EMBO/SourceData huggingface dataset:
Figure 3(A) MEFs transiently co‐expressing Myc‐PAT4 and mStr‐Rab12 were immunostained with anti‐Myc antibody.(B) MEFs transiently expressing Myc‐PAT4 were immunostained with the antibodies indicated. Scale bars, 20 μm.(C) Total cell lysates and surface biotinylated proteins from MEFs stably expressing HA‐PAT4 were analysed by immunoblotting with the antibodies indicated.(D) Lysates of MEFs stably expressing HA‐PAT4 that had been transfected with control or Rab12 siRNAs were analysed by immunoblotting with the antibodies indicated.(E) Quantification of (D). ***P0.005.(F) PAT4 mRNA levels from MEFs transfected with control or Rab12 siRNA as revealed by reverse‐transcription PCR analyses. Gapdh was used as an internal control.(G) Total cell lysates and surface biotinylated proteins from MEFs stably expressing HA‐PAT4 that had been transfected with control or Rab12 siRNA were analysed by immunoblotting with the antibodies indicated. Note that the amounts of plasma‐membrane‐localized PAT4 protein and TfR protein were higher in Rab12‐knockdown MEFs, whereas the total amounts of EGFR protein and plasma membrane‐localized EGFR protein in these cells were slightly lower, but the mechanisms responsible for these changes are unknown. GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; MEFs, mouse embryonic fibroblasts; PAT4, proton‐coupled amino‐acid transporter 4; siRNA, short interfering RNA.
Figure 4(A) Lysates of MEFs transfected with the siRNAs indicated were analysed by immunoblotting with the antibodies indicated.(B) Quantification of the phospho‐S6K levels shown in (A).(C) MEFs transfected with the siRNAs indicated were cultured under nutrient‐rich (N) or starved (S) conditions and were immunostained with anti‐LC3 antibody. The mean numbers of LC3‐positive dots per cell are shown.
Figure 1A) BW5147 reporter cells stably expressing human FcγR-ζ chain chimeras or BW5147 parental cells (grey/dashed) were stained with FcγR specific conjugated mAbs as indicated and measured for surface expression of FcγRs via flow cytometry. Bar graphs show means of three independent experiments performed in technical duplicates. Error bars = SD.C) Immobilized IC, immobilized IgG and IgG opsonized cells represent qualitatively similar ligands for FcγRs. Response curves of human FcγRs activated by opsonized cells (293T cells stably expressing CD20 + Rituximab [Rtx]), immobilized IC (rec. soluble CD20 + Rtx) and immobilized IgG (Rtx). sICs formed using monovalent antigen (rec. soluble CD20 + Rtx) do not activate human FcγRs. X-Axis shows sample concentration determined by antibody molarity. Y-Axis shows FcγR activation determined by reporter cell mouse IL-2 production (OD 450nm). Two independent experiments performed in technical duplicates. Error bars = SD.
Figure 3A) Negatively selected primary NK cells purified from PBMCs of three healthy donors were tested for activation markers using flow cytometry. NK cells were incubated with immobilized IgG (mepolizumab, Mpz), soluble IC (Mpz:IL-5 = 1:1), soluble Mpz or soluble IL-5 (all at 200 nM, 106 cells). Incubation with PMA and Ionomycin (Iono) served as a positive control. Incubation with medium alone served as a negative control. Each dot represents one donor measured in technical duplicates. Error bars = SD. One-way ANOVA (Tukey); *p<0.05, ****p<0.0001.B) Negatively selected primary neutrophils purified from whole blood of four healthy donors were tested for adhesion and activation markers using flow cytometry. Neutrophils were incubated with immobilized IgG (Mpz), soluble IC (Mpz:IL-5 = 1:1), soluble Mpz or soluble IL-5 (all at 200 nM, 2*105 cells). Incubation with PMA or immobilized rituximab served as positive controls. Incubation with medium served as a negative control. Immobilized FcγRII and FcγRIII specific mAbs served as functional controls. Mean florescence intensity (MFI) values at t=30 minutes of incubation are presented as increase over t=0 min. Each dot represents one donor measured in technical duplicates. Error bars = SD. Two-way ANOVA compared to medium (Dunnett); *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Figure 4B) sICs of different size were generated by cross-titration according to the AF4 measurement. Reporter cells were incubated with fixed amounts of either mAb (infliximab, left) or Ag (rhTNF-α, right) and titrated amounts of antigen or antibody, respectively. X-Axis shows titration of either antigen or antibody, respectively (TNF-α calculated as monomer). Two independent experiments performed in technical duplicates. Error bars = SD.C) Purified primary NK cells from three different donors were incubated with cross-titrated sICs as in B. NK cells were measured for MIP-1β expression (% positivity). Incubation with PMA and Ionomycin served as a positive control. Incubation with medium alone served as a negative control. Each dot represents one experimental replicate, the dashed line represents the mean values.
