EMBO/sd-character-level-ner huggingface dataset:
circHIPK3 sponges miR-29b and miR-30c(A,B) qPCR analysis of TREx 0 and 24 post-induction (n=3) using GAPDH as a housekeeper, analysis is of levels of circHIPK3 (A) and HIPK3 (B).Figure 2(A,B) qPCR analysis of TREx 0 and 24 post-induction (n=3) using GAPDH as a housekeeper, analysis is of levels of circHIPK3 (A) and HIPK3 (B). Data information: data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological. (C) FISH analysis of TREx cells reactivated for 16 hours with probes against circHIPK3 (red). DAPI was used as nuclei stain (blue), scale bars represents 5 μm on upper panel and 10 μm on lower panel. The lower panel shows a wider field of view. (D) qPCR analysis of Ago2 RIPs in TREx cells transfected with scr, miR-29b or miR-30c showing circHIPK3 enrichment over GAPDH, with each repeat normalised to an IgG scr RIP (n=3). Data information: data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological. (E) qPCR analysis of Streptavidin RIPs of circHIPK3 or GAPDH enrichment in HEK-293T cells transfected with either biotinylated miR-29b or miR-30c over scr (n=4). Data information: data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological.
Figure 2( Images of HeLa GFP-Parkin cells expressing mCherry-NDP52 (A under valinomycin treatment. The images are from Movie EV 2 and 3, respectively. Cyan, GFP-Parkin; magenta, mCherry-NDP5 Scale bars, 10 µm. Images are representative of five independent experiments (B) Time-lapse images of HeLa GFP-Parkin cells expressing mCherry-NDP52 under valinomycin treatment. The images are representative of five independent experiments and correspond to the boxed regions of Figure 2A and Movie EV 2. Cyan, GFP-Parkin; magenta, mCherry-NDP52. The white arrowheads indicate colocalization of GFP-Parkin and mCherry-NDP52. The yellow arrowheads indicate mCherry-NDP existing inside GFP-Parkin ring structures Images o HeLa GFP-Parkin cells expressin mCherry-OPTN (C) under valinomycin treatment. The images are from Movie EV 2 and 3, respectively. Cyan, GFP-Parkin; magenta OPTN. Scale bars, 10 µm. Images are representative of five independent experiments (D) mCherry-NDP52 or OPTN signals colocalized with Parkin-ring structures and existing inside of Parkin-ring structures (irrupted) were quantified. Data were from five independent experiments in cells incubated with valinomycin for 2 h. Values are the means ± SEM. **p < 0.01 compared with mCherry-OPTN expressing cells, determined with one-way ANOVA followed by the Student"s t (E) Mitophagic flux in NDP52 KD cells expressing siRNA-resistant NDP52 or NDP52 mutants. Cells were incubated with valinomycin for 3 h. Results are the summary of three independent experiments. Values are the means ± SEM. *p < 0.05, **p < 0.01 vs scrambled oligo-transfected cells; †p < 0.05, ††p < 0.01 vs empty vector-transfected cells, determined with one-way ANOVA followed by the Tukey-Kramer post hoc test (F) HeLa-GFP-Parkin cells expressing mCherry-NDP52 mutants were incubated with 1 µM valinomycin and 100 nM bafilomycin A1 for 3 h. Cell lysates were subjected to immunoprecipitation using anti-RFP magnetic beads. Immunoprecipitates were analyzed by western blotting. Data are representative of three independent experiments (G) Images of HeLa GFP-Parkin cells expressing mCherry-NDP52∆LIM-L or mCherry-NDP52∆SKICH treated with valinomycin for 2h. The images are from Movie EV 4. Cyan, GFP-Parkin; magenta, mCherry-NDP52 mutant. Scale bars, 10 µm. Images are representative of three independent experiments
