EMBO/SourceData huggingface dataset:
Figure 7Loss of cyclin F induced E2F7/8-dependent Homologous Recombination deficiency. HeLa cells that were stably transformed with pDR-GFP were transfected with siRNA as indicated.48h after the initial transfection, cells were harvested for Flow-cytometry. GFP positive cells were gated (Fig EV5A). Relative HR efficiency were adjusted to the scramble siRNA condition. Data represent averages ± SEM (n=3); **P<0.01 (Student's t-test). n.s.: not significant.Loss of cyclin F induced E2F7/8-dependent γ-H2AX accumulation. HeLa cells were transfected with indicated siRNA for 24 hours, and then treated with nocodazole for 16 hours before fixation for immunofluorescence staining of γ-H2AX. DAPI was used to stain the cell nucleus. Relative intensity of γ-H2AX was quantified by Image J software, and 150 cells were quantified for each condition. Red bars represent averages; **P<0.01 (Student's t-test). n.s.: not significant. Scale bar 20 μm.Loss of cyclin F increased DNA damage recovery time before cell division. RPE cells integrated with the 53BP1 construct were transfected with indicated siRNA for 24 hours, and then treated with HU for 16 hours. At the beginning of the imaging, only the single cells with at least one 53BP1 foci were traced, till the time frame that no 53BP1 foci was observed. The mitotic progression of the cells was defined as the duration from damage recovery to cell division. Histogram shows the damage recovery time (green) and the mitotic progression (black) of 50 cells for each condition. Chi-square analysis was performed to test the statistical significance (P<0.01).Knockdown of cyclin F caused a delay in DNA lesion recovery that is dependent on E2F7/8. The cumulative curves represent the add-up number of cells that overcome the DNA damage lesions, for a time frame of 24 hours. For each condition, 50 cells were quantified. Log-rank tests were performed to analyze the statistical significance.
Figure 1Wild-type and LGP2-KO HEK293 cells in 24-well plates were transfected with 200 ng/ml of short poly I:C IFN-β and IP-10 mRNA expression was determined by RT-qPCR. The data represent the mean ± SD (n = 3, t-test, *p < 0.05). LGP2 KO2 and KO3 are independently isolated LGP2 KO clones. Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.Wild-type and LGP2-KO HEK293 cells in 24-well plates were infected with SeV. IFN-β and IP-10 mRNA expression was determined by RT-qPCR. The data represent the mean ± SD (n = 3, t-test, *p < 0.05). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.(F) HEK293 cells in 24-well plates were transfected with LGP2 (0.1, 0.3, or 0.6 μg/well) and RIG-I (0.1 μg/well) expression vectors as indicated. 24 h after transfection, cells were stimulated with 200 ng of short poly I:C for 4 hr. Total RNAs were extracted, and the expression of IFN-β mRNA was determined by RT-qPCR and normalized to GAPDH (n = 3, t-test, *p < 0.05). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.(G) HEK293FT cells were transfected with FLAG-tagged LGP2 and HA-tagged ubiquitin expression vectors. Whole cell extracts (WCE) were prepared 24 h after transfection. Immunoprecipitation was performed with anti-FLAG antibody (IP: FLAG) and the proteins were detected by western blot analysis with the indicated antibodies.(H) FLAG-tagged LGP2 was transfected into HEK293 cells. Cells were then infected with SeV at MOI = 10 for 24 h. Immunoprecipitation was performed with anti-FLAG antibody, and the proteins were detected with the indicated antibodies.
