EMBO/SourceData huggingface dataset:
Figure legend: "Depletion of RBM14 induces ectopic formation of centriolar protein complexesA-E Mitotic control U2OS cells or U2OS cells treated with RBM14 siRNA were stained with antibodies against centrin-2 (green) and C-Nap1 (magenta) (A), Centrobin (green) and C-Nap1 (magenta) (B), centrin-2 (green) and CPAP (magenta) (C), acetylated-tubulin (green) and CP110 (magenta) (D), and centrin-2 (green) and γ-tubulin (magenta) (E). DNA is shown in blue. Insets show approximately twofold magnified views of fluorescent foci around the centrosome. Scale bar, 5 μm. Histograms on the right represent frequency of mitotic cells with excess foci of the indicated proteins at spindle poles in each condition. Values are mean percentages ± standard error of mean (SEM) from three independent experiments (n = 30 for each condition). *P &lt; 0.05, **P &lt; 0.01, n.s., not significant (one-tailed t-test). Note that we counted the number of C-Nap1 foci after anaphase in mitosis when C-Nap1 signals recover from the reduction in prometaphase."
NCBI gene IDs:
"RBM14: 10432"
Figure legend: "A U2OS cells or U2OS cells expressing FLAG-RBM14 full length (FL, aa1-669), N-terminal fragment (RBM14[N], aa1-150) or C-terminal fragment (RBM14[C], aa151-669) and treated with control siRNA or siRNA against 3′ UTR targeting endogenous RBM14 were stained with antibodies against FLAG as well as centrin-2. Histograms represent frequency of mitotic cells with excess centrin foci at spindle poles in each condition. Values are mean percentages ± SEM from three independent samples (n = 30 for each condition). **P &lt; 0.01, n.s., not significant (one-tailed t-test)."
NCBI gene IDs:
"RBM14: 10432"
Figure legend: "C, D Cytoplasmic RBM14 could suppress centriole amplification in HU-treated cells. (C) U2OS cells or U2OS cells expressing FLAG-RBM14 FL or [C] and treated with or without HU were stained with antibodies against centrin-2 (green) and FLAG (magenta). (D) U2OS cells or U2OS cells expressing FLAG-RBM14 FL, FLAG-RBM14-NES or GFP-RBM14-PACT and treated with or without HU were stained with antibodies against FLAG (magenta, left) or GFP (magenta, right) as well as centrin-2 (green). Insets show approximately twofold magnified views of fluorescent foci around the centrosome. Scale bar, 10 μm. Histograms represent frequency of cells in interphase with excess centrin foci in each condition. The percentages of U2OS cells with centrosomal localization of the RBM14 full-length protein or mutants are shown below the immunofluorescence images. We counted only cells that had adequate intensity of FLAG or GFP signals and did not find any significant difference in the total expression levels of the exogenous RBM14 proteins. Values are mean percentages ± SEM from three independent experiments (n = 30 for each condition). **P &lt; 0.01 (one-tailed t-test). Please note that cytoplasmic expression levels of GFP-RBM14-PACT are less than those of FLAG-RBM14 FL (˜0.5-fold) or FLAG-RBM14-NES (˜0.7-fold)."
NCBI gene IDs:
"RBM14: 10432"
Figure legend: "D Mitotic U2OS cells treated with control or RBM14 siRNA were stained with antibodies against centrin-2 (green) and STIL (magenta). Insets show approximately twofold magnified views of fluorescent foci around the centrosome. Scale bar, 5 μm. Histograms represent frequency of mitotic cells with excess STIL foci co-localized with centrin foci. Values are mean percentages ± SEM from three independent samples (n = 30 for each condition). **P &lt; 0.01 (one-tailed t-test)."
NCBI gene IDs:
"RBM14: 10432"
Figure legend: "F Interaction between STIL and GFP-CPAP in the presence of FLAG-RBM14 FL or fragments. U2OS cells expressing GFP-CPAP were transfected with empty vector as a control, FLAG-RBM14[N], FLAG-RBM14[C] or FLAG-RBM14 FL constructs, immunoprecipitated with STIL antibodies, and the resulting IPs were analyzed by Western blotting using STIL, CPAP or FLAG antibodies. Quantification of relative protein amounts of co-immunoprecipitated GFP-CPAP in each STIL-IP fraction, normalized with the amount of STIL precipitated, is shown below the panels. Means ± SEM were calculated from three independent experiments. **P &lt; 0.01 (one-tailed t-test). Note that we did not find any significant effect of exogenous expression of RBM14 full-length protein or deletion mutants on the expression levels of GFP-CPAP or endogenous STIL proteins in this experiment."
