EMBO/soda-nel huggingface dataset:
The MMS22L-TONSL heterodimer directly promotes RAD51- dependent recombination upon replication stress (2016)
Figure 5

Figure 5. MMS22L interacts physically and functionally with RAD51.

A Amylose pulldown of MBP (control) or MBP-tagged MMS22L-TONSL and RAD51. Eluate proteins were detected by silver staining. MT: MBP-MMS22L-GST-TONSL.

B Quantitative analysis of RAD51 binding to MMS22L-TONSL such as shown in Figure EV6A. Averages shown, n = 2; error bars, SEM.

C, D Yeast two-hybrid analysis of interactions between LexA DNA binding domain-tagged MMS22L and indicated proteins fused to GAL4 activating domain (GAD) assessed by survival on media containing 3-AT in the absence of histidine and by X-Gal assays.

E, F Streptavidin pulldowns of indicated recombinant proteins and biotinylated ssDNA. The proteins in the input and eluates were detected by western blotting.

G Streptavidin pulldown of HSS-tagged MMS22L variants stably integrated and overexpressed in U2OS cells. Input and co-precipitated proteins were detected by immunoblotting.

H Representative immunofluorescence images of CPT-induced foci in stable U2OS cell lines depleted of endogenous MMS22L and overexpressing indicated siRNA-resistant variants of HSS-MMS22L treated with 50 nM CPT for 1 hour. Scale bar: 5 μm.

I-K Quantification of the effect of indicated MMS22L mutations on RAD51 foci in U2OS cells treated with 50 nM CPT for 1 h (I; n = 3), 50 nM CPT for 18 h (J; n = 3) or 5 µM ETP for 1 h followed by 3 hours incubation without the drug (K; n = 3). Graphs represent averaged median values; error bars indicate SEM. Statistical analysis according to one-way ANOVA with Bonferroni post-test; *** P ≤ 0.001; ** P ≤ 0. 01; ns, not significant. Representative immunofluorescence images are shown in (H), Figures EV6G and EV6H, respectively.
The MMS22L-TONSL heterodimer directly promotes RAD51- dependent recombination upon replication stress (2016)
Figure 6

Figure 6. MMS22L-TONSL promotes DNA strand exchange by reducing RAD51 binding to dsDNA.

A Schematic representation of the DNA strand exchange assay with ssDNA.

B-D Strand exchange assay with ssDNA and RAD51, and with (C) or without (B) the addition of MMS22L-TONSL (75 nM).

D Quantification shows averages, n = 2; error bars, SEM.

E Averages of quantified strand exchange assays with ssDNA, RAD51 (270 nM) and varying concentrations of MMS22L-TONSL or MMS22L. n = 2; error bars, SEM.

F Quantification of electrophoretic mobility shift assays with dsDNA or ssDNA, RAD51 and MMS22L-TONSL or MMS22L. Averages shown, n = 2; error bars, SEM.

G A scheme of streptavidin pulldown assay performed in (H, I).

H A representative immunoblot showing the effect of increasing amounts of dsDNA on RAD51 binding to ssDNA (1 nM).

I A representative immunoblot showing effects of MMS22L-TONSL and MMS22L titration into reactions containing RAD51, ssDNA (1 nM) and dsDNA (10 nM), as indicated.
Identification and characterization of novel factors that act in the nonsense-mediated mRNA decay pathway in nematodes, flies and mammals. (2015)
Figure 2

Newly identified NMD factors

RNAi was induced with an empty vector as a negative control (panel I), whereas a smg-2 clone (panel II) was used as a positive control. Panels i and ii show brightfield images of the PTCxi strain treated with the negative and positive controls, respectively. Depletion of five genes (panels III to VII) resulted in increased GFP expression. Panels iii to vii show brightfield images of the phenotypes of the affected worms. The scale bars correspond to 100 μm.

