EMBO/SourceData huggingface dataset:
Figure 3A COS‐1 cells were treated with EHD1 siRNAs for 72 h. Cell lysates were prepared and then immunoblotted with EHD1 antibody. α‐tubulin was used as a loading control.B, C Cells were treated with EHD1 siRNA1 for 72 h. Cells were pulsed with Alexa 488‐Tfn and chased in medium with unlabeled Tfn. During the chase, cells were imaged every 3 min for 57 min. In (B), representative images of Alexa 488‐Tfn in control or EHD1‐depleted cells are shown. In (C), the fluorescence intensity of Alexa 488‐Tfn is shown as percentage of that at 0 min (mean ± SEM, n > 3 cells from three independent experiments). ***P 0.001; two‐tailed Student's t‐test. Scale bars, 10 μm.B, C Cells were treated with EHD1 siRNA1 for 72 h. Cells were pulsed with Alexa 488‐Tfn and chased in medium with unlabeled Tfn. During the chase, cells were imaged every 3 min for 57 min. In (B), representative images of Alexa 488‐Tfn in control or EHD1‐depleted cells are shown. In (C), the fluorescence intensity of Alexa 488‐Tfn is shown as percentage of that at 0 min (mean ± SEM, n > 3 cells from three independent experiments). ***P 0.001; two‐tailed Student's t‐test. Scale bars, 10 μm.D COS‐1 cells were treated with EHD1 siRNA1 for 72 h. Cells were pulsed with Alexa 594‐CTxB for 5 min and chased for 60 min. Cells were then fixed and stained for GM130. The Pearson's coefficient between CTxB and GM130 is shown (mean ± SD, n > 30 cells from two independent experiments). ***P 0.001; two‐tailed Student's t‐test. See Supplementary Fig S7A for the representative images.E COS‐1 cells were treated with EHD1 siRNA1 for 72 h and stained for TfnR. The image represents the maximum intensity z‐projection of four confocal slices. Scale bar, 10 μm.F COS‐1 cells co‐transfected with GFP‐ATP8A1 and myc‐CDC50A were stained for EHD1. Scale bar, 10 μm.G Cells were treated with ATP8A1 siRNA1 for 72 h and stained for EHD1. For the rescue experiments, siRNA‐resistant GFP‐ATP8A1 (WT or E191Q) and myc‐CDC50A were transfected 48 h after siRNA transfection. Asterisks indicate cells with GFP‐ATP8A1 expression. The graph represents percentage of cells with perinuclear localization of EHD1 as mean ± SD of three independent experiments (at least 141 cells were counted for each condition). Scale bars, 10 μm.
Figure 4A The effect of various lipids on the ATPase activity of the purified ATP8A1‐CDC50A complex. The ATPase activity was measured in the presence of 5 mM ATP and 10 mol % of one of the following lipids: 1,2‐dioleoyl‐phosphatidycholine (PC), 1,2‐dioleoyl‐PS, 1,2‐dioleoyl‐PE, 1,2‐dioleoyl‐phosphatidylglycerol (PG), 1,2‐dioleoyl‐phosphatidylinositol (PI), 1,2‐dioleoyl‐phosphatidic acid (PA), sphingomyelin (SM), cholesterol (Chol), or brain polar lipid (BPL) and 90% PC. The activity of the ATP8A1 (E191Q)‐CDC50A in the presence of 1,2‐dioleoyl‐PS was also determined.B NBD‐labeled PS or PE transport (flippase) activity of ATP8A1 (WT and E191Q)‐CDC50A complex. The percentage of NBD‐lipid flipped was shown.D, E PSA‐3 and R‐41 cells were transiently transfected with GFP‐EHD1 and then cultured under the indicated conditions for 72 h. Cells were then fixed, and the nuclei were strained with DAPI. Arrowheads indicate the perinuclear RE localization of GFP‐EHD1. Scale bars, 10 μm.