NCBI gene IDs:
"CPAP: 55835
RBM14: 10432"
Figure legend: "G U2OS cells treated with the indicated siRNAs were stained with antibodies against centrin-2 (green) and C-Nap1 (magenta). Histograms represent frequency of mitotic cells with excess centrin foci at spindle poles in each condition. Values are mean percentages ± SEM from three independent experiments (n = 30 for each condition). **P &lt; 0.01 (one-tailed t-test). Representative mitotic cells treated with siRNAs against RBM14 alone, RBM14 + STIL or RBM14 + CPAP are shown below. Insets show approximately twofold magnified views of fluorescent foci around the centrosome. Scale bar, 5 μm."
NCBI gene IDs:
"CPAP: 55835
RBM14: 10432
STIL: 6491"
Figure legend: "A U2OS cells treated with control siRNA, RBM14 siRNA alone or RBM14 + HsSAS-6 siRNAs were stained with antibodies against centrin-2 (green) and HsSAS-6 (magenta). DNA is shown in blue. Insets show approximately twofold magnified views of fluorescent foci around the centrosome. Scale bar, 5 μm. Histograms represent frequency of mitotic cells with excess centrin foci in the indicated conditions. Values are mean percentages ± SEM from three independent experiments (n = 30 for each condition). n.s., not significant (one-tailed t-test)."
NCBI gene IDs:
"RBM14: 10432
SAS-6: 163786"
Figure legend: "B Live imaging of cycling RBM14-depleted HeLa cells expressing GFP-centrin1 (green) and RFP-H2B (magenta). Insets show approximately 1.5-fold magnified images of fluorescent foci. Time is denoted in hh:min, and scale bar is 10 μm. Time zero corresponds to the onset of excess centrin foci formation. Quantification of the number and total signal intensity of centrin foci in HeLa cells treated with control or RBM14 siRNA over time. Means ± SEM are shown (n = 5)."
NCBI gene IDs:
"RBM14: 10432"
Figure legend: "C Ectopic formation of centriolar intermediates likely occurs from late G1 to S phase. Time-lapse recording across the cell cycle of HeLa cells expressing GFP-centrin (green) and RFP-H2B (magenta) and treated with RBM14 siRNA. Time is denoted in hh:min, and scale bar is 10 μm. Time zero corresponds to the metaphase onset."
NCBI gene IDs:
"RBM14: 10432"
Figure legend: "E Amorphous electron-dense structures containing microtubules are formed in RBM14-depleted cells (see also Supplementary Fig S5I). Electron micrographs from U2OS cells depleted of RBM14. Squares indicate the ectopic electron-dense structures, whereas circles indicate the pre-existing centrioles. Scale bar, 500 nm and 200 nm (magnified views). Average diameter of pre-existing centrioles and the amorphous electron-dense structures are 225 ± 8 nm (n = 10) and 130 + 11 nm (n = 16), respectively. Insets show magnified views of the squares. Note that microtubules (black arrowheads) are observed within the electron-dense structures (white arrowheads)."
NCBI gene IDs:
"RBM14: 10432"
Figure legend: "G U2OS cells expressing GFP-CPAP (green) and treated with RBM14 siRNA were analyzed using CLEM. Separate z-plane images of the cell with GFP-CPAP foci and the threefold magnified images are aligned from bottom to top along z-axis (left LM panels). White broken lines represent cell shapes, and blue broken lines represent nuclear shapes. Corresponding regions of EM images are shown on the right. White squares (a-d) represent the region around GFP-CPAP foci. Note that other than pre-existing centrioles (a, 1-4), ectopic small CPAP foci are recognizable as amorphous electron-dense structures (b-d). One of pre-existing centrioles (1) is elongated because of exogenous GFP-CPAP expression. Scale bar, 10 μm (LM), 2 μm (EM, low magnified image), 500 nm [magnified images of (a) and (d)), 200 nm (magnified images of (b) and (c)]."