Figure 5A) Experiments shown for an exemplary group of 8 SLE patients and two healthy individuals. Dose-dependent reactivity of FcγRs IIIA and IIB/C was observed only for SLE patient sera and not for sera from healthy individuals. Each titration represents one donor measured in technical duplicates.B) Activation of FcγRs IIB/C and IIIA by patient serum is mediated by serum derived sICs. Patient serum samples were depleted of sICs by PEG precipitation and the supernatant (SN) was compared to untreated serum regarding FcγR activation (left). Shown is one exemplary experiment performed in technical duplicates. Error bars indicate reproducibility as range. IgG concentration in the precipitate (PC), supernatant (SN) or unfractionated serum respectively is shown in the bar graph (right).C-D) Correlation of FcγR activation data from A with (C) patient SLEDAI or (D) conventional SLE disease markers (α-dsDNA levels indicated as IU/ml or C3d concentrations indicated as mg/L). Left panel: FcγRIIIA; right panel: FcγRIIB/C. FcγR activation from a dose-response curve as in A was calculated as area under curve (AUC) for each SLE patient represented by individual dots. AUC from SLE patients are expressed as fold change compared to the healthy control mean (n=4). SLE patients with α-dsDNA levels below 50 IU/ml and C3d values below 6 mg/L were excluded. One-tailed Spearman's. Dotted lines show 95% confidence bands.
Figure 6A) Reporter cells expressing mFcγRIII, mFcγRIV or parental BW5147 cells were incubated with titrated amounts of synthetic sICs generated from rhTNF-α and mouse-anti-hTNF-α at a 1:1 ratio by mass. One experiment performed in technical duplicates. Error bars indicate reproducibility as range.B) Titrations of three mouse sera per group (C57BL/6, K/BxN or NZB/WF1) were incubated with mFcγR reporter cells and FcγR activation was assessed as described above. Sera from BL/6 mice served as negative control. Each symbol represents mean values of one mouse measured in technical duplicates. Error bars = Mean with SD.
Figure 1D, E Immunostaining of MCT2 and SMCT1 in tumors of KIC mice treated with βOHB (100mg/kg/day, i.p.) or 0.9% NaCl (i.p.) (n=5 mice/group). Areas of MCT2 (upper panel) and SMCT1 (lower panel) stainings (D) are expressed as mean of percentage of total tissue area ± SEM. Significance was defined by Mann-Whitney test. *: p<0.05, **p<0.01. Representative images of MCT2 and SMCT1 stainings (E) in tumors from KIC mice treated with βOHB or NaCl. Scale bar: 100 µm.
Figure 2B-D [U-13C]Glucose (n=3 mice) (B), [U-13C]Glutamine (n=5 mice) (C) and [U-13C]Acetate (n=6 mice) (D) tracing into βOHB in poorly differentiated PDA explants from KIC mice. Data are expressed as mean ± SEM.
Figure 3G Volcano plot illustrating proteins significantly under- or over-represented in sg-CTRL and sg-HMGCL PANC-1 spheroids (n=3 independent experiments). Significance was defined by one-tailed Student's t-test. Protein levels with a q-value<0.05 (horizontal axis) and a fold change <-1.5 or >+1.5 (vertical axis) are considered as significantly down or up-regulated in sg-HMGCL PANC-1 spheroids.
