EMBO/SourceData huggingface dataset:
Figure 7C) Left panel: representative images of ORO staining of cardiac sections of Pkp2+/- mice, fed a 3-month HFD plus atorvastatin. Right panel: quantification of the percentage of ORO positive area (n=9) is compared to that in HFD (as in Figure 6C; Two-tailed Student's t-test). D) Representative images of PPARγ immunostaining (green) on cardiac sections of Pkp2+/- mice fed a 3-month HFD plus atorvastatin (n=9). Quantification is compared to the values of Pkp2+/- in HFD and relative to WT in CD (as in Figure 6D; Two-tailed Student's t-test). E) Representative images of MDA immunostaining (green) on cardiac sections of Pkp2+/- mice fed a 3-month HFD plus atorvastatin (n=9). Quantification is compared to the values of Pkp2+/- in HFD and relative to WT in CD (as in Figure 6E; Two-tailed Student's t-test). F) Representative images of CD36 immunostaining (green) on cardiac sections of Pkp2+/- mice fed a 3-month HFD plus atorvastatin (n=9). Quantification is compared to the values of Pkp2+/- HFD and relative to WT in CD (as in Figure 6F; Two-tailed Student's t-test). Nuclei are counterstained with Hoechst33342 (blue). Data information: mean ± SEM. * p<0.05; ** p<0.01; *** p<0.001.
Figure 1(A) E7.0 whole embryo and transverse section (TSs) (dashed lines) through (a1) extra-embryonic tissue showing expression in a ring of extra-embryonic ectoderm (ee) and extraembryonic mesoderm (em) and absence of transcripts in the allantois (al); (a2) revealing expression in epiblast (ep) and in mesendoderm (me) emerging from the primitive streak (ps), dashed box (a2') defines higher magnification region shown in a2. (B) E8.5 whole embryo dorsal view and TSs (dashed lines) showing expression in: (b1,b2,b3) the open anterior neural plate (future fore, mid and hindbrain) including (b3) neural crest (nc) emerging from hindbrain; (b4) in the dorsal neural tube (prospective neural crest) and dorsal somites (s); (b5) in closing preneural tube neural (pnt) and epithelising somites; (b6) in caudal lateral epiblast (cle) adjacent to the node (n) at the anterior tip of the primitive streak, but not in presomitic mesoderm (psm). White arrows indicate open anterior and posterior neural plate . (C) E9.5 whole embryo side view and TSs (white dashed lines) showing expression in: (c1) forebrain (fb) and hindbrain , including optic vesicles (opv), (c1') seen at high magnification (and indicated by box grey dashed line) and otic vesicles (ov) (c1'') seen at high magnification (box grey dashed line); (c2) high expression in dorsal hindbrain (hb) and (c3) spinal cord (sc) and dermamyotome (dm) of the differentiating somite; (c5) throughout open posterior neural plate. (D) High magnification of E9.5 whole embryo showing expression in first branchial arch (ba) and forming cranial ganglia, which are just becoming apparent, (V), (VII), (VIII) (IX) and (X). OV, otic vesicle. (E) Expression in mesenchyme cells underlying the apical ectodermal ridge (aer) in the progress zone (pz) and in the zone of polarising activity (zpa) of the forelimb (fl). Data information: Images representative of n≥ 4 embryos for each stage. Scale bars 200 µm, except sections 100 µm.