Figure 5A His‐tagged EHD1 was mixed with PC‐based liposomes containing the indicated % (mol/mol) of PS or PE. After 15 min, the mixture was spun at 100,000 g for 30 min, and the resultant supernatant (S) and pellet (P) were subjected to SDS-PAGE. The proteins and lipids were then stained with Coomassie blue.B, C The intensities of individual bands in (A) were quantified with ImageJ, and the percentage of bound proteins was calculated. In (B), the data represent the mean ± SD of n = 3 independent experiments and were analyzed with one‐way analysis of variance (ANOVA) followed by Tukey-Kramer post hoc test. *P 0.01; NS, not significant. In (C), each point represents the mean of two independent experiments with an error bar showing the range of values obtained. The sigmoidal fitting was applied.D, E His‐tagged EHD1 was mixed with PC‐based liposomes (20, 40, or 60% PS) of decreasing size produced by extrusion through filter pores of the size indicated. After 15 min, the mixture was spun at 100,000 g for 30 min, and the resultant supernatant (S) and pellet (P) were subjected to SDS-PAGE. The proteins and lipids were then stained with Coomassie blue. In (E), the intensities of individual bands were quantified (mean ± SEM of three independent experiments). See Supplementary Fig S8 for the size distributions of the prepared liposomes.
Figure 6A His‐tagged 2xPH was mixed with liposomes that were composed of 75% (mol/mol) PE, 20% (mol/mol) PC, and 5% (mol/mol) of PS, PA, PI, PIPs, or sulfatide. In the case of PE/PC, liposomes of 80% (mol/mol) PE and 20% (mol/mol) PC were used. After 15 min, the mixture was spun at 100,000 g for 30 min, and the resultant supernatant (S) and pellet (P) were subjected to SDS-PAGE. The gels were stained with Coomassie blue.B COS‐1 cells were incubated in medium containing Alexa 488‐Tfn for 40 min, washed, fixed, and stained with recombinant 2xPH. Magnified image is shown in the right panel. Scale bar, 10 μm.C COS‐1 cells were transiently transfected with GFP‐lact‐C2 and stained for Rab11. Scale bar, 10 μm.D, E COS‐1 cells were transiently transfected with GFP‐lact‐C2 and stained with recombinant 2xPH and anti‐GM130 antibody. The asterisk indicates a cell expressing GFP‐lact‐C2, and the arrowhead indicates a non‐expressing cell. The graph in (E) shows fluorescence intensity per pixel of recombinant 2xPH stain in RE area, which was delimited by GM130. Data are normalized to the 2xPH intensity in GFP‐lact‐C2 (−) cells (mean ± SD, n = 13 cells for each condition). Scale bar, 10 μm.F-H COS‐1 cells stably expressing GFP‐lact‐C2 were treated with ATP8A1 siRNA1 or EHD1 siRNA1 for 72 h and stained with recombinant 2xPH and anti‐GM130 antibody. The intensities of 2xPH staining and GFP‐lact‐C2 in RE area were measured. The intensity of 2xPH (G) and the ratio of 2xPH to GFP‐lact‐C2 intensities (H) are shown. In (H), data are normalized to that of control cells. Data represent mean ± SD and were analyzed with two‐tailed Student's t‐test (n > 26 cells from three independent experiments). **P 0.01; ***P 0.001; NS, not significant. Scale bars, 10 μm.
Figure 7A COS‐1 cells co‐transfected with GFP‐ATP8A2 (WT) and myc‐CDC50A were stained for myc‐tag and TfnR. Scale bar, 10 μm.B The effect of increasing levels of PS on the ATPase activity of ATP8A2 (WT and I376M)‐CDC50A complex.C NBD‐labeled PS transport (flippase) activity of ATP8A2 (WT and I376M)‐CDC50A complex. The percentage of NBD‐lipid flipped was shown.D Cells were treated with ATP8A1 siRNA1 for 72 h. ATP8A2‐1D4 (WT or mutants) and myc‐CDC50A were transfected 48 h after siRNA transfection. The cells were then stained for EHD1 and 1D4‐tag. Scale bars, 10 μm.E Quantification of percentages of cells with perinuclear EHD1 localization. Data represent mean ± SD of three or four independent experiments. At least 20 cells were counted per experiment.F Cells were treated with ATP8A1 siRNA for 72 h. GFP‐ATP8A2 (WT or mutant) and myc‐CDC50A were transfected 48 h after siRNA transfection. The cells were then stained for TfnR. Data represent mean ± SD of three or four independent experiments. At least 20 cells were counted per experiment.G, H Surface biotinylation assay with Atp8a2+/+ or Atp8a2−/− cortical neurons. The ratio of surface to total TfnR was quantified and normalized to Atp8a2+/+. Data represent mean value (2 mice for each genotype).
