Journal: PLOS Genetics

Article Title: Fibroblast mechanoperception instructs pulmonary developmental and pattern specification gene expression programs

doi: 10.1371/journal.pgen.1011924

Figure Lengend Snippet: ( A ) Western blots for HOXA5 in WT and Thy-1 KO iHLFs cultured on 5kPa PDMS hydrogel or TCP following stimulation with 20μM SRC specific inhibitor KB SRC 4 or 10μM pan-SFK inhibitor PP2 for 6h. Quantification of HOXA5 normalized to loading control was plotted as a ratio of normalized signal relative to DMSO vehicle control. Mean ± S.D. plotted; N = 3 (Thy-1 KO -5kPa and WT-1GPa) or N = 4 (WT-5kPa and Thy-1 KO -1GPa) independent experiments; WT-5kPa and WT-1GPa = 1-way ANOVA with post-hoc uncorrected Fisher’s LSD test, Thy-1 KO -5kPa and Thy-1 KO -1GPa = non-parametric Kruskal-Wallis test with post-hoc uncorrected Dunn’s test. Tests chosen based on data distribution and variance. ( B ) Western blots for HOXA5 in WT and Thy-1 KO iHLFs cultured on 5kPa PDMS hydrogel or TCP substrates coated with αV-promoting (Fn 4G ) or α5-promoting (Fn 9*10 ) peptide fragments of the 9–10FnIII integrin binding site. Quantification of HOXA5 normalized to loading control was plotted as a ratio of normalized signal relative to full length human Fn (Fn Full ) control. Mean ± S.D. plotted; N = 3 (WT-5kPa, WT-1GPa, and Thy-1 KO -5kPa) or N = 4 (Thy-1 KO -1GPa) independent experiments; WT-5kPa = 1-way ANOVA with post-hoc uncorrected Fisher’s LSD test, Thy-1 KO -5kPa = Brown-Forsythe and Welch ANOVA with post-hoc unpaired t-tests with Welch’s correction, WT-1GPa and Thy-1 KO -1GPa = non-parametric Kruskal-Wallis test with post-hoc uncorrected Dunn’s test. Tests chosen based on data distribution and variance. For all statistical tests: ns = p > 0.05; p < 0.05 (*); p < 0.01 (**); p < .001 (***); p < .0001 (****).

Article Snippet: After allowing for adhesion, cells were stimulated with 1% FBS DMEM containing 20μM SRC inhibitor KB SRC 4 (Tocris) or 10μM pan-Src Family Kinase (SFK) inhibitor PP2 (Tocris) for periods of 3h or 6h.

Techniques: Western Blot, Cell Culture, Control, Binding Assay

Journal: bioRxiv

Article Title: Fibroblast mechanoperception instructs pulmonary developmental and pattern specification gene expression programs

doi: 10.1101/2024.12.26.630418

Figure Lengend Snippet: (A) Western blots for HOXA5 in WT and Thy-1 KO iHLFs cultured on 5kPa PDMS hydrogel or TCP following stimulation with 20μM SRC specific inhibitor KB SRC 4 or 10μM pan-SFK inhibitor PP2 for 6hr. Quantification of HOXA5 normalized to loading control was plotted as a ratio of normalized signal relative to DMSO vehicle control. Mean ± S.D. plotted; N=3 (Thy-1 KO -5kPa and WT-1GPa) or N=4 (WT-5kPa and Thy-1 KO -1GPa) independent experiments; WT-5kPa and WT-1GPa = 1-way ANOVA with post-hoc uncorrected Fisher’s LSD test, Thy-1 KO -5kPa and Thy-1 KO -1GPa = non-parametric Kruskal-Wallis test with post-hoc uncorrected Dunn’s test. Tests chosen based on data distribution and variance. (B) Western blots for HOXA5 in WT and Thy-1 KO iHLFs cultured on 5kPa PDMS hydrogel or TCP substrates coated with αV-promoting (Fn 4G ) or α5-promoting (Fn 9*10 ) peptide fragments of the 9-10FnIII integrin binding site. Quantification of HOXA5 normalized to loading control was plotted as a ratio of normalized signal relative to full length human Fn (Fn Full ) control. Mean ± S.D. plotted; N=3 (WT-5kPa, WT-1GPa, and Thy-1 KO -5kPa) or N=4 (Thy-1 KO -1GPa) independent experiments; WT-5kPa = 1-way ANOVA with post-hoc uncorrected Fisher’s LSD test, Thy-1 KO -5kPa = Brown-Forsythe and Welch ANOVA with post-hoc unpaired t-tests with Welch’s correction, WT-1GPa and Thy-1 KO -1GPa = non-parametric Kruskal-Wallis test with post-hoc uncorrected Dunn’s test. Tests chosen based on data distribution and variance. For all statistical tests: ns = p > 0.05; p < 0.05 (*); p < 0.01 (**); p < .001 (***); p < .0001 (****).