Figure 2(A-H) Live wildtype littermate and Slc7a5-null embryos imaged shortly after dissection. (A-D) Wildtype and (E-H) Slc7a5-null E9.75 embryos from lateral (A, D, E, H), frontal (B, F) or dorsal (C, G) views; (e1-h1) higher magnification images of the Slc7a5-null embryo. White arrowheads indicate the smaller limb bud (compare B and F), open/reduced forebrain (compare D and H), open neural tube at posterior (compare C and E, e1) and anterior (compare B and F, f1) regions. Black arrowheads indicate kinked neural tube (compare C and G, g1) and apparently missing optic and smaller otic vesicles (ov) (compare D and h1). Data information: Scale bars 200 µmmRNA in situ hybridization and immunofluorescence in E9.5 or E10.5 wildtype and Slc7a5-null embryos for key marker genes. (I-L) Fgf8 mRNA transcripts were detected in wildtype (I-j1) and in Slc7a5-null (K-l1) E10.5 embryos (n=4 each) (white arrowheads indicate limb buds in K and the isthmus in L) with (j1, l1) sections through isthmus at midbrain/hindbrain border and (i1, k1) lateral views of forelimb buds. Data information: Scale bars 200 µm, except sections 100 µm.mRNA in situ hybridization and immunofluorescence in E9.5 or E10.5 wildtype and Slc7a5-null embryos for key marker genes. (M-N) Wholemount Tubulin-β-III (Tuj1) immunofluorescence performed on (M, m1) wildtype and (N, n1) Slc7a5-null E9.5-E10.5 embryos (n=5 each condition). Dorsal root ganglia (DRG) and Sympathetic Chain Ganglia (SCG) are indicated with open arrowheads together with cranial ganglia IX, X and XI. Images in (m1, n1) show higher magnification of the cranial ganglia V, VII/VIII (open arrowheads). Data information: Scale bars 200 µmmRNA in situ hybridization and immunofluorescence in E9.5 or E10.5 wildtype and Slc7a5-null embryos for key marker genes. (O-P) Neurog2 expression in wildtype (O-o4) and Slc7a5-null embryos (P-p4) (n= 5 and n=6 each), showing reduced expression in forebrain (o2, p2), cranial ganglia (o1, o3, p1, p3, arrowheads) and in spinal cord at level of forelimb (o4, p4, arrowheads indicate position of neural crest). Data information: Scale bars 200 µm, except sections 100 µm.
Figure 3(A-B) Sox10 in situ hybridization in (A) wildtype and (B) Slc7a5-null E9.5 embryos (n=4 each) with TSs of (a1, b1) the hindbrain at level of trigeminal ganglion V, (a2, b2) otic vesicles and the neural tube at more posterior levels (a3, a4, b3, b4). Scale bars 200 µm, except sections a1 - b4, 100 µm. Closed white arrowheads in (a3, a4) indicate neural crest, open white arrowheads indicate depleted neural crest in (b3) and position where neural crest should be in (b4).(C-G) Immunofluorescence on TSs caudal spinal cord of E9.5 wildtype littermates and Slc7a5-null embryos; Pax3 and FoxA2 were used as indicators for dorso-ventral organisation in (C, F) wildtype, n=2 (for FoxA2) and n= 4 (For Pax3) and (D, G) Slc7a5-null, n=3 (for FoxA2 ) and n = 3 for Pax3) neural tube. Arrowheads indicate border of Pax3 expression domain. Scale bars 50 µm. (E) Percentage of Pax3 expressing cells was determined by counting these cells and all DAPI labelled nuclei in the neural tube and comparison made between wildtype (4 embryos, 6 sections each) and Slc7a5-null (3 embryos, 6 sections each), each dot represents the average for one embryo, unpaired t-test, *p = 0.018 (see original source data). Error bars indicate SEM.