Figure 1c and d. Representation and quantification of cell length in epidermal (e) and cortical (c) cell files. Optical, longitudinal sections of 5 DAG old Col-0 roots 12 HAT to ammonium (C) or nitrate (D) supplemented media. The first 20 epidermal (e) and cortex (c) cells (from quiescent center (QC)) are highlighted in grey and in red on ammonium (C), and in blue and green on nitrate (D), respectively. Scale bar=30 µm. Column bars denote the geometric mean of the cell lengths at the respective positions. Lines represent a polynomial regression fit, with calculated slopes between cells 10 and 20 of 3.32639+0.17172 (ammonium, epidermis), 1.22033+0.08754 (ammonium, cortex) and 1.70502+0.09532 (nitrate, epidermis), 0.82342+0.06973 (nitrate, cortex). Data are derived from 3 biological replicates, n=18 roots in each case.
Figure 2a and b. Maximum intensity Z-stack projection images of 5 DAG old roots expressing the R2D2 auxin signaling reporter 12 HAT to ammonium (a) or nitrate (b) supplemented media. White arrows mark the position of the 10th and 20th cells from QC; "e" and "c" mark epidermis and cortex, respectively. Scale bar=50 µm. Graphs denote normalized relative auxin levels at the respective positions. Lines represent polynomial regression fit with 95% confidence band. Data are derived from 5 roots per condition from 3 biological replicates.
Figure 3b and c. Representation and quantification of cell length in epidermal (e) and cortical (c) cell files. Optical, longitudinal sections of 5 DAG eir1-4 roots 12 HAT to ammonium (B) or nitrate (C) supplemented media. The first 20-20 epidermal and cortex cells (from quiescent center (QC)) are highlighted in grey and in red on ammonium (B) and in blue and green on nitrate (C), respectively. Scale bar=30 µm. Column bars denote the geometric mean of cell length at the respective positions. Lines represent a polynomial regression fit, with calculated slopes between cells 10 and 20 of 0.75884+0.02624 (ammonium, epidermis), 1.13088+0.08446 (ammonium, cortex) and 2.06912+0.10341 (nitrate, epidermis), 0.99878+0.07278 (nitrate, cortex). Data are derived from 3 biological replicates, n=9 (ammonium) and 8 (nitrate) roots.
Figure 4b. Higher magnification of pseudo-colored confocal images of 5 DAG old roots expressing PIN2-GFP 12 HAT to ammonium or nitrate supplemented media. "e" denotes epidermis and "c" cortex, respectively. Color code represents GFP intensity from low (blue) to high (white) values. Scale bar=12 µm. White stars mark PIN2-GFP protein localization on the lateral membranes. Box plots display lateralization index (fluorescent signal detected on apical/basal membranes divided by the signal value at inner/outer membranes) of roots on ammonium (n=31 cells from 6 roots) or nitrate (n=24 cells from 6 roots) supplemented medium. The statistical significance was evaluated with ANOVA at p<0.05. The box chart components are defined as; box (25%-75%), central band (median line), central box (mean) and the range is within 1.5IQR.d. Microscopic images showing PIN2-Dendra fluorescent signal five hours after photoconversion of PIN2-Dendra into its red form. Depletion of the red signal and recovery of the green signal over a 6 hours period was followed in parallel in 5 DAG old roots12 HAT to ammonium or nitrate supplemented media. Note the increase in the intensity of the green signal in roots transferred to nitrate. Graph represents the mean signal + SD (n=6 roots per condition, 20 cells per root analyzed). The experiment was repeated 3 times. Scale bar=20 µm.
