EMBO/soda-nel huggingface dataset:
MIWI Prevents Aneuploidy during Meiosis by Cleaving Excess Satellite RNA (2020)
Figure 6

Figure 6. The effects of removing MIWI and Dicer on the progression and fidelity of meiotic division.

A Frequency of overlapping, adjacent, and separated inner kinetochores. Spermatocytes from Miwi+/-;Dicerf/+;Stra8-Cre, Miwi-/-;Dicerf/+;Stra8-Cre, Miwi+/-;Dicerf/-;Stra8-Cre, and Miwi-/-;Dicerf/-;Stra8-Cre mice were used to examine sister kinetochore configuration at meiosis I as shown in Fig 3A and 3B. n=3 mice for each genotype. The total numbers of counted cells in the four types of spermatocytes were 101, 117, 129 and 115, respectively. The total numbers of counted sister kinetochore pairs in the four types of spermatocytes were 4,040, 4,680, 5,160 and 4,600, respectively.

B Frequency of the different sister kinetochore configurations. The four types of spermatocytes in A were used to examine sister kinetochore configurations in meiosis I metaphase with O-O, S-S, O-S, or S-O as defined in the text. n=3 mice for each genotype. The total numbers of counted cells in the four types of spermatocytes were 134, 140, 140 and 130, respectively. The total numbers of counted sister kinetochore pairs in the four types of spermatocytes were 5,360, 5,600, 5,600 and 5,200, respectively.

C Frequency of abnormal arrangement in the meiotic metaphase I plate. The four types of spermatocytes in A and B were used to perform immunofluorescence as shown in Fig 1B and C. n=3 mice for each genotype. The total numbers of counted metaphase I plates in the four types of spermatocytes were 221, 181, 421 and 240, respectively.

D Frequency of meiosis I metaphase cells with chromosomal abnormalities. The four types of spermatocytes in A-C were used to perform a metaphase spread as shown in Fig 1H and 1I. n=3 mice for each genotype. The total numbers of counted MI cells in the four types of spermatocytes were 89, 73, 132 and 104, respectively.

E Frequency of meiosis II metaphase cells with chromosomal abnormalities. The four types of spermatocytes in A-D were used to perform a metaphase spread as shown in Fig 1K and L. n=3 mice for each genotype. The total numbers of counted MII cells in the four types of spermatocytes were 74, 87, 85 and 94, respectively.

Data information: The results represent the mean ± SD of at least three independent experiments. The statistical test was assessed using one-way ANOVA test. (*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; n.s., P>0.05)
Alu element‐containing RNAs maintain nucleolar structure and function (2015)
Figure 1

Pol II transcription is essential for nucleolar structure and function

Confocal laser scanning fluorescence microscopy (CLSM) images showing DNA staining (blue in the merged images), rDNA FISH (green) and NCL immunofluorescence (red) in untreated HeLa cells or in cells treated with α‐amanitin (50 μg/ml), AMD (50 ng/ml) or the Pol III inhibitor ML‐60218 (200 μM) for 5 h.
CLSM images of propidium iodide‐stained RNA after microinjection of buffer or nucleolar RNA into α‐amanitin‐treated HeLa cells. Nucleoli were visualized by immunofluorescence of nucleophosmin (NPM). The graph represents the average number of nucleolar domains (± 95% CI) based on the analysis of 90, 87, 80 and 86 cells, respectively. **P‐value 0.01, t‐test.

Data information: Scale bars, 10 μm.

See also Appendix Figs S1 and S2.
Alu element‐containing RNAs maintain nucleolar structure and function (2015)
Figure 3

aluRNA is localized to nucleoli and partially rescues α‐amanitin‐induced nucleolar dispersion

CLSM images showing nucleolar co‐localization of NCL (immunofluorescence) with aluRNA (RNA FISH) but not with L1‐repeat RNA. Cells were pre‐treated in situ with RNase A or an RNase inhibitor. Nuclei were counterstained with DAPI. The signal intensity of nucleolar aluRNA was two‐fold higher compared to nucleoplasmic signal (n = 92, P‐value 0.05, t‐test).
Graphs representing the average number of nucleolar bodies after microinjection of in vitro transcribed RNA into HeLa cells that were pre‐treated with α‐amanitin (50 μg/ml) for 5 h or left untreated (control) (± 95% CI. **P‐value 0.01, n = 90, 87, 86, 83 or 86 cells, respectively). Representative CLSM images of propidium iodide‐stained RNA are shown on the right side.