Figure 4(A-D) Proliferation was assessed in the spinal cord (forelimb level) (A, B) using a phospho-Histone3 (phospho-H3) antibody (green) to identify mitotic cells and DAPI (blue) to label nuclei; cells were counted in 3 wildtype (18 sections) and 3 Slc7a5-null embryos (18 sections) and mitotic index calculated; (C, D) Proliferation was also assessed in the forebrain in 3 wildtype embryos (17 sections) and 3 Slc7a5-null embryos (16 sections). White dashed lines indicate midline. Data information All scale bars 50 µm.(E, F) Comparison of mitotic index in wildtype and Slc7a5-null (E) spinal cord, p = 0.872 ,and (F) forebrain *p = 0.049, each dot represents the average for one embryo, unpaired t-test Data information: *p<0.05, **p<0.01, ***p<0.001(G, H) mTORC1 activity in the spinal cord (hindlimb level) was assessed by immunofluorescence using an antibody against the phospho-ribosomal protein S6 (phosho-S6 Ser 235/236). Data information All scale bars 50 µm.(I) Labelling intensity was measured and plotted relative to DAPI intensity; 6 wildtype embryos (18 sections) and 5 Slc7a5-null embryos (19 sections). Each dot represents the average for one embryo, (*p = 0.0457) unpaired t-test with Welch correction Data information: *p<0.05, **p<0.01, ***p<0.001(J) Western blot of individual E9.5 wildtype (n=3) and Slc7a5-null (n=4) embryo lysates immunoblotted using an antibody against phospho-S6K (Thr389) and total P70S6K for loading control. Band intensities were measured with FIJI and an unpaired t-test was performed for statistical analysis Data information: *p<0.05, **p<0.01, ***p<0.001(K-L) (K) Validation by qPCR of the targets identified by RNAseq; qPCR was performed on individual E8.5 embryos of each genotype (wildtype n=5, heterozygous n=9 and Slc7a5-null n=7). (L) qPCR was performed on individual E9.5 embryos (wildtype n=5, heterozygous n=3 and Slc7a5-null n=4) using primers specific for genes associated with the integrated stress response. A one-way Anova test with a Turkey post-test was performed. Data information: *p<0.05, **p<0.01, ***p<0.001 Error bars indicate SEM.
Figure 5mRNA in situ hybridization was performed at E9.5 to detect Chac1 in (A-a6) wildtype and (B-b6) Slc7a5-null littermate embryos (n=3 for each condition); Chac1 expression at E10 in emerging limb buds assessed in (C) wildtype and (D) Slc7a5-null littermate embryos (n=2 for each condition) Scale bars 200 µm for whole embryo images and 100 µm for sections.mRNA in situ hybridization was performed at E9.5 to detect Trib3 mRNA detected in (E-e6) wildtype and (F-f6) Slc7a5-null littermate embryos (n=3 for each condition) and in emerging limb buds at E10.5 assessed in (G) wildtype and (H) Slc7a5-null littermate embryos (n=2 for each condition). Scale bars 200 µm for whole embryo images and 100 µm for sections.mRNA in situ hybridization was performed at E9.5 Chac1 and Trib3 transcripts were also detected in wildtype E9.5 embryos after >3 days in situ hybridisation reaction (n=3 and n=6 respectively). Lateral view (I and K), optic vesicles (white arrowheads), branchial arches (ba) and otic vesicles (ov). Dorsal view (J and L), neural tube (nt) and limb buds (white arrowheads). Scale bars 200 µm for whole embryo images and 100 µm for sections.
Figure 6Western blot of individual E9.5 wildtype (n=4) and Slc7a5-null (n=4) embryo lysates immunoblotted using an antibody against phospho-GCN2 (Thr899) and total GCN2 (loading control), ***p = 0.0001 ; band intensities measured with FIJI and analysed with unpaired t-test Data information: All error bars indicate SEM.Western blot of individual E9.5 wildtype (n=4) and Slc7a5-null (n=4) embryo lysates immunoblotted using an antibody against phospho-eIF2α (Ser51) and total eIFα (loading control), *p = 0.0482, band intensities measured with FIJI and analysed with unpaired t-test Data information: All error bars indicate SEM.(C-F) phosphorylated eIFα was assessed by immunofluorescence in E9.5 embryos in the forebrain, wildtype (C,c1) (n=2 embryos, 0/14 sections) and Slc7a5-null (D, d1) (n=2 embryos 16/16 sections), and in hindbrain/anterior spinal cord, wildtype (E-e2) (n= 4 embryos, 0/62 sections) and Slc7a5-null (F-f2) (n= 4 embryos, 54/54 sections) including in the otic vesicle (compare e2, f2). Scale bars in (C-e1, D-f1) 100 µm and in (e2 and f2) 25µm. Arrowheads indicate ventral midline.(G) An increase in Trib3 protein was detected by Western blot in Slc7a5-null embryos, each lane represents an E9.5 embryo lysate (wildtype n=3, Slc7a5-null, n=4) with α-Tubulin loading control, unpaired t-test *p = 0.0153 Data information: All error bars indicate SEM.(H-L) TUNEL assay to detect apoptotic cells in wildtype (n=3 embryos, 32 sections) or Slc7a5-null (n=5 embryos, 47 sections); (H, I) transverse sections through the head at level of the forebrain and (J, K) the spinal cord, (H-K) scale bars 50 µm, arrowheads indicate ventral midline. (L) Quantification of TUNEL positive cells within spinal cord sections was performed Each dot represents the average apoptosis cell count for a single embryo, Welch's correction unpaired t-test, p = 0.250, F-test to compare variances, p = 0.0003 Data information: All error bars indicate SEM.
