atp competitive fak pyk2 inhibitor vs 6063  (Selleck Chemicals)

Journal: Cell Communication and Signaling : CCS

Article Title: Integrin αVβ1-activated PYK2 promotes the progression of non-small-cell lung cancer via the STAT3-VGF axis

doi: 10.1186/s12964-024-01639-1

Figure Lengend Snippet: PYK2 and p -PYK2(Tyr402) are highly expressed in NSCLC tissues and PTK2B mRNA is associated with a poor prognosis. A PYK2 protein expression level of NSCLC tissues is higher than paired normal lung tissues. B IHC indicate the higher expression of p -PYK2(Tyr402) in NSCLC tumor than adjacent nontumor tissue, and higher expression in later TNM stage than earlier TNM stage. Scale bar, 100 μm. C PTK2B expression level is negatively related with the overall survival of NSCLC patients. The expression and prognostic data were downloaded from the TCGA database. D-E PTK2B mRNA and PYK2, p -PYK2(Tyr402) protein levels in NSCLC cell lines. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001

Article Snippet: The first group received intraperitoneal injections of the PYK2 inhibitor PF4618433 (HY-18,312, MCE, USA; 2 mg/kg), and the second group received the STAT3 inhibitor Stattic (HY-13,818, MCE, USA; 2 mg/kg) [ ] once every two days until the end of the experimental period.

Techniques: Expressing

Journal: Cell Communication and Signaling : CCS

Article Title: Integrin αVβ1-activated PYK2 promotes the progression of non-small-cell lung cancer via the STAT3-VGF axis

doi: 10.1186/s12964-024-01639-1

Figure Lengend Snippet: Inhibition of NSCLC cell proliferation and cell cycle G1-S arrest by knockdown of PYK2. A-B PYK2 mRNA and protein levels were knockdown in A549 and H460 sh-PYK2 cells. C-D CCK-8 assay was used to detect cell viability in A549 and H460 cells; cell viability was determined at 24, 48, and 72 h. PYK2-knockdown inhibited growth of A549 and H460 cells. E PYK2-knockdown inhibited the clonogenic ability of A549 and H460 cells. F-G EdU further confirmed that PYK2 knockdown inhibited cell proliferation. Scale bar, 200 μm. H-I Knockdown of PYK2 caused cell cycle G1-S arrest of A549 and H460 cells. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001

Article Snippet: The first group received intraperitoneal injections of the PYK2 inhibitor PF4618433 (HY-18,312, MCE, USA; 2 mg/kg), and the second group received the STAT3 inhibitor Stattic (HY-13,818, MCE, USA; 2 mg/kg) [ ] once every two days until the end of the experimental period.

Techniques: Inhibition, Knockdown, CCK-8 Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Integrin αVβ1-activated PYK2 promotes the progression of non-small-cell lung cancer via the STAT3-VGF axis

doi: 10.1186/s12964-024-01639-1

Figure Lengend Snippet: PYK2-overexpression promoted the proliferation of H1299 cells, knockdown of PYK2 attenuates tumor growth in a murine xenograft model. A-B PYK2 mRNA and protein levels increased in H1299 PYK2 overexpressed cells. C CCK-8 assay to detect cell viability in H1299 cells; PYK2-overexpression promoted the clonogenic ability of H1299 cells. D EdU further confirmed that PYK2 overexpression enhanced cell proliferation. Scale bar, 200 μm. E Overexpression of PYK2 caused cell cycle G1 stage transfer to S stage in H1299 cells. F PYK2-knockdown in A549 cells xenografts in nude mice ( n = 6) at the experimental endpoint. Tumors were dissected and photographed as shown. G Tumor growth curves in mice ( n = 6 in each group). H Each tumor formed was weighted. I-J WB and IHC detected p -PYK2 expression and the total PYK2 expression in tumor cell. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001

Article Snippet: The first group received intraperitoneal injections of the PYK2 inhibitor PF4618433 (HY-18,312, MCE, USA; 2 mg/kg), and the second group received the STAT3 inhibitor Stattic (HY-13,818, MCE, USA; 2 mg/kg) [ ] once every two days until the end of the experimental period.

