EMBO/SourceData huggingface dataset:
Figure 5A, B. Effect of ectopic LITATS1 expression (A) or LITATS knockdown (B) on TβRI expression in MDA-MB-231 cells. Right panel: quantification of relative TβRI protein levels. Vinculin or GAPDH, loading control. Representative blots from a minimum of three independent experiments are shown.C, D. Analysis of TβRI protein stability (as measured by western blotting) in MDA-MB-231 cells with ectopic LITATS1 expression (C) or LITATS knockdown (D). Cells were treated with CHX (50 µg/mL) for the indicated durations. Quantification of the relative TβRI protein level is shown in the lower panel. GAPDH, loading control. Representative blots from a minimum of three independent experiments are shown.E. TβRI expression in MDA-MB-231 cells with ectopic LITATS1 expression in the absence or presence of lysosome or proteasome inhibitors. Cells were incubated with vehicle control DMSO (-), the lysosome inhibitor BafA1 (20 nM) or HCQ (20 μM), or the proteasome inhibitor MG132 (5 μM) for 8 h. Vinculin, loading control. Representative results from a minimum of three independent experiments are shown. F. Effect of LITATS1 on TβRI ubiquitination. HEK293T cells were transfected with ectopic expression constructs for HA-Ubiquitin (HA-Ub), caTβRI-FLAG and/or LITATS1. TβRI polyubiquitination was analyzed by western blotting. Representative blots from a minimum of three independent experiments are shown. D
Figure 6B. Interactions between LITATS1 and TβRI or SMURF2 in MDA-MB-231 cells were detected by the CARPID approach. Cells with stable expression of TurboID-dCasRx were transduced without (Co.) or with (#1 and #2) LITATS1 targeting gRNAs. Cells were stimulated with or without TGF-β (2.5 ng/mL) for 2 h and were then stimulated with biotin (500 μM) for 30 min. Western blotting was performed to detect SMURF2 and TβRI expression in whole-cell lysates (Input) and immunoprecipitates (IP). GAPDH and HA-dCasRx expression levels were measured for equal loading of Input samples and as the negative control or positive control, respectively, for proximity biotinylation in immunoprecipitate (IP) samples. Representative results from a minimum of three independent experiments are shown.C. Interactions between LITATS1 and TβRI (left) or SMURF2 (right) were analyzed by RIP. MDA-MB-231 cells were stimulated with or without TGF-β (5 ng/mL) for 4 h before RIP. RT‒qPCR was performed to detect LITATS1 expression in immunoprecipitates from MDA-MB-231 cells. IgG was included as the control for immunoprecipitation. Representative results from two independent experiments are shown.D. Interactions between LITATS1 and caTβRI or SMURF2 were analyzed by RNA pull-down. Biotinylated 25x poly(A), antisense LITATS1 (LITATS1-AS) or LITATS1 was incubated with lysates from HEK293T cells transfected with the caTβRI-HA or MYC-SMURF2 expression construct. Western blot analysis was performed to detect HA or MYC expression in whole-cell lysates (Input) and immunoprecipitates (IP). Representative blots from a minimum of three independent experiments are shown.E. In vitro RNA pull-down assays were performed to evaluate the interactions between LITATS1 and SMURF1/2. In vitro-transcribed Antisense LITATS1 (LITATS1-AS) or LITATS1 (LITATS1-S) was incubated with recombinant FLAG-tagged SMURF1 or SMURF2 protein. Western blotting analysis was performed to evaluate FLAG expression in input and IP samples. The amounts of RNA probes used for RNA pull-down were evaluated by agarose gel electrophoresis. Representative results from a minimum of three independent experiments are shown.G. Effect of LITATS1 overexpression on SMURF2-mediated TβRI polyubiquitination. HEK293T cells were transfected with expression constructs for HA-Ubiquitin (HA-Ub) and caTβRI-FLAG and ectopic expression constructs for SMURF2 and/or LITATS1. Polyubiquitination of TβRI was evaluated by western blotting. Representative blots from a minimum of three independent experiments are shown.H. Effect of SMURF2 knockdown on LITATS1-mediated TβRI polyubiquitination. MDA-MB-231 cells with stable HA-Ub expression were transduced with expression constructs for LITATS1 and/or two different SMURF2 shRNAs, as indicated. Polyubiquitination of TβRI was evaluated by western blotting. Representative blots from a minimum of three independent experiments are shown.
