EMBO/SourceData huggingface dataset:
Figure 6RPE-specific overexpression of VEGF-A in VMD2Cre+/WTROSA-STOPfl/fl-VEGF-A164mice (VEGF-ARPEmice) results in CNV at sites of VEGF-A overexpression (Cre+ patches (white nuclei); red arrow), while Cre- areas with normal VEGF-A expression show no CNV (white arrow). Neovessels are CD31+ (red). VEGF-A-induced CNV lesions and RPE barrier breakdown in VMD2Cre+/WT ROSA-STOPfl/fl-VEGF-A164mice resemble those seen in VEGF-Ahypermice (B.-C., arrows; phalloidin staining in green). Scale bars 100 μm.D. Genetic inactivation of Flk1 specifically in the RPE in VEGF-Ahypermice (VMD2Cre+/WTFlk1fl/flVEGF-Ahypermice) prevents RPE barrier breakdown and CNV lesion formation. Choroidalflat mounts of 5 months old VEGF-Ahypermice and VMD2Cre+/WTFlk1fl/flVEGF-Ahypermice are shown. β-gal shows uniform VEGF-A expression in these mice (green). Crestaining (red) shows co-localization of β-gal and Crestaining in the nuclei of RPE cells in VMD2Cre+/WTFlk1fl/flVEGF-Ahypermice (red areas of staining occur in CNV lesions of VEGF-Ahypermice due to labeling of anti-mouseIgG and not Cre, as the anti-mouseIgG secondary antibodies were used to detect the mouse anti-Cre antibody). Phalloidinstaining (white) reveals extensive RPE barrier breakdown and CNV lesions (arrow) in VEGF-Ahypermice, which is not observed in VMD2Cre+/WTFlk1fl/flVEGF-Ahypermice. Scale bar 100 μm.
Figure 7A. NLRP3 is expressed in RPE cells at sites of CNV lesions (arrow; white).B. Lack of NLRP3 staining in VEGF-Ahyper/NLRP3-/- mice (control). Scale bars 100 μm.C. Expression of NLRP3 [*p-value: 0.0478] and IL-1β [*p-value: 0.0398], but not of IL-18 [p-value: 0.3529] is increased in RPE/choroid lysates from VEGF-Ahyper mice when compared to matched control littermate mice. N=7 mice/group (3 independent experiments). Graphs show mean ± SEM.D.-G. Complement pathway activation (immunolabeling for the product of complement pathway activation C5b-9, an inducer of the NLRP3 inflammasome) is observed in CNV lesions of VEGF-Ahypermice (white arrow in E.) but not in age-matched control mice (D.) Co-localization of C5b-9 in the same CNV lesion is observed with complement C1qA (the initiator of the classical complement pathway and an inducer of the NLRP3 inflammasome) (F.), and with TLR2 (G.) (white arrows). Autofluorescence in red in D. and E. CD31+vessels in red in I. and J. Yellow arrows show site of choroid. Scale bars 50 μm.