Data information: Scale bars, 10 μm.

See also Appendix Fig S5, Appendix Table S4.
Alu element‐containing RNAs maintain nucleolar structure and function (2015)
Figure 4

Knockdown or overexpression of aluRNA impact size of nucleoli and rDNA transcription

CLSM images showing NPM (immunofluorescence, red) in HeLa cells transfected with control ASO, aluRNA ASO, or treated with transfection reagent only (mock). Nuclei were counterstained with DAPI (blue). A fraction of 80 ± 8% of the cells treated with aluRNA ASO (n = 143) showed irregular nucleoli or dispersed nucleoli (53% of the cells). Only 26 ± 9% of the cells treated with control ASO presented abnormal nucleoli, with none of them showing a dispersion of nucleolar domains (n = 123), P‐value 0.01, t‐test.
CLSM images showing NCL localization (immunofluorescence), aluRNA (RNA FISH) and DNA (DAPI). Transfection of HeLa cells was done as in (A).
HeLa cells were transfected as in (A), and nascent RNAs were visualized by pulse labeling with ethynyl uridine (EU) for 30 min. Nuclei were counterstained with DAPI (DNA).
Levels of 47S pre‐rRNA in mock‐, control ASO‐ and aluRNA ASO‐treated HeLa cells. Top: Northern blot. Center: Agarose gel electrophoretic analysis of RNA. Bottom panel: Quantification from RT–qPCR levels of 47S pre‐rRNA normalized to 18S rRNA. Error bars represent the standard deviation (n = 6). **P‐value 0.01, t‐test.

Data information: Scale bars, 10 μm. See also Appendix Figs S1C, S6, S7 and S8, Appendix Table S4.

Source data are available online for this figure.
Alu element‐containing RNAs maintain nucleolar structure and function (2015)
Figure 5

aluRNA overexpression induces an increase in nucleolar volume and activity

HeLa cells were transfected with an empty vector or plasmids expressing aluRNA or L1‐repeat RNA and GFP from a separate promoter. Transfected cells were identified by GFP fluorescence, and nucleoli volumes were evaluated based on the immunofluorescence signal of the nucleolar marker NPM. The graphs show that the volume of nucleoli is increased by 30% in the presence of ectopic aluRNA (± 95% CI, n = 20). **P‐value 0.01, t‐test. Scale bar, 10 μm.
CLSM live‐cell imaging of RFP‐NCL in cells transfected with a plasmid expressing aluRNA and imaged for 10 h. The arrows indicate nucleolar domains displaying time‐dependent increase in the nucleolar size or fusion of two domains into one. Scale bars, 10 μm.
HeLa cells were transfected as in (A) and incubated for 24 h. Nascent RNA was pulse‐labeled for 30 min, and nucleoli were visualized by immunofluorescence of NPM. Scale bars, 10 μm.
Graphs represent the intensity of nucleolar fluorescence signals as percentage of the intensity of nuclear signals and reveal a 27% increase in the presence of ectopic aluRNA (± 95% CI, n = 30). **P‐value 0.01, *P‐value = 0.01, t‐test.
Alu element‐containing RNAs maintain nucleolar structure and function (2015)
Figure 6

Nucleoli disruption in mouse cells by depletion of B1‐containing RNAs

CLSM images of UBF immunofluorescence in mouse NIH 3T3 cells treated for 5 h with α‐amanitin to inhibit Pol II transcription show the dispersion of nucleoli, as previously also imaged via RNA staining (Caudron‐Herger et al, ).
CLSM images showing the localization of NPM (immunofluorescence, red) in mouse NIH 3T3 cells treated with ASO as indicated and counterstained with DAPI (blue).