Figure 7A. An in vitro RNA pull-down assay was performed to evaluate the interaction between LITATS1 truncation mutants and SMURF2. Recombinant FLAG-SMURF2 protein was incubated with antisense LITATS1 (LITATS1-AS), LITATS1 (LITATS1-S), or LITATS1 truncation mutants (T1-T4). Western blot analysis was performed to evaluate FLAG expression in immunoprecipitates (IP). The amounts of RNA probes used for RNA pull-down were evaluated by agarose gel electrophoresis. Representative results from a minimum of three independent experiments are shown.E. SMURF2 expression and localization (as measured by immunofluorescence) upon LITATS1 depletion in A549 cells. DAPI staining was performed to visualize nuclei. Scale bar=23.2 μm. Representative results from two independent experiments are shown.F. Effect of LITATS1 knockdown on SMURF2 localization in A549 cells. After subcellular protein fractionation, western blotting was performed to detect SMURF2 expression in whole-cell lysates (Total) and the cytoplasmic (Cyto) and nuclear (Nuc) fractions. The levels of the cytoplasmic marker GAPDH and the nuclear marker Lamin A/C are included to demonstrate subcellular protein fractionation. Representative results from two independent experiments are shown.
Figure 1B. Representative image of primary microglia simultaneously treated with Aβ oligomer-bound SN (oAβ-SN; in pHrodo red (magenta)) and control SN (Ctrl-SN; in pHrodo deep red (cyan)). Scale bar, 50 μm.F. Time-lapse images of primary Homer1-eGFP neurons (green) treated with 50 nM oAβ versus vehicle control. Yellow arrows indicate increasing PSVue550 (magenta) signal on dendritic spines with Aβ oligomer treatment over 45 min. Scale bar, 2 μm.I. Quantification of colocalized Homer1-eGFP with PSVue among total Homer1. 3 ROIs per neuron, 4-5 neurons, n=3 independent experiments. J. Quantification of colocalized PSVue with Homer1-eGFP among total PSVue. 3 ROIs per neuron, 4-5 neurons, n=3 independent experiments. Data information: Data shown as mean ± SD Each shaded point represents 1 ROI and each open point represents the mean unpaired (I and J) t-test. P-values shown as *P<0.05; **<0.01; ***P<0.001; ****P<0.0001.
Figure 2A. Time-lapse images of microglia (cyan, labelled with IB4-647, 3D rendered) internalizing (yellow arrowheads) PSVue+ Homer1-eGFP dendritic spines. Note PSVue- Homer1-eGFP dendritic spines are left behind (empty yellow arrowheads). Scale bar, 5 μm.C. SRM images of Homer1-eGFP and CD68 (magenta) lysosomes in microglia. Inset shows orthogonal view of colocalized Homer1-eGFP and CD68. Scale bar, 5 μm.G. (Top panel) Representative traces of spontaneous GCaMP7 signals at 48 h post-treatment with Aβ oligomer in dendritic spines from neuron-only culture (cyan), neuron-microglia co-culture (magenta), and neuron-microglia co-culture treated with AnnxV (yellow). (Bottom panel) number of calcium transients per minute before (0 h), at 24 h and 48 h post-treatment of Aβ oligomer. ∼10 spines per neuron, ∼10 neurons per experiment, from 3 independent experiments. Data information: Kruskal-Wallis test followed by Dunn's multiple comparisons test (G) P-values shown ns P>0.05; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. J-K. Number of PSVue puncta per 100 μm2 (J) and mean intensities of PSVue signal (K) in neuron-only culture, and neuron-microglia co-culture with or without AnnxV. 16 ROIs per experiment, n=3 independent experiments. Data information: Data shown as box plots (J and K). The top and the bottom of the boxes represent the 75th and 25th percentiles, respectively, and the lines in the middle represent the median. The whiskers represent the highest and lowest values that are not outliers. one-way ANOVA followed by Bonferroni post-hoc test (J and K). P-values shown ns P>0.05; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.