Figure 5d. Box plots display the distribution of the cell membrane derived PIN2-GFP fluorescence intensity (FI) values (in arbitrary units, a.u.) in roots transferred to ammonium ((grey, epidermis (ep) and red, cortex (co) and to nitrate (blue, epidermis (ep) and green, cortex (co)). 5 cells per roots were analyzed in at least 9 roots per genotype per treatment. The statistical significance was evaluated with ANOVA at p<0.05. The box chart components are defined as; box (25%-75%), central band (median line), central box (mean) and the range is within 1.5IQR.g. Box plots display lateralization index (fluorescent signal detected on apical/basal membranes vs inner/outer membranes) of P2wt, P2D and P2A roots transferred to ammonium (grey) or nitrate (red) supplemented medium. At least 24 cells from 5 roots were analyzed per genotype per treatment. The statistical significance was evaluated with ANOVA at p<0.05. The box chart components are defined as; box (25%-75%), central band (median line), central box (mean) and the range is within 1.5IQR.
Figure 1Immunofluorescence staining of RANKL during in vitro adipogenesis of BMSCs from AdipoqCre; ranklfl/fl mice (Rankl-/-) and ranklfl/fl mice (Rankl+/+). Scale bar=50 μm.
Figure 2Micro-CT and statistical analyses of BMD, BV/TV, Tb.N and Tb.Sp in trabecular bone from AdipoqCre; ranklfl/fl (Rankl-/-) and ranklfl/fl (Rankl+/+) mice at 4 weeks, 8 weeks and 16 weeks (n=5 independent biological replicates). Scale bar=1 mm. Data were compared using an unpaired t-test (** indicates P < 0.01), error bars are standard deviations.
Figure 3Representative images of TRAP staining of femur from AdipoqCre; ranklfl/fl (Rankl-/-) and ranklfl/fl (Rankl+/+) mice at 8 weeks. Scale bar=200 μm. Panel a1 and a4 indicate trabecular bone, a2 and a5 stand for growth plate, a3 and a6 denote cortical bone.Immunofluorescence staining of osteocalcin from 8-week-old mice (n=6 mice in Rankl-/- group and 4 mice in Rankl+/+ group). Scale bar=50 μm. TB: trabecular bone. Statistical analyses of Ocn+ cells (n=6 independent microscopic vision fields from random mice samples). Data were compared using an unpaired t‐test (** indicates P < 0.01), error bars are standard deviations.Calcein staining of trabecular bone at 8 weeks. Scale bar=50 μm. TB: trabecular bone. Images are representative of 6 independent biological replicates in Rankl-/- and 5 replicates in Rankl+/+ group. Mineral apposition rate (MAR) of trabecular bone at 8 weeks.(n=6 mice in Rankl-/- group and 5 mice in Rankl+/+ group). Data were compared using an unpaired t‐test ( ** indicates P < 0.01), error bars are standard deviations.Serum CTX-1, OCN and bone marrow RANKL levels from Rankl-/- and Rankl+/+ mice at 4 weeks (n=5 and 5), 8 weeks (n=5 and 6) and 16 weeks (n=5 and 5). Data were compared using an unpaired t‐test (* indicates P < 0.05, ** indicates P < 0.01), error bars are standard deviations..
Figure 4Micro-CT analyses of trabecular bone from Grounded and Tail suspended model (n=5 independent biological replicates). Scale bar=1 mm. Data were compared using an unpaired t‐test (* indicates P < 0.05, ** indicates P < 0.01), error bars are standard deviations.
Figure 5Micro-CT analyses of sham and OVX model of AdipoqCre; ranklfl/fl (Rankl-/-) and ranklfl/fl (Rankl+/+) mice (n=5 independent biological replicates). Scale bar=1 mm. Data were compared using an unpaired t‐test (** indicates P < 0.01), error bars are standard deviations.TRAP staining and statistical analyses of numbers of osteoclasts in Sham and OVX model (Images are representative of 5 independent biological replicates in (C), and n=5 independent microscopic vision fields in (D)). Scale bar=100 µm. Data were compared using an unpaired t‐test (** indicates P < 0.01), error bars are standard deviations.Immunofluorescence staining of OCN and statistical analyses of the number of osteoblasts (Images are representative of 5 independent biological replicates in (E), and n=5 independent microscopic vision fields in (F)). Scale bar=50 µm. TB: trabecular bone. Data were compared using an unpaired t‐test (** indicates P < 0.01), error bars are standard deviations.