NCBI gene IDs:
"RBM14: 10432"
Figure legend: "B-E Control U2OS cells or U2OS cells treated with siRNA targeting RBM14 were cold-treated for 30 min, followed by 30-60 min incubation at 37°C and stained with antibodies against centrin (magenta in (B) and (D)) or γ-tubulin (magenta in (E)) as well as α-tubulin (green). DNA is shown in blue. Insets show approximately twofold magnified views of fluorescent foci around the centrosome. Scale bar, 5 μm. Histograms represent the percentages of mitotic cells showing the indicated phenotype at each time point. Values are mean percentages ± standard error of mean (SEM) from three independent experiments (n = 30 for each condition). *P &lt; 0.05, **P &lt; 0.01 (one-tailed t-test)."
NCBI gene IDs:
"RBM14: 10432"
Figure legend: "A U2OS cells treated with RBM14 siRNA and stained with antibodies against centrin-2 (green) as well as HsSAS-6 (magenta). DNA is shown in blue. Insets show approximately twofold magnified views of fluorescent foci around the centrosome. Scale bar, 5 μm. Histograms represent the frequency of mitotic cells with the indicated phenotype. Values are mean percentages ± SEM from three independent experiments (n = 60 for each condition)."
NCBI gene IDs:
"RBM14: 10432"
Figure legend: "B Control U2OS cells or U2OS cells treated with siRNA targeting RBM14 were stained with antibodies against HsSAS-6 as well as centrin-2. Immunofluorescence micrographs are shown in Supplementary Fig S6D. When we counted HsSAS-6 foci in this paper, we only counted cells before metaphase/anaphase transition considering the disappearance of HsSAS-6 from anaphase in mitosis. Histograms represent frequency of mitotic cells with excess foci of the indicated proteins at spindle poles. Values are mean percentages ± SEM from three independent experiments (n = 30 for each condition). *P &lt; 0.05 (one-tailed t-test)."
NCBI gene IDs:
"RBM14: 10432"
Figure legend: "D RBM14-depleted HeLa cells expressing GFP-centrin (green) were stained with antibodies against CP110 (magenta) and HsSAS-6 (blue). Scale bar, 1 μm. A representative stained procentriole-like structure as well as endogenous centrioles is shown together with a schematic. Histograms represent length between the centers of the foci of the indicated proteins. Values are mean percentages ± SEM from seven individual cells."
NCBI gene IDs:
"RBM14: 10432"
Figure legend: "E, F Ectopic DsRed-centrin-2 foci in RBM14-depleted cells recruit HsSAS-6-GFP. (E) Live imaging of RBM14-depleted U2OS cells expressing HsSAS-6-GFP (green) and DsRed-centrin-2 (magenta). Time is denoted in hh:min, and scale bar is 10 μm. Time zero corresponds to the onset of recording. Pre-existing HsSAS-6-GFP foci (white arrowheads) and ectopic HsSAS-6-GFP focus (red arrowhead) were traced throughout the time-lapse recording. White broken lines represent cell shapes, and blue broken lines represent nuclear shapes. (F) Histograms represent the percentages of ectopic DsRed-centrin-2 foci with HsSAS-6-GFP recruitment (n = 69 foci for control, and n = 67 foci for RBM14 siRNA)."
NCBI gene IDs:
"RBM14: 10432"
Figure legend: "G Histograms represent frequency of mitotic cells with excess foci of HsSAS-6 in the indicated conditions. In this experiment, cells were treated with double amount of RBM14 siRNA (20 pmol) compared to that in the other experiments. Values are mean percentages ± SEM from three independent experiments (n = 30 for each condition). *P &lt; 0.05 (one-tailed t-test)."
NCBI gene IDs:
"RBM14: 10432"
Figure legend: "H HeLa cells expressing GFP-centrin (green) and RFP-H2B (magenta) and treated with RBM14 siRNA were analyzed using Live CLEM. Pre-existing GFP-centrin foci (white arrowheads) and ectopic GFP-centrin foci (red arrowheads) were traced throughout the time-lapse recording. Z-stacked confocal images spanning the entire height of the cells (&lt; 30 μm) are shown (upper panels). Time is denoted in hh:min. Time zero corresponds to the onset of ectopic formation of GFP-centrin foci. The cells fixed after live imaging were also analyzed for confirming the local correlation between LM and EM images (lower panels). Red square (ectopically formed GFP-centrin foci) represents the region corresponding to that of the EM images acquired from serial sections. Note that ectopic GFP-centrin foci that form a spindle pole include not only electron-dense structures (1, 3) but also a morphologically recognizable centriole-like structure (2). Scale bars, 10 μm (live imaging) and 500 nm (EM)."