Data information: Scale bars, 10 μm.
Alu element‐containing RNAs maintain nucleolar structure and function (2015)
Figure 7

Ectopically expressed aluRNA accumulates in the nucleolus together with associated genomic loci

CLSM images of MS2‐tagged aluRNA or MS2‐tagged control RNA (a fragment from the transcript of the CDV3 gene). MS2‐loop‐containing RNAs were visualized by RNA FISH using MS2‐loop‐specific FISH probes (green); nucleoli were visualized by immunofluorescence of NCL (red).
The top panel depicts the experimental approach used to tether MS2‐loop‐containing RNAs to lacO arrays via a GFP‐tagged LacI‐MS2 coat fusion protein. The lacO arrays were stably integrated in the genome of U2OS cells. In the bottom panel, CLSM images of U2OS cells are shown that were transfected with MS2‐aluRNAR. They reveal the localization of LacI‐GFP‐MS2 and NCL (immunofluorescence, red). The arrow indicates the lacO array, which is associated with a nucleolar domain.
MS2‐loop‐containing forward aluRNA or aluRNAL or aluRNAR were recruited to the single stably integrated lacO array in the U2OS F4IIB8 cell line. The propensity for nucleolar localization was evaluated as the average number of lacO arrays with tethered RNA detected in nucleoli (± 95% CI) and is plotted in the bar chart. Calculations are based on analysis of more than 100 cells. **P‐value 0.01, t‐test, from the analysis of two independent biological replicates. As controls, transcripts of the MS2 loops only, the MS2‐CDV3, MS2‐RepA, MS2‐STARD7 and MS2‐CORO1C RNAs were used. The inset shows a CLSM image with MS2‐GFP‐LacI (green) and NPM (red, immunofluorescence) after recruitment of MS2‐aluRNA.

Data information: Scale bars, 10 μm.
Alu element‐containing RNAs maintain nucleolar structure and function (2015)
Figure 8

NCL and NPM interact with aluRNA and target genomic loci to the nucleolus

Top: Scheme illustrating the experimental approach used to tether GFP‐tagged LacI fusion proteins via a GFP‐binding protein (GBP) to lacO arrays. The lacO arrays are stably integrated in the genome of U2OS cells. Bottom: CLSM images showing the localization of GBP‐LacI‐RFP, GFP‐NCL, GFP‐NPM or GFP‐TIP5‐RBD fusion proteins relative to aluRNA that was visualized by RNA FISH. The insets contain an enlarged image of one of the lacO loci.
CLSM images showing GBP‐LacI‐RFP and GFP‐NCL (green) recruited to lacO arrays as indicated by arrows. Immunofluorescence of endogenous NPM (red) marks nucleoli. The inset shows decondensation of lacO arrays in the nucleolus. The graph at the bottom depicts the average number of lacO loci tethered to nucleoli (± 95% CI) by the indicated proteins (n = 72). NCL‐∆RNA is an NCL deletion mutant lacking the RBD and GAR domains required for RNA binding. **P‐value 0.01, t‐test.
CLSM images of UBF (immunofluorescence, green) or rDNA (DNA FISH, green) and DNA (DAPI, blue) after knockdown of NCL and NPM by siRNA in HeLa cells.

Data information: Scale bars, 10 μm.

See also Appendix Fig S9.
Ceramide targets autophagosomes to mitochondria and induces lethal mitophagy (2012)
figf1

