EMBO/soda-nel huggingface dataset:
Drug-microenvironment perturbations reveal resistance mechanisms and prognostic subgroups in CLL (2022)
Figure 5

Figure 5. Microenvironmental stimuli influence ex vivo drug response.

(A) Graphical representation of the four drug-stimulus interaction categories. Categories are defined according to the nature of the interaction (synergistic or antagonistic), and whether the viability is increased or decreased by the stimulus (positive or negative). x-axis shows treatment type, y-axis shows viability with each treatment. Red and blue points and lines depict a representative treatment response pattern for given interaction type. Blue horizontal lines represent the expected viability for combinatorial treatment in the absence of an interaction between drug-stimulus (i.e. additive effects), black horizontal lines represent the measured viability after combinatorial treatment. The difference between the black and blue lines indicates the impact of the interaction on expected viability.

(B) Bar plot of the number of interactions in each category where the p-value for the interaction term βint <0.05.

(C) Heatmap of all βint values with associated p-value <0.05, annotated with interaction category (I-IV). Rows and columns indicate drugs and stimuli, ordered according to hierarchical clustering. Scale indicates size and sign of βint for given drug-stimulus combination.

I: Positive βint and antagonistic (Microenvironmental stimulation reduces drug effect)

II: Negative βint and antagonistic (Drug reduces stimuli effect)

III: Positive βint and synergistic (Microenvironmental stimulation and drug have synergistic pro-survival effect)

IV: Negative βint and antagonistic (Microenvironmental stimulation and drug show synergistic

toxicity)

(D - I) Examples of drug-stimulus interactions, for each category. Plots show the natural logarithm of the relative viability of 192 CLL samples after treatment with the respective drug and stimulus. Each line represents one patient sample linked across treatments. Black horizontal lines in each single treatment indicate the expected viability based on linear modelling. Black and blue horizontal lines in the combinatorial treatment indicate the expected viability based on the additive effect of the drug and stimulus (blue), and the expected viability accounting for the additive effect and the interaction (black). (D+E) Ibrutinib, a clinically used BTK inhibitor, is blocked by IL4 and IFNγ. (F) The JAK inhibitor pyridone-6 inhibits the pro-survival effect of sCD40L + IL4 stimulation. (G) The p38 inhibitor ralimetinib and IFNγ show a synergistic pro-survival effect not observed in either single treatment. (H) TLR agonists, including CpG ODN (shown) increase sensitivity to BTK inhibition by ibrutinib, despite increasing viability as single treatments. (I) Soluble anti-IgM sensitises CLL samples to HSP90 inhibition by luminespib.
Drug-microenvironment perturbations reveal resistance mechanisms and prognostic subgroups in CLL (2022)
Figure 7

Figure 7. IL4 and TLR signalling are upregulated in CLL-infiltrated lymph nodes.

(A + B) Mean (A) pSTAT6 and (B) pIRAK4 staining intensity in CLL-infiltrated (n=100 for pSTAT and pIRAK4) and tumour-free lymph node biopsies (n=98 for pSTAT6 and n=100 for pIRAK4) after background subtraction (y axis), p-values from Student's t-test. Each dot represents the mean of all cells in the tissue microarray cores per patient sample. The central bar, boxes and whiskers of the plot represent the median, first and third quartiles, and 1.5-times IQR, respectively.

(C - F) Example images of immunohistochemistry sections: (C + D) pSTAT6 levels in (C) CLL-infiltrated and (D) tumour-free samples. (E + F) pIRAK4 levels in (E) CLL-infiltrated and (F) tumour-free samples. Scale bar defines 20µm in all images.

(G + H) Kaplan-Meier plots for time to next treatment stratified by levels (high / low) of pSTAT6 (G) and pIRAK4 (H). P-values from univariate Cox proportional hazard models.
Activation of focal adhesion kinase by Salmonella suppresses autophagy via an Akt/mTOR signaling pathway and promotes bacterial survival in macrophages. (2014)
Figure 1

FAK is selectively recruited to LAMP-1-positive SCVs.

