative images of hippocampi from mice treated with 2F-, 3F- and Tg2576- derived seeds after visualization for Aβ deposits using the 4G8 antibody (A-D) or ThS (E-H). CA, cornu ammonis 1; DG, dentate gyrus. Scale bar in (B), (D), (F) and (H) represents 500 µm and applies to all panels. Arrows represent regions of interest (alveus, DG).(I, J) Aβ (I) and ThS (J) burden quantification in hippocampus. Statistical analyses were performed using One-way ANOVA (*p<0.05, **p<0.001). Data in graphs is represented as averages depicting standard errors. Measurements considered five tissue slices per animal, and 4-7 animals were included per group.
Figure 3(A-H) Representative pictures of hippocampi from mice treated with different injectate after visualization for micro- and astro-glial associated signals using anti-GFAP (A-D) and anti-Iba-1 (E-H) antibodies. Scale bar in (D) and (H) represents 500 µm and applies to all panels. CA1-3, cornu ammonis 1-3; DG, dentate gyrus; so, striatum oriens; sp, striatum pyramydale; sr, striatum radiatum.(I-N) Anti-GFAP burden in hippocampus (I), dentate gyrus (J) and alveus (K) for all animal groups was quantified for image analysis. Similarly, anti-Iba-1 burdens for all experimental and control groups were calculated in hippocampus (L), dentate gyrus (M) and alveus (N). Statistical analyses were performed by One-way ANOVA (*p<0.05, **p<0.001).. Data in graphs is represented as averages depicting standard errors. Measurements considered five tissue slices per animal, and 4-7 animals were included per group.(O-R) Correlations between Aβ burden and glial burden were calculated in the alveus for mice injected with 2F (O) and 3F (P) fibrils. In the same way, the relationship between glial activation and Aβ burden in the hippocampus was calculated for mice treated with 2F (Q) and 3F (R) aggregates. Statistical analyses were performed by Pearson correlation coefficients test in which p<0.05 was considered significant.
Figure 4A) Panels showing representative epifluorescence microscopy images of the dentate gyrus of mice treated with experimental and control materials and immunostained with both 4G8 (green) and anti-GFAP (red) antibodies. The third column of panels from left to right depict merged images. Right panels are insets obtained from the merged images (white punctuated squares). Scale bars at the left panels represent 200µm and are applicable to the pictures labeled as "4G8", "GFAP" and "Merge". Scale bars on the right images (inset) represent 50µm. g, granular layer; h, hilar region; m, molecular layerB) A similar analysis as explained in A) was performed for the alveus. Images were arranged in the same manner as described above. Scale bars at the left panels represent 200µm and are applicable to the pictures labeled as "4G8", "GFAP" and "Merge". Scale bars on the right images (inset) represent 50µm. Alv, alveus ; so, striatum oriens. One tissue slice per animal was used for these analyses.
Figure 5A) Panels showing representative pictures of the dentate gyrus of mice treated with experimental and control Aβ seeds and stained with both 4G8 (green) and anti-Iba-1 (red) antibodies. The third column of panels from left to right depict merged images. Right panels are insets obtained from the merged images (white punctuated squares). : Scale bars at the left panels represent 200µm and are applicable to the pictures labeled as "4G8", "Iba-1" and "Merge". Scale bars on the right images represent 50µm. ; g, granular layer; h, hilar region; m, molecular layer;B) A similar analysis as explained in A) was performed for the alveus. Images were arranged in the same manner as described above. Scale bars at the left panels represent 200µm and are applicable to the pictures labeled as "4G8", "Iba-1" and "Merge". Scale bars on the right images represent 50µm. Alv, alveus; g, granular layer ; so, striatum oriens. One tissue slice per animal was used for these analyses.
Figure 6A) 1D 13C ssNMR spectra of fibrils that were prepared in vitro by seeded growth from amyloid-containing extracts of mouse brain homogenates. Mice had been treated with 2F-, 3F-, or Tg2576-derived seeds. Aβ40 was 15N,13C-labeled at F19, V24, G25, S26, A30, I31, L34, and M35. Vertical dashed lines indicate some of the ssNMR peaks that vary among the three samples. Asterisk indicates a peak from residual sodium dodecyl sulfate in the 2F sample.