NCBI gene IDs:
"RBM14: 10432"
Figure legend: "(A) Growth assay of the indicated cells with and without doxycycline-inducible overexpression of PLK4. Experiments were performed in wild-type or SAS6 monoclonal knockout cells. Data acquired across n = 3 biological replicates. Mean ± s.e.m."
NCBI gene IDs:
"PLK4: 10733
SAS6: 163786"
Figure legend: "(G) Top: Graph showing the efficiency of frameshifting INDELs measured using TIDE. N.D. = Not determined. Data shown is from n = 1 biological replicate. Bottom: Graph showing the relative growth of doxycycline treated PLK4Dox cells expressing an sgRNA targeting the indicated genes. Each dot displays measurements from a single experiment. Experiments were performed in polyclonal knockout cells. Data acquired across n ≥ 3 biological replicates. Mean ± s.e.m."
NCBI gene IDs:
"PLK4: 10733"
Figure legend: "(H) Quantification of centrosome number in PLK4Dox cells expressing an sgRNA targeting the indicated genes. Experiments were performed in polyclonal knockout cells. Data acquired across n ≥ 3 biological replicates. Mean ± s.e.m."
NCBI gene IDs:
"PLK4: 10733"
Figure legend: "(D) Quantification of the fraction of cells with PIDD1 localized to the mature mother centriole in PLK4Dox cells expressing an sgRNA targeting the indicated genes. A dot displays measurements from each experiment. Experiments were performed in PLK4Dox polyclonal knockout cells. Data acquired across n = 3 biological replicates. Mean ± s.e.m."
NCBI gene IDs:
"PLK4: 10733"
Figure legend: "(F) Graph showing the relative growth of doxycycline treated PLK4Dox cells that were knocked out for the indicated genes. Each dot displays measurements from a single experiment. Experiments were performed in PLK4Dox monoclonal knockout cells. Data acquired across n ≥ 3 biological replicates. Mean ± s.e.m."
NCBI gene IDs:
"PLK4: 10733"
Figure legend: "(G) Quantification of the fraction of cells with PIDD1 localized at the mature mother centriole. Experiments were performed in PLK4Dox monoclonal knockout cells. Data acquired across n = 3 biological replicates. Mean ± s.e.m."
NCBI gene IDs:
"PLK4: 10733"
Figure legend: "(A) Quantification of the fraction of cells with ANKRD26 localized at the mature mother centriole. Experiments were performed in PLK4Dox monoclonal knockout cells. Data acquired across n = 3 biological replicates. Mean ± s.e.m."
NCBI gene IDs:
"PLK4: 10733"
Figure legend: "(B-D) Representative images of WT or ANKRD26-/- PLK4Dox cells immunostained with the indicated antibodies. Scale bar = 5 µm."
NCBI gene IDs:
"ANKRD26: 22852
PLK4: 10733"
Figure legend: "(E) Quantification of the fraction of WT or ANKRD26-/- hTERT RPE1 cells with cilia. Each dot displays measurements from a single experiment. Data acquired across n = 5 biological replicates. Mean ± s.e.m."
NCBI gene IDs:
"ANKRD26: 22852"
Figure legend: "(F) Growth assay of the indicated cells with and without doxycycline-inducible overexpression of PLK4. Experiments were performed in PLK4Dox monoclonal knockout cells. Data acquired across n = 3 biological replicates. Mean ± s.e.m."
NCBI gene IDs:
"PLK4: 10733"
Figure legend: "(G) Quantification of centrosome number in PLK4Dox cells expressing an sgRNA targeting the indicated genes. Experiments were performed in PLK4Dox monoclonal knockout cells. Data acquired across n = 3 biological replicates. Mean ± s.e.m."