(a) C18-pyridinium ceramide. (b) Effects of C18-Pyr-Cer (C18PC, lanes 2 and 4) on the lipidation of LC3B (LC3B-II) were examined by western blotting in the absence or presence of a pan-caspase inhibitor Z-VAD (lanes 3 and 4) and compared to vehicle-treated controls (lanes 1 and 3). Full blots can be found in Supplementary Figure 13. (c) Formation of double-membrane autophagosomal vesicles (black arrows) in the absence (control) or presence of C18PC was visualized by TEM (left and right, respectively). Higher magnification of TEM visualization is shown in lower panels. Scale bars, 10 μm (top) and 500 nm (bottom). (d) Effects of siRNA-mediated knockdown of Atg3 or Atg7 on cell death in the absence or presence of C18PC were determined and compared to effects of transfection with Scr siRNAs (controls) using Trypan blue exclusion assay. (e) Roles of C18PC in the regulation of lethal autophagy were assessed after treatment of MEFs isolated from wild-type (ATG5+/+) and ATG5−/− knockout mice with C18PC. Control cells were treated with vehicle. (f) Effects of C18PC on caspase-dependent or caspase-independent cell death were determined in MEFs isolated from Bax−/− Bak−/− or Casp3−/− Casp7−/− mice and from wild-type (Bax+/+ Bak+/+) or Casp3+/− Casp7+/− mice, used as controls. Data shown are an average of at least three experiments ± s.d. (*P 0.05).
Ceramide targets autophagosomes to mitochondria and induces lethal mitophagy (2012)
figf3

(a) Effects of expression of wild-type (WT) CerS1 and its catalytically inactive V5-tagged mutant (Mut), which cannot generate C18-ceramide, on the formation of LC3B-II were determined by western blotting (+/− tet). Successful induction of wild-type and mutant CerS1 expression was confirmed using anti-V5. β-actin was used as a loading control. Full blots can be found in Supplementary Figure 13. (b) Generation of endogenous C18- and C18:1-ceramides in response to wild-type CerS1 and mutant CerS1 induction was measured by LC/MS/MS. (c) Effects of wild-type CerS1 and C18-ceramide induction (+ tet) on GFP and LC3B-GFP lipidation were visualized using confocal microscopy and compared to effects of noninduced controls (− tet). Scale bars, 10 μm. (d) The effects of induction of wild-type CerS1 and C18-ceramide and catalytically inactive mutant CerS1 (+ tet) in the regulation of mitochondrial function were assessed by measuring OCR using the SeaHorse and compared to roles of noninduced controls (− tet). (e) Targeting mitochondria with autophagolysosomes in the absence (− tet) or presence (+ tet) of wild-type CerS1 and C18-ceramide was visualized by colocalization of MTG and LTR using confocal microscopy. Scale bars, 10 μm. (f) Effects of wild-type and mutant CerS1 (−/+ tet) on ATP generation. (g) Effects of wild-type CerS1 (−/+ tet) on ATP generation in the absence (control shRNA) or presence of shRNA-mediated knockdown of LC3B. Data shown are an average of at least three experiments ± s.d. (*P 0.05).
Ceramide targets autophagosomes to mitochondria and induces lethal mitophagy (2012)
figf4

(a) Subcellular localization of ceramide, generated by wild-type CerS1 induction (+ tet), was visualized in mitochondria by colocalization of ceramides and mitochondria with anti-ceramide and anti-Tom20, respectively, using confocal microscopy. (b,c) Mitochondrial (Mito) targeting of LC3B-II and C18-ceramide, generated by induction of wild-type CerS1 (b), and that of the products of the catalytically inactive mutant CerS1 (+ tet) (c) was visualized by colocalization of ceramide (Cer; green), CFP-LC3B (blue) and MTR using anti-ceramide and confocal microscopy. Noninduced cells (− tet) were used as controls. Scale bars, 10 μm.
Ceramide targets autophagosomes to mitochondria and induces lethal mitophagy (2012)
figf5

(a) Binding of Flag-tagged wild-type LC3B and LC3BI35A, LC3BF52A or LC3BG120A with endogenous C18-ceramide (C18-Cer), generated in response to wild-type CerS1 induction (left) or exogenous C18-Pyr-Cer (right), was measured using LC/MS/MS after pulldown using anti-Flag–conjugated beads. Data shown are an average of at least three experiments ± s.d. (*P 0.05). (b) Effects of CerS1 and C18-ceramide induction on the lipidation of Flag-tagged wild-type, LC3BI35A, LC3BF52A and LC3BG120A proteins were examined by western blotting. Noninduced cells (− tet) were used as controls. β-actin was used as a loading control. Full blots can be found in Supplementary Figure 13.
Ceramide targets autophagosomes to mitochondria and induces lethal mitophagy (2012)
figf6