Depletion of the novel NMD genes in C. elegans leads to upregulation of the PTCx NMD reporter mRNA, which was monitored by quantitative RT–PCR relative to the expression of ama-1 reference gene. The values shown are the average fold-change (mean ± SEM) from at least three independent experiments relative to empty vector-depleted worms. Statistical analysis was performed using the Mann–Whitney U-test for non-parametric distributions. *P < 0.05.
Identification and characterization of novel factors that act in the nonsense-mediated mRNA decay pathway in nematodes, flies and mammals. (2015)
Figure 3

<italic>Drosophila nompA</italic> is required for NMD in <italic>Drosophila</italic> embryos

A–C Embryos expressing a GFP-NMD sensor (green) under the control of nompA regulatory sequences show signal in support cells linked to the embryonic peripheral nervous system (PNS) [labeled by 22C10 signal (magenta)]. DAPI signal is shown in blue. Expression of UAS-RNAi constructs against Upf1 (B) or nompA (C) genes using a nompA-Gal4 driver leads to upregulation of the GFP-NMD sensor when compared to wild-type (no RNAi) (A), revealing a reduction in NMD activity in the knockdown conditions. Scale bars represent 100 μm.

D–F Higher magnification (40×) of the areas marked by a rectangle in (A–C) further illustrates the upregulation of the GFP-NMD sensor in Upf1- (E) and NompA-depleted cells (F). Optical fields include embryonic abdominal segments A4–A5. Scale bars represent 10 μm.

G Quantification of GFP signal in cells marked by an arrow in panels (D–F) shows a significant upregulation of NMD sensor expression upon downregulation of Upf1 (dark gray) and NompA (black) compared to wild-type (no RNAi) (light gray). Results represent the average of five biological replicates (mean ± SEM). Pair-wise comparisons were performed using a one-tailed t-test (non-parametric) between treatments and wild-type. ***P < 0.001.
Identification and characterization of novel factors that act in the nonsense-mediated mRNA decay pathway in nematodes, flies and mammals. (2015)
Figure 4

GNL2 and SEC13 are required for NMD in human cells

A, B HeLa cells stably expressing a wild-type β-globin reporter (A) or a β-globin NS39 NMD reporter (B) were mock-depleted or depleted of UPF2, GNL2, SEC13, RAB27A, RAB27B or both RAB27A and RAB27B. The level of the β-globin mRNA was monitored by quantitative RT–PCR relative to two reference genes (POLR2J and ACTB). The values shown are the average fold-change (mean ± SEM) from four independent experiments relative to mock-depleted cells (control). Statistical analysis was performed using the Mann–Whitney U-test for non-parametric distributions. *P < 0.05. The level of depletion of NMD factors is shown in Supplementary Fig S3C.

C, D Analysis of the half-life of β-globin reporters. Samples were collected at the indicated time points, and the mRNA levels of the HBB reporters were monitored by qRT–PCR and normalized to POLR2J and ACTB reference genes. The values shown are the average fold-change (mean ± SEM) from three independent experiments relative to the first time point.

E Depletion of GNL2 and SEC13 leads to a significant upregulation in the mRNA levels of endogenous NMD substrates. Samples were analyzed as described in (A, B) *P < 0.05; ***P < 0.01, ***P < 0.001.

F GNL2 and SEC13 contribute to the negative NMD feedback loop, regulating the levels of transcripts encoding NMD factors. RT–qPCR analysis of total cellular RNA from HeLa cells depleted of GNL2 (in green) and SEC13 (in purple) is shown. The graph shows distribution of relative fold-change from eight independent experiments relative to mock-depleted cells (control). Statistical analysis was performed using Student's t-test. *P < 0.05.
A THEMIS:SHP1 complex promotes T‐cell survival (2014)
Figure 1

