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: International Journal of Biological Sciences

Article Title: Breast cancer-derived CAV1 promotes lung metastasis by regulating integrin α6β4 and the recruitment and polarization of tumor-associated neutrophils

doi: 10.7150/ijbs.94153

Figure Lengend Snippet: CAV1 Promotes BC Lung Metastasis by Activating the Src/FAK/α6β4 Pathway in MDA-MB-231 Cells and Simultaneously Activating Src/PI3K Signaling Downstream of α6β4 in Lung Epithelial Cells. a: CCK8 assay was used to detect the optimal acting concentration of the Src inhibitor PP2. b-e: Western blot analysis was performed to detect the expression of CAV1 and integrin Src/FAK/α6β4 signaling pathway after overexpression, knockdown, and addition of PP2. f: Western blot was used to detect the activation of integrin α6β4 downstream signaling in lung epithelial cells. g,h: IHC staining was used to detect the activation of integrin α6β4 downstream signaling PI3K/Src in mice lungs. Bar=100um. Data ware shown as mean ± SD and assessed with One-way ANOVA test. (n=3) (ns stands for non-significant difference; *p<0.05; **p<0.01; ***p<0.001).

Article Snippet: Caveolin1 Y14 phosphorylation was inhibited by the SRC family kinase inhibitor PP2, which was obtained from Abmole Bioscience Inc (Houston, USA).

Techniques: CCK-8 Assay, Concentration Assay, Western Blot, Expressing, Over Expression, Knockdown, Activation Assay, Immunohistochemistry

Journal: The journal of pain

Article Title: Microglial P2Y12 Signaling Contributes to Cisplatin-induced Pain Hypersensitivity via IL-18-mediated Central Sensitization in the Spinal Cord.

doi: 10.1016/j.jpain.2023.01.005

Figure Lengend Snippet: Figure 5. Spinal blockade of the P2Y12/Src family kinase (SFK)/p38 pathway suppresses the cisplatin-induced IL-18 production. (A) IL-18 immunoreactivity was colocalized with IBA-1, (B) but not with NeuN or GFAP in the spinal dorsal horn. (C) IL-18 immunoreac- tivity was colocalized with P2Y12, p-SFK, and p-p38 in the spinal dorsal horn. Tissues were harvested on day 15 post administration. Scale bars: 50 mm and 20 mm (zoom). (D−F) Real-time qPCR analyses showed that MRS2395 (D), PP2 (E) or SB239063 (F) significantly attenuated the cisplatin-induced upregulation of IL-18 mRNA expression in the spinal dorsal horn. (G−I) Western blotting analyses showed that MRS2395 (G), PP2 (H) or SB239063 (I) significantly attenuated the cisplatin-induced upregulation of IL-18 protein expression in the spinal dorsal horn. MRS2395 (2 mg, intrathecally), PP2 (10 mg, intrathecally), PP3 (10 mg, intrathecally) or SB239063 (10 mg, intrathecally) was injected daily once, on days 12, 13, 14, and 15 after the first cisplatin injection. Tissues were harvested 4 h following the last injection on day 15. Data are shown as the mean § SEM. **P < .01, in comparison with the saline + DMSO (or PP3) group; ##P < .01, compared with the cisplatin + DMSO (or PP3) group, n = 4 per group, one-way ANOVA with the post hoc Dunnett test. Abbreviations: ANOVA, analysis of variance; DMSO, dimethyl sulfoxide; GFAP, glial fibrillary acidic protein; IBA-1, ionized cal- cium-binding adapter molecule 1; IL-18, interleukin-18; NeuN, neuronal nuclei; qPCR, quantitative polymerase chain reaction; SEM, standard error of the mean; SFK, Src family kinase.TagedEnd

Article Snippet: Src family kinase inhibitor PP2 (S7008) and P38 inhibitor SB239063 (S7741) were obtained from Selleck Chemicals (China).

Techniques: Expressing, Western Blot, Injection, Comparison, Saline, Binding Assay, Real-time Polymerase Chain Reaction

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Trogocytosis Results in Sustained Intracellular Signaling in CD4 + T Cells

doi: 10.4049/jimmunol.1201507

Figure Lengend Snippet: TCR-proximal signaling is sustained by trogocytosed molecules in trog+ cells. (A) Top panel, T cells were incubated for 30 min after recovery from standard trogocytosis assay before fixation and staining. In untreated cells, pZAP-70 (red) and the TCR (blue) colocalize with the trogocytosed MHC:peptide on the T cell surface. Bottom panel, The TCR and MHC:peptide colocalize on T cells treated with the Src inhibitor PP2 following the 30-min incubation period, but the there is minimal phospho-ZAP-70 detected. Middle panel, For cells with a 10-min PP2 treatment to extinguish signaling and before incubation for 20 min after PP2 removal, the pZAP-70 level rebounds and it colocalizes with the trogocytosed MHC:peptide and TCR. More than 100 individual T cell were imaged using a ×60 objective. Left image for each group is a fluorescence composite image to show the position of the T cell. Scale bars, 10 μm. (B) Integrated intensity of pZAP70 ≥6-fold above background in images as in (A) (top panels). Thirty-minute untreated T cells (green bar), T cells incubated with PP2 for 30 min (orange bar), or for treated for 10 min then incubated for 20 min after removal of PP2 (purple bar) are shown. (C) The frequency of pZAP-70 colocalizing with trogocytosed MHC:peptide for each treatment group is shown. (D) The frequency of TCR colocalizing with the TCR. Bars represent mean ± SEM. Horizontal lines indicate statistical comparison between indicated groups, *p ≤ 0.05.

Article Snippet: Cells incubated at very low density (10 4 /ml) were treated for 10 or 30 min with Src family tyrosine kinase inhibitor PP2 (Life Technologies) ( 61 ).

Techniques: Incubation, Trogocytosis Assay, Staining, Fluorescence