(a,b) Effects of G120A and F52A mutations, which perturbed C18-ceramide binding of LC3B, on targeting of mitochondria by LC3B-containing autophagosomes (visualized by confocal microscopy using anti-Flag and anti-Tom20) (a) or mitochondrial function (measurement of OCR) (b). V, vector. (c) Effects of siRNA-mediated knockdown of p62, NIX and Drp1 on OCR in the absence or presence of CerS1 and C18-ceramide induction were measured using the SeaHorse. Data shown are an average of at least three experiments ± s.d. (*P 0.05). (d) Effects of siRNA-mediated knockdown of Drp1 (lower left and right) on the localization of ceramide within mitochondrial membranes were visualized using the colocalization of anti-ceramide (red) and anti-Tom20 (green) under confocal microscopy, in the absence (upper and lower left) or presence (upper and lower right) of CerS1 and C18-ceramide induction (−/+ tet) compared to Scr siRNA–transfected controls (upper left and right). Scale bars, 10 μm in a and d. Enlarged images are shown in boxed areas in d.
Ceramide targets autophagosomes to mitochondria and induces lethal mitophagy (2012)
figf7

(a,b) Roles of shRNA-mediated stable knockdown of endogenous LC3B expression in the regulation of mitochondrial function (a) or UM-SCC-22A xenograft-derived tumor growth (b) in the absence or presence of wild-type CerS1 and C18-ceramide induction (−/+ tet) were determined using the SeaHorse or measurement of tumor volumes in the flanks of SCID mice (n = 6 per group), respectively. Data shown are an average of at least three experiments ± s.d. (*P 0.05). (c) Mechanism by which C18-ceramide (Cer) induces lethal autophagy involves, at least in part, the lipidation of LC3B, forming LC3B-II (LC3-PE), and interaction of LC3B-II and ceramide on mitochondrial membranes upon Drp1-mediated mitochondrial fission, which targets autophagolysosomes to mitochondria for lethal mitophagy.
Phosphorylation of myelin regulatory factor by PRKG2 mediates demyelination in Huntington's disease (2019)
Figure 1

Figure 1. LAQ increased the expression of myelin associated proteins and reduced the binding of MYRF to mutant HTT in PLP-150Q mice

Electron microscopy revealed a number of demyelinated or degenerated axons in PLP-150Q mice at the age of 5 months. LAQ (5 mg/kg) treatment at 3 months of age for 2 months improved myelination in PLP-150Q mice. Scale bars: 2μm (Low magnification) and 0.5 μm (High magnification).

G-ratios, which were calculated and plotted against axon diameter with linear regression, were shown beneath the micrographs and were significantly decreased in PLP-150Q mice after LAQ treatment (G=0.6418±0.0191), compared with age-matched vehicle group (G=0.7727±0.0203). One-way ANOVA with Tukey's test. *P=0.0166. At least 182 axons per genotype were examined from 3 mice in each group.

Western blotting showing that LAQ (5 or 25 mg/kg) treatment for 2 months up-regulates multiple myelin proteins (MBP, MOBP and MOG) in the corpus callosum in PLP-150Q mice, which were treated from 3 months of age. Ratios of MBP, MOBP or MOG to vinculin obtained from 3 independent experiments were presented on the right. One-way ANNOVA followed with Tukey's test. MBP: ***P=0.0009; MOG: ***P=0.0003; MOBP: **P=0.0067. Data are mean ±SEM

Quantitative PCR of the transcript expression of myelin associated genes (MBP and MOG) in PLP-150Q mouse corpus callosum at 5 months of age after LAQ (5 or 25 mg/kg) treatment for 2 months. Student's t-test. MBP: ***P=0.0007; MOG: ***P=0.0009. Data are mean ±SEM (n=3).

The MBP DNA promoter was inserted into the pGL4.1 luciferase report vector and was co-expressed with N-terminal MYRF (nMYRF) and mHTT to assess its transcription activity via the luciferase assay. MYRF markedly enhanced the MBP promoter activity. N-terminal mutant HTT significantly inhibited the reporter activity, ***P< 0.001; which was reversed by LAQ (5 μM) treatment, **P< 0.01. The ratios were obtained from 3 independent experiments. One-way ANNOVA followed with Tukey's test. Data are mean ±SEM.