Figure 7(A-B) (A,a1) Wnt β-catenin target Axin2 strongly expressed in littermate, (a2, a3) in TS, and (B, b1) in Scl7a5-null embryos (b2, b3) in TS (n=2 littermates, n=6/6 Slc7a5-null embryos). Data information: White dashed lines indicate position of sections. Scale bars 200 µm(C, d1) Axin2 transcripts in embryo trunk explant exposed to DMSO (C, c1, n=12/12) were reduced in Wnt-c59 exposed explants (D, d1, n=14/14). Images in (c1, d1) show TS through explants (data from 3 independent embryo explant experiments). (E, F) Slc7a5 transcripts in embryo trunk explant exposed to DMSO (E, e1, n=9/10) or Wnt-c59 (F, f1, n=15/15). Images in (e1, f1) show TS through explants (data from 3 independent embryo explant experiments). Data information: Scale bars = 100 µm(G, H) Slc7a5 expression in control T-Cre;Ctnnb1flLOF/Δ heterozygous littermate embryo (G, g1) and in TS (g2, g3), and in T-Cre;Ctnnb1flLOF/Δ mutant embryo where Slc7a5 transcripts are absent in posterior neural tube (the region of T-cre recombination, indicated with white arrowhead in H) and shown in TS in (h2, h3) (n= 0/6 littermate and n=6/6 mutant embryos). Data information: White dashed lines indicate position of sections. Scale bars, 200 µm(I, J) Trib3 is lacking in T-Cre;Ctnnb1flLOF/Δ heterozygous control embryos at E7.75 (I), caudal region dissected from the same embryo (white dashed box in I) (i1) and viewed in TS (i2); Trib3 detected in homozygous T-Cre;Ctnnb1flLOF/Δ embryo (J), in caudal region dissected from the same embryo (white dashed box in J) (j1) and shown in TS in caudal epiblast and remnant primitive streak (ps) (j2) (n= 3/3 T-Cre;Ctnnb1flLOF/Δ embryos and 14 littermate controls). Trib3 was also detected in the more rostral forming neural tube at this early stage (seen in J) and so in cells not directly experiencing β-catenin loss; this may reflect the failure to form paraxial mesoderm, which provides signals that promote and support neurogenesis. Data information: White dashed lines indicate position of sections. Scale bars all sections 50 µm.(K-M) At E9.5 Trib3 was not detected in T-Cre;Ctnnb1flLOF/Δ heterozygous littermate embryos (K, k1) and in TS ( k2, k3), but was detected in homozygous T-Cre;Ctnnb1flLOF/Δ mutant embryos (L-m3) in patches of cells in neural tube (nt) (L - l2), gut (g) (l3) and somites (s) (m2, arrowheads) (n=9/9 T-Cre;Ctnnb1flLOF/Δ embryos and 12 littermate controls). Data information: White dashed lines indicate position of sections. Scale bars, all sections 50 µm.