Figure 3A. Trem2 CV and Trem2 R47H KI primary microglia treated simultaneously with oAβ-bound synaptosomes (SN) conjugated with pHrodo red (magenta) and control SN, conjugated with pHrodo deep red (cyan). Scale bar, 50 μm. B. pHrodo fluorescence with time shown as AUC at 3 h. AUC of oAβ-SN is higher compared to Ctrl-SN in Trem2 CV but not in Trem2 R47H KI microglia. ∼40 microglia per ROI, 2 ROIs per well, 2-3 wells per experiment, n=3 independent experiments. C. Quantification of the percent of C1qa area covered normalized to WT showing an increase in the NL-F but not NL-F KI; Trem2 R47H KI. N=3-4 animals per genotype. Data information: Data shown as mean ± SEM. Each shaded point represents 1 ROI, and each open point represents the mean of each independent experiment. Two-way ANOVA followed by Bonferroni's post-hoc test. P-values shown as ns P>0.05; *P<0.05; ***P<0.001; ****P<0.0001.Hippocampal CA1 SR of 6 mo WT, Trem2 R47H KI, NL-F KI and NL-F KI; Trem2 R47H KI mice immunostained for P2Y12 (red), CD68 (magenta) and Homer1 (green). G. Representative 3D surface rendering reconstructions showing increased Homer1 in microglia in NL-F KI compared to WT, Trem2 R47H KI, and NL-F KI; Trem2 R47H KI mice. Scale bar, 10 μm.I. SRM images from the hippocampal CA1 SR of 6 mo WT, Trem2 R47H KI, NL-F KI and NL-F KI; Trem2 R47H KI mice immunostained for Synaptotagmin (Syt1/2, magenta) and Homer1 (green), pre- and post-synaptic puncta, respectively. Scale bar, 1 μm.
Figure 4B. Percentage of synaptic Homer1-immunoreactive puncta found co-localized with PSVue in NL-F KI; Trem2 R47H compared to NL-F KI. 3 ROIs per animal, n=3-4 animals per genotype. Data information: Data shown as mean ± SEM. Unpaired t-test (B) P-values shown as ns P>0.05; *P<0.05; **P<0.01, ****P<0.0001.D. Number of spontaneous calcium transients per minute at 48 h post-treatment of Aβ oligomer in neuron-only culture (cyan), neuron- Trem2 CV microglia co-culture (magenta) or neuron- Trem2 R47H KI microglia co-culture (purple). ∼10 spines per neuron, ∼10 neurons per experiment, from 3-4 independent experiments. Data information: Data shown as mean ± SEM. Each shaded point represents 1 ROI, and each open point represents the mean (or median for GCaMP studies) of each independent experiment. Central bands of the violin plot (D) represent median and quartiles. Kruskal-Wallis test followed by Dunn's test (D), P-values shown as ns P>0.05; *P<0.05; **P<0.01, ****P<0.0001.F. SRM 3D images from the hippocampal CA1 SR of 6 mo WT, Trem2 R47H KI, NL-F KI and NL-F KI; Trem2 R47H KI mice immunostained for Bassoon (cyan), a presynaptic marker enriched in functional active zones. Scale bar, 1 μm.G. Representative western blots comparing levels of cleaved caspase-3 (17/19 kDa) and procaspase-3 (35 kDa) with respect to GAPDH (38 kDa) loading control in synaptosomes isolated from human NDC, AD, NDC TREM2 and AD TREM2 brains. 1 lane represents 1 patient.H. Western blot densitometry analysis showing cleaved caspase-3 levels (left graph) and the ratio of cleaved caspase-3/procaspase-3 (right graph) in synaptosomes (SN) isolated from human NDC, AD, TREM2 NDC and TREM2 variants. N= 9 NDC, 11 AD, 5 TREM2 NDC, 10 TREM2 AD cases. Data information: Data shown as mean ± SEM. The top and the bottom of the box plot (H) represent the 75th and 25th percentiles, respectively, and the line represents the median. The whiskers represent the highest and lowest values that are not outliers. One-way (H) ANOVA followed by Bonferroni's post-hoc test. P-values shown as ns P>0.05; *P<0.05; **P<0.01, ****P<0.0001.