Figure 4A, B Quantification of volume with representative images (A) and weight (B) of sg-CTRL (n=15 for A and n=9 for B), sg HMGCL #2 (n=19 for A and n=8 for B), #3 (n=7 for A and B) pancreatic tumors. Data are expressed as mean of tumor volume or weight ± SEM. Significance was defined by Mann-Whitney test. *: p<0.05, **p<0.01, ***p<0.001.C Extracellular matrix quantification following trichrome staining in sg-CTRL and sg-HMGCL #2 pancreatic tumors sections (n=8 and 9 mice/group respectively, left panel). Data are expressed as mean of percentage of total tissue area ± SEM. Significance was defined by Mann-Whitney test. **: p<0.01. Representative images of trichrome staining in sg-CTRL and sg-HMGCL pancreatic tumors. Scale bar: 100 µm (right panel).D, E Quantification of volume (D) and weight (E) of sg-CTRL pancreatic tumors treated with 0.9% NaCl (i.p.) (n=4), and pancreatic tumors from two different clones of sg-HMGCL treated with 0.9% NaCl (i.p.) or βOHB (100mg/kg/bi-weekly, i.p.) (n=10/group). Data are expressed as mean of tumor volume or weight ± SEM. Significance was defined by Mann-Whitney test. ns: not significant, **p<0.01.H [U-13C]βOHB tracing into TCA intermediate: citrate in sg-CTRL and sg-HMGCL #2 and #3 PANC-1 cells cultured in indicated glucose concentrations. Data are expressed as mean ± SEM. (n=2 independent experiments). Significance was defined by one-way ANOVA followed by a Bonferroni's multiple comparisons test, only significances between sg-HMGCL #2, #3 PANC-1 cells and sg-CTRL PANC-1 cells under the same culture condition are mentioned. ***p<0.001.
Figure 5(B) Box-and-whisker plots of ketone bodies (KB) metagene score defined as first component of PCA of genes in primary tumors (n=728), in all metastases (n=76), and in liver metastases specifically (n=35). Box-and-whisker plot were defined with default parameters by median value (central band at the 50th percentile), interquartile ranges (IQR, box limited by 25th and 75th percentile) and whisker boundaries defined at 1.5x IQR. Significance was defined by Student's t-test.D-F Effect of βOHB treatment on liver metastatic incidence, size and status. Representative HPS staining of liver lobes from metastatic mice treated with βOHB (100mg/kg/day, i.p.) or 0.9% NaCl (i.p.) (n=8 mice/group). Metastatic areas are separated from liver by yellow dotted lines. Scale bar: 1mm and 500 µm for insets (D). Number and size of metastasis per lobe and classified in small, medium and large size (E). Pathological status of metastasis from mice treated with βOHB or NaCl (n=13 or 5 lobes/group, respectively). Data are expressed as percentage of total metastasis in all liver lobes presenting metastasis (F).