(A) FAK immunoblotting was performed on macrophages isolated from the peritoneum of WT and FAKΔmyeloid mice. (B) WT and FAK−/− PEMs were incubated with S. typhimurium strain ΔinvG for 0–5 hours before immunoblotting with the indicated antibodies. (C, E and G) WT PEMs expressing GFP-FAK were incubated with either ΔinvG Salmonella (C), LPS-coated beads (E) or E. coli (G) for a total of 5 hours before analysis by immunofluorescence. Cells were co-stained with antibodies recognizing LAMP1 (red). Dapi was used to visualize nuclei and bacteria (blue). Bars represent 10 µm. Arrowheads indicate bacteria or beads in enlarged panel where bars represent 2 µm. (D, F and H) The percent of LAMP1-positive (gray bars) and LAMP1+GFP+ (black bars) ΔinvG Salmonella (D), LPS-coated beads (F) and E. coli (H) was quantified. At least 100 bacteria were counted per condition; N = 3.
Activation of focal adhesion kinase by Salmonella suppresses autophagy via an Akt/mTOR signaling pathway and promotes bacterial survival in macrophages. (2014)
Figure 2

<italic>Salmonella</italic> specifically targets FAK for activation.

(A) TLR4−/− PEMs were incubated with ΔinvG Salmonella for 0–5 hours before immunoblotting with the indicated antibodies. (B) WT PEMs were incubated with LPS-coated beads for 0–5 hours before immunoblotting for the indicated antibodies. (C) Levels of phosphorylated proteins were quantified by densitometry, normalized to the amount of total protein present in each sample, and expressed relative to the basal level in uninfected cells. Values are means ± SEM, N = 3, *p<0.05. Dashed line drawn at 1 indicates basal levels of activation. (D) WT PEMs were incubated with the orgA::tet; spiA::kan Salmonella strain deficient in both the SPI1 and SPI2 T3SSs (ΔSPI1ΔSPI2) for 0–5 hours before immunoblotting with the indicated antibodies. (E) WT PEMs expressing GFP-FAK were incubated with ΔSPI1ΔSPI2 Salmonella for a total of 5 hours before analysis by immunofluorescence. Cells were co-stained with antibodies recognizing LAMP1 (red). DAPI was used to visualize nuclei and bacteria (blue). Bars represent 10 µm or 2 µm in enlarged inset.
Activation of focal adhesion kinase by Salmonella suppresses autophagy via an Akt/mTOR signaling pathway and promotes bacterial survival in macrophages. (2014)
Figure 3

Akt activation in response to <italic>Salmonella</italic> requires FAK.

(A) WT and FAK−/− PEMs were incubated for 0–5 hours with ΔinvG Salmonella before immunoblotting with the indicated antibodies. (B) WT PEMs were pretreated for 1 hour with the FAK-kinase inhibitor PF-228 (0.5 µm) before incubation with ΔinvG Salmonella for 5 hours. Lysates were immunoblotted with the indicated antibodies. (C–D) WT or FAK−/− macrophages were incubated with either the ΔΣΠΙ1ΔΣΠΙ2 strain (C) or LPS-coated beads (D) for 0–5 hours before immunoblotting with the indicated antibodies. In panels C and D, WT PEMs were also incubated with ΔinvG (5 h) for comparison of Akt activation. Vertical white lines in panel D indicate non-contiguous lanes generated from a single exposure. (E) Levels of phosphorylated proteins were quantified by densitometry, normalized to the amount of total protein present in each sample, and expressed relative to the basal level in uninfected cells. Values are means ± SEM, N = 3, *p<0.05. Dashed line drawn at 1 indicates basal levels of activation. (F) WT PEMs expressing HA-Akt were either left untreated or incubated with ΔinvG Salmonella for 5 hours. Lysates were subjected to immunoprecipitation (IP) with anti-HA antibodies before immunoblotting for FAK and HA. In parallel, lysates were immunoblotted for FAK, HA and ERK1/2 to control for the level of expression. (G) WT and FAK−/− macrophages expressing HA-Akt were stained with anti-HA and anti-LAMP1 before analysis by confocal microscopy. White boxes show enlarged regions in lower panel. Arrows indicate HA+LAMP+ Salmonella evident in WT PEMs. (H) The percentage of ΔΣΠΙ1ΔΣΠΙ2 (first grouping) or ΔinvG (second grouping) Salmonella co-localizing with HA and LAMP-1 5 h post-infection. At least 100 bacteria were counted per condition. Values are means ± SEM, N = 3, *p<0.05.
Activation of focal adhesion kinase by Salmonella suppresses autophagy via an Akt/mTOR signaling pathway and promotes bacterial survival in macrophages. (2014)
Figure 4

FAK-dependent Akt-mTOR signaling inhibits the recruitment of LC3 to SCVs.