Article Snippet: After allowing for adhesion, cells were stimulated with 1% FBS DMEM containing 20μM SRC inhibitor KB SRC 4 (Tocris) or 10μM pan-Src Family Kinase (SFK) inhibitor PP2 (Tocris) for periods of 3hr or 6hr, respectively.

Techniques: Western Blot, Cell Culture, Control, Binding Assay

Journal: bioRxiv

Article Title: Targeting NRP1 with EG00229 induces neurovascular permeability

doi: 10.1101/2023.11.07.564946

Figure Lengend Snippet: (A) Quantification of sulforhodamine B fluorescence changes in the ex vivo retina permeability assay after treatment with SU1498, SB203580 or PP2 (inhibitors) alone, compared to VEGF164 or EG00229 treatement with or without inhibitor pre-treatment (n = 3 treatments of 3 independent retinas each). Data are shown as mean ± SD; each data point represents the value for one retina from one mouse. Significant P-values are shown for inhibitor versus baseline (asterisks) and EG00229 ± inhibitor (hashtags): *** P, < 0.001; ###, P < 0.001; ns, not significant (P > 0.05). Repeated measures two-way ANOVA. ( B,C ) Freshly dissected retinae from wild type mice were incubated in Krebs solution (vehicle) with or without EG00229, or preincubated with SB203580 or PP2 for 15 min before adding EG00229. Ex vivo retinae were then fixed and immunostained with the vascular EC marker isolectin B4 (IB4, green) and an antibody against P-p38 (magenta). Epifluorescent images ( B , scale bars: 10 μm) were used for quantification of pixel intensity for P-p38 in the IB4-positive vascular area ( C ). Significant P-values for P-p38 staining in the IB4+ area for inhibitors (SB203580 or PP2 versus SB203580) versus EG00229 are indicated with asterisks or after pre-treatment with the inhibitors, indicated with hashtags: *, P < 0.05; ***, P < 0.001; ###, P < 0.001; ns, not significant (P > 0.05). One-way ANOVA.

Article Snippet: The NRP1 inhibitor EG00229, the VEGFR2 inhibitor SU1498, the P38 inhibitor SB203580 and the SFK inhibitor PP2 were all purchased from MERCK (Dorset, United Kingdom) and used at a concentration of 10 μM unless otherwise specified.

Techniques: Fluorescence, Ex Vivo, Permeability, Incubation, Marker, Staining

Journal: bioRxiv

Article Title: Targeting NRP1 with EG00229 induces neurovascular permeability

doi: 10.1101/2023.11.07.564946

Figure Lengend Snippet: (A) Schematic of the transendothelial flux assay with brain microvascular ECs. Primary rat brain microvascular ECs were grown on permeable transwell inserts until they reached a TEER greater than 200 Ω/cm2. 4 kDa FITC dextran was then added to the apical chamber. Baseline FITC flux was recorded as the time-dependent accumulation of fluorescence in the basal chamber before adding EG00229 or VEGF164 and continued recording. ( B,C ) EG00229 increases the permeability of brain microvascular EC monolayers. Linear regression analysis of flux over time before and after adding VEGF164 or EG00229, defined as time 0. ( B ) Data are shown as mean ± SD flux change from 4 independent experiments over time. ( C ) Data are also shown as mean fold change of flux following EG00229 or VEGF164 treatment in comparison to the baseline. Asterisks indicate statistical significance (one-way ANOVA): **, P < 0.01; ***, P < 0.001; ns, not significant (P > 0.05). ( D ) EG00229 activates p38 and SFK in human ECs. Confluent hCMEC/D3 were treated with EG00229 for the indicated times. Cell lysates were used for immunoblotting with the indicated antibodies, followed by quantification of pixel intensities for P-P38 and P-SFK relative to total P38 and SRC pixel intensities, respectively. GAPDH was used as a loading control. Data are shown as mean fold change ± SD. P38 and SFK phosphorylation levels after EG0229 treatment were quantified at different time points relative to 0 min. Asterisks indicate significant P-values for phosphorylation induction after EG00229 treatment (one-way ANOVA): *, P < 0.05; **, P < 0.01; ns, not significant (P > 0.05).