Figure 1(A) Mouse embryos were immunostained with anti-neurofilament antibody (blue), showing mesencephalic projection. Dispersed axon projections (arrows point to the area of the superior colliculus) and decreased axon fibers (arrowheads point to the area of the pretectal commissure) were observed in Plcg1f/f;Nes-Cre mice at E11.5 (n = 20). The areas of the superior colliculus is shown magnified in each inset (scale bar = 200 μm)(B) Representative images between the posterior and anterior borderlines of the superior colliculus (a and b). The red boxes were drawn as rostrocaudal region of interest (ROI) based on the upper part of the pretectal commissure in the embryonic mesencephalon (scale bar = 100 μm)(C and D) The axons passing through the ROI was quantified by measuring the fluorescence intensity in the ROI, and the distribution of axons was statistically analyzed (n = 5; t-test; **P < 0.005)(E) Whole-mount immunostaining images show neurofilament (red), netrin-1 (purple), and DCC (green), respectively. An E11.5 Plcg1f/f;Nes-Cre embryo showed shortened dorsal ramus and opthalmic nerves (white and yellow boxes). Scale bar: 200 μm(F - G) Enlarged views of the opthalmic nerve (yellow arrow heads) and dorsal ramus (white arrow heads) (scale bar: 50 μm)(H) Nerve length measurements; Op: ophthalmic nerve; DR: dorsal ramus (n = 3; t-test; **P < 0.005). Data are presented as the mean ± s.e.m(I) Double immunostaining images. The pattern of expression and colocalization of DCC (green)- and TH (red)-positive axon bundles extending toward the alar thalamus. Scale bar: 100 μm
Figure 2(A) In primary mesencephalic neurons, the phosphorylation of PLCγ1 on Y783 was increased proportionally to the netrin-1 concentration. The lower graph presents quantification of the band intensities in (A) (n = 3)(B) Phosphorylation of PLCγ1 on Y783 was decreased after knockdown of the DCC receptor by siRNA (siDcc) (N = 3)(C) Western blot for the NTN1 secretion test. Lane #1: transfection-free lysate, #2: transfection-free supernatant, #3: NTN1-transfected cell lyasate, #3 and #4: NTN-1- transfected supernatant(D) Ventral mesencephalic (A9) cultures (daggers) derived from control or Plcg1f/f;Nes-Cre mice, were co-cultured with netrin-1-releasing 293T cell aggregates (asterisks; scale bar = 100 μm). The areas delineated by the white boxes between the mesencephalon tissue and 293T cell aggregates are magnified in the respective insets (n = 4)(F) Mean angle of the axon extension(G) Quantification of the ventral mesencephalic axons. The bar graphs indicate the percentages of proper targeting of ventral mesencephalic axons correctly targeting the netrin-1-releasing cell aggregates (n = 200). Each experimental set was performed in quadruplicate(H) Anti-tyrosine hydroxylase (TH) immunostaining in ventral mesencephalic (VM) tissue. (scale bar = 200 μm)(I) Primary mesencephalic neuron culture in the absence or presence of NTN1-conditioned media (n = 100; scale bar: 500μm)(K) Mean length of neurites (F-value:1.82, t-test, P**<0.005)
Figure 3(A) Co-immunoprecipitation data showed that PLCγ1 did not interact with the DCC receptor (n = 2, N = 3).(B) Immunoprecipitation assay showed that Src kinase interacted with PLCγ1. Rac1 is a positive interacting control for PLCγ1 (N = 3).(C) In vitro kinase assay confirmed that Src kinase directly phosphorylated Y783 of PLCγ1. (D) Quantification of the band intensities in (C) (N = 3).(E) Src kinase inhibitor reduced the specific phosphorylation of PLCγ1 on Y783 (arrow; Src I IC50 conc = 44 nM; N = 3).(F) Rac1 activity assay (N = 3). (G) Quantification of Rac1 activity levels in (F).(H) Representative immunoblot of cultured Plcg1f/f (n = 2, N = 3) and Plcg1f/f;Nes-Cre (n = 2, N = 3) mesencephalic neurons stimulated with conditioned media containing Netrin-1.
Figure 4(B) Immunohistochemical staining of control and Plcg1 conditional knockout (cKO) sagittal brain sections. The deletion of Plcg1 caused structural changes in SI and OT projections in the Plcg1f/f;Nes-Cre (d) mice compared to the controls (a) (n = 4; scale bar = 1 mm). The areas delineated by the white boxes (insets) are magnified images of the OT (b and e), and the yellow boxes are magnified images of the SI (c and f). (C) Quantification of the axon bundle distribution. Green boxes in (b) and (e) were selected as tegions of interest (ROIs) using the ZEN software (n = 4) (one-way ANOVA, F-value: 118.5745, P**<0.005).(D) Representative images of tyrosine hydroxylase (TH)- positive neurons (scale bar = 1 mm). The areas delineated by the white boxes are magnified in the insets. The yellow arrow indicated misoriented TH-positive axons (magenta: TH).(E) Fluorescence intensities in the ROIs were measured according to distance (n = 4) (one-way ANOVA, F-value: 271.8, **P<0.005). Cumulative values of fluorescence intensities per section are shown in the lower graph (S: section).(F) In situ hybridization of PLCγ1 in mDA neurons (scale bar = 50 μm) (green: TH, violet: DIG-labeled probe).(G) Representative images of the CC axon bundles in control (left) and a Plcg1f/f;Nes-Cre (right) mice (n = 4). The tip of the CC was triangular in the control mouse, but oval in the Plcg1f/f;Nes-Cre mouse. The areas delineated by the white boxes are magnified in the insets (red: neurofilament, green: glial fibrillary acidic protein, blue: Hoechst; scale bar = 1 mm).(H) The area of the CC in the Plcg1f/f;Nes-Cre mouse was 36% smaller than that in the control mouse (scale bar = 500 μm) (I) Quantitative comparison of the CC areas in the control and Plcg1f/f;Nes-Cre mice (n = 4; t-test; **p < 0.005). Data are presented as the mean ± s.e.m.