Figure 1A. Spinning disk time-lapse microscopy of WT and UBAP2L KO HeLa cells synchronized with double thymidine block and release (DTBR) in mitosis. The selected frames of the movies are depicted and the corresponding time is indicated in minutes. SiR-DNA was used for DNA staining.I, J. The time of mitotic entry (I) and from prophase to anaphase (J) was quantified. At least 50 cells per condition were analyzed for each experiment (n=3). Red bars represent the mean. K-M. The percentages of cells with abnormal mitosis (chromosome misalignments and/or DNA bridges) (K), polylobed or multinucleated daughter cells (L) and mitotic cell death (M) were quantified. At least 50 cells per condition were analyzed. N. The nuclear size of at least 1000 control and UBAP2L-downregulated interphasic cells was measured. Red bars represent the mean.
Figure 2A-C. Immunofluorescence (IF) representative pictures of WT or UBAP2L KO cells synchronized in G1/S with DTB or in early mitosis with DTBR (A) and quantification of the percentage of cells with normal (2) or abnormal number of centrosomes in interphase (B) or in early mitosis (C). At least 50 cells per condition were analyzed for each experiment. Graphs represent the mean of three biological replicates ± SD (two sample two-tailed t-test ns=non-significant).D-F. IF representative pictures of HeLa cells transfected with siCTL or siUBAP2L for 48h and synchronized in G1/S with DTB or in early mitosis with DTBR (D) and quantification of the percentage of cells with normal (2) or abnormal number of centrosomes in interphase (E) or in early mitosis (F). At least 50 cells per condition were analyzed for each experiment. Graphs represent the mean of three biological replicates ± SD (two sample two-tailed t-test ns=non-significant).G, H. IF representative pictures of WT or UBAP2L KO cells synchronized in mitosis with DTBR (G) and quantification of the percentage of cells with bipolar or non-bipolar mitotic spindle (H). At least 50 cells per condition were analyzed for each experiment. The graph represents the mean of three biological replicates ± SD (two sample two-tailed t-test ns=non-significant).
Figure 3D. WB analysis of WT or UBAP2L KO HeLa cell lysates from G1/S synchronized cells using DTB. Proteins MW is indicated in kDa. WB is representative of three independent replicates.E, F. IF representative pictures of G1/S synchronized WT or UBAP2L KO HeLa cells using DTB and quantification of PLK1 nuclear intensity (A.U.) ROIs are shown in the corresponding numbered panels. Scale bar, 5µm. At least 250 cells were quantified per condition for each replicate. Each dot of graphs (F) represents PLK1 nuclear intensity in a single nucleus where PLK1 signal was detectable. Interphasic cells which do not express PLK1 were excluded from the quantification. The measurements of four biological replicates are combined, red bars represent the mean (Kruskal-Wallis test with Dunn's correction **P<0,01, ****P<0,0001).H. WB analysis of HeLa cell lysates from unsynchronized cells treated with the indicated siRNAs. Proteins MW is indicated in kDa. WB is representative of three independent replicates. I. Quantification of the relative protein levels of BubR1. Graphs represent the average ratio of BubR1 (lower band) or phospho-BubR1 (upper band)/GAPDH (A.U.) from three biological replicates ± SD (two sample two-tailed t-test *P<0,05, ns=non-significant).
Figure 4B. WB analysis of control or UBAP2L-silenced HeLa lysates from mitotic cells synchronized with 1µM paclitaxel and treated with 100µg/mL CHX for the indicated times. Proteins MW is indicated in kDa. WB is representative of three independent replicates.C. WB analysis of WT or UBAP2L KO HeLa lysates from G1/S synchronized (DTB) cells treated with 25µM of the proteasomal inhibitor MG132 for the indicated times. Proteins MW is indicated in kDa. WB is representative of three independent replicates.D. Representative IF pictures of WT or UBAP2L KO HeLa cells synchronized in G1/S using DTB, in S using 2mM hydroxyurea (HU) for 20h or in G2 using 10µM of CDK1 inhibitor RO 3306 for 20h.