AID expression in pancreas promotes proliferation and NKG2D ligand expression.Figure 4Representative images of Ki67 staining in pancreas from aged (75-week-old) R26AID+/+p48CRE+/KI (top) or R26AID+/KIp48CRE+/KI mice (bottom). Detail is shown on the right. Scale bar: 100 μm. Graph shows quantification of Ki67-positive epithelial cells per mm2 of tissue (n = 8). *P = 0.0266. Representative immunofluorescence staining of 20-week-old R26AID+/KIp48CRE+/KI mice: blue, DAPI; red, Ki67; green, CK8. Scale bar: 50 μm. Detail is shown on the bottom. Quantitative FACS analysis of RAE expression in pancreatic explants from R26AID+/+p48CRE+/KI and R26AID+/KIp48CRE+/KI mice. Left: Graph shows mean fluorescence intensity. Each dot represents an individual mouse. n = 9 (R26AID+/+p48CRE+/KI); 8 (R26AID+/KIp48CRE+/KI). P = 0.077. Right: Representative FACS staining for RAE in explants from R26AID+/+p48CRE+/KI (red) and R26AID+/KIp48CRE+/KI mice (black). Analysis of RAE expression by ddPCR in aged (75-week-old) mice. Data are presented as the percentage of positive drops normalized to the mean control value. Each point represents an individual mouse and shows the mean amplification from two independent experiments n = 7 (R26AID+/+p48CRE+/KI); 9 (R26AID+/KIp48CRE+/KI). *P = 0.012. Analysis of killing activity. Primary explants of pancreatic cells from R26AID+/+p48CRE+/KI or R26AID+/KIp48CRE+/KI mice were cultured with primary NK cells, and killing activity was assessed as described in Materials and Methods (n = 4). P = 0.19.
Figure 3Human osteosarcoma U2OS cells were treated with different concentrations of dactinomycin (DACT) (0.25, 0.5 or 1 µM) for 6 h. Thapsigargin (THAPS) at 3 µM was used as a positive control. After fixation, cells were stained with phospho-eIF2α (Ser51) specific antibody followed by an AlexaFluor-647 secondary antibody, nuclei were counterstained with Hoechst 33342 and phosphorylation was assessed by fluorescence microscopy. Images were segmented, analyzed and the red cytoplasmic fluorescence intensity was measured. Images are shown for untreated control cells (Ctr), THAPS and DACT at 1 µM U2OS cells stably expressing ATF4-reporter were treated as described above for 12 h. The expression and nuclear translocation of ATF4 was assessed by fluorescence microscopy and the nuclear green fluorescence intensity was quantified. Images are shown for untreated control cells (Ctr), THAPS and DACT at 1 µM U2OS stably expressing ATF6-GFP were treated as described above and nuclear translocation of ATF6 was represented as the ratio of nuclear versus cytoplasmic green fluorescence intensity. Images are shown for untreated control cells (Ctr), THAPS and DACT at 1 µM U2OS cells stably expressing venus in frame with alternatively spliced XBP1 (sXBP1) were treated as described above for 12 h. De novo expressed venus was measured intracellular. Images are shown for untreated control cells (Ctr), THAPS and DACT at 1 µM U2OS wild-type and knock out for eIF2α kinases 1, 2, 3 and 4 cells were treated with 3 µM THAPS as a positive control for EIF2AK3-mediated eIF2α phosphorylation or with 1 µM DACT for 6 h. After fixation, cells were stained for peIF2α as described above and cytoplasmic intensity was quantified. Images are shown for untreated control cells (Ctr), THAPS and DACT at 1 µM U2OS CALR-RFP were injected subcutaneously (s.c) in the flank of nu/nu mice. Tumors were further injected with PBS (Ctr) (n=5), 0.5 mg/kg tunicamycin (TM) for 6 h (n=3) or 0.5 mg/kg DACT for 6 h (n=4) or 24 h (n=3). Tumor slices were cut and stained with a peIF2α antibody followed by an AlexaFluor-647 secondary antibody and counterstained with Hoechst 33342. Two slices per tumor were imaged for their DAPI, RFP and Cy5 signals. Out-of-focused images were removed from the dataset leading to the analysis of the following conditions: 8 x Ctr, 5 x TM, 8 x DACT 6 h and 6 x for DACT 24 h. PeIF2α was quantified measuring Cy5 intensity in the cytoplasm (K, L) and CALR translocation by measuring the coefficient variation (CV) of the RFP signal in the cytoplasm (M, N). Representative images of peIF2α are shown for Ctr, TM and DACT at 6 h (K), whereas images of CALR are shown for Ctr and DACT at 24 h