Figure 8A. CNV lesion numbers per eye in 6-week old VEGF-Ahypermice compared with VEGF-Ahypermice lacking CASP1/CASP11, NLRP3, IL1R1, IL-18, TLR2, or those heterozygous for beclin-1 or homozygous for hypomorphic ATG16L1. CASP1/CASP11, NLRP3 or IL-1R1 deficiency results in significantly fewer CNV lesions. Each value represents the total CNV lesion number/eye for an individual mouse. Mean ± SEM is shown. P-values are based on comparison with the VEGF-Ahypermouse group. Absolute numbers of mice per group are indicated in ( ). P-values were calculated with a two-tailed unpaired Student's t-test. Separately, p-values are shown between VEGF-Ahyper/CASP1-/-/CASP11-/- and VEGF-Ahyper/NLRP3-/- or VEGF-Ahyper/IL1R1-/-mouse groups. WT mice developed no CNV lesions.B. Average CNV lesion area (in μm2/lesion) in 6-week old VEGF-Ahypermice compared with VEGF-Ahypermice lacking CASP1/CASP11, NLRP3, IL1R1, IL-18, TLR2, or those heterozygous for beclin-1 or homozygous for hypomorphic ATG16L1. Lack of TLR2 or CASP1/CASP11 significantly reduces CNV lesion size, while reduced autophagy moderately increases CNV size. Each value represents the average CNV lesion size/eye for an individual mouse. Mean ± SEM is shown. P-values are based on comparison with the VEGF-Ahypermouse group. Absolute numbers of mice per group are indicated in ( ). P-values were calculated with a two-tailed unpaired Student's t-test. Separately, p-values are shown between VEGF-Ahyper/CASP1-/-/CASP11-/- and VEGF-Ahyper/NLRP3-/- or VEGF-Ahyper/IL1R1-/-mouse groups.C.-E. Representative choroidal flat mount images show multifocal CNV lesions in VEGF-Ahyper mice and a significant reduction in CNV numbers in those VEGF-Ahyper mice that lack either CASP1/CASP11 or IL1R1 (and therefore IL-1β-signaling). Staining for phalloidin in green and for CD31 in red. Scale bars 200 μm.. Laser-induced CNV experiments demonstrate that treatment with a caspase-1 inhibitor (compared to DMSO treated controls) or a neutralizing TLR2 antibody (compared to isotype matched IgG treated controls) inhibits CNV lesion growth. P-values are shown. N=15 mice/group. Representative CNV lesions in choroidal flat mounts are shown. Scale bars 100 μm.
Figure 9Figure 9: VEGF-A-induced non-exudative AMD-like pathologies can be prevented by targeting NLRP3, IL1R1 or caspase-1/caspase-11.A. No significant abnormalities of the choroid, RPE or retina are seen in control wild-type littermate mice. Representative image of a 28-months old control eye is shown. White arrow indicates normal Bruch's membrane. Chor: choroid; RPE; PR: photoreceptor; ONL: outer nuclear layer.B. Aged VEGF-Ahypermice develop a progressive age-dependent degeneration of the RPE (red arrow) and the photoreceptors (yellow arrow) with accumulation of thick sub-RPE basal laminar deposits (black arrow). These non-exudative AMD-like pathologies occur also at sites devoid of CNV lesions.C.-E. Genetic inactivation of CASP1/CASP11, NLRP3, or IL1R1 strongly inhibits the manifestation of RPE and photoreceptor degeneration in aged VEGF-Ahypermice.F.-G. VEGF-Ahyper/IL18-/-mice show significant RPE and photoreceptor degeneration, with shortened and irregular photoreceptor outer segments (white arrows). Thick sub-RPE deposits that protrude into the RPE plane are observed as well (black arrows).H. VEGF-Ahyper/Becn1+/-mice show significant RPE and photoreceptor degeneration.I. In contrast, age-matched VEGF-Ahypomice that are hypomorphic for VEGF-A and express β-galactosidase from the endogenous VEGF-A locus (as do VEGF-Ahypermice) do not show these eye pathologies.B-I. 21-months old mice. Scale bars 20 μm.
Figure 10A. Graphs show means of a- and b-waves of dark-adapted and light-adapted ERG responses (ERG intensities in μV) for both the left and the right eyes from 21-months old VEGF-Ahypermice, VEGF-Ahyper/CASP1-/-/CASP11-/-mice, CASP1-/-/CASP11-/-mice, VEGF-Ahyper/IL1R1-/-mice, IL1R1-/-mice, VEGF-Ahyper/IL18-/-mice, and IL18-/-mice. None of these mice had cataracts. Graphs show mean ± SEM.B. Representative ERG tracings are shown for VEGF-Ahyper mice and for VEGF-Ahyper/CASP1-/-/CASP11-/- mice. X-axes show milliseconds (ms). Y-axes show ERG intensities (in μV).