(A) WT and FAK−/− PEMs were incubated with S. typhimurium strain ΔinvG for 0–24 hours before immunoblotting with the indicated antibodies. (B) WT PEMs were incubated with Akt inhibitor AKTV/triciribine (10 µm) for 30 minutes before infection with S. typhimurium strain ΔinvG for 5 hours. Lysates were immunoblotted with the indicated antibodies. (C) WT and FAK−/− PEMs were incubated with S. typhimurium strain ΔinvG for a total of 5 hours before analysis with antibodies recognizing LC3. DAPI was used to visualize nuclei and bacteria. Bars represent 10 µm. White boxes show enlarged regions in inset panels, bars represent 2 µm. White arrowheads indicate LC3-negative bacteria, yellow arrow heads indicate LC3-positive bacteria (panels c, f). Yellow arrowhead in panel f denotes an LC3+ cluster of bacteria. (D) The percentage of ΔinvG (first grouping) or ΔΣΠΙ1ΔΣΠΙ2 (second grouping) Salmonella co-localizing with LC3 at the indicated time points is displayed. At least 100 bacteria were counted per condition per time point. Values are means ± SEM, N = 3, *p<0.05. (E) WT and FAK−/− PEMs were incubated with S. typhimurium strain ΔinvG for 5 h with or without Bafilomycin A1 (300 ng) before immunoblotting with the indicated antibodies. Vertical white line indicate non-contiguous lanes generated from a single exposure. (F) FAK−/− PEMs were incubated with DQ-BSA-red for 1 hour before infection with S. typhimurium strain ΔinvG for 5 h. Cells were co-stained for LC3 and DAPI was used to visualize nuclei and bacteria. White boxes show enlarged regions in inset panel. (G) WT PEMs were pretreated with the mTORC1 inhibitor rapamycin (4 µm) for 1 hour before incubation with ΔinvG Salmonella for a further 5 hours. Cells were lysed before immunoblotting with the indicated antibodies. (H) WT and FAK−/− PEMs were pretreated with rapamycin (4 µm), Akt inhibitor AKTV/triciribine (10 µm) or left untreated (-Tx) or before infection with S. typhimurium strain ΔinvG for a further 5 hours. WT macrophages were also pretreated with the FAK inhibitor PF228 (0.5 µm) for 1 hour prior to incubation with ΔinvG Salmonella for 5 hours. Cells were then assessed for the percentage of ΔinvG Salmonella co-localizing with LC3. At least 100 bacteria were counted per condition. Values are means ± SEM, N = 3, *p<0.05. N.s. not significant.
Activation of focal adhesion kinase by Salmonella suppresses autophagy via an Akt/mTOR signaling pathway and promotes bacterial survival in macrophages. (2014)
Figure 5

SPI-1-deficient <italic>Salmonella</italic> are targeted by autophagy in the absence of FAK.

(A) WT and FAK−/− PEMs were incubated with LPS-coated beads (50∶1; beads∶cell) for 1 hour before analysis with antibodies recognizing LC3. DAPI was used to visualize nuclei. Bars represent 10 µm. The percentage of LPS-beads co-localizing with LC3 is displayed to the right. At least 100 beads were counted per condition. Values are means ± SEM, N = 3. (B) PEMs were treated with Atg5, ULK1 or control siRNAs for 48 hours before immunoblotting with the indicated antibodies. (C–D) FAK−/− PEMs treated with Atg5, ULK1 or control siRNA for 48 hours were incubated with LPS-coated beads or ΔinvG Salmonella for 1 or 5 hours before analysis with antibodies recognizing LC3. The percentage of LPS-beads or Salmonella co-localizing with LC3 is displayed to the right. At least 100 beads or bacteria were counted per condition. Values are means ± SEM, N = 3. (E) WT and FAK−/− PEMs were infected with ΔinvG for 5 hours before preparation and examination by transmission electron microscopy (TEM). Arrowheads indicate double membrane.
Activation of focal adhesion kinase by Salmonella suppresses autophagy via an Akt/mTOR signaling pathway and promotes bacterial survival in macrophages. (2014)
Figure 6

FAK deficiency improves control of <italic>Salmonella</italic> infection.