Techniques: Over Expression, Knockdown, CCK-8 Assay, Expressing

Journal: Cell Communication and Signaling : CCS

Article Title: Integrin αVβ1-activated PYK2 promotes the progression of non-small-cell lung cancer via the STAT3-VGF axis

doi: 10.1186/s12964-024-01639-1

Figure Lengend Snippet: Knockdown of Integrin αVβ1 could inhibit the tyrosine 402 site phosphorylation of PYK2. A Transient transfection of siRNAs of Integrin α3, α5, αV, α6, and β1 to screen which one could affect the activation of PYK2 and WB to detect p -PYK2(tyr402) in knockdown of each Integrin in A549 cells; B CO-IP demonstrated the interaction of PYK2 and Integrin αV, β1. C-D Colocalization of PYK2 and Integrin αV, β1 (PYK2, SANTACRUZ, sc-393,181, Mouse for A and PYK2, abcam, ab32571, Rabbit for B) by IF. Scale bar, 20 μm

Article Snippet: The first group received intraperitoneal injections of the PYK2 inhibitor PF4618433 (HY-18,312, MCE, USA; 2 mg/kg), and the second group received the STAT3 inhibitor Stattic (HY-13,818, MCE, USA; 2 mg/kg) [ ] once every two days until the end of the experimental period.

Techniques: Knockdown, Transfection, Activation Assay, Co-Immunoprecipitation Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Integrin αVβ1-activated PYK2 promotes the progression of non-small-cell lung cancer via the STAT3-VGF axis

doi: 10.1186/s12964-024-01639-1

Figure Lengend Snippet: p -PYK2(Tyr402) interacts with STAT3 and can regulate the tyrosine 705 phosphorylation of STAT3 and p -STAT3(Tyr705) nuclear accumulation. A GSEA analysis showed a positive relation between PYK2 and the JAK-STAT signaling pathway. B Level of total STAT3 and p -STAT3(Tyr705) in A549, H460 stable PYK2 knockdown stable cell lines and H1299 stable PYK2 overexpressed cell line. C p -PYK2(Tyr402) and STAT3 colocalized in A549 and H460 cells. D Coimmunoprecipitation assay of exogenously expressed PYK2 and STAT3 plasmid in HEK293T cell demonstrated the interaction of these two proteins. E p -PYK2(Tyr402) and STAT3 could be directly bound to each other. F-G Nucleocytoplasmic separation and immunofluorescence experiments showed more p -STAT3(Tyr705) nuclear accumulation in PYK2 overexpressed compared to the vector

Article Snippet: The first group received intraperitoneal injections of the PYK2 inhibitor PF4618433 (HY-18,312, MCE, USA; 2 mg/kg), and the second group received the STAT3 inhibitor Stattic (HY-13,818, MCE, USA; 2 mg/kg) [ ] once every two days until the end of the experimental period.

Techniques: Knockdown, Stable Transfection, Co-Immunoprecipitation Assay, Plasmid Preparation, Immunofluorescence

Journal: Cell Communication and Signaling : CCS

Article Title: Integrin αVβ1-activated PYK2 promotes the progression of non-small-cell lung cancer via the STAT3-VGF axis

doi: 10.1186/s12964-024-01639-1

Figure Lengend Snippet: PYK2 regulates VGF expression through p -STAT3(Tyr705). A VGF in volcano plot of RNA sequencing based on H1299 PYK2 overexpressed cells compared to vector. B VGF mRNA reduced in two stable knockdown cell lines. C IHC of VGF; its scores were higher in tumor than paired adjacent normal lung tissue; higher in advanced TNM stage than earlier stage; IHC scores of p -PYK2(Tyr402) and VGF have a linear correlation. Scale bar, 100 μm. D Serum VGF of healthy control was lower than that in NSCLC patients. E Negative effect of VGF expression on overall survival prognosis of NSCLC patients from TCGA database. F Treatment with the p -PYK2(Tyr402) inhibitor PF4618433 20µMand p -STAT3(Tyr705) inhibitor Stattic 5µM reversed the increase in VGF mRNA. G The luciferase assay confirmed STAT3 transcriptionally regulated VGF. H Protein of VGF in stable knockdown cells and H1299 PYK2 OE cell lines with PF4618433 20µM and Stattic 5µM treated or not. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001

Article Snippet: The first group received intraperitoneal injections of the PYK2 inhibitor PF4618433 (HY-18,312, MCE, USA; 2 mg/kg), and the second group received the STAT3 inhibitor Stattic (HY-13,818, MCE, USA; 2 mg/kg) [ ] once every two days until the end of the experimental period.

Techniques: Expressing, RNA Sequencing Assay, Plasmid Preparation, Knockdown, Control, Luciferase

Journal: Cell Communication and Signaling : CCS

Article Title: Integrin αVβ1-activated PYK2 promotes the progression of non-small-cell lung cancer via the STAT3-VGF axis

doi: 10.1186/s12964-024-01639-1

Figure Lengend Snippet: PYK2 inhibitor PF4618433 and STAT3 inhibitor Stattic reversed the enhancing effect of PYK2-overexpressing for xenograft subcutaneous tumor growth in vivo. A-B The rescue phenotypic experiments with Stattic 5µM attenuated the pro-cancer effect of PYK2 overexpression in CCK-8 and colony formation. C Flow chart of this animal experiment. D-F Tumor photos and growth curves, tumor weight at execution of vector, PYK2 OE, PYK2 OE + PF4618433, PYK2 OE + Stattic. G WB of each group for PYK2, p -PYK2(Tyr402), STAT3, p -STAT3(Tyr705) and VGF. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001

Article Snippet: The first group received intraperitoneal injections of the PYK2 inhibitor PF4618433 (HY-18,312, MCE, USA; 2 mg/kg), and the second group received the STAT3 inhibitor Stattic (HY-13,818, MCE, USA; 2 mg/kg) [ ] once every two days until the end of the experimental period.