NCBI gene IDs:
"PLK4: 10733"
Figure legend: "(H) Western blot showing expression of pro-CASP2 and P21 following treatment with dox for the specified number of days. Experiments were performed in PLK4Dox monoclonal knockout cells. (I) Quantification of pro-CASP2 levels following treatment with dox for the specified number of days. Experiments were performed in PLK4Dox monoclonal knockout cells. Each dot displays measurements from a single experiment. Data acquired across n = 6 biological replicates. Mean ± s.e.m. "
NCBI gene IDs:
"PLK4: 10733"
Figure legend: "(C) Western blot showing the expression of ANKRD26. Experiments were performed in monoclonal ANKRD26-/- PLK4Dox cells expressing the indicated ANKRD26 transgenes."
NCBI gene IDs:
"ANKRD26: 22852
PLK4: 10733"
Figure legend: "(D) Quantification of the fraction of cells with ANKRD26 (left) and PIDD1 (right) localized to the mature mother centriole in monoclonal ANKRD26-/- PLK4Dox cells expressing the indicated transgenes. Each dot displays measurements from a single experiment. Data acquired across n = 3 biological replicates. Mean ± s.e.m."
NCBI gene IDs:
"ANKRD26: 22852
PLK4: 10733"
Figure legend: "(E) Graph showing the relative growth of doxycycline-treated, monoclonal ANKRD26-/- PLK4Dox cells expressing the indicated ANKRD26 transgenes. Each dot displays measurements from a single experiment. Data acquired across n = 3 biological replicates. Mean ± s.e.m."
NCBI gene IDs:
"ANKRD26: 22852
PLK4: 10733"
Figure legend: "(F) Western blot showing expression of pro-CASP2 and P21 following treatment with dox for 4 days. Experiments were performed in monoclonal ANKRD26-/- PLK4Dox cells expressing the indicated transgenes."
NCBI gene IDs:
"ANKRD26: 22852
PLK4: 10733"
Figure legend: "B Volcano plot showing proteins enrichment (fold change in log10 scale) after affinity purification in Jurkat T cells expressing ARHGAP45OST molecules compared to affinity purification in control Jurkat T cells expressing similar levels of WT (untagged) ARHGAP45 proteins prior to (unstimulated) and at 120 s after (stimulated) TCR stimulation. ARHGAP45 interacting proteins with a greater than 2-fold enrichment and a p-value &lt; 0.01 were selected as specific ARHGAP45 interactors (Dataset EV1) and some of them are specified in red. Red lines represent the thresholds set on P-value and enrichment to identify specific ARHGAP45 interactors."
NCBI gene IDs:
"ARHGAP45: 23526"
Figure legend: "B The ratio of CTV/CMTPX-labeled cells present in each organ (Ro) was determined and normalized by dividing it with Ri, the ratio of CTV/CMTPX-labeled cells present in the cell mixtures prior to injection. Ro/Ri ratio corresponding to each of the specified mice are shown. Data shown for Arhgap45-/-mice correspond to 4 independent experiments (E1 to E4) involving a total of 15 individual mice whereas data shown for WT mice correspond to 2 independent experiments (E1 and E2) involving a total of 5 individual mice. The Arhgap45-/- mice analyzed on the same day as the control mice corresponded to the E1 and E2 experiments. Mean and SD are shown. ns, non-significant, **** P ≤ 0.0001; multiple unpaired T tests."
NCBI gene IDs:
"Arhgap45: 70719"
Figure legend: "C Expression of CCR7, and LFA-1 on naïve CD4+ and CD8+ T cells found in LNs of WT and Arhgap45-/- mice, analyzed by flow cytometry. Gray shaded curves, isotype control staining. D Quantification of data shown in (C). The MFI corresponding to each analyzed mouse is shown together with mean and SD. Each dot represents an individual mouse and CD4+ T cells from at least 4 mice have been analyzed for each condition. Mice analyzed on the same day are represented by similar dots (see key in bottom right corner). ns, non-significant, ** P ≤ 0.002; multiple unpaired T tests."