Figure 11Figure 11: Lack of caspase-1/caspase-11 inhibits CNV lesion formation, retinal degeneration, fundus abnormalities and fundus autofluorescence in aged VEGF-Ahyper mice.A. OCTs in aged VEGF-Ahypermice show retinal degeneration with a thinning of the retina and CNV lesions extending from the choroid into the subretinal space (arrows). OCTs appear normal in age-matched VEGF-Ahyper/CASP1-/-/CASP11-/-mice and CASP1-/-/CASP11-/-mice.B. Fundus autofluorescence imaging shows areas of autofluorescence in the fundus of aged VEGF-Ahypermice, but not in age-matched VEGF-Ahyper/CASP1-/-/CASP11-/-mice and CASP1-/-/CASP11-/-mice.C. Abnormal pigment loss and degenerative changes are observed in the fundus of aged VEGF-Ahypermice, while the fundus of age-matched VEGF-Ahyper/CASP1-/-/CASP11-/-mice and CASP1-/-/CASP11-/-mice does not show these abnormalities.Representative images of 21-months old mice are shown.
Figure 2A-C Viability counts of the indicated V. parahaemolyticus (A-B) or E. coli (C) prey strains before (0 h) and after (4 h) co-incubation with the indicated V. parahaemolyticus attackers on media containing 3% NaCl at 30°C. In A and B, media also contain 0.1% arabinose to induce expression from plasmids. In A, prey strains contain either an empty plasmid (pEmpty) or a plasmid for arabinose-inducible expression of VP1389 (pVP1389). In B and C, prey strains contain an empty plasmid that provides a selection marker, and the attackers are derivatives of a ∆hns mutant (parental). In B, the attackers contain an empty plasmid, or plasmids for the arabinose-inducible expression of VP1388 (pVP1388) or VP1390 (pVP1390). Data are shown as the mean ± SD, n = 3 technical replicates. Statistical significance between samples at the 4 h timepoint by an unpaired, two-tailed Student's t-test is denoted above. A significant difference was considered as P < 0.05. DL, assay detection limit. ∆hcp1 was used as a T6SS1- control strain. The experiment was performed at least three times with similar results; results of a representative experiment are shown.
Figure 3A VP1388 binds VP1390. Immunoprecipitation using α-FLAG antibodies from E. coli cells co-expressing the indicated C-terminal FLAG- and Myc-tagged proteins from arabinose-inducible plasmids.B VP1388 and VgrG1 co-precipitate with VP1390. Immunoprecipitation using α-FLAG antibodies from V. parahaemolyticus ∆hns/∆hcp1/∆vp1390 derivatives harboring plasmids for the arabinose-inducible expression of FLAG-tagged sfGFP or VP1390. Cells were grown in MLB media supplemented with chloramphenicol to maintain the plasmids, and 0.1% arabinose. Endogenous VP1388 and VgrG1 were detected using α-VP1388 and α-VgrG1 antibodies, respectively.C Expression (cells) and secretion (media) of VP1388, VP1390, and VgrG1 from the indicated V. parahaemolyticus ∆hns-derived strains harboring an empty plasmid (pEmpty) or plasmids for the arabinose-inducible expression of VP1388 (pVP1388) or VP1390 (pVP1390). Samples were grown in media containing 3% NaCl and supplemented with 0.1% arabinose at 30°C. RNA polymerase β (RNAp) was used as a non-secreted protein loading control. Arrows denote the bands at the expected size of VP1388 or VP1390, as indicated in the appropriate panels.