Figure 3(A) Wild-type (WT) K562 cells and indicated deletion isolates were treated with 100nM Bafilomycin A1 (BafA1) or DMSO for 18h and analyzed by flow cytometry for red:green ratio of tf-NBR1. Median values for each sample are identified by a black line within each violin. The red dotted line across all samples corresponds to the red:green ratio in wild-type cells. The red solid line across all samples corresponds to the ratio observed under maximally inhibited conditions (ATG9AKO cells). (n > 10,000 cells). (B) Representative confocal micrograph (as maximum intensity projections) of ATG7KO/TAX1BP1KO K562 cells expressing tf-NBR1. Selected region (white box) of micrograph is shown as single and merged channels from fluorescence microscopy. RFP, magenta; GFP, green; merged, white; Hoechst, blue. Scale bars: large panel, 5 µm; small panels, 1 µm. (C) Correlative light and electron microscopy (CLEM) of K562 ATG7KO/TAX1BP1KO cells expressing tf-NBR1 under basal conditions. Analysis workflow is indicated by green arrows. White arrow indicates a structure of interest. White box demarcates zoomed area in bottom images. NBR1, green; Hoechst, blue.
The Q623H caPOT1 mutation does not induce a DNA damage response at telomeres Schematic of the POT1 locus and editing strategy used to engineer hESCs with the Y223C and Q623H caPOT1 mutations.Figure 1Schematic of the POT1 locus and editing strategy used to engineer hESCs with the Y223C and Q623H caPOT1 mutations. Representative images for the TIF analysis shown in (C). Shown are POT1Y223C/- hESCs and an unedited wild-type hESC clone (WT/WT) isolated in parallel, as well as a POT1 WT/Q623H hESC and a contemporaneously isolated hECS clones (WT/WT). Scale bars = 10µm.   Box and whisker plot (showing min, max and average) quantifying telomere-dysfunction induced foci (TIF) in two POT1 Y223C/- hESC lines, one POT1 Q623H/Q623H, and one POT1WT/Q623H hESC lines compared to wild-type unedited control hESCs. Cells were analyzed 3 months post targeting. Cells were stained for TRF1 and γ-H2Ax. POT1Y223C/- cells have increased TIFs compared to an unedited wild-type cell clone, isolated and cultured in parallel (P<0.01, two-tailed Mann-Whitney test, one biological replicate).   Images of telomere and centromere FISH on metaphase spreads of cell lines as in (B). Cy3 Tel-C PNA probes were used. Arrows indicate chromosomal abnormalities. E) Quantification of chromosomal abnormalities as determined by FISH analysis in (D).
Atg3 recruitment to membranes is dependent on Atg16 and Atg12.Figure 3(A) Coomassie‐stained gels showing co‐sedimentation assays using Folch liposomes and the indicated proteins. Atg5-Atg12/Atg16 was most efficient in recruiting Atg3 to the pellet fraction. (B) Quantification of Atg3 in the pellet fraction in the presence of the indicated proteins. The quantification is based on three independent experiments. (C) Coomassie‐stained gels showing the results of Atg8-PE conjugation assays using the indicated proteins and E. coli lipids derived liposomes. Atg8-PE conjugation was detected as characteristic band shift as the conjugated form runs faster on urea‐SDS gels. The numbers above the gels indicate the time in minutes. Note that at least under these conditions almost no Atg8-PE conjugation was detected in the absence of the Atg5-Atg12 conjugate or the Atg5-Atg12/Atg16 complex. The numbers next to the gels indicate the molecular weight in kDa. The averages and the error bars representing the standard deviations are shown. P‐values were calculated using Student's t‐test. NS, non‐significant; P, pellet; S, supernatant. Figure source data can be found with the Supplementary data.