NCBI gene IDs:
"Arhgap45: 70719"
Figure legend: "The cells specified below were loaded into the upper chamber of a Transwell migration device without (no) or with the specified chemokines in the lower chamber. The percentage of cells that migrated to the bottom chamber after 2 h (T cells) or 4 h (B cells) is shown. A Chemotaxis of naïve CD4+ T cells from WT and Arghap45−/− mice in response to CCL19, CCL21 and CXCL12. B Chemotaxis of naïve CD8+ T cells from WT and Arghap45−/− mice in response to CCL19, CCL21 and CXCL12. C Chemotaxis of naïve B cells from WT and Arghap45−/− mice in response to CCL19 "
NCBI gene IDs:
"Arghap45: 70719"
Figure legend: "D Adding CCL19 in both the upper and lower chambers (CCL19 UL) prevented migration of WT and Arghap45−/− naïve T cells whereas the use of Transwell membrane inserts precoated with ICAM-1 (CCL19 + ICAM-1) did not change the pattern of chemotaxis observed using 'bare' membrane inserts (CCL19). E Chemotaxis of naive CD4+ T cells and B cells from WT, Arghap45−/−, and Arhgap45∆T/∆T mice in response to CCL19. F Chemotaxis of activated CD4+ T cells from WT and Arghap45−/− mice in response to CCL19. Data information: In (A) to (F) each dot represents an individual mouse and as shown CD4+ T cells from 3 to 9 mice have been analyzed for each condition. Mice analyzed on the same day are represented by similar dots (see key in bottom right corner). Mean and SD are shown. *P ≤ 0.033, ** P ≤ 0.002, *** P ≤ 0.001, **** P ≤ 0.0001; A, B, C, D, F multiple unpaired T test; E two-way ANOVA test. "
NCBI gene IDs:
"Arghap45: 70719
Arhgap45: 70719"
Figure legend: "Analysis of migration patterns of WT and Arghap45−/− activated T cells on 2D surface coated with ICAM-1. Each track represents the migratory path of individual WT and Arghap45−/− activated T cells recorded over 466 s in a single field of view, Trajectories were plotted to a common starting point, and &gt; 200 T cells were recorded per plot using time-lapse microscopy at 10 x magnification. Analysis of migration patterns of WT and Arghap45−/− naive T cells on 2D surface coated with ICAM-1 and CCL21. Same conditions as in (A)."
NCBI gene IDs:
"Arghap45: 70719"
Figure legend: "A, B Representative kymographs of WT (A) and Arghap45−/− (B) naive CD4+ T cells on 2D surface coated with ICAM-1 and CCL21."
NCBI gene IDs:
"Arghap45: 70719"
Figure legend: "A Irradiated H-2 Kb- positive spleen cells isolated from T cell-deficient mice were pulsed for 2 hours with the N4 agonist OVA peptide and cultured with CD8+ T cells purified from WT OT-I (WT) or Arhgap45−/− OT-I (Arhgap45−/−) mice. Cell proliferation was measured by luminescence after 48 h. Data are representative of 2 independent experiments with 3 mice per genotype (mean and SEM of triplicate are shown)."
NCBI gene IDs:
"Arhgap45: 70719
Arhgap45: 23526
H-2 K: 14964"
Figure legend: "E. Double  immunofluorescent staining of Fn12230, fadA-deletion mutant US1  (∆fadA) and spontaneous mutant  lam in log and stationary phases using mAb 7H7 at 1:800 dilution and anti-human β-amyloid polyclonal antibody A11 at 1:500 dilution, followed by incubation with Alexa Fluor 680-conjugated donkey anti-rabbit  and Alexa Fluor 555-conjugated goat anti-mouse, both at 1:1,000 dilution. Co-staining of FadA and A11 was observed in Fn in  stationary phase, not in log phase, or in US1  or lam. The imagines were taken under 60X objective. Scale bar equals 5 μm."
NCBI gene IDs:
"fadA: 45633877"
Figure legend: "F. Scanning electron microscopy  of Fn 12230, US1 (∆fadA) and lam in log and  stationary phase at 10,000x magnification. Note the fibrous structure coating  Fn in stationary  phase, pointed by the clear arrows, but not in log phase. Scale bar equals 1 μm."
NCBI gene IDs:
"fadA: 45633877"
Figure legend: "G. Analysis of amyloid-like  FadA  produced by  Fn 23726 and its ∆fap2  mutant. An aliquot of 50 μg detergent-resistant pellets was loaded onto each lane, followed by SDD-AGE and Western blot analysis as described above. The mutant produced significantly less  amyloid-like polymers than the wild type."
NCBI gene IDs:
"fap2: "