Figure 4A Toxicity of periplasm-targeted proteins in E. coli. E. coli strains containing plasmids for the arabinose-inducible expression of sfGFP (used as a control), VP1388 or VP1390 fused to an N-terminal PelB signal peptide (perisfGFP, periVP1388, and periVP1390, respectively) were spotted at 10-fold serial dilutions onto LB agar plates supplemented with kanamycin (to maintain plasmids) and either 0.2% glucose, to repress protein expression, or 0.001% arabinose, to induce protein expression. Plates were incubated over-night at 30°C or 37°C, as indicated.B VP1389 interacts with VP1390. Co-immunoprecipitation of FLAG-tagged VP1390 or BC3020 using Myc-tagged VP1389 when co-expressed in V. parahaemolyticus Δvp1389 (input). Precipitated proteins (output) were detected by immunobotting using α-Myc and α-FLAG antibodies. Source data are available online for this figure.C VP1390 induces cell lysis in E. coli. Time-lapse microscopy of E. coli cells expressing periplasm-targeted sfGFP, VP1388, or VP1390 (perisfGFP, periVP1388, and periVP1390, respectively) from an arabinose-inducible vector, grown on LB agarose pads supplemented with kanamycin (to maintain the plasmid), 0.2% arabinose (to induce expression), and propidium iodide (PI; pink). Merging of the phase contrast and PI channels are shown. Scale bar = 2 µm.
Figure 5Time-lapse microscopy of competition between V. parahaemolyticus ∆hns (T6SS+/VP1390+), ∆hns/∆hcp1 (T6SS-/VP1390+), or ∆hns/∆vp1390 (T6SS+/VP1390-) attackers and V. parahaemolyticus ∆vp1389 prey that express GFP. Attacker and prey were mixed (2:1 ratio) and spotted on LB agarose pads supplemented with propidium iodide (PI; pink). Merging of the phase contrast, GFP (green), and PI (pink) channels, as well as the PI channel alone are shown. Scale bar = 5 µm. The experiment was performed three times with similar results; results of a representative experiment are shown.
Figure 2(A-C) Telophase GSCs marked by H3S10p (gray) expressing old (green) and new (red) histone H3 show non-overlapping patterns (A). Telophase GSCs expressing old (green) and new (red) histone H4 show moderate levels of overlap (B). GSCs expressing old (green) and new (red) histone H2A show high levels of overlap (C). (D) A control where the old and new fluorescent tags have been switched shows the non-overlapping patterns of old (red) versus new (green) H3Rev histone in mitotic telophase GSCs marked by H3S10p (gray). (E) Quantification of log2 ratios of total old and new histone inherited by each future daughter cell of the GSC division, where a value of 0 is equal inheritance at exactly a 1:1 ratio. Data gathered over 9 separate experiments for H3Rev GSCs, H2A GSCs, and H4 GSCs. Data gathered over 16 separate experiments for H3 GSCs. Values are mean + 95% CI. (F-H) Telophase CBs marked by H3S10p (gray) expressing old (green) and new (red) histone H3 also show non-overlapping patterns. (F) Telophase CBs expressing old (green) and new (red) histone H4 (G) and H2A (H), which depict more overlap like GSCs. (I) A control with switched old and new fluorescent tags shows the non-overlapping old old (red) versus new (green) H3Rev histone patterns in mitotic telophase CBs marked by H3S10p (gray).
Figure 3(B-C) Non-overlapping patterns of old (green) versus new (red) histone H3 (B) and overlapping patterns for H2A (C) in mitotic prometaphase bam mutant cells marked by anti-H3S10p (gray), similar to WT GSCs. Scale bars: 2μm.(F-H) Examples showing old (green) versus new (red) H3 distribution patterns and percentage of signal strength at a single genomic locus labeled with fluorescent probes (gray) for dad gene (F), bgcn gene (G), and ss gene (H) in bam mutant GSC-like cells at prometaphase marked by anti-H3S10p (blue). Scale bars: 1μm. Zoomed-in images of each probe are displayed to the right of each figure panel. Scale bars: 0.1μm.