Figure 5IF analysis of G1/S synchronized WT or UBAP2L KO HeLa cells using DTB and transfected with the indicated flag-tagged UBAP2L protein fragments for 48h (A) Scale bar, 5µm. At least 100 cells per condition were quantified for each experiment. Graphs represent the mean of three replicates ± SD (one-way ANOVA with Sidak's correction *P<0,05, **P<0,01, ns=non-significant).IF representative pictures of WT or UBAP2L KO HeLa cells transfected with the indicated flag-tagged UBAP2L protein fragments for 48h and synchronized in mitosis using monastrol release (MR) (C) Scale bar, 5µm. At least 50 cells per condition were quantified for each experiment.Colony formation assay of WT or UBAP2L KO HeLa cells transiently transfected with the indicated flag-tagged UBAP2L protein fragments after 7 days of culture. Graphs represent the mean of three replicates ± SD (one-way ANOVA with Sidak's correction *P<0,05, ****P<0,0001, ns=non-significant).
Figure 6Representative IF images of WT or UBAP2L KO HeLa cells synchronized in G1/S using DTB and transfected with the indicated flag-tagged UBAP2L constructs and control or G3BP1/2 siRNAs for 48h (A)Representative IF images of WT or UBAP2L KO HeLa cells synchronized in G1/S using DTB and treated with vehicle (DMSO) or 25µM of MG132 for 4h (C)
Figure 7Representative IF images of control or UBAP2L-downregulated cells synchronized in prometaphase using 1mM monastrol and released at the indicated time points.E. Spinning disk time-lapse microscopy of PLK1-eGFP Knock-In (KI) HeLa cells synchronized with DTBR in mitosis. The selected frames of the movies are depicted and the corresponding time is indicated in minutes. SiR-DNA was used for DNA staining.
Figure 8A, B. Representative IF pictures of WT or UBAP2L KO HeLa cells synchronized in mitosis with DTBR (A) and quantification of the relative PLK1 intensity at the inner centromere (A.U.) (B). ROIs are shown in the corresponding numbered panels. Scale bar, 5µm. At least 50 cells were quantified per condition for each experiment. Each dot represents PLK1/INCENP intensity ratio at a single centromere. The measurements of three biological replicates are combined, red bars represent the mean (Mann-Whitney test ****P<0,0001).C, D. Representative IF pictures of WT or UBAP2L KO HeLa cells synchronized in mitosis with DTBR (C) and quantification of the relative PLK1 intensity at the centrosomes (A.U.) (D). ROIs are shown in the corresponding numbered panels. Scale bar, 5µm. At least 50 cells were quantified per condition for each experiment. Each dot represents PLK1/Pericentrin intensity ratio at a single centrosome. The measurements of three biological replicates are combined, red bars represent the mean (Mann-Whitney test ****P<0,0001).E, F. Representative IF pictures of WT or UBAP2L KO HeLa cells synchronized in mitosis with DTBR (E) and quantification of the relative PLK1 intensity at the midbody (A.U.) (F). ROIs are shown in the corresponding numbered panels. Scale bar, 4µm. At least 50 cells were quantified per condition for each experiment. Each dot represents PLK1/PRC1 intensity ratio at the midbody. The measurements of three biological replicates are combined, red bars represent the mean (Mann-Whitney test **P<0,01).G. Representative IF pictures of WT or UBAP2L KO HeLa cells synchronized in mitosis with DTBR (n=3) to qualitatively assess the localization of PLK1 at the mitotic spindle with α-tubulin staining. ROIs are shown in the corresponding numbered panels. Scale bar, 5µm.
Figure 9D. WB analysis of control (siCTL) or siUBAP2L 48h-treated HeLa cells were synchronized with 1mM monastrol for 16h, treated with DMSO or with 10nM of the PLK1 inhibitor BI2536 for 45min, subsequently washed out from monastrol for the indicated time and collected for protein extraction. This moderate treatment is sufficient to restore the aberrant PLK1 catalytic activity observed in UBAP2L KO cells to the levels of the WT, enabling correct mitotic progression but prevents control cells to progress through mitosis. Proteins MW is indicated in kDa. WB is representative of three independent replicates.DAPI staining of the experiment described in (D) showing different mitotic stages (E).