SHP1 and SHP2 binding to THEMIS is GRB2 dependent

SHP1 immunoprecipitation (IP) from Jurkat 1G4‐CD8 cells. Upper panel of immunoblots shows expression levels in the input lysates, and isolated protein complexes are shown at the bottom. Rabbit IgG IP (isotype) is shown as control.
Lentiviral knock‐down/re‐expression constructs for THEMIS‐wt‐Strep and THEMIS‐dPRR1‐Strep in 1G4‐CD8 cells. Knock‐down/re‐expression constructs are based on shTHEMIS‐128476. Red arrowheads indicate the specific signal for endogenous THEMIS and re‐expressed THEMIS‐Strep. The higher molecular weight band observed in all lanes is a non‐specific signal.
Streptactin pull‐downs (PDs) of THEMIS‐Strep from 1G4‐CD8 cells. Cells as described in (B) were stimulated with 6V tetramers, followed by Streptactin PDs of THEMIS and immunoblot analysis of isolated protein complexes. Separate experiments for SHP1 and SHP2 are shown.
SHP1 IP in the presence and absence of SHP1 tail phosphorylation. SHP1 was immunoprecipitated from J.CaM1.6 LCK‐Tet cells. Where indicated, LCK expression was induced with doxycycline prior to the experiment or Src‐kinase inhibitor PP2 was used to abolish residual SHP1‐pY564. The upper panel of immunoblots shows the effect of LCK activity on SHP1‐pY564 levels. The red arrowhead indicates residual pY564. Isolated protein complexes are shown in the lower panels.
In vitro phosphatase treatment of the THEMIS:GRB2:SHP1 complex. Bead‐bound THEMIS‐Strep complexes from 1G4‐CD8 cells were incubated in alkaline phosphatase (AP) buffer in the absence (lane 1) or presence of AP (lane 2), or left completely untreated (lane 3), prior to washing and elution.
Anti‐HA IP from HEK293 cells transfected with HA‐SHP1 and GRB2‐Myc constructs. GRB2 mutants used: W36K, N‐SH3 mutant; W193K, C‐SH3 mutant. Upper panels show expression levels in the input; isolated proteins complexes are shown at the bottom. Relative amounts of GRB2‐Myc are normalized to the bait HA‐SHP1.
Far‐Western blot of GRB2‐SH3 domains binding to full‐length SHP1. HA IPs from empty vector or HA‐SHP1‐transfected HEK293 cells were subjected to far‐Western blotting using recombinant GST‐tagged N ‐or C‐SH3 domains of GRB2. GST alone served as a background control. Blots were re‐probed for SHP1 loading for normalization of anti‐GST signals. Data shown are representative of three independent experiments.
A THEMIS:SHP1 complex promotes T‐cell survival (2014)
Figure 2

THEMIS knock‐down increases TCR‐induced phospho‐ERK in Jurkat 1G4‐CD8 cells

Lentiviral knock‐down (KD) of THEMIS expression in 1G4‐CD8 cells. The efficiency of the THEMIS KD was assessed using immunoblotting.
Phospho‐flow analysis of pERK responses in THEMIS KD cells. 1G4‐CD8 control and THEMIS KD cells were stimulated with NY‐ESO‐1 pMHC tetramers and analysed for ERK phosphorylation using flow cytometry. Data shown are representative of three independent experiments.
Quantification of pERK‐positive cells from (B). Data from three independent experiments were used in the analysis. n = 3, means ± SEM are shown; two‐tailed unpaired Student's t‐test, *P 0.05, **P 0.01.
Phospho‐flow analysis of pERK responses against titrated concentrations of 9V tetramer. Time point was fixed at 60 s. Data shown are representative of three independent experiments.
Quantification of pERK‐positive cells from (D). Data from three independent experiments were used in the analysis. n = 3, means ± SEM are shown.
A THEMIS:SHP1 complex promotes T‐cell survival (2014)
Figure 3