Figure 1A, Unsupervised cluster analysis of patients with cutaneous malignant melanoma based on global gene expression (RNA-seq) in metastases (n=367). Partitioning around medoids clustering algorithm was used (k=2).B, Heat-map displaying the expression of bromodomain genes in metastases from patients with cutaneous malignant melanoma (n=367). BRD4, SMARCA4, TRIM28 and BRPF1 are highlighted by a black bar. Gene expression is represented by z-score.C, Kaplan-Meier analysis of overall survival of patients with stage III melanoma in cluster C1 (median survival 79.5 months) and C2 (median survival 25.9 months), and of patients with stage III melanoma with high (median survival 61.5 months) or low (median survival 107 months) TRIM28 expression. The log-rank test was used for statistical testing of survival data.
Figure 2C, GSEA plot of RAS signature genes in in TRIM28high and TRIM28low metastatic tumors (n=367), and in A375 cells transduced with shT28-1, shT28-2 or shNTC (shLUC and shSCR) (n=3 per construct).
Figure 3C, Metagene profiles after CUT&RUN sequencing of TRIM28 in untransduced A375 cells and of RNAPII in A375 cells transduced with shT28-1 or shSCR lentivirus. The lower panel is focused on the TRIM28 metagene profile to clearer depict its binding profile around TSS.E, TRIM28 and RNAPII occupancy at EGR1 and JUNB in A375 as determined by CUT&RUN sequencing (PI = length-normalized pause index).F, Immunoblots against TRIM28 and JUNB in A375, A2058 and SK-MEL-28 cells transduced with shSCR, shT28-1 or shT28-2 lentivirus. Results are expressed as mean ±SEM from three biological replicates (n=3). G, Densitometry of JUNB and TRIM28 protein levels (relative to β-actin) in A375, A2058 and SK-MEL-28 melanoma cells based on the immunoblots in F. One-way ANOVA with Dunnett's post hoc test was used for statistical testing.
Figure 4A, Immunoblotting to determine the overexpression of JUNB in A375 cells transduced with pBABE-JUNB.B, Volcano plot displaying differentially expressed genes after RNA-seq analysis of JUNB overexpressing A375 cells (pBABE-JUNB) and control A375 cells transduced with empty pBABE vector (EV) (n=4). Genes induced after JUNB overexpression are highlighted in yellow, and gene suppressed after JUNB overexpression are highlighted in blue.D, Heatmap displaying the induction of CXCL1 and CXCL8, and suppression of YAP1 target genes, in A375 cells overexpressing JUNB.E, Expression of YAP1 signature genes in A375 cells after transduction with dCas9-KRAB lentiviruses expressing gRNA targeting JUNB (gJUNB-1 or gJUNB-2) or control gRNA (gEGFP). qRT-PCR was used to determine expression levels, and results are expressed as mean ±SEM from three biological replicates (n=3).F, Quantification of Matrigel invasion experiments with A375 cells transduced with pBABE-JUNB (JUNB) or pBABE empty vector (EV) retrovirus. Results are expressed as mean ±SEM from three biological replicates (n=3) and the two-sided unpaired t-test was used. G, Representative images from Matrigel invasion assays in (F). Scale bar is 60 μm.H, Quantification of Matrigel invasion experiments with A375 cells transduced with dCas9-KRAB lentiviruses expressing gRNA targeting JUNB (gJUNB-1) or control gRNA (gEGFP). Results are expressed as mean ±SEM from three biological replicates (n=3) and the two-sided unpaired t-test was used. I, Representative images from Matrigel invasion assays in (H). Scale bar is 60 μm.J, Quantification of Matrigel invasion with A375 cells transduced with dCas9-KRAB lentiviruses expressing gRNA targeting JUNB (gJUNB-1) or a control gRNA (gEGFP), and then transfected with non-targeting siRNA (siNTC) or siRNA targeting YAP1 (siYAP1) and TAZ (siTAZ). Results are expressed as mean ±SEM from three biological replicates (n=3). One-way ANOVA and the Tukey post hoc test were used for statistical testing.K, Tumor growth after subcutaneous injection of 2.0x106 A375 cells transduced with pBABE-JUNB (JUNB) or pBABE empty vector (EV) retrovirus (n=10 mice per group). Results are expressed as mean ±SEM. Repeated-measures ANOVA was used for the statistical test of tumor growth. L, Tumor weight after the subcutaneous injection of A375 cells as shown in (K). Tumor weight was analyzed 20 days after subcutaneous injection. Results are expressed as mean ±SEM. The two-sided Mann-Whitney U-test was used for the statistical test of tumor weight.