Figure 1Selected BioID REV7 results, shown as dot plots. The spectral counts for each indicated prey protein are shown as AvgSpec. Proteins were selected based on and iProphet probability of >0.95, BFDR of <0.05 and ≥10 peptide count. The circle size represents the relative abundance of preys over baits.U2OS EJ5-GFP cells were transfected with the indicated siRNAs. At 24hr post-transfection, cells were transfected with the I-SceI expression plasmid, and the GFP+ population was analyzed 48 hr post-plasmid transfection. The percentage of GFP+ cells was determined for each individual condition and subsequently normalized to the non-targeting condition (siCTRL).
Figure 2Quantification of the Neutral Comet assay. U2OS cells stably expressing shCtrl, shREV7 or shSHLD2 were exposed to IR (10 Gy) and run in low melting agarose under neutral conditions. Immunofluorescence against DNA stained with SYBR Gold was performed to measure the tail moment.U2OS cells were transfected with the indicated siRNA. 48h post-transfection the cells were treated with NCS to induce DNA damage and the cells were harvested at 0, 1, 2, 4 and 24 hr post NCS treatment. Flow Cytometry analysis of phosphorylated-H2AX signal was used to measure γ-H2AX endogenous signal.Sensitivity to IR monitored by colony formation assay. MCF-7 cells stably expressing the the indicated shRNAs were exposed to increasing doses of IR. 24 h post-irradiation, the cells were re-seeded to allow colony formation, fixed and stained with 0.4% crystal violet. Shown is the quantification of colonies per condition which possessed more than 50 colonies.MCF-7 cells stably expressing the the indicated shRNAs 24 h post-irradiation, the cells were re-seeded to allow colony formation, fixed and stained with 0.4% crystal violet. Shown is the quantification of colonies per condition which possessed more than 50 colonies. cells were exposed to increasing doses of UV radiation.
Figure 3U2OS cells stably expressing HA-REV7 (Top) or HA-SHLD2 (Bottom) were pre-sensitized with 10ug/mL Hoescht 33342 before exposed to UV micro-irradiation. Immunofluorescence against endogenous HA and γ-H2AX epitope was subsequently performed to monitor REV7 and SHLD2 accumulation at sites of damage. Shown are representative micrographs. Scale bar = 5µm.U2OS LacR-Fok1 cells were transfected with GFP or GFP-SHLD2 and 24 h later DNA damage was induced using Shield-1 and 4-OHT. The cells were then processed for GFP and mCherry immunofluorescence. Shown are representative micrographs. Scale bar = 5µm. Quantification of the experiments shown in (C). Data are represented as the mean ± SD (n=3). At least 100 cells per condition were counted.Quantification of the experiments shown in (C). Shown is the quantification of the GFP signal at the mCherry-LacR-Fok1 focus.293T cell lines expressing ER-AsiSI with Flag-SHLD2 and treated with 1 µM of 4-OHT. 6h later the cells were processed and immunoprecipitated with Anti-FLAG Magnetic Beads and anti-γ-H2AX.x/Protein A/G magnetic beads. DNA was purified and subjected to qPCR detection. Shown is the quantification of IP efficiency as the percentage of DNA precipitated from input (Bottom).U2OS cells stably expressing HA-SHLD2∆720-904 (Left) or HA-SHLD2∆1-680 (Right) were processed as in (B). Immunofluorescence against endogenous HA and RPA32 epitope was subsequently performed to monitor RPA32 and SHLD2 accumulation at sites of damage. Shown are representative micrographs. Scale bar = 5µm.