THEMIS knock‐down increases TCR‐induced pY142‐CD3‐ζ phosphorylation

1G4‐CD8 control and THEMIS KD cells were stimulated with NY‐ESO‐1 pMHC tetramers and analysed for pY142‐CD3‐ζ phosphorylation by flow cytometry. Data shown are representative of three independent experiments.
Quantification of pY142‐CD3‐ζ responses of cells from (A). Data from three independent experiments were used in the analysis. Responses are expressed as percentage of the positive control sodium pervanadate. n = 3, means ± SEM are shown; two‐tailed unpaired Student's t‐test, **P 0.01.
A THEMIS:SHP1 complex promotes T‐cell survival (2014)
Figure 4

THEMIS knock‐down increases TCR signalling in peripheral CD4+ T cells

Lentiviral knock‐down of THEMIS expression in primary human CD4+ T cells. Following transduction, cells were expanded on CD3/CD28 beads in the presence of exogenous IL‐2 and analysed for THEMIS KD by immunoblotting on day 4 post‐transduction. Data shown are representative of three independent experiments.
pERK response in THEMIS KD human CD4+ T cells. Transduced CD4+ cells were removed from beads, rested overnight and stimulated with CD3 mAb for the indicated time points. ERK phosphorylation was assessed by immunoblotting. Data shown are representative of three independent experiments.
Expression of CD25 and CD69 surface markers of THEMIS KD human CD4+ T cells was assessed on day 4 post‐transduction by flow cytometry. Data shown are representative of three independent experiments.
CD69 up‐regulation on re‐stimulated THEMIS KD CD4+ T cells. CD3/CD28 beads were removed on day 1 post‐transduction, and cells were cultured in IL‐2 and IL‐7. After removal of cell debris at day 4, cells were restimulated with CD3/CD28 beads at the indicated bead to cell ratio. CD69 surface expression was assessed by flow cytometry 24 h later. Data shown are representative of two independent experiments. n = 3, means ± SD are shown; two‐tailed unpaired Student's t‐test, *P 0.05, ***P 0.001.
A THEMIS:SHP1 complex promotes T‐cell survival (2014)
Figure 5

Increased TCR‐induced apoptosis in THEMIS KD T cells

Donor‐derived human CD4+ T cells transduced as in Fig A were analysed for apoptosis by Annexin‐V surface staining on day 4 post‐transduction. Data from three independent experiments were used in the analysis. n = 3, means ± SD are shown; two‐tailed unpaired Student's t‐test, **P 0.01.
Annexin‐V staining of THEMIS KD 1G4‐CD8 cells. THEMIS KD and control 1G4‐CD8 cells were stimulated for 24 h with plate‐bound 6V tetramers. Annexin‐V surface expression was analysed by flow cytometry and is shown as fold‐increase over non‐stimulated. Data from three independent experiments were used in the analysis. n = 3, means ± SD are shown; two‐tailed unpaired Student's t‐test, *P 0.05, **P 0.01, ***P 0.001; ns, not significant.
Poly‐caspase activity in THEMIS KD 1G4‐CD8 cells. Cells were stimulated for 24 h with NY‐ESO‐1 pMHC tetramers. Camptothecin was used as a positive control for caspase activation. A FAM‐FLICA detection probe was used to assess poly‐caspase activity by flow cytometry. Data shown are a representative example of three independent experiments.
Quantification of poly‐caspase activity of cells from (C). Data from three independent experiments were used in the analysis. n = 3, means ± SEM are shown; two‐tailed unpaired Student's t‐test, *P 0.05, **P 0.01; ns, not significant.
A THEMIS:SHP1 complex promotes T‐cell survival (2014)
Figure 6