Figure 6Three-dimensional CT analyses of BMD, BV/TV, Tb.N, and Tb.Sp in trabecular bone from Vehicle (n=5 independent biological replicates) and ROS (n=6 independent biological replicates) model of AdipoqCre; ranklfl/fl (Rankl-/-) and ranklfl/fl (Rankl+/+) mice. Scale bar=1 mm. Data were compared using an unpaired t‐test (** indicates P < 0.01), error bars are standard deviations.  TRAP staining and statistical analyses of numbers of osteoclasts in the trabecular bone from Vehicle (n=6 and 5 independent biological replicates) and ROS (n=6 and 6 independent biological replicates) treatment. Scale bar=100 µm. Data were compared using an unpaired t‐test (** indicates P < 0.01), error bars are standard deviations. n=5 independent microscopic vision fields in (D).OCN staining and statistical analyses of the number of osteoblasts in femurs from Vehicle (n=6 and 5 independent biological replicates) and ROS (n=6 and 6 independent biological replicates) treatment. Scale bar=50 µm. TB: trabecular bone. Data were compared using an unpaired t‐test (** indicates P < 0.01), error bars are standard deviations. n=5 independent microscopic vision fields in (F).
Figure 1(B) Numbers of surface tumors per liver were counted. (C) The cumulative diameter of all surface tumors per liver per mouse reflects the overall tumor load. (D) Weight of the livers including tumor mass relative to the body weight (LW/BW ratio) was recorded.(E) Representative pictures of total livers with tumors of the indicated genotypes.
Figure 2(A) Immunoblots of murine wt non-tumorous and tumor tissue lysates were probed for CASP2, RAIDD, and p53. HSP90 and PonceauS protein staining served as loading controls.(B) Proliferation of tumor tissue was determined by the percentage of Ki67-positive nuclei analyzed by flow cytometry. T: wt n=17, Casp-2-/- n=11, Raidd-/- n=10, Pidd1-/- n=8; NT: wt n=12, Casp-2-/- n=11, Raidd-/- n=7, Pidd1-/- n=5. N-numbers refer to biological replicates.(D) Immunoblots of the experiment described in (C) at t(8h), t(48h), and t(weaning+5d) for CASP2, RAIDD, p53, γH2AX, and HSP90 as loading control.(E) The number of binucleated hepatocytes per field was counted on H&E stained paraffin sections of PBS treated wt and Pidd1-/- mice at t(0h) and at t(48h) after PBS or DEN treatment. N-numbers refer to biological replicates.(F) Immunofluorescence of the same livers was perfomed for the distal centriolar protein CP110 to stain centrioles, β-catenin for cell borders, and DAPI to stain DNA. Representative images of a mononucleated hepatocyte with 2 centrioles and a binucleated hepatocyte with 4 centrioles is shown.(G) Centriole number was quantified with respect to the number of nuclei per cell in wt and Pidd1-/- livers at the time points described in (E); n=3 biological replicates.
Figure 3(A) Binucleation was assessed blinded in H&E stained sections of murine non-tumor (NT) and tumor tissue (T) T: wt n=14, Casp-2-/- n=11, Raidd-/- n=10, Pidd1-/- n=10; NT: wt n=8, Casp-2-/- n=10, Raidd-/- n=8, Pidd1-/- n=9. N-numbers refer to biological replicates.Binucleation was assessed blinded in H&E stained sections of murine non-tumor (NT) and tumor tissue (T) also shown in (B). T: wt n=14, Casp-2-/- n=11, Raidd-/- n=10, Pidd1-/- n=10; NT: wt n=8, Casp-2-/- n=10, Raidd-/- n=8, Pidd1-/- n=9. N-numbers refer to biological replicates.C, D (C) Representative histograms of nuclei stained for DNA, which were isolated from non-tumor and tumor tissue of wt and Casp2-/- mice. These animals are marked as red "x" in the quantification shown in (D) were the degree of polyploidy is reflected as percentage of octaploid nuclei (T: wt n=17, Casp-2-/- n=11, Raidd-/- n=10, Pidd1-/- n=8; NT: wt n=12, Casp-2-/- n=11, Raidd-/- n=7, Pidd1-/- n=5). N-numbers refer to biological replicates.