Figure 6(A) Female C57BL/6J and ADX mice were put in normoxia and hypoxia for 6h and 24h. Plasma was isolated for metabolomics analysis. The heatmap represents log10 of metabolites which are significantly increased of the plasma of C57BL/6J mice (C57BL/6J P ≤ 0.05 and ADX P > 0.05, LFC > 1), significantly increased in the plasma of C57BL/6J and ADX mice (C57BL/6J P ≤ 0.05 and ADX P ≤ 0.05, LFC > 1), and significantly increased in the plasma of ADX mice only (C57BL/6J P > 0.05 and ADX P ≤ 0.05, LFC > 1). (B) Metabolomics analysis identifying the presence of FFAs in the plasma of C57BL/6J and ADX mice 6h after hypoxia. N=6 per group, two independent experiments. Data information: All bars represent mean ± SEM. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. ****P<0.0001, ***P<0.001, **P<0.01, *P≤0.05. (C) PPARα gene response in the liver of C57BL/6J mice 24h after hypoxia via RT-qPCR. N=6 per group, two independent experiments. Data information: All bars represent mean ± SEM. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. ****P<0.0001, ***P<0.001, **P<0.01, *P≤0.05. (D-G) FFA levels and stress GRE genes were determined in the plasma and iWAT of female C57BL/6J mice injected with 5 mg RU486 or vehicle (DMSO) (D, F), and in GRdim/dim mice and their wild-type littermates (E, G) after 6h of normoxia and hypoxia. N=4-9 per group. Data information: All bars represent mean ± SEM. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. ****P<0.0001, ***P<0.001, **P<0.01, *P≤0.05. (H-J) Blood ketone body levels 6h after normoxia and hypoxia in female C57BL/6J and ADX mice (H), in female C57BL/6J mice injected with 5 mg RU486 or vehicle (DMSO) (I), and in GRdim/dim mice and their wild-type littermates (J). N=5-9 per group. Data information: All bars represent mean ± SEM. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. ****P<0.0001, ***P<0.001, **P<0.01, *P≤0.05. (K) iWAT weight of C57BL/6J and ADX mice 6h after normoxia or hypoxia. N = 5-6 per group. Data information: All bars represent mean ± SEM. P-values were calculated using two-way ANOVA followed by post-hoc Šídák's multiple comparisons test to correct for multiple testing during the pairwise multiple comparisons, except if otherwise stated. ****P<0.0001, ***P<0.001, **P<0.01, *P≤0.05.
Figure 2A Schematic diagram of constructs used to express patient-derived pure and interrupted SCA8 repeat tracts with predicted protein products and C-terminal epitope tags. * Due to TAG encoded stop codons polyGln proteins do not express an epitope tag. CGG interruptions and the encoded amino acid interruptions are indicated in red. B, C Cell death measured by lactase dehydrogenase (LDH) (B) and cell viability measured by 3-(4,5- dimethyl-thiazol-. 2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (C) in T98 cells 42 hrs post-transfection of pure and interrupted SCA8 repeat tracts; LDH n=8, MTT n=12, n=independent experiments, * p < 0.05, NT not transfected, EV: empty vector; unpaired t test; mean ± SEM. D, E qRT-PCR showing transcript levels of Pure 96 and Int.95 (D, n=6; p=0.0187), and Pure 104 and Int.102 (E, n=6; **** p<0.0001) repeats; n=independent experiments; unpaired t test; mean ± SEM.