(A) WT and FAK−/− PEMs were incubated with S. typhimurium strain ΔinvG for 1.5, 3 and 5 hours. Bacterial survival was then assayed using a standard gentamycin resistance assay described in the Materials and Methods. Values are means ± SEM, N = 6, *p<0.05. (B) WT and FAK−/− PEMs were pretreated with IFN-γ (50 ng/ml), rapamycin (4 µm), or left untreated before incubation with ΔinvG Salmonella for 3 hours. Bacterial survival was then assayed as described in (A). Values are means ± SEM, N = 6, *p<0.05. (C) PEMs were treated with control siRNA or depleted of Atg5 before incubation with ΔinvG Salmonella for 3 hours. Bacterial survival was then assayed as described in (A). Values are means ± SEM, N = 3, *p<0.05 vs WT siControl. (D) Bacterial loads in WT (black circles) and FAKΔmyeloid mice (white circles) in the indicated tissues 5 days after oral infection with S. typhimurium strain SL1344. Each point indicates data from an individual mouse. *p<0.05. (E) Hematoxylin and eosin (H&E) staining of spleen isolated from WT and FAKΔmyeloid mice 5 days post-infection with S. typhimurium strain SL1344. Arrows indicate areas of leukocyte infiltration. Lesion indicated by yellow arrow is outlined in the higher magnification panel to the right. Bars represent 200 µm and 50 µm. (F) Gr-1 immunostaining of spleens isolated from WT and FAKΔmyeloid mice 5 days post-infection with S. typhimurium strain SL1344. Panels b and d are enlarged sections indicated by black boxes in panels a and c. Gr-1-positive cells appear red-brown. (G) Quantitation of flow cytometric data collected from the spleens of infected mice. *p<0.05.
Beclin1-binding UVRAG targets the class C Vps complex to coordinate autophagosome maturation and endocytic trafficking (2008)
figf1

(a) UVRAG interaction with C-Vps. 293T cells were co-transfected with Flag–UVRAG and HA–Vps16, HA–Vps11, HA–Vps18, HA–Vps33 or HA–Vps39. Whole-cell lysates (WCLs) were used for immunoprecipitation (IP) with an anti-Flag antibody, followed by immunoblotting (IB) with an anti-HA antibody. 1% WCL was used as the input. (b) Interaction between endogenous UVRAG and C-Vps subunits. WCLs of 293T cells were used for IP with control serum (control) or an anti-UVRAG antibody, followed by IB with the indicated antibodies. The right panel shows endogenous protein expression. (c) Confocal microscopy analysis of the colocalization of endogenous UVRAG and Vps16 or Vps18 in HeLa cells. Insets highlight colocalization. (d) Confocal microscopy analysis of the colocalization of UVRAG with C-Vps subunits in HeLa cells transfected with Flag–UVRAG, and epitope-tagged C-Vps subunits as indicated. (e) Colocalization of UVRAG–C-Vps in early endosomes. HeLa cells were co-transfected with EGFP–Vps16 and Flag–UVRAG, stained with antibodies to LAMP1, LAMP2, EEA1 and Rab5, followed by confocal microscopy. All images are representative of at least three independent experiments. Scale bars, 5 μm. The raw data for a and b are shown in Supplementary Information, Fig. S6
Beclin1-binding UVRAG targets the class C Vps complex to coordinate autophagosome maturation and endocytic trafficking (2008)
figf2

(a) The N-terminal C2 domain or the C-terminal region of UVRAG interacts individually with Vps16. 293T cells were co-transfected with HA–Vps16, together with vector, Flag–UVRAG or Flag–UVRAG mutants. WCLs were immunoprecipitated with anti-Flag followed by immunoblotting with an anti-HA antibody. (b) HeLa cells were transfected with EGFP–Vps16 together with Flag–UVRAG mutants as indicated and stained with anti-Flag (red), followed by confocal microscopy. Scale bars, 5 μm. (c) Interactions of the UVRAG C-terminal truncated mutants with Vps16. At 48 h post-transfection with HA–Vps16 and Flag–UVRAG C-terminal mutants, 293T WCLs were immunoprecipitated with anti-Flag, followed by immunoblotting with anti-HA. (d) UVRAG C2 domain or C-terminal region deletion abolishes Vps16 binding. At 48 h post-transfection with HA–Vps16 together with Flag–UVRAG or its mutants, 293T WCLs were immunoprecipitated with anti-Flag followed by immunoblotting with an anti-HA antibody. (e) Schematic representation of UVRAG and its deletion mutants. ++, strong binding; +, weak binding; −, no binding. The raw data for a, c and d are shown in Supplementary Information, Fig. S6
Beclin1-binding UVRAG targets the class C Vps complex to coordinate autophagosome maturation and endocytic trafficking (2008)
figf3