Figure 4(A-B) Old (red) versus new (green) histone H3 distribution patterns and percentage of signal strength with a single genomic locus labeled with fluorescent probes (cyan) for dad (A) and bam (B) in WT telophase female GSCs, marked by anti-H3S10p (gray). (C) Quantification of the probe association ratios with old versus new H3 for each candidate gene in WT female GSCs. Data gathered over 8 separate experiments. D
Figure 2B Body weight of Uqcrh -/- mice and wild-type littermates at 1-2 weeks (n = 5 to 10), during the post-weaning period from 5-6 weeks of age (n = 3 to 7) and weekly from 6 to 10 weeks (n = 13 to 66), data are shown as mean ± SD. **** p < 0.0001, 2-Way ANOVA, from 7.5wk of age for Uqcrh-/- males and females versus sex-matched controls. Data are shown as single data points and median, or categorical data, males and females pooled, since genotype-related effects were comparable for both sexes from different cohorts. * p < 0.05, *** p <0.001, Wilcoxon rank-sum test.C, D Clinical chemistry plasma concentrations of parameters related to acid-base balance (Lactate and Ammonia) and metabolic state (Glucose) n = 15 Uqcrh-/- and 16 WT pups 1-2-week-old (C); n = 15 f, 15 m Uqcrh-/- and 15 f,15 m WT mice aged 8-9 weeks (D); sex not differentiated. Data shown as scatter dot plots with line at median. * p < 0.05, ** p < 0.01, **** p < 0.0001, Wilcoxon rank sum test.F Measures related to behaviour and neurological function, data generated by the SHIRPA (9 weeks old, F), n = 7f, 8m Uqcrh-/- and 8f, 9m WT animals. Prepulse inhibition of the acoustic startle as a parameter associated with sensorimotor gating (10 weeks, E right panel), n = 7 f/5 m Uqcrh-/- and 8 f/ 9 m WT mice. Sex is not differentiated. Data are shown as scatter dot plots with a line representing the median. *** p < 0.001, **** p < 0.0001, Wilcoxon rank-sum test.G Parameters related to heart function generated by Echocardiography (ECHO) and Electrocardiography (ECG), n = 15 f, 15 m Uqcrh-/- and 15 f, 15 m WT mice, 6 weeks old.
Figure 3A Respiratory chain enzyme activities from human control (green) and patient (red) fibroblasts. Mean activities of controls (n = 8) are set to 100% and error bars represent 1 standard deviation. Data are normalised to citrate synthase (CS) activity. B Respiratory chain enzyme activities in heart, brain and liver, of wild-type and Uqcrh-/- mice. Data are normalised to citrate synthase (CS) activity. Values are given as mean ± SD, n = 4 WT, 5 Uqcrh-/- (Heart), 3 WT, 3 Uqcrh-/- (Brain), 1 WT and 2 Uqcrh-/- (Liver). * p < 0.05, **** p < 0.0001, 2-way ANOVA (Multiple comparison). C Western blot analysis of OXPHOS components in the patient (P) and control (n = 2, C1-2) fibroblasts. D BN-PAGE analysis of OXPHOS complex assembly in enriched mitochondria from patient and control fibroblasts (n = 2, C1-2) solubilised with DDM. Immunoblotting was performed using antibodies to a subunit of each complex (CI [NDUFB8], CII [SDHA], CIII [UQCRC2], CIV [MT-CO1], and CV [ATP5A]) F Immunohistochemical staining of UQCRC2 was performed on liver, heart and kidney derived from wild-type and Uqcrh-/- mice at 9 weeks of age. Staining of VDAC1/porin was also carried out G Graphs indicate score values of immunohistochemical staining intensity of both VDAC1/porin and UQCRC2. Data are given as mean ± SEM. * p < 0.05; n = 3, Student's t-test (unpaired samples).