Figure 6A, Western blot of BT463NS showing phosphorylation of ATM (pATM) and Chk2 (pChk2), and accumulation of RAD51 at the indicated time points after IR in the absence (5 Gy) or in the presence (5 Gy + HGF) of HGF (10 ng/ml). Total ATM is also shown. H3 was used as loading control. B, Western blot of BT308NS showing phosphorylation of ATM (pATM) and Chk2 (pChk2), and accumulation of RAD51 at the indicated time points after IR in the absence (5 Gy) or in the presence (5 Gy + JNJ) of the MET inhibitor JNJ38877605 (500 nM). Total ATM is also shown. β-actin was used as loading control. Veh: non-irradiated vehicle-treated cells. C, Densitometric analysis of ATM phosphorylation shown in (B). *: t-test, p<0.01. D, Western blot of BT308NS showing phosphorylation of ATM, Chk2, and AKT (pAKT), and accumulation of RAD51 24 h after IR (5 Gy) in the absence (vehicle, veh) or in the presence of MAPK (PD98059, 20 M), AKT (Ly294002, 20 M), MET (JNJ38877605), or ATM (CGK733, 10 M) inhibitors. Total ATM and AKT are also shown. β-actin was used as loading control. Ctrl: non-irradiated cells. E, Western blot of BT463NS showing phosphorylation of ATM, Chk2, and Aurora Kinase A (pAurora A) at the indicated time points after IR in the absence (5 Gy) or in the presence (5 Gy + Ly) of the AKT inhibitor Ly294002. Total ATM and Aurora Kinase A are also shown. H3 was used as loading control. Veh: non-irradiated vehicle-treated cells. F, Western blot of BT308NS showing phosphorylation of Aurora Kinase A at the indicated time points after IR in the absence (5 Gy) or in the presence (5 Gy + JNJ) of JNJ38877605. Total Aurora kinase A is also shown. H3 was used as loading control. Veh: non-irradiated vehicle-treated cells. G, Western blot of BT463NS showing phosphorylation of Aurora Kinase A at the indicated time points after IR in the absence (5 Gy) or in the presence (5 Gy + HGF) of HGF. Total Aurora kinase A is also shown. H3 was used as loading control (the same control applies to both panel A and G). H, Western blot of BT308NS showing phosphorylation of ATM and Aurora Kinase A at the indicated time points after IR in the absence (5 Gy) or in the presence (5 Gy + MLN) of the Aurora Kinase A inhibitor MLN8237 (20 M). Total ATM and Aurora kinase A are also shown. H3 was used as loading control. Veh: non-irradiated vehicle-treated cells. I, Cell viability of BT308NS, measured 72 h after IR (5 Gy) in the absence (vehicle) or in the presence of MET (JNJ38877605), Aurora Kinase A (MLN8237), or AKT (Ly294002) inhibitors (fold vs. non-irradiated vehicle-treated cells, mock). *: t-test (5 Gy + inhibitor) vs. (5 Gy + vehicle), p<0.01.
Mfn2 loss‐of‐function decreases autophagy in response to ER stress.Figure 3(A, upper panel) WT (black circles) or Mfn2 KO cells (white circles) were treated with 1 μM Tg for a range of times. (A, lower) Densitometric quantification. Data are mean±s.e.m. (n=3). *P0.05 versus WT group; #P0.05 versus WT non‐Tg‐treated group. (B, upper panel) WT or Mfn2 KO cells were treated with Tg for 0, 6 or 12 h, in the presence or absence of Bafilomycin (Baf, 100 nM). LC3b‐I and LC3b‐II expression was measured by western blot. (B, lower panel) Densitometric quantification of LC3b‐II levels (relative to tubulin). Data are mean±s.e.m. (n=3). *P0.05 versus WT+Baf+Tg group. (C) WT or Mfn2 KO cells stably expressing mCherry‐GFP‐LC3b were treated with 1 μM Tg, 100 ng/ml Brefeldin (Bref), or 0.5 μg/ml tunicamycin (Tm) for 24 h and examined by confocal microscopy. Scale bar: 10 μm. (D) Quantification of mCherry+/GFP‐ (mCherry‐LC3) red puncta per cell (a measure of acidic autophagosomes) is shown. Data are mean±s.e.m. (n=3; 100 cells were analyzed per experiment and group). *P0.05 versus WT group. (E) Acidic compartments of WT or Mfn2 KO cells were stained with Lysotracker Green and analyzed by flow cytometry. Data are given as mean±s.e.m. (n=3). *P0.05 versus WT group. (F) LAMP1 expression was immunodetected in WT and Mfn2 KO cells in basal conditions. (G) Expression of Beclin‐1 or LC3b transcripts in WT or Mfn2 KO cells treated with or without 1 μM Tg for 24 h. Data are mean±s.e.m. (n=3). *P0.05 versus WT group.Source data for this figure is available on the online supplementary information page.