(a) HeLa.Vec and HeLa.UVRAG cells transfected with HA–Vps16 and GFP–LC3 were treated with 2 μM rapamycin for 2 h, and processed for confocal microscopy (left panel; scale bars, 5 μm). The percentage of GFP–LC3 punctae positive for HA–Vps16 staining was quantified (right panel; data are mean ± s.e.m., n = 60, *P 0.01). (b) Effects of Vps16 on UVRAG-mediated autophagosome formation. Light microscopic quantification of autophagosomes in HeLa.Vec and HeLa.UVRAG cells (upper left panel) or in HCT116.Vec and HCT116.UVRAG cells (bottom left panel) when transfected with GFP–LC3 together with control siRNA or Vps16 siRNA (data are mean ± s.e.m., n = 60 for HeLa cells; n = 450 for HCT116 cells, *P 0.01; **P 0.001). Immunoblotting of Vps16 and tubulin are shown in the right panel.
Beclin1-binding UVRAG targets the class C Vps complex to coordinate autophagosome maturation and endocytic trafficking (2008)
figf4

(a) UVRAG enhances the colocalization efficiency of GFP–LC3 with LAMP1. HeLa.Vec and HeLa.UVRAG cells transfected with GFP–LC3 were either untreated or treated with rapamycin (2 μM) in the absence or presence of bafilomycin A1 (0.1 μM) and stained for LAMP1. Insets highlight the colocalization. The percentage of LAMP1-positive autophagosomes was calculated in each setting (data are mean ± s.e.m., n = 200; 5 independent experiments, **P 0.01; *P 0.05). (b) UVRAG enhances the acquisition of CD63 by autophagosomes. The GFP–LC3-transfected HeLa.Vec and HeLa.UVRAG cells were treated as described in a and stained for CD63 and the percentage of CD63-positive autophagosomes was calculated (data are mean ± s.e.m., n = 200, **P 0.01; *P 0.05). (c) UVRAG promotes autophagic degradation of long-lived proteins. HeLa.Vec and HeLa.UVRAG cells were incubated for 16 h with L-3H-Leu (1 μCi ml−1). The degradation of long-lived proteins was measured at the indicated time points in complete medium (NC), EBSS alone (Starv.), or EBSS + 0.1 μM bafilomycin A1 (Starv.+BalfA1). Results (data are mean ± s.e.m. of triplicates) are representative of 2 independent experiments. Scale bars, 5 μm.
Beclin1-binding UVRAG targets the class C Vps complex to coordinate autophagosome maturation and endocytic trafficking (2008)
figf5

(a) Colocalization of GFP–LC3 and LAMP1. HeLa cells expressing wild-type or mutant UVRAG were transfected with GFP–LC3 and incubated under normal conditions or treated with rapamycin (2 μM). Autophagosome fusion with LAMP+ structures was analysed by confocal microscopy (left panel; rapamycin-treated) and quantified (right upper panel; data are mean ± s.e.m., n = 100). Arrows and insets show the LAMP1+ autophagosomes. The number of GFP–LC3-positive dots per cell was counted using a fluorescence microscope (right bottom panel; data are mean ± s.e.m., n = 60, *P 0.05; **P 0.0001). (b) Vps16 or Vps18 knockdown inhibits UVRAG-mediated autophagosome maturation. HeLa.Vec (left) and HeLa.UVRAG (right) cells were transfected with GFP–LC3 together with control siRNA, Vps16 siRNA, or Vps18 siRNA, followed by confocal microscopy. Arrows in the top panel denote the autophagosomes with LAMP1 staining. Immunoblotting of Vps16, or Vps18 and tubulin are shown in the middle panel. The percentage of LAMP1+ autophagosomes was calculated (data are mean ± s.e.m., n = 50, *P 0.05). Scale bars, 5 μm. The raw data of the immunoblots are shown in Supplementary Information, Fig. S6.
Beclin1-binding UVRAG targets the class C Vps complex to coordinate autophagosome maturation and endocytic trafficking (2008)
figf6