Immunoprecipitation of transgenic mHTT from 3-month-old PLP-150Q mouse brains revealed that 5 or 25 mg/kg LAQ treatments for 2 months reduced the interaction between transgenic mutant HTT and MYRF. fMYRF: full-length MYRF; nMYRF: N-terminal MYRF. The ratio of immunoprecipitated MYRF to input obtained from 3 independent experiments was shown on the right. One-way ANNOVA followed with Tukey's test. **P=0.009. Data are mean ±SEM.
Phosphorylation of myelin regulatory factor by PRKG2 mediates demyelination in Huntington's disease (2019)
Figure 2

Figure 2. LAQ dephosphorylated N-terminal MYRF at pS259 to affect its interaction with mutant HTT

Western blotting of the phosphorylated- NF-kB, -Akt, -JNK in PLP-150Q mouse corpus callosum at 3 months of age showing that LAQ (5 or 25 mg/kg) treatment for 2 months could dephosphorylate these proteins. Ratios of the phosphorylated proteins to their total proteins obtained from 3 independent experiments were presented on the right. One-way ANNOVA with Tukey's test. NF-kB: ***P=0.00067(left) and 0.00086(right); JNK: ***P=0.00013(left) and 0.00047(right); Akt: **P=0.0014 and ***P=0.00012. Data are mean ±SEM.

In vitro phosphorylation assay of N-terminal MYRF (nMYRF). GST fusion proteins containing nMYRF were incubated overnight with brain lysates from PLP-23Q or PLP-150Q mice that were treated with vehicle or 5 mg/kg LAQ (left panel). GST fusion proteins containing wild type N-terminal MYRF, S259A, or S261A were incubated with PLP-150Q mouse tissue lysates (right panel). The beads were then centrifuged and analyzed by western blotting with anti-GST (lower panels) and anti-phosphor-serine (upper panels). Note that LAQ treatment could eliminate mutant HTT (PLP-150Q)-mediated MYRF phosphorylation and that S259A substitution prevents MYRF phosphorylation.

Co-immunoprecipitation of nMYRF and N-terminal HTT (1-212 aa) containing 150Q in HEK293 cells. Five μM LAQ treatment decreased the phosphorylation of immunoprecipitated MYRF and the amount of co-immunoprecipitated HTT. Λ-PPase served as the positive control for the dephosphorylation of MYRF. The ratios of the pho-serine MYRF or precipitated HTT to immunoprecipitated MYRF are shown under the blots, which were obtained from 3 independent western blotting experiments. One-way ANNOVA with Tukey's test. ***P=6.51x10-5. Data are mean ±SEM.

Co-transfection of nMYRF with N-terminal mutant HTT in HEK293 cells and immunoprecipitation of nMYRF with or without LAQ (5 μM) treatment. Coomassie blue staining confirms the presence of immunoprecipitated N-terminal MYRF bands (arrow).

Transcriptional activity of wide type N-terminal MYRF, S259A, or S261A with the MBP promoter reporter in HEK293 cells was detected using a luciferase assay. Compared to wide type MYRF or S261A, S259A (non-phosphorylated) remained transcriptional activity that was not inhibited by N-terminal mutant HTT. The ratios were obtained from 3 independent experiments. One-way ANNOVA with Tukey's test. ***P< 0.001. Data are mean ±SEM.

Co-transfection of wide type N-terminal MYRF, S259A, or S261A with N-terminal mutant HTT in HEK293 cells and immunoprecipitation of MYRF. Compared to wide type MYRF and S261A, less S259A (non-phosphorylated) was precipitated with mutant HTT. The ratios of the pho-serine MYRF or precipitated HTT to immunoprecipitated MYRF are shown under the blots and were obtained from 3 independent experiments. One-way ANNOVA with Tukey's test. ***P=2.30x10-5 . Data are mean ±SEM.