Figure 4Copy number variations were determined using whole genome sequencing of 30 nuclei "mini-bulks" isolated from Casp2-/- or wt tumors (n=11 per genotype). Diploid and tetraploid pools of tumor cell nuclei were analyzed separately. A, B (A) Representative dot plots show the degree of aneuploidy and heterogeneity and are quantified in (B, empty symbols). Data are represented as median, statistical significance determined by one-way ANOVA with multiplicity correction (Sidak-Holm); * p≤0.05, ** p≤0.01. N-numbers refer to biological replicates.
Figure 5(A) Matched human patient non-tumor (NT) and tumor tissue (T) samples were analyzed by immunoblotting for CASP2, PIDD1 and RAIDD on membranes a and b. PonceauS protein staining serves as loading control. The asterisk refers to an unspecific band, "C" and "CC" indicate the functionally active fragments of PIDD1 generated by autoprocessing, PIDD-C and PIDD-CC. Densitometric quantification of these blots is shown in Fig. EV4A.(B) mRNA expression analysis of the PIDDosome components in the TCGA Provisional LIHC data set across disease stages. Patients are divided based on the p53 mutation/deletion (mut/del) status. Statistical significance was determined using two-way ANOVA with Tukey's post-hoc analysis. The central band represents the median, the boxes indicate the 25th and 75th percentile, the whiskers are the 5th and 95th percentiles, statistical significance was defined as * p≤0.05, ** p≤0.01.(C) The same data set was analyzed for recurrence-free survival based on high or low expression of CASP2, RAIDD or PIDD1 divided at the median. Statistical significance was determined using a Log-rank test.
Figure 6(B) E2F target genes expression was analyzed for correlation with expression of CASP2, RAIDD and PIDD1 using Spearman's correlation coefficient. The central band represents the median, the boxes indicate the 2nd and 3rd quartile, and the whiskers are 1.5 times the interquartile range (n=200 genes).Correlation (Pearson) of CASP2 mRNA with transcript levels of Ki67 in the TCGA data set.Correlation (Pearson) of RAIDD and PIDD1 mRNA with transcript levels of Ki67 in the TCGA data set.
Figure 7(A) The ploidy index is inferred as 1/cell density of tumorous (T) and non-tumorous (NT) tissue of HCC patients with different liver diseases. ALD - alcoholic liver disease (n=65); HBV - hepatitis B virus infection (n=24); HCV - hepatitis C virus infection (n=85); NASH - non-alcoholic steatohepatitis (n=33). Statistical significance was determined by one-way ANOVA with multiplicity correction (Sidak-Holm); * p≤0.05, ** p≤0.01, *** p≤0.001. n-numbers refer to the number of patient samples analyzed.(B) All patients were grouped according to HCC tumor cell density as surrogate for tumor ploidy. Data were divided at the median and exact patient numbers are shown in the graphs. Median recurrence-free survival (RFS) for a period of 10 years for patients with low tumor ploidy was 4.8 years. For patients with high tumor ploidy the median RFS could not be defined as more than 50% of the patients survived without relapse. Patient characteristics are shown in Appendix table S1. A Log-rank test was used to determine statistical significance.
Figure 1A Baseline blood counts of granulocytes. One-way ANOVA; n = 8 mice per ZT; *P = 0.0001 ZT5 vs. ZT13.B Flow cytometry quantification of neutrophils in digested hearts. The representative dot plots show the gating strategy for cardiacneutrophils (CD45+Ly6G+CD11b+) at ZT5. One-way ANOVA; n = 5 mice per ZT; *P = 0.0001 ZT5 vs. ZT13.C Representative immunostainings for neutrophils in the myocardium (left ventricle), identified as Ly6G-positive (20x magnification).D Cardiac mRNA expression levels normalized to HPRT. One-way ANOVA; n = 5 mice per ZT; ZT5 vs. ZT17: *P = 0.0064 (CXCL1), *P = 0.0007 (CXCL2), *P = 0.0007 (CXCL5), *P = 0.0001 (ICAM-1), *P = 0.0009 (VCAM-1), *P = 0.0181 (CCL3), *P = 0.0360 (CCL5).E Mean fluorescence intensity (MFI) of CXCR2 expression by neutrophils in blood and heart. One-way ANOVA; n = 3 mice for ZT1, ZT17, ZT21 and n = 5 for ZT5, ZT9, ZT13; ZT5 vs. ZT13: *P = 0.0425 (blood), *P = 0.0078 (heart).