Figure 2A) HEK293 cells were transfected with FLAG-RIG-I or FLAG-LGP2 expressing vector. Cells were then infected with SeV, and whole cell extracts were collected at indicated time points. Immunoprecipitation was performed with anti-FLAG antibody. The proteins were subjected to SDS-PAGE and detected by western blotting.B) A549 cells transfected with HA-tagged ubiquitin were stimulated with 200 ng/ml of short poly I:C for 6 h. PLA was performed with anti-HA, anti-RIG-I, and anti-LGP2 antibodies. The number of PLA signals were counted (t-test, *p < 0.05, n = 20). Each dot represents the number of PLA signals of each cell. Data information: Scale bars represent 10 μm. All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.C) HEK293 cells were transfected with empty and LGP2 expression vectors for 24 hr. PLA was performed with anti-LGP2 and anti-K63-Ub antibodies. The number of PLA signals were counted (t-test, *p < 0.05, n = 33). Each dot represents the number of PLA signals of each cell. Data information: Scale bars represent 10 μm. All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.D-F) HEK293 cells were transfected with 200 ng/ml of short poly I:C. Cells were fixed at the indicated time points, and PLA was performed with anti-RIG-I, anti-LGP2, and anti-K63-Ub antibodies (D). The number of PLA signals were counted (E, F) (t-test, *p < 0.05, n = 30). Each dot represents the number of PLA signals of each cell. Data information: Scale bars represent 10 μm. All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.
Figure 3A) HEK293FT cells were transfected with FLAG-tagged LGP2 and HA-tagged Riplet expression vectors. 24 h after transfection, cells were lysed, and WCE were prepared. Immunoprecipitation was performed with anti-FLAG antibody, and the proteins were detected by western blotting with the indicated antibodies.B) HEK293FT cells were transfected with FLAG-tagged LGP2 expression vector. 24 h after transfection, immunoprecipitation was performed with anti-FLAG antibody and control IgG. Immunoprecipitates were subjected to SDS-PAGE, and the proteins were detected by western blotting with indicated antibodies.C) HEK293FT cells were transfected with FLAG-tagged LGP2, Riplet, and HA-tagged ubiquitin expression vectors, and immunoprecipitation was performed with anti-FLAG antibody.D) HEK293FT cells were transfected with FLAG-tagged LGP2, Riplet, and myc-tagged K63-only-Ub expression vectors. 24 h after transfection, immunoprecipitation was performed with anti-FLAG antibody.E) WT and Riplet KO HEK293 cells were transfected with FLAG-tagged LGP2 and myc-tagged K63-only-Ub expression vectors, and immunoprecipitation was performed with anti-FLAG antibody.(F, G) WT and Riplet KO HEK293 cells were transfected with 200 ng/ml of short poly I:C for 6 h, and PLA was performed with anti-LGP2 and anti-K63-Ub antibodies. The number of PLA signals were counted (t-test, *p < 0.05, n = 100). Each dot represents the number of PLA signals in each cell. Scale bars represent 10 μm. Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.
Figure 4(A) Liquid chromatography-tandem mass spectrometry (LC-MS/MS) of LGP2 in Riplet-expressing cells. LC-MS/MS data was quantified using LFQ method. The chromatographic profiles for each ubiquitination site are shown in the right panels. Schematic representation of ubiquitination sites of RIG-I and LGP2 are shown in the upper panel.(B) FLAG-tagged LGP2 fragments and HA-tagged Riplet expression vectors were transfected into HEK293FT cells. 24 h after transfection WCE were prepared. immunoprecipitation was performed with anti-FLAG antibody. Immunoprecipitates were subjected to SDS-PAGE, and the proteins were detected by western blotting with indicated antibodies. The data is a representative of two independent experiments.(C) FLAG-tagged LGP2 and FLAG-tagged LGP2-4KR expression vectors were transfected into HEK293FT cells. 24 h after transfection, WCE were prepared. Immunoprecipitation was performed with anti-FLAG antibody. The data is a representative of two independent experiments.