Figure 4A Western blot analysis of control (n = 2, C1-2) and patient fibroblasts transduced with a lentiviral vector (pLVX) containing wild-type UQCRH. Expression of wild-type UQCRH was induced using various concentrations of doxycycline (dox) up to 20 ng/ml for 72 hours. B BN-PAGE analysis of control (n = 2, C1-2) and patient fibroblasts transduced with a lentiviral vector (pLVX) containing wild-type UQCRH. Transduced fibroblasts were either uninduced or induced with 20 ng/ml doxycycline (dox) for 72 hours. C Immunofluorescence for UQCRC2 (green), VDAC1/porin (red) and nuclei (DAPI, blue), in control (NHDF) fibroblasts. D Immunofluorescence for UQCRC2 (green), VDAC1/porin (red) and nuclei (DAPI, blue), in patient fibroblasts. E Immunofluorescence for UQCRC2 (green), VDAC1/porin (red) and nuclei (DAPI, blue), in control (NHDF) fibroblasts transduced with wild-type UQCRH. F Immunofluorescence for UQCRC2 (green), VDAC1/porin (red) and nuclei (DAPI, blue), in patient fibroblasts transduced with wild-type (WT) UQCRH. G Immunofluorescence for UQCRC2 (green), VDAC1/porin (red) and nuclei (DAPI, blue), in control (NHDF) fibroblasts transduced with the pseudogene UQCRHL. H Immunofluorescence for UQCRC2 (green), VDAC1/porin (red) and nuclei (DAPI, blue), in patient fibroblasts transduced with the pseudogene UQCRHL. I Graph indicates the staining intensity of UQCRC2 in control (NHDF) fibroblasts (green) and patient fibroblasts (red), those respective cell lines transduced (T) with wild-type UQCRH (diagonal lines) and those cell lines transduced with the pseudogene UQCRHL (vertical lines) measured using Image J, Data are given as mean ± SEM. One-way ANOVA (Kruskal-Wallis test), * p < 0.05; n = 3 macroscopic field (10-14 cells for macroscopic field were measured).
Figure 5Mitochondrial membranes from fibroblasts of control (WT) and patient fibroblasts (i, immortalised) were solubilised with digitonin and separated on native gradient gels. A BN-PAGE gel stained with Coomassie showing molecular weight marker (BHM) and samples from control and patient (primary fibroblasts, (I)=immortalised fibroblasts) B BN-PAGE gel stained with NADH:NTB reductase activity stain showing molecular weight marker (BHM) and samples from control and patient (primary fibroblasts, (I)=immortalised fibroblasts) C Further complexome analysis of the BN-PAGE gels shown in A and B. Each native lane was cut into even fractions from high to low molecular mass. Proteins in each slice were digested with trypsin and analysed by quantitative mass spectrometry. Shown here are average IBAQ values of all identified subunits from complexes III, I and IV D Profile of complex III (averaged IBAQ values) in the patient (dashed line) and control (solid line) fibroblasts. E Profile of complexes III (red), I (yellow) and IV (green) to visualise and assess supercomplex composition and stability in control (solid lines) and patient (dashed lines) fibroblasts. heart tissue from wild-type (WT) and Uqcrh-/- mice were solubilised with digitonin and separated on native gradient gels. F BN-PAGE gel stained with Coomassie (left) and NADH:NTB reductase activity stain (right) showing molecular weight marker (BHM) and heart tissue samples from wild-type (WT) and Uqcrh-/- mice. G BN-PAGE and immunoblot using the antibody to CIII subunit UQCRC2 in heart tissue samples from wild-type (WT) and Uqcrh-/- mice. H Further complexome analysis of the BN-PAGE gels shown in F, shown here are average IBAQ values of subunits from complexes III, I and IV I Profile of complex III (averaged IBAQ values) in wild-type (+/+, solid line) and Uqcrh-/- (-/-, dashed line) heart tissue. J Profile of complexes III (red), I (yellow) and IV (green) to visualise and assess supercomplex composition and stability in wild-type (+/+, solid line) and Uqcrh-/- (-/-, dashed line) heart tissue.