Article Snippet: The NRP1 inhibitor EG00229, the VEGFR2 inhibitor SU1498, the P38 inhibitor SB203580 and the SFK inhibitor PP2 were all purchased from MERCK (Dorset, United Kingdom) and used at a concentration of 10 μM unless otherwise specified.

Techniques: Flux Assay, Fluorescence, Permeability, Comparison, Western Blot

Journal: bioRxiv

Article Title: Targeting NRP1 with EG00229 induces neurovascular permeability

doi: 10.1101/2023.11.07.564946

Figure Lengend Snippet: Confluent primary rat brain ECs were treated with EG00229 for the indicated times. Cell lysates were used for immunoblotting with the indicated antibodies, followed by quantification of pixel intensities for P-P38 and P-SFK relative to total P38 and SRC pixel intensities, respectively. GAPDH was used as a loading control. In ( A ), p38 and SFK phosphorylation levels after EG0229 treatment were quantified at different time points relative to 0 min. In ( B ), EG00229 treatment was preceded by incubation with the SFK inhibitor PP2 an EG00229-induced p38 phosphorylation after PP2 treatment was compared to EG00229 alone at each time point. Data are shown as mean fold change ± SD. Asterisks indicate significant P-values for phosphorylation induction after EG00229 treatment (one-way ANOVA): *, P < 0.05; **, P < 0.01; ns, not significant (P > 0.05).

Article Snippet: The NRP1 inhibitor EG00229, the VEGFR2 inhibitor SU1498, the P38 inhibitor SB203580 and the SFK inhibitor PP2 were all purchased from MERCK (Dorset, United Kingdom) and used at a concentration of 10 μM unless otherwise specified.

Techniques: Western Blot, Incubation

Journal: bioRxiv

Article Title: Targeting NRP1 with EG00229 induces neurovascular permeability

doi: 10.1101/2023.11.07.564946

Figure Lengend Snippet: VEGF164 binds to VEGFR2 and NRP1 to induce complex formation, whereas EG00229 binds to NRP1 alone and impairs its interaction with VEGF164. VEGF164 or EG00229 binding to their receptors activates SFK and downstream p38 signalling, which in turn induces CDH5 rearrangements and opening of adherens junctions. Created with BioRender.

Article Snippet: The NRP1 inhibitor EG00229, the VEGFR2 inhibitor SU1498, the P38 inhibitor SB203580 and the SFK inhibitor PP2 were all purchased from MERCK (Dorset, United Kingdom) and used at a concentration of 10 μM unless otherwise specified.

Techniques: Binding Assay

Journal: Science Advances

Article Title: Piezo1 activates noncanonical EGFR endocytosis and signaling

doi: 10.1126/sciadv.adi1328

Figure Lengend Snippet: ( A and D ) Schematic of the experimental setups. ( B , E , and H ) Representative maximum intensity projections of pY1173-EGFR (B and E) or pSer1046/1047-EGFR (H) stainings in HeLa cells treated for 15 min with vehicle, 10 μM Yoda1, or EGF (10 ng/ml) with or without 30-min preincubations with 200 nM PP2 (SFK inhibitor, B), or 10 μM SB202190 (p38 inhibitor, E). ( C and F ) Counts of pY1173-EGFR puncta per cell after the indicated treatments. Each symbol represents a cell, with n : vehicle, 1143; Yoda1, 717; EGF, 32; PP2, 1694; Yoda1 + PP2, 887; and EGF + PP2, 829; six experiments (C); vehicle, 867; Yoda1, 1125; EGF, 1544; SB, 948; Yoda1 + SB, 1358; and EGF + SB, 1016; three experiments (F). ( G ) Representative Western blots of pS1046/1047-EGFR. Vinculin used as loading control. ( I ) Counts of pSer1046/1047-EGFR puncta per cell after the indicated treatments. Each symbol represents an independent experiment ( N = 4), with ≥20 cells per picture and n = 4 pictures per experiment. ( J ) Manders colocalization coefficient for pSer1046/1047-EGFR and EEA1. Each symbol represents a picture ( n for each = vehicle, 13; Yoda1, 14; and EGF, 12), from N ≥ 4 independent experiments with ≥20 cells per picture. Scale bars, 20 μm. Error bars = median ± interquartile range. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 one-way ANOVA followed by Kruskal-Wallis post hoc test with Dunn’s correction for multiple comparisons.