Figure 5(A) Cumulative histograms based on fractional anisotropy (FA) values. (B), show the CC volume relative to FA values. As FA values increased, the structural (CC volume differences increased between the control and Plcg1f/f;Nes-Cre mice. (n = 4; Shapiro-Wilk and t-test; *p < 0.05). Data are presented as the mean ± s.e.m.(C), Cumulative histograms based on fractional anisotropy (FA) values. (D), show the number of tensors relative to FA values. As FA values increased, the structural number of tensors) differences increased between the control and Plcg1f/f;Nes-Cre mice. (n = 4; Shapiro-Wilk and t-test; *p < 0.05). Data are presented as the mean ± s.e.m.(E) Cumulative histograms based on fractional anisotropy (FA) values. (F) show the CC volume and the number of tensors relative to FA values. As FA values increased, the structural (CC volume and the number of tensors) differences increased between the control and Plcg1f/f;Nes-Cre mice. Depending on the number of tensors, the length in the CC appeared to be shortened (E, F). (n = 4; Shapiro-Wilk and t-test; *p < 0.05). Data are presented as the mean ± s.e.m.
Figure 1(A) (B) CPT sensitivity assayed by 10-fold serial dilutions of different mutant combinations on YPAD plates. Four biological replicates have been performed.(C) Colony-forming unit assays to increasing CPT concentrations of the same mutant combinations as in (B). Mean values ± SD are plotted. Four biological replicates have been performed.(D) UV sensitivity assayed by 10-fold serial dilutions of different mutant combinations with rad3-102 allele on YPAD plates after exposure to the indicated UV-C doses. Five biological replicates have been performed.(F) BIR frequencies (Lys+ survivors among total cells) for the WT and indicated mutant strains are plotted on a logarithmic scale. Data represent the mean ± SD from 3 to 6 independent experiments. ns, non-significant difference, * P-value = 0.0134, Mann-Whitney unpaired t test.
Figure 2(A) (B) CPT sensitivity assayed by 10-fold serial dilutions of different mutant combinations on YPAD plates. Four biological replicates have been performed.(C) UV sensitivity assayed by 10-fold serial dilutions of different mutant combinations with rad3-102 allele on YPAD plates after exposure to the indicated UV-C doses. Four biological replicates have been performed.(D) Synthetic combinations of rad3-102 mus81∆ with rad51∆ and pol32∆. Tetrads dissected on YPAD medium are shown. Triangles indicate either a severe growth defect or lethality.
Figure 3(A) Analysis of DNA content by flow cytometry of G1 phase synchronized wild type, rad52∆, rad51∆, mus81∆ and pol32∆ cells and further released into S phase. Cells were synchronized in G1 with α -factor, treated with DMSO or 100 µM CPT, let in G1 for 1 h, and released into S phase. Three biological replicates have been performed.(B) (C) Analysis of DNA content by flow cytometry of G1 phase synchronized wild type, rad3-102, rad3-102 rad51∆, rad3-102 mus81 and rad3-102 mus81-dd cells and further released into S phase. Cells were synchronized in G1 with α-factor, untreated or irradiated with 20 J/m2 UV-C, let in G1 for 2h, and released into S phase. Three biological replicates have been performed.