Increased TCR‐induced apoptosis in SHP1 KD T cells

Lentiviral KD of SHP1 expression in 1G4‐CD8 cells. KD efficiency was assessed using immunoblotting.
Annexin‐V staining of THEMIS and SHP1 KD 1G4‐CD8 cells. Control, THEMIS and SHP1 knock‐down Jurkat 1G4‐CD8 cells were stimulated for 24 h with plate‐bound 6V tetramers. Annexin‐V surface expression was analysed by flow cytometry and is shown as fold‐increase over non‐stimulated. Data from three independent experiments were used in the analysis. n = 3, means ± SD are shown; two‐way ANOVA, Bonferroni post‐test, *P 0.05, **P 0.01, ***P 0.001.
Lentiviral knock‐down of THEMIS and SHP1 expression in primary human CD4+ T cells. Following transduction, cells were expanded on CD3/CD28 beads in the presence of exogenous IL‐2. Knock‐down efficiency was analysed by immunoblotting on day 4 post‐transduction.
Expression of CD25, CD69 and Annexin‐V surface markers of cells from (C) was assessed by flow cytometry. Data shown are representative of three independent experiments.
Annexin‐V staining of 1G4‐CD8 cells re‐expressing a SHP1 tail tyrosine mutant. Jurkat 1G4‐CD8 cells were transduced with Knock‐down/re‐expression constructs for HA‐SHP1 wild‐type (wt) or Tyr536/564 to Phe double mutant (YYFF). Cells were stimulated and analysed by flow cytometry as in (B). Data shown are representative of three independent experiments. n = 3, means ± SD are shown.
A THEMIS:SHP1 complex promotes T‐cell survival (2014)
Figure 7

T‐cell activation in OT‐I TCR transgenic mice expressing LCKS59A molecules

CD8+ T cells from OT‐I Rag2−/− mice expressing wild‐type (LCK) or LCKS59A proteins were stimulated with H‐2Kb‐positive antigen‐presenting cells pulsed with serial dilutions of the OVA (257–264) peptide, the OVA peptide variants Q4 and T4, and VSV, a peptide that is not recognized by the OT‐I TCR. CD69 up‐regulation (left) and TCR down‐regulation (right) were analysed. Data are representative of three independent experiments.
CD8+ T cells from OT‐I Rag2−/− mice expressing wild‐type (LCK) or LCKS59A proteins were labelled with CFSE and stimulated with serial dilution of H‐2Kb‐OVA or H‐2Kb‐Q4R7 peptide‐MHC tetramers. After 3 days, the absolute numbers of divided T cells were determined. Data are representative of three independent experiments.
MIWI Prevents Aneuploidy during Meiosis by Cleaving Excess Satellite RNA (2020)
Figure 1

Figure 1. Increased frequency of cell death and chromosome segregation defects in Miwi-/- spermatocytes.

A TUNEL assay comparing apoptotic cells in Miwi+/- and Miwi-/- testis during diakinesis. Blue, DAPI; Green, TUNEL labeling. Asterisk indicates TUNEL-positive stained cells in the meiotic metaphase stage. Scale bars, 10 μm.

B, C Meiotic metaphase I plate in FACS-sorted Miwi heterozygous (B) and knockout (C) spermatocytes. Green: phospho-histone H3; red: α/β-tubulin. Asterisk indicates misaligned chromosome. Scale bars, 10 μm.

D Frequency of abnormal arrangement in the meiotic metaphase I plate. White, Miwi heterozygous; Light gray, Miwi knockout. Three mice were used for each genotype, total numbers of counted metaphase I plate in Miwi heterozygous and knockout are 113 and 181, respectively.

E, F Meiotic metaphase II spread preparations of Miwi heterozygous (E) and knockout (F) spermatocytes. Scale bars, 5 μm.

G Frequency of meiotic metaphase II (MII) cells with chromosomal abnormalities. Chromosome counts were defined as the number of paired sister chromatids per MII metaphase cell. The frequency was calculated as the number of cells with given pairs of sister chromatids (20, <20, or >20) divided by the total number of cells. Three mice were used for each genotype, total numbers of counted MII cells in Miwi heterozygous and knockout are 204 and 302, respectively.

H, I Meiotic metaphase I spread preparations of Miwi heterozygous (H) and knockout (I) spermatocytes. Scale bars, 5 μm.

J Frequency of meiotic metaphase I (MI) cells with chromosomal abnormalities. Chromosome counts were defined as the number of paired homologous chromosomes per MI metaphase cells. The frequency was calculated in the same way as in (G), except for that the number of paired chromosomes was counted instead. Three mice were used for each genotype, total numbers of counted MI cells in Miwi heterozygous and knockout are 265 and 358, respectively.