Figure 5(A) Colony formation of WT and TM induced Gata6 iKO keratinocytes stained with hematoxylin. Primary keratinocytes were isolated from induced iKO newborn mice, plated on feeder cells, and stained with hematoxylin at day 7 after plating (top). Phase contrast images of cells are shown below. Scale bars: 30 m. (B) Quantification of colony formation from (A), average SD from n=3 mice in each genotype. (C) Western blot of WT and iKO cultured keratinocytes after 0 or 6 hours of tamoxifen induction, blotted with actin and Gata6 antibodies, show a decrease in Gata6 protein levels after 6 hours. (D) Phase contrast images of WT and Gata6 iKO keratinocytes 3 days after induction with TM (n > 3 per genotype). (E-H) Images and quantification of DNA damage in cultured keratinocytes based on staining for DNA damage marker H2A.X (E and G) and comet assay (F and H). Arrowheads in (F) indicate DNA damaged cell nuclei with comet shape. Quantification of H2A.X (G) represents average SD from at least 300 cells per condition from 3 experiments using different cell lines . *Unpaired t-test P-values: 6h=0.01 and 24h=0.004. (H) Quantification of comet assay represents average SD (n=3). (I-J) FACS cell cycle profiles and quantification show that Gata6 iKO keratinocytes show decreased proliferation and G2-M arrest. (I-J) FACS cell cycle profiles and quantification show that Gata6 iKO keratinocytes show decreased proliferation and G2-M arrest. (J) Quantification of FACS cell cycle analysis represent average SD (n=3 per genotype). *Unpaired t-test P-values: G1 = 0.03, S = 5x10-4, G2-M = 7x10-4. (K) Quantification of the frequency of H2A.X+ staining in keratinocytes within S phase (EdU+) or another cell cycle stage (EdU-) following a 20 minute EdU pulse labeling (average SD, n=5). *Unpaired t-test P-values WT=0.1, iKO= 0.05. Mann-Whitney U test P-values: WT=0.09, iKO=0.03. (L-M) Cultured keratinocytes were made quiescent by gradual serum depletion. (L) Quantification of EdU+ cells in proliferative and serum depleted quiescent cells (average SD, n=4). (M) Quantification of H2A.X+ keratinocytes during proliferative and quiescent conditions (average SD, n=4). *Unpaired t-test P-value = 0.04. Mann-Whitney U test P-value=0.06.
Loss of keratin results in defective ExE region expansion.Figure 4A. Z-projections of whole-mount C57BL/6J wild-type (WT, Left) and KtyII-/- (Right) embryos at LB stage stained for vimentin (green) and F-actin (Phalloidin, white). B. Sagittal sections of C57BL/6J wild-type and KtyII-/- E7.5 embryos immunostained for Krt8 (n= 15 and n=5, respectively), F-actin (n=11 and 2), Vimentin (n=5 and 1), E-cadherin (n=1 and 1), T (n=4 and 3), Dsg2 (n=15 and 5) and Pecam-1 (n=4 and 3). Data information: White arrows point toward allantois and scale bars represent 25 µm. C. Scheme for measurements. Quantification of embryo morphology at LB stage in C57BL/6J wild-type (white boxes/ black dots, n=22 including 16 in which allantois could be measured), and KtyII-/- embryos (grey boxes/ black triangles, n=9 including 8 in which allantois could be measured). (D) Embryonic region length. Ratios of whole embryo and ExE cavity lengths (E) , over embryonic region length. Quantification of embryo morphology at LB stage in C57BL/6J wild-type (white boxes/ black dots, n=22 including 16 in which allantois could be measured), and KtyII-/- embryos (grey boxes/ black triangles, n=9 including 8 in which allantois could be measured). allantois length and width (F) Quantification of embryo morphology at LB stage in C57BL/6J wild-type (white boxes/ black dots, n=22 including 16 in which allantois could be measured), and KtyII-/- embryos (grey boxes/ black triangles, n=9 including 8 in which allantois could be measured). amnion length and thickness (G) H. Z-projections of C57BL/6J wild-type (Top) and KtyII-/- (Bottom) embryos at LB stage stained for F-actin (Phalloidin, white). Amnion is delimited by yellow dashed lines. Scale bar represents 25 µm. I-I''. SEM images of opened C57BL/6J wild-type (n=3) and KtyII-/- (n=2) LB stage embryos with zooms on the allantois region (I'-I''). Scale bars represent 200 (I), 20 (I') and 10 (I'') µm.