Figure 6A) Microarray analysis was performed as described in materials and methods. A heatmap of the microarray data of LGP2 and LGP2-4KR stably expressing cells stimulated with short poly I:C for 12 h.B, LGP2 and LGP2-4KR stably expressing HEK293 cells were stimulated with 200 ng/ml of short poly I:C, and the expression of the genes at the indicated time points were determined by RT-qPCR and normalized to GAPDH (n = 3, two-way ANOVA, *p < 0.05). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.C) LGP2 and LGP2-4KR stably expressing HEK293 cells were stimulated with 200 ng/ml of short poly I:C, and the expression of the genes at the indicated time points were determined by RT-qPCR and normalized to GAPDH (n = 3, two-way ANOVA, *p < 0.05). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.LGP2 and LGP2-4KR stably expressing A549 cells were infected with influenza A virus (Flu) at MOI = 10, and the expression of the genes at the indicated time points were determined by RT-qPCR and normalized to GAPDH (n = 3, two-way ANOVA, *p < 0.05). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.LGP2 and LGP2-4KR stably expressing A549 cells were infected with influenza A virus (Flu) at MOI = 10, and the expression of the genes at the indicated time points were determined by RT-qPCR and normalized to GAPDH (n = 3, two-way ANOVA, *p < 0.05). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.H, I) WT and indicated mutant HEK293 cells were stimulated with 200 ng/ml of short poly I:C for 8 h. WCEs were prepared and subjected to SDS-PAGE. The proteins were detected with indicated antibodies.J, K) LGP2 and LGP2-4KR expressing vectors were transected into HeLa cells with Riplet expressing vector. 18 hr after transfection, cells were fixed, and then the PLA signals of LGP2 and endogenous MAVS were detected. Peroxisome was visualized using CellLight Peroxisome-GFP reagent. Colocalization of GFP (peroxisome) and the PLA signals (red) were observed by confocal microscopy. Scale bars represent 10 μm (J). The colocalization area was normalized to the area of the PLA signals (n = 25, t-test, *p < 0.05) (K). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.
Figure 7WT, and Riplet KO HEK293 cells were stimulated with 200 ng/ml of short poly I:C. The expression of SP100, PML, and ANKRD1 mRNA was determined by RT-qPCR and normalized to GAPDH (n = 3, two-way ANOVA, *p <0.05). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.B) WT, LGP2 KO, HEK293 cells were stimulated with 200 ng/ml of short poly I:C. The expression of SP100, PML, and ANKRD1 mRNA was determined by RT-qPCR and normalized to GAPDH (n = 3, two-way ANOVA, *p <0.05). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.C, WT and Riplet KO A549 (C) cells were stimulated with 200 ng/ml of short poly I:C. The expression of SP100 was determined by RT-qPCR (n = 3, two-way ANOVA, *p < 005). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.D) WT and Riplet KO MEF (D) cells were stimulated with 200 ng/ml of short poly I:C. The expression of SP100 was determined by RT-qPCR (n = 3, two-way ANOVA, *p < 005). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.E) WT and Riplet KO BMM were infected with influenza A virus (Flu) at MOI = 10. PML and SP100 mRNA expression was determined by RT-qPCR (n = 3, two-way ANOVA, *p < 0.05). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.F) WT and Riplet KO A549 cells were infected with influenza A virus infection at MOI = 10. WCE was prepared at the indicated time points and subjected to SDS-PAGE. Proteins were detected by western blotting with the indicated antibody.G, LGP2 or LGP2-4KR stably expressing WT or Riplet KO HEK293 cells were infected with SeV (G) IFN-β mRNA levels at indicated time points were determined by RT-qPCR. (n = 3, two-way ANOVA, *p < 0.05, ns: not significant (p > 0.05)). Right graph in panel G is an enlargement of Y-axis of the left graph (dotted line). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.H) LGP2 or LGP2-4KR stably expressing WT or Riplet KO HEK293 cells were stimulated with 200 ng/ml of short poly I:C (H). IFN-β mRNA levels at indicated time points were determined by RT-qPCR. (n = 3, two-way ANOVA, *p < 0.05, ns: not significant (p > 0.05)). Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.