Figure 1Expression profile of zRbm14a during early embryonic development. Lysates from 5 embryos were loaded in each lane and subjected to immunoblotting (B). α-tubulin served as an internal control. The relative levels of zRbm14a (B, C) are shown as band intensities normalized to those of α-tubulin. Quantification results from three independent experiments are shown in (C).Subcellular localizations of zRbm14a. The representative confocal micrographs (D) were acquired from the animal pole of zebrafish embryos. Exposures were kept the same during the imaging for comparison. DAPI labels nuclear DNA. Framed regions were magnified to show details. Arrows denote representative cytoplasmic puncta, whereas arrowheads point to perinuclear condensates.Subcellular localizations of zRbm14a. Subcellular localizations (E) were scored from the indicated number of cells from 4 to 6 embryos.F. Spindle-pole enrichment of zRbm14a condensates (arrowheads) in mitotic cells. Exposures were kept the same during the imaging for comparison.G. zRbm14a condensates colocalized with γ-tubulin puncta. The framed blastomeres at the indicated cell cycle stages were magnified to show details. Arrows point to spindle poles.H. Subcellular localizations of zRbm14b resembled those of zRbm14a. Zebrafish embryos were microinjected with 400 pg of GFP-Rbm14b mRNA per egg at the 1-cell stage and fixed at 4 hpf.I. Asymmetric segregations of zRbm14a condensates in mitotic blastomeres by associating with centrosomal γ-tubulin puncta (arrows). A typical 8-cell mitotic embryo was entirely represented, with the two blastomeres in the framed region magnified to show details. The cartoon inset illustrates relative positions of the three blastomeres in the 16-cell embryo. Numbers in yellow are relative fluorescent intensities of spindle pole-associated zRbm14a condensates, smaller ones set to 1. Refer to Movies EV1 and EV2 for 3D-reconstructed images of the two representative cells in 2-hpf embryos.J. Quantification results on the ratio of zRbm14a condensates associated with spindle pole pairs in mitotic blastomere.
Figure 4Phenotypes of zRbm14 morphants. Totally 8 ng of ctrl-MO, 14-tMOs, or 14-MOs were co-injected with 100 pg of in-vitro transcribed GFP mRNA (as a tracer) into 1-cell embryos to generate control, maternal zRbm14, and zygotic zRbm14 morphants, respectively. Representative embryos, imaged under a dissecting microscope, are shown in (A)Phenotypes of zRbm14 morphants. Totally 8 ng of ctrl-MO, 14-tMOs, or 14-MOs were co-injected with 100 pg of in-vitro transcribed GFP mRNA (as a tracer) into 1-cell embryos to generate control, maternal zRbm14, and zygotic zRbm14 morphants, respectively. phenotyping results in (B). Total numbers of embryos analyzed are listed over histograms. Sample dots are included for quantification results with no error bars.Maternal zRbm14 morphants displayed reduced cell proliferation in MZT. Embryos stained with Hoechst 33342 and Pyronin Y to mark the nucleus and the cytoplasm, respectively, were imaged from the animal pole to cover a 10-μm z-depth with a confocal microscope (C). Maximum intensity-projected images (C)Maternal zRbm14 morphants displayed reduced cell proliferation in MZT. 30 embryos in each condition were used to quantify cell number (D), cell size (E), and nuclear size (F). Each sample dot represents the average value from each embryo.
Figure 5Phenotypes of embryos. Typical embryos, cropped from representative experimental groups in , are shown in (B). Only GFP-positive embryos viable at 2.5 hpf were collected and imaged for analyses.Phenotypes of embryos. Their phenotypes served as criteria for quantifications in (C). Only GFP-positive embryos viable at 2.5 hpf were collected and imaged for analyses. Total embryo numbers analyzed are listed over histograms.D. Hexanediol (Hex) treatment from 2.5 hpf blocked the blastula development. Representative images are presented for each group of embryos, with phenotypes indicated by insets of colored boxes. Total embryo numbers analyzed are listed over histograms.