Figure 4U2OS mCherry-LacR-Fok1 cells were treated with the indicated siRNA and subsequently transfected with a GFP-SHLD2 construct. 24 h post-transfection DNA damage was induced using Shield-1 and 4-OHT. The cells were then fixed and analyzed for the intensity of the GFP-SHLD2 signal at mCherry-LacR-Fok1 focus. Shown is the quantification of the GFP-SHLD2 signal at the Fok1 focus. Data are represented as a box-and-whisker plot where the whiskers represent the 10-90 percentile. At least 75 cells were counted per condition.293T cells were transfected with Flag-REV7 and GFP-SHLD2 expression vectors as indicated. 24h post-transfection cells were treated with DMSO or with 10uM of ATM inhibitor KU-60019 for 1 h prior to irradiation. 1h post-irradiation (10 Gy) nuclear extracts were prepared and REV7 complexes were immunoprecipitated using anti-Flag (M2) Resin and then analyzed by immunoblotting using GFP, REV7 and p-Chk1 antibodies.U2OS EJ5-GFP cells were transfected with the indicated siRNAs. At 24hr post-transfection, cells were transfected with the I-SceI expression plasmid, and the GFP+ population was analyzed 48 hr post-plasmid transfection. The percentage of GFP+ cells was determined for each individual condition and subsequently normalized to the non-targeting condition (siCTRL).CH12F3-2 cells stably expressing the indicated shRNAs were stimulated with a cocktail of cytokines (CIT) to induce class switching to IgA. The percentage of IgA+ cells was monitored 24 and 48h post-stimulation by staining with an anti-IgA antibody followed by flow cytometry analysis.U2OS DR-GFP cells were transfected with the indicated siRNAs. At 24hr post-transfection, cells were transfected with the I-SceI expression plasmid, and the GFP+ population was analyzed 48 hr post-plasmid transfection. The percentage of GFP+ cells was determined for each individual condition and subsequently normalized to the non-targeting condition (siCTRL).U2OS cells were transfected with the indicated siRNAs and then treated with CldU, IdU and NCS 48h post-transfection as indicated. The slides were stained, dehydrated, mounted and visualized and shown is the quantification of CldU/IdU tract length in order to visualize DNA end-resection (Right). At least 500 DNA tracks were measured per condition.
Figure 5Genes significantly enriched or dropped out after a 14-day treatment with Doxorubicin were identified by plotting as a Log2 fold change compared to untreated. Ranking was determined based on the Log2 fold score (Left). The top ten Doxorubicin sensitizers are indicated on the right with their respective fold change (Fc) in Log2.293T cells were transfected with Flag-REV7 and GFP-SHLD1 (Left) or Flag-SHLD2 and GFP-SHLD1 (Right) expression vectors as indicated. 24h post-transfection cells were treated with DMSO or with 10uM of ATM inhibitor KU-60019 for 1 h prior to irradiation. 1h post-irradiation (10 Gy) nuclear extracts were prepared and REV7 or SHLD2 complexes were immunoprecipitated using anti-Flag (M2) Resin and then analyzed by immunoblotting using GFP and REV7 antibodies.U2OS EJ5-GFP cells were transfected with either siCTRL, siSHLD1 #1 or siSHLD1 #2. At 24hr post-transfection, cells were transfected with the I-SceI expression plasmid, and the GFP+ population was analyzed 48 hr post-plasmid transfection. The percentage of GFP+ cells was determined for each individual condition and subsequently normalized to the non-targeting condition (siCTRL).CH12F3-2 cells stably expressing either shCTRL, shSHLD1#1 or shSHLD1#2 were stimulated with a cocktail of cytokines (CIT) to induce class switching to IgA. The percentage of IgA+ cells was monitored 24 and 48h post-stimulation by staining with an anti-IgA antibody followed by flow cytometry analysis.U2OS DR-GFP cells were transfected with the indicated siRNAs. At 24hr post-transfection, cells were transfected with the I-SceI expression plasmid, and the GFP+ population was analyzed 48 hr post-plasmid transfection. The percentage of GFP+ cells was determined for each individual condition and subsequently normalized to the non-targeting condition (siCTRL).
Figure 6Survival analysis of low and high expressers of SHLD2 in a cohort of 24 patients affected by Triple Negative Breast cancer (TNBC). Data are represented as Kaplan-Meyer curves with expression classified as low and high.Relapse-free Survival (Basal Subtype) of low and high expressers of SHLD2 obtained from the KM-plotter database. Data are represented as Kaplan-Meyer curves with expression classified as low and high.