Figure 1A-F (A, B, D-F) RNA levels determined by RT-qPCR. Results are presented as mean ± SD,n = 10. P-values are indicated on the top of each plot. (C) Spearman correlation suggests an in vivo positive correlation between the expressions of H19 and Igf1r in a statistically significant manner. Spearman correlation coefficient, P-values, and sample numbers are marked on the top left of the plot.
Figure 2A-D Endometrial stromal cells from patients #166 and #80 were each transfected with a mixture of siCon (control siRNA) and microRNA inhibitor control (iCon), siH19 and iCon, or siH19 and iLet7 (let-7-specific inhibitor). RNA and proteins were analyzed at 48 h post-transfection. Combined results from the two patient cells are presented.A-D Endometrial stromal cells from patients #166 and #80 were each transfected with a mixture of siCon (control siRNA) and microRNA inhibitor control (iCon), siH19 and iCon, or siH19 and iLet7 (let-7-specific inhibitor). Western blot gels from #166 cells are shown in (C). Quantitation of Western blots combining both patient cells are shown in (D).E-H Endometrial stromal cells from patients #98 and #212S were each transfected with empty vector or pH19. RNA and proteins were analyzed 48 h post-transfection. Combined results from the two patient cells are presented.E-H Endometrial stromal cells from patients #98 and #212S were each transfected with empty vector or pH19. RNA and proteins were analyzed 48 h post-transfection. Combined results from the two patient cells are presented. Western blot gels from #98 cells are shown in (G). Quantitation of Western blots combining both patient cells are shown in (H).
Figure 3A-F The indicated cells were transfected with siCon, siH19, empty vector, or pH19. RNA, cell viability (as indicated by viable cell numbers), and caspase activity (as a readout for apoptosis) were analyzed 48 h post-transfection. Combined results from two patient cells in each group are presented.G E2 stimulates H19 expression in endometrial stromal cells. Endometrial stromal cells #166 and #98 were stimulated with E2 (+) or vehicle (−) for 48 h, followed by RT-qPCR analysis of RNA extracted from the cells. Results combining two patient cells are shown.
Figure 1(B) P2 MRI images (axial, sagittal and coronal plane) showing ventriculomegaly (red asterisk), slightly thickened cortex and bilateral enlarged gyri (white asterisk).(D) FIBCD1 expression in various human visceral tissues and brain regions (inset). Expression is plotted relative to the tissue with lowest detectable expression (trachea; inset, choroid plexus). n represents technical replicates (n = 2).
Figure 2(A) Immunofluorescent images of control and neuronal (Nsyb) CG10359 (dFibcd1) RNAi-mediated knockdown D. melanogaster, 3rd instar larvae NMJ (NMJ6/7) stained with anti-horseradish peroxidase antibodies. Empty control and RNAi-mediated knockdown of CG10359 (dFibcd1-i) lines #1, 2 and 3 shown. Scale bar = 20µm. Representative images of 3 independent experiments. Quantification of (A), control and CG10359 knockdown lines NMJ neuron bouton number (B) n(empty) = 12; n(line #1) = 20; n(line #2) = 11; n(line #3) = 14. Data information: each data point represents an individual NMJ Data is represented as mean and error bars represent SEM. p values were calculated using 2-way ANOVA (panels B * = p≤0.05; ** =p≤0.01; *** =p≤0.001; **** p≤0.0001.Quantification of NMJ neuron axon branch points (C). n(empty) = 12; n(line #1) = 20; n(line #2) = 11; n(line #3) = 14. Data information: each data point represents an individual NMJ Data is represented as mean and error bars represent SEM. p values were calculated using 2-way ANOVA (panels C), * = p≤0.05; ** =p≤0.01; *** =p≤0.001; **** p≤0.0001.(E) Representative coronal section images of Golgi-Cox staining of Fibcd1 WT and KO hippocampi (left), Neurolucida tracing of hippocampal CA1 pyramidal neurons (middle) and apical dendrites with spines (right). Scale bars as indicated.(F-I) Quantifications of (F) dendritic spine density, (G) total length of apical as well as basal dendrites, (H) dendritic nodes in apical as well as basal dendrites and (I) number of dendrites (n = 5). Data information: for panels F-I, each data point represents an individual mouse. Data is represented as mean and error bars represent SEM. p values were calculated using unpaired Student's t-test (panels F-I, * = p≤0.05; ** =p≤0.01; *** =p≤0.001; **** p≤0.0001.