Article Snippet: Experiments involving inhibitors included a 30-min pretreatment with EGFR kinase inhibitor 1 μM PD153035 (Sigma-Aldrich, SML0564), SFK inhibitor 200 nM PP2 (Sigma-Aldrich, P0042), or p38 inhibitor 10 μM SB202190 (Abcam, ab120638), all reconstituted in DMSO.

Techniques: Western Blot

Journal: Frontiers in Immunology

Article Title: Integrated signaling and transcriptome analysis reveals Src family kinase individualities and novel pathways controlled by their constitutive activity

doi: 10.3389/fimmu.2023.1224520

Figure Lengend Snippet: SFK overexpression suffices to drive ligation-independent positive and negative signaling pathways. (A) Lck-expressing cells have more robust signaling responses on a whole cell population basis. Resting Lck- or Lyn-expressing cells were stained for pCD79a, pSyk (Y525/526) and pAKT (Ser473) and analyzed by FACS. Graphs display the frequency of cells double-positive for GFP and pCD79a, pSyk and pAKT respectively (Gating as in Figure 1A , % of cells in the upper right quadrants) (n≥4, Unpaired Student t test; mean +/- standard deviation [SD]; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant). (B) Lyn is more efficient than Lck in igniting phosphporylation of BCR signaling mediators on an equal protein expression level. Same as in (A) but for equivalent levels of SFK expression ( Figure S3E for description of equal GFP gating strategy). (C) Enhanced CD22 tyrosine phosphorylation in response to SFK overexpression. CD22 was immunoprecipitated from the Lck- and Lyn-BJAB cell lines and their -Dox counterparts in the absence or presence of stimulation with 10 µg/mL a-IgM for 10min. IPs were analyzed by western blotting with a-pY 4G10 (upper panel) and a-CD22 (lower panel) antibodies. HEK293T cells (devoid of CD22 expression) were used as a negative control for the IPs. (D) SFK overexpression ignites the CD22/SHP-1 inhibitory pathway and this depends on their intact kinase activity. Left-hand panels. CD22 IPs as in C, but with additional samples treated for 10 min with 100 µM of the pan-SFK inhibitor PP2, as indicated. IPs were analyzed by western blotting with a-pSHP-1 Y564 (upper panel), total a-SHP-1 (middle panel) and a-CD22 (lower panel) antibodies. Right-hand panels. Total cell lysates from the same experiment, analyzed by western blotting with a-pSHP-1 Y564 (upper panel) and a-pY416 (middle panel) to verify the PP2-induced reduction in SFK activity. Actin blot (lower panel) attests equal sample loading. Shown are representative blots of two independent experiments.

Article Snippet: The pan-SFK inhibitor PP2 (Calbiochem) was used at 30 μM for 18h in the humidified incubator (for treatment of samples stained with ER tracker) and at 100 μM for 10 min at 37°C (for transient SFK inhibition).

Techniques: Over Expression, Ligation, Protein-Protein interactions, Expressing, Staining, Standard Deviation, Phospho-proteomics, Immunoprecipitation, Western Blot, Negative Control, Activity Assay

