Journal: Molecular medicine (Cambridge, Mass.)

Article Title: Targeting PYK2, entrectinib allays anterior subcapsular cataracts in mice by regulating TGFβ2 signaling pathway.

doi: 10.1186/s10020-024-00921-9

Figure Lengend Snippet: Fig. 3 Entrectinib retards the migration and proliferation of human lens epithelial cells. (A) Cells were exposed to different doses of Entrectinib(0–64µM). IC50 = 10.06µM. (B) The effects of Entrectinib (0.25, 0.5, 1, 2µM) on proliferation induced by TGFβ2(10ng/ml) were assessed by CCK8 assay. (C) The EdU assay illustrated the effects of Entrectinib(0.25, 0.5, 1, 2µM) on proliferation induced by TGFβ2(10ng/ml). The proportion of EdU-positive cells was quanti fied and statistically analyzed (Scale bar = 200 μm). (D) Cell migration was recorded at 0, 6, 12, and 24 h after administrated TGFβ2(10ng/ml) and different doses of Entrectinib (0.25, 0.5, 1, 2µM). Black straight lines showed the wound edges. The migration rates were quantified and statistically analyzed (Scale bar = 200 μm). (E) After being seeded for 24 h, cells that moved vertically and stuck to the polycarbonate membrane’s exterior were stained with crystal violet and recorded. The number of cells that migrated through the membrane was quantified and statistically analyzed (Scale bar = 200 μm). Data was expressed as means ± SD, n = 3. #P < 0.05, ##P < 0.01, ###P < 0.001 and ####P < 0.0001versus control group. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 versus TGFβ2 group

Article Snippet: Recombinant human TGFβ2 was purchased from MedChemExpress Co., ltd. (America).

Techniques: Migration, CCK-8 Assay, EdU Assay, Staining, Membrane, Control

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: Targeting PYK2, entrectinib allays anterior subcapsular cataracts in mice by regulating TGFβ2 signaling pathway.

doi: 10.1186/s10020-024-00921-9

Figure Lengend Snippet: Fig. 4 Entrectinib suppressed the EMT of the human lens epithelial cells. (A) Relative mRNA expression of α-SMA, collagen I, fibronectin, and E-cadherin were analyzed by quantitative real-time PCR. GAPDH served as a reference gene. (B-C) Western blot was applied to analyze the relative protein expres sion of α-SMA, collagen I, fibronectin, and E-cadherin in cells. GAPDH served as a reference protein. (D) Immunofluorescence staining of α-SMA (red) and nuclei (blue) (Scale bar = 50 μm). Data was expressed as means ± SD, n = 3. ####P < 0.0001 versus control group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus TGFβ2 group

Article Snippet: Recombinant human TGFβ2 was purchased from MedChemExpress Co., ltd. (America).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence, Staining, Control

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: Targeting PYK2, entrectinib allays anterior subcapsular cataracts in mice by regulating TGFβ2 signaling pathway.

doi: 10.1186/s10020-024-00921-9

Figure Lengend Snippet: Fig. 5 Entrectinib inhibits the activation of Smad and non-Smad signaling pathway induced by TGFβ2. (A-B) The phosphorylation levels of Smad2, Smad3, AKT, JNK, ERK, and P38 in cells treated by TGFβ2(10ng/ml) and different doses of Entrectinib (0.25, 0.5, 1, 2µM) were analyzed by western blot. GAPDH served as a reference protein. Data was expressed as means ± SD, n = 3. #P < 0.05, ##P < 0.01 and ####P < 0.0001 versus control group. **P < 0.01, ***P < 0.001 and ****P < 0.0001 versus TGFβ2 group

Article Snippet: Recombinant human TGFβ2 was purchased from MedChemExpress Co., ltd. (America).

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Control

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: Targeting PYK2, entrectinib allays anterior subcapsular cataracts in mice by regulating TGFβ2 signaling pathway.

doi: 10.1186/s10020-024-00921-9

Figure Lengend Snippet: Fig. 6 PYK2 is a potential target of Entrectinib. (A) The binding model between Entrectinib (pink) and the crucial residues of PYK2 (purple). (B) The bind ing affinity between PYK2 and Entrectinib was analyzed by Microscale thermophoresis (MST). (C) Cellular Thermal Shift Assay and Western blot were performed to evaluate the stability of PYK2 after incubation with or without Entrectinib at different temperatures. (D) Drug Affinity Responsive Target Sta bility and Western blot were performed to evaluate the resistance of PYK2 to enzymatic hydrolysis. (E) The phosphorylation levels of PYK2 in cells treated by TGFβ2(10ng/ml) and different doses of Entrectinib (0.25, 0.5, 1, 2µM) were analyzed by western blot. GAPDH served as a reference protein. Data was expressed as means ± SD, n = 3. ###P < 0.001 versus control group. ****P < 0.0001 versus TGFβ2 group

Article Snippet: Recombinant human TGFβ2 was purchased from MedChemExpress Co., ltd. (America).

Techniques: Binding Assay, Microscale Thermophoresis, Thermal Shift Assay, Western Blot, Incubation, Phospho-proteomics, Control

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: Targeting PYK2, entrectinib allays anterior subcapsular cataracts in mice by regulating TGFβ2 signaling pathway.

doi: 10.1186/s10020-024-00921-9

Figure Lengend Snippet: Fig. 7 The knockdown of PYK2 inhibits the TGFβ2-induced EMT through a non-Smad signaling pathway. (A) The knockdown efficiency of PYK2 was evaluated by western blotting. (B) The knockdown of PYK2 inhibited the EMT induced by TGFβ2. The efficacy of Entrectinib is influenced by PYK2 knock down. (C) PYK2 knockdown inhibited the activation of AKT, JNK, ERK, and P38. GAPDH served as a reference protein. Data was expressed as means ± SD, n = 3. #P < 0.05 and ##P < 0.01 versus the control group

Article Snippet: Recombinant human TGFβ2 was purchased from MedChemExpress Co., ltd. (America).

Techniques: Knockdown, Western Blot, Activation Assay, Control