Figure 4(B) Immunoblot analysis (left) and quantification of signal intensity (right) of Fibcd1 WT (blue) versus Fibcd1 KO littermates (red) adult hippocampi with antibodies against CS-0S, CS-4S, CS-6S and actin as a loading control. Each lane represents an independent animal (n = 3). Protein marker sizes are indicated. Data information: For panel B, each data point represent hippocampal protein isolates from an individual mouse, Data are shown as mean values +/- SEM. p values were calculated using paired Student's t-test (panels B * = p≤0.05; ** =p≤0.01; *** =p≤0.001; **** p≤0.0001.(F) Confocal images depicting internalisation of FITC-tagged CS-4S by FIBCD1-overexpressing HEK293T lines compared to untransduced cells and unstained cells. Left, representative images; right, quantification. Data is plotted as total puncta per condition (n = 5). Cells are further stained with CellMask Orange (cellular membrane) and Hoechst (nuclei). Scale bar = 50µm. Data information: for panels F, each data point represents an individual cell preparation. Data are shown as mean values +/- SEM. p values were calculated using 1-way ANOVA (panels F, * = p≤0.05; ** =p≤0.01; *** =p≤0.001; **** p≤0.0001.
Figure 5(A) Left, representative image of immunofluorescent staining (MAP2, red; DAPI, blue) of primary hippocampal cultures at 2 days in vitro (DIV), plated on +/- CSPG coating with and without prior digestion with ChABC, as indicated. Right, quantification of DIV2 images, showing the number of protruding cells per field normalised to untreated condition. n(Fibcd1 WT) = 4; n(Fibcd1 KO) = 3. Scale bar = 250µm. (B) Left, representative images of DIV14 neurons, same conditions as in (A). Right, quantification of DIV14 images, representing the percentage of cells per field that are clumped. n(Fibcd1 WT) = 3; n(Fibcd1 KO) = 2. Scale bar = 250µm. Data information: For panels A and B each data point represents an individual preparations of primary cell culture. Data are represented as mean and error bars represent SEM. p values were calculated by 1-way ANOVA. * = p≤0.05; ** =p≤0.01; *** =p≤0.001.
Figure 1(A) Live imaging of MTOC dynamics during meiosis I. Z-projection images (top) show MTOCs (mNG-Cep192, green) and chromosomes (H2B-mCherry, magenta). Central MTOCs are magnified (squares). 3D-reconstructed spindles are aligned with the spindle axis (middle). Central MTOCs are indicated with arrowheads. Time in h:mm. The kymograph (bottom) shows projected signals on the spindle axis for all time points.(B) Density maps of MTOCs (left) and chromosomes (right) along the spindle axis in a representative oocyte. The color represents the percentage of MTOCs or chromosome volumes, respectively, coded from dark (0%) to white (50% or more of the total volume). (C) Average density maps of 21 oocytes mapped as in (B). (D) Dynamics of MTOC sorting and chromosome congression. Ratio of MTOCs (left) or chromosomes (right) in the polar region versus those in the middle region of the spindle are plotted over time.(E) Dynamics of spindle elongation. The spindle was visualized with EGFP-Map4 The length/width ratio of the spindle was measured after 3D reconstruction (n=13 oocytes from 3 independent experiments).(F) Percentage of oocytes carrying one or more MTOCs in the middle region of the spindle. Oocytes were fixed and stained for MTOCs (pericentrin, green), chromosomes (Hoechst33342, magenta) and microtubules (α-tubulin, grey). n=21, 28, 24 oocytes. A representative oocyte fixed at 5 hours after NEBD is shown. Arrowheads indicate central MTOCs.