Figure 1(C) HeLa cells were harvested at indicated time points and processed for western blotting probed for GBP1 and 5 protein levels, α-Tubulin (Tubulin) as loading control.
Figure 2(B) Top: Experimental outline of CRISPRa targeting of GBP1 and ASCL1 (control) by transient transfection with plasmids containing dCAS9-SAM. Bottom: HeLa cells were transfected with dCas9-SAM technology with gRNA for indicated genes, either alone or in combination with a 24h exposure to IFNγ. RNA was isolated and GBP1 mRNA expression was measured by RT-qPCR after 48h of transfection and normalized to ACTB expression. Statistical significance was determined using two-way ANOVA. Data are shown as mean. Error bars, SD; n=3 biological replicates.(C) Top: Experimental outline of CRISPRa targeting of GBP1 and ASCL1 (control) by transient transfection with plasmids containing dCAS9-SAM, followed by induction with IFNγ. Bottom: RNA was isolated, GBP1 mRNA level was determined as indicted in experimental outline in top, by RT-qPCR and normalized to ACTB mRNA level. Statistical significance was determined using Ordinary one-way ANOVA. Data are shown as mean. Error bars, SD; n=3 biological replicates.(D) Experiment as in C but with addition of IFNγ during priming. Statistical significance was determined using Ordinary one-way ANOVA. Data are shown as mean. Error bars, SD; n=3 biological replicates.
Figure 3(B) Stable CRISPR knockouts were generated for indicated genes in HeLa cells. Knockout (KO) cells or their parental controls (WT) were induced with IFNγ for 24 hours or left untreated and prepared for SDS-PAGE and immunoblotting. Blots incubated with GBP5 antibody assess gene expression. α-Tubulin (Tubulin) was used as a loading control. Note that GBP5 and Tubulin blot of parental cells is as in 1C.HeLa cells were primed with IFNγ for 24 hours, followed with IFNγ washout. After 48h, naïve and primed cells were induced by IFNγ for 1h and 3h. Cells were harvested at indicated time points and processed for CUT&RUN. Representation of processed data of CUT&RUN for STAT1 (D) and IRF1 (E) occupancy at GBP5 and GBP4 genes. Sequenced reads were mapped to the human genome (hg38), coverage data are displayed as reads per million (RPM) at equal scaling.
Figure 5(A) Single-cell RNA-seq from HeLa cells for STAT1 Each dot represents STAT1 expression in one cell in naïve (n=91), induction (n=90) and reinduction (n=92). Error bars, SEM.(C) Blot probing for STAT1 before and after IFNγ induction in STAT1 knockout (STAT1KO), STAT1 rescued (STAT1, constitutive) and parental control (WT), to confirm knockout and rescue status. α-Tubulin (Tubulin) was used as a loading control.(D) STAT1 rescue cells and their parental control were subjected to IFNγ induction and reinduction regime as outlined in Figure 1A. Cell extracts were processed for western blotting and probed for GBP5 expression before and after induction and reinduction as indicated in Figure 1A. α-Tubulin (Tubulin) was used as a loading control.(G) Cells constitutively expressing STAT1 (as in B) were primed with IFNγ, followed by IFNγ washout. After 48h, naïve and primed cells were induced by IFNγ for different time points (5, 15, 30, 60, 180 min). Cell extracts were prepared at indicated timepoints and processed for western blotting. Immunoblot of pSTAT1-Y701, and α-Tubulin (Tubulin) as a loading control.