Journal: Frontiers in Immunology

Article Title: Integrated signaling and transcriptome analysis reveals Src family kinase individualities and novel pathways controlled by their constitutive activity

doi: 10.3389/fimmu.2023.1224520

Figure Lengend Snippet: Role of SFKs in the regulation of ER homeostasis. (A) SFK overexpression suffices to induce ER expansion in an enzymatic activity-related fashion. Lck- or Lyn-expressing cells and the -Dox counterparts were stained with ER tracker Blue-White DPX at the indicated time points after Dox addition to the cell cultures and analyzed by FACS. ER expansion measured by increases of ER tracker dye fluorescence (lower panel histograms) is correlated to increases in SFK protein levels (GFP fluorescence, upper panel histograms). 18h incubation with 30μM of the pan SFK inhibitor PP2 reduced ER expansion to baseline (-Dox) levels. The correspondent reduction of pY416 after PP2 incubation for each SFK is shown in the bottom-panel histograms. Treatment with 50 µM of the ER-stress inducer Chloroquine (CQ) for 12h was used a positive control for the expected function of the ER-tracker reagent. Graph shows the ratio of ER tracker dye MFI from Lck- or Lyn-expressing cells to -Dox treated cells. Since essentially there was no difference between the Lck and Lyn samples, statistics were calculated for the mean values between each time point (n=4, Unpaired Student t test; mean +/- SD; **P < 0.01, ***P < 0.001; ns, not significant). (B) SFK-driven ER expansion is not concurrent with upregulation of UPR mediators. RT-qPCR of the indicated UPR modulators. Each time point corresponds to samples stained with ER tracker shown in (A) RT-qPCR data are shown log 2 FC of the gene expression between Lck-BJAB (black)/-Dox and Lyn-BJAB (grey)/-Dox (n=2, Multiple unpaired Student t test; mean +/- SD; *P < 0.05, **P < 0.01, ***P < 0.001; ns: not significant). (C) SFK overexpression suffices to induce FAM134B oligomerization in an enzymatic activity-related fashion. Lck- or Lyn-expressing cells and their -Dox counterparts were mixed at a ratio of 1:1, immobilized on poly-D-lysine coated microscope slides, stained for FAM134B (white), and visualized by confocal microscopy. GFP (green) denotes the SFK-expressing cells. Sample sets include: i) untreated cells (left-hand side panels) ii) SFK-expressing cells treated with 30μM PP2 for 18h, mixed with untreated -Dox cells (right-hand side panels) to provide a comparative visualization of SFK activity influence on the cargo receptor clustering and iii) -Dox cells treated with 50 µM CQ for 12h (indicative image shown in lower panels of D, below). FAM134B oligomers within single cells, appearing as distinctive puncta, were quantified for each sample. Collective quantitation results from three independent experiments (for the untreated samples) and two independent experiments (for PP2 and CQ treatments) are displayed on the adjacent graph. Number of cells for each sample -Dox/untreated n=239, -Dox/CQ n=79, Lck-BJAB/untreated n=164, Lck-BJAB/PP2 n=98, Lyn-BJAB/untreated n=144, Lyn-BJAB/PP2 n=84. Scale bar, 5 µm (n=2, Unpaired Student t test; mean +/- standard deviation [SD]; ****P < 0.0001). (D) SFK-driven FAM134B oligomerization does not coincide with LC3 colocalization. Lck- or Lyn-expressing cells and their -Dox counterparts mixed at a ratio of 1:1 (upper panels), or -Dox cells treated with 50 µM CQ for 12h (lower panels) were immobilized on poly-D-lysine coated microscope slides, stained for FAM134B (white) and LC3B (red), and visualized by confocal microscopy. GFP (green) denotes the SFK-expressing cells. Colocalization of FAM134B and LC3 was quantified by the coloc2 pre-installed plugin of Image (J) Collective colocalization analysis results from two independent experiments are displayed on the adjacent graph. Number of cells for each sample -Dox/untreated n=116, -Dox/CQ n=116, Lck-BJAB/untreated n=76, Lyn-BJAB/untreated n=73. Scale bar, 5 µm (n=2, Unpaired Student t test; mean +/- standard deviation [SD]; ****P < 0.0001).

Article Snippet: The pan-SFK inhibitor PP2 (Calbiochem) was used at 30 μM for 18h in the humidified incubator (for treatment of samples stained with ER tracker) and at 100 μM for 10 min at 37°C (for transient SFK inhibition).

Techniques: Over Expression, Activity Assay, Expressing, Staining, Fluorescence, Incubation, Positive Control, Quantitative RT-PCR, Gene Expression, Microscopy, Confocal Microscopy, Quantitation Assay, Standard Deviation