Figure 4(A) Analysis of replicated DNA tracks length by single-molecule DNA combing in WT, rad52∆, rad51∆, mus81∆ and pol32∆ cells exposed to CPT. Exponentially growing cells were treated with DMSO or 50 µM CPT for 2h and then pulse-labeled with 50 µM EdU for 20min. DNA fibers were combed on silanized coverslips and EdU-labeled DNA was detected by Click chemistry. Graph depicts the distribution of EdU tracks length in kb. Box and whiskers indicate 25-75 and 10-90 percentiles, respectively. Median EdU tracks length is indicated in kb (values from two biological replicates were pooled). Asterisks indicate the P-value of the Mann-Whitney unpaired t test, **: p=0.0015. The percentage of EdU track length decrease between the DMSO and CPT conditions is indicated between parentheses for each strain. Representative images of DNA fibers are shown. Red and white: EdU, green: DNA.(C) Analysis of replication intermediates by 2D gel electrophoresis. Replication intermediates were monitored at early origin ARS305 and region C in WT, rad3-102, mus81∆ and rad3-102 mus81∆ cells. Cells were synchronized in G1 with α-factor and collected at the indicated time points after release into S phase. A scheme of the studied chromosomal region is shown (drawn to scale). Relevant probes are indicated by gray bars, and coordinates of ARS and restriction sites are indicated in bp. Two biological replicates have been performed.
Figure 5(A) Analysis of nuclear Rad52-GFP foci formation. An illustrative image of the experimental setup is shown. DIC, differential interference contrast; yellow, Rad52-GFP; red, mCHERRY-Pus1 (nuclear compartment marker). Scale bar = 5 µm.(B) (C) Kinetic analysis of Rad52 foci formation. Wild-type and mus81∆ cells containing Rad52-YFP and mCHERRY-Pus1 were synchronized in G1 with α-factor, treated with DMSO or 100 µM CPT, let in G1 for 30 min, and released into S phase. Cells were collected at the indicated time points and visualized by fluorescence microscopy. Mean ± SEM of cells with Rad52 foci are shown for each time point. Flow cytometry profiles corresponding the experimental setup are shown. Four biological replicates have been performed.(D) Mms4-Flag10 phosphorylation analyzed by immunoblot in wild type cells exposed to CPT. Wild-type cells were synchronized in G1 with α-factor, treated with DMSO or 50 µM CPT, let in G1 for 1 h, and released into S phase. Cells were collected at the indicated time points and Mms4 was immunodetected with anti-Flag antibodies. Clb2 immunodetection serves as a marker for G2 phase entry. FACS profiles corresponding the experimental setup are also shown. Two biological replicates have been performed.(E) CPT sensitivity assayed by 10-fold serial dilutions of S-MUS81 and G2-MUS81 alleles compared to the wild type, mus81, mms4∆ and cdc5-2 mutants. Three biological replicates have been performed.(F) UV sensitivity assayed by 10-fold serial dilutions of S-MUS81 and G2-MUS81 alleles in combination with rad3-102 on YPAD plates after exposure to the indicated UV-C doses. Three biological replicates have been performed.
Figure 6(A) (B) Pulsed-field gel electrophoresis (PFGE) analysis of wild type, mus81∆, cdc5-2 and G2-MUS81 cells in response to CPT. Cells were synchronized in G1 with α-factor, treated with DMSO or 100 µM CPT, let in G1 for 1 h, and released into S phase. Cells were collected at the indicated time points. DNA contents was analyzed by flow cytometry and the DNA extracted in agarose plugs was analyzed by PFGE. Upper panel: agarose gel stained with ethidium bromide; lower panel: Southern blot using a chromosome IV specific probe. JMs, joint molecules accumulated in the gel wells. The mean value of JMs relative to the total amount of DNA is indicated for each time point. Two to three biological replicates have been performed.(C) (D) PFGE analysis of rad52∆ and rad52∆ mus81∆ cells in response to CPT performed as in (A) (B). The gel has been stained with Ethidium bromide and densitometry profiles corresponding to the +CPT 140 min time points in rad52∆ and rad52∆ mus81∆ cells are shown. Two biological replicates have been performed.