K, L Mitotic metaphase spread preparations of Miwi heterozygous (K) and knockout (L) testes. Scale bars, 5 μm.

M Frequency of mitotic metaphase cells with chromosomal abnormalities. Chromosome counts were defined as the number of chromosomes per mitotic metaphase cell. Three mice were used for each genotype, total numbers of counted mitotic metaphase cells in Miwi heterozygous and knockout are 52 and 71, respectively.

Data Information: In (D), (G), (J), and (M), the results are presented as the mean ± SD of three independent experiments. The statistical test was assessed using two-tailed unpaired t test. (**P<0.01; ***P<0.001; n.s.: P>0.05). +/-, Miwi heterozygous; -/-, Miwi knockout.
MIWI Prevents Aneuploidy during Meiosis by Cleaving Excess Satellite RNA (2020)
Figure 5

Figure 5. MIWI works with Dicer to ensure proper satellite RNA processing.

A RNA pull-down was performed using in vitro transcribed biotinylated transcripts and whole cell extracts from Miwi+/-, Miwi-/-, Miwi-/ADH, and Mov10l1f/-;Stra8-Cre testes. Mov10l1f/-;Stra8-Cre mice represent a pachytene piRNA-deficient condition. In vitro transcribed RNAs corresponding to approximately one repeat of major and minor satellite sequences were used as baits. Precipitated protein was visualized by Western blotting using anti-Dicer antibody.

B Equal amounts of total RNA isolated from spermatocytes of Miwi+/-;Dicerf/+;Stra8-Cre, Miwi-/-;Dicerf/+;Stra8-Cre, Miwi+/-;Dicerf/-;Stra8-Cre and Miwi-/-;Dicerf/-;Stra8-Cre mice, were resolved on a 1% agarose gel. The gel was subjected to Northern blot analysis using probes specific for the sense and antisense sequences of major and minor satellites. The blot was re-hybridized with a 28S rRNA-complementary probe to demonstrate equal loading (bottom on the left).

C in vitro RNA processing assay with nuage-enriched nuclear fraction from Miwi+/-;Dicerf/+;Stra8-Cre, Miwi-/-;Dicerf/+;Stra8-Cre, and Miwi+/-;Dicerf/-;Stra8-Cre mice, and 5'-end radioactively labelled RNA targets corresponding to approximately one and two repeats of sense and antisense major satellite sequences. Arrow indicates the cleavage products of In vitro transcribed sense major satellite RNAs by MIWI-piRNA complex.

D Equal amounts of total RNA isolated from spermatocytes of Miwi+/-;Dicerf/+;Stra8-Cre, Miwi-/-;Dicerf/+;Stra8-Cre, Miwi+/-;Dicerf/-;Stra8-Cre and Miwi-/-;Dicerf/-;Stra8-Cre mice, were digested with RNase III (RNase III) or nuclease S1 (S1), and further resolved on a 1% agarose gel. The gel was subjected to northern blot analysis using probes specific for the sense (S) and antisense (As) strands of major (Maj) and minor (Min) satellite transcripts. The blot was re-hybridized with 18S and 28S-complementary probes to demonstrate equal loading and the same efficiency of RNase III or nuclease S1 digestion.

E Sequential in vitro RNA processing assay using 5'-end radioactively labelled RNA targets corresponding to approximately one repeat of sense and antisense major satellite sequences. The nuage-nuclear fraction from Miwi+/- testes is indicated as N. Total cellular extracts (T) from Miwi+/- testes were subjected to immunodepletion using anti-Dicer antibodies (α-Dicer), with IgG (IgG) as a negative control. The incubation time (Incub. t) was 0.5 and 1 hour. The arrow indicates the product of MIWI-mediated cleavage of the in vitro transcribed sense major satellite RNA.