Figure 1B. Internalization (cytoplasm) and recycling (plasma membrane) of FGF7- and FGF10-stimulated endogenous FGFR2 (green) for 0, 15, 40, and 120 min. in T47D. TRITC-Tf is a marker of recycling (red). Nuclei are stained in blue. *, cells with receptor recycled to the plasma membrane. Scale bar, 5 μm. C. Red and green pixels overlap fraction representing the co-localization of FGFR2b with the recycling endosomes marker Tf (above) and the proportion of green over total pixels representing FGFR2b in the cytoplasm (below) upon stimulation with FGF7 (dark green) or FGF10 (burgundy) for 15, 40, or 120 min.. Values represent the median ± SD of at least 3 independent experiments. Representative pictures are shown in B (cytoplasm). *, p value<0.005 (Student's t-test). D H. Internalization (cytoplasm) and recycling (plasma membrane) of FGF10-stimulated HA-FGFR2b (green) transfected in BT549 stimulated for 0, 40 and 120 min.. Cells were depleted or not of TTP or RCP by a pool of siRNAs. TRITC-Tf is a marker of recycling (red). Nuclei are stained in blue. *, cells with receptor recycled to the plasma membrane. Scale bar, 5 μm.
Figure 2(J) control or 40 min. FGF10- or TGFα-stimulated T47D left untreated or depleted of TTP, followed or not by transfection with siRNA resistant Flag-TTP (Flag-TTP*) or depleted of RCP followed or not by transfection with siRNA resistant RCP-GFP (RCP-GFP*) were immunoblotted with the indicated antibodies. **, non-specific band (J).
Figure 3F, G Lysates from T47D or BT20 treated with DMSO, PD173074, AG1478, or the MEK inhibitors U0126 and MEK162 and stimulated or not with either FGF10 or TGFα (F); T47D or BT20 treated with DMSO, or the p38 inhibitor BMS582949 and stimulated or not with either FGF10 or TGFα (G) were immunoblotted with the indicated antibodies.
Figure 4Lysates from T47D stimulated or not with FGF10, EGF for different time periods or treated with PD173074 (C), MEK162 (D), AG1478 (E) before stimulation were immunoblotted with the indicated antibodies.G, H Percentage of EdU incorporation in T47D pre-treated or not with FGF10 for 40 min. and stimulated or not with FGF10 or EGF (G) or incubated with MEK162, PD173074, or AG1478 before pre-treatment (H). N= 6. P value = <0.05 *, <0.01 **, <0.001*** (one-way ANOVA with Tukey test).
Figure 5A. Co-localization of FGFR2 (red), EGFR (green), and the recycling marker Tf (blue) in T47D stimulated or not with FGF10 or TGFα for 40 min. Scale bar, 5 μm. C. Co-localization of EGFR (green), T693 phosphorylated EGFR (blue), and the recycling marker Tf (red) in T47D stimulated or not with FGF10 or TGFα for 40 min. Scale bar, 5 μm.E. Co-localization of FGFR2 (red), EGFR (green), and the recycling marker Tf (blue) in T47D depleted of EGFR by siRNA followed by transfection with wt or T693A and stimulated or not with either FGF10 or TGFα for the indicated time periods. Scale bar, 5 μm.
Figure 6E, F Lysates from T47D (E) and BT20 (F) depleted of EGFR, transfected with wt or T693A, and stimulated or not with either FGF10 or TGFα for the indicated time intervals were immunoblotted with the indicated antibodies.
Figure 7A-D Percentage of EdU incorporation in T47D (A, C) or BT20 (B, D) depleted of EGFR or not, transfected with wt or T693A, and stimulated or not with FGF10 (A, B) or with TGFα (C, D) and pre-treated (C, D) or not (A, B) with FGF10 for 40 min. Values represent the median ± SD of N= 4. P value =<0.05 *, <0.01 **, <0.001*** (one-way ANOVA with Tukey test).