(a) UVRAG expression promotes the recruitment of Rab7 GTPase to autophagosomes. HeLa.Vec and HeLa.UVRAG cells transfected with GFP–Rab7 and RFP–LC3 were maintained either under normal conditions (NC) or treated with rapamycin, followed by confocal microscopy. Insets highlight the Rab7 acquisition of autophagosomes. The percentage of Rab7+-autophagosomes was quantified (right panel; data are mean ± s.e.m., n = 60, ***P 0.001; **P 0.01). Scale bars, 5 μm. (b) Rab7 GTPase activity is increased with UVRAG expression. At 48 h post-transfection with GFP–Rab7 together with vector, UVRAG or UVRAGΔN(1–147) vector, 293T WCLs were used for immunoprecipitation with anti-GFP, followed by the Rab7 GTPase activity assay. Left panel: Autoradiographs of the GTP hydrolysis products analysed by TLC. Right panel: Quantification of the percentage of GTP hydrolyzed by Rab7 (data are mean n = 2 independent experiments). (c) Increased Rab7–RILP interaction UVRAG is expressed. HCT116.vector, HCT116.UVRAG and HCT116.UVRAGΔN(1–147) cells were transfected with GFP–Rab7 together with the GST-tagged Rab7 binding domain of RILP (GST–RILPRBD, residues 243–318). WCLs were used for GST pulldown, followed by immunoblotting with an anti-GFP antibody. The raw data of the immunoblots are shown in Supplementary Information, Fig. S6.
Beclin1-binding UVRAG targets the class C Vps complex to coordinate autophagosome maturation and endocytic trafficking (2008)
figf7

(a) EGF-stimulated endocytosis and post-endocytic trafficking. Uptake of Alexa Fluor 488–EGF (green) by HCT116.Vec (left panel) and HCT116.UVRAG (right panel) cells was followed by confocal microscopy. At the indicated times, cells were fixed and stained with antibodies to LAMP1 (red), and Topro-3 (blue, to stain the nucleus). Magnified images of the insets (fourth row) highlight trafficking of EGF towards LAMP1-positive structures. (b) EGFR degradation. HCT116.Vec, HCT116.UVRAG and HCT116.UVRAGΔN(1–147) cells were treated with EGF (200 ng ml−1) at 37 °C for the indicated times. WCLs were subjected to immunoblotting with an anti-EGFR antibody. EGFR at each time point was quantified and indicated as a percentage relative to that at time zero (right panel; data are mean ± s.e.m., n = 3). (c) DQ-green BSA dequenching assay. Representative confocal microscopy images of DQ-BSA proteolysis (left panel) and flow cytometry analysis (right panel) of RAW264.7 cells expressing an empty vector (RAW264.7.Vec; dotted black line), wild-type UVRAG (red line) or UVRAGΔN(1–147) mutant (green line) loaded with 10 μg ml−1 DQ green–BSA for the indicated times. Background (grey line) represents samples without the BSA load. (d) Effect of UVRAG on endosome fusion. RAW264.7.Vec and RAW264.7.UVRAG cells were pulse-labelled with Oregon green 514–avidin and dextran-568 for 10 min and chased for 30 min. Cells were then labelled with biotin–BSA for 10 min, and processed for confocal microscopy (left panel). In the absence of biotin–BSA, Oregon green 514–avidin showed very low fluorescence (first row of confocal image). The percentage of dextran-568-labelled endosomes with visible Oregon green staining (denoted by arrows in the left panel) was calculated from approximately 20 different cells in 2 independent experiments (mean, right panel). Scale bars, 5 μm. The raw data of the immunoblots in b are shown in the Supplementary Information, Fig. S6.
PLK1 REGULATES CENTROSOME MIGRATION AND SPINDLE DYNAMICS IN MALE MOUSE MEIOSIS (2021)
Figure 1

Figure 1. Distribution of PCNT and SYCP3 during the first meiotic division.

A-G Double immunolabelling of PCNT -PCNT- (green) and SYCP3 (red) on squashed WT mouse spermatocytes on (A) zygotene, (B) pachytene, (C) diplotene, (D) diakinesis, (E) prometaphase I, (F) metaphase I and (G) telophase I. Chromatin has been stained with DAPI (blue). Scale bar in G represents 10 μm.