Techniques: In Vivo, Over Expression, CCK-8 Assay, Plasmid Preparation

Journal: Journal of neuroinflammation

Article Title: Enhanced phagocytosis associated with multinucleated microglia via Pyk2 inhibition in an acute β-amyloid infusion model.

doi: 10.1186/s12974-024-03192-7

Figure Lengend Snippet: Fig. 1 Inhibition of Pyk2 signaling induces multinucleation formation in microglia. A Morphological changes between mononuclear and multinucleated microglia. Immunocytochemistry of MG6 cells stained with anti-Iba1 (green) and anti-β-tubulin (magenta). Nuclear DNA was labeled with DAPI (white). Scale bar is 20 μm. B Cells were treated with Pyk2-Inh for 24 h. Phase contrast images (upper panel) and DAPI stained images (lower panel) were shown. Circles are indicated as multinucleated cells. C The quantification of the number of multinucleated cells/well (control: n = 4, Pyk2-Inh-500 nM: n = 4, Pyk2-Inh-1000 nM). D Cell proliferation was assessed by CCK-8. MG6 cells were treated with 100 μg/mL CSF1 and indicated concentration of Pyk2-Inh (n = 6 per group, One-way ANOVA, p < 0.05). E Representatives immunoblot images. The ratio of p-Pyk2/ Pyk2, pFAK/FAK, p-ERK/ERK, p-Src/Src and Mannose receptor/Tubulin were described under the blot. Pyk2-Inh was administered as a pre-treatment two hours before stimulation with CSF1 (100 μg/mL). F Image quantification and analysis were performed. The same experiments were repeated three times. All full-length uncropped original western blots are included in a Sup. Fig. 4. Values are mean ± SD. #: negative control (without CSF1) vs control (with CSF1), ##: p < 0.01; *: control (with CSF1) vs Pyk2-Inh (with CSF1), *: p < 0.05; **: p < 0.01; ***: p-value < 0.001; ****: p-value < 0.0001

Article Snippet: Pyk2 inhibitor (Pyk2-Inh, PF-431396, CAS no. 717906- 29-1) was purchased from TOCRIS Biosciences/Biotechne (Minneapolis, MN) and dissolved in dimethyl sulfoxide (DMSO) (Sigma, MO), then diluted to corresponding concentrations.

Techniques: Inhibition, Immunocytochemistry, Staining, Labeling, Control, CCK-8 Assay, Concentration Assay, Western Blot, Negative Control

Journal: Journal of neuroinflammation

Article Title: Enhanced phagocytosis associated with multinucleated microglia via Pyk2 inhibition in an acute β-amyloid infusion model.

doi: 10.1186/s12974-024-03192-7

Figure Lengend Snippet: Fig. 2 Multinucleated cells are generated from abscission failure in cytokinesis, not cell fusion. A Dynamics of cell division by time-lapse video microscopy. The phase contrast images of the microglia’s mitosis at different times. Nuclei were observed with Hoechst 33,342 staining. Arrowheads indicate representative images from anaphase to cytokinesis (control: n = 4, Pyk2-Inh: n = 4). B Cell division was quantified, categorizing it as either appropriate or inappropriate, in both control (n = 4) and Pyk2-Inh treated groups (n = 4). C Fluorescence images of microglia cells showing the stages of cell division. Immunocytochemistry of MG6 cells stained with anti-Pericentrin (red), anti-β-tubulin (green) and phalloidin (white). Nuclear DNA was labeled with DAPI (blue). Independent experiments were performed at least three times

Article Snippet: Pyk2 inhibitor (Pyk2-Inh, PF-431396, CAS no. 717906- 29-1) was purchased from TOCRIS Biosciences/Biotechne (Minneapolis, MN) and dissolved in dimethyl sulfoxide (DMSO) (Sigma, MO), then diluted to corresponding concentrations.

Techniques: Generated, Microscopy, Staining, Control, Fluorescence, Immunocytochemistry, Labeling

Journal: Journal of neuroinflammation

Article Title: Enhanced phagocytosis associated with multinucleated microglia via Pyk2 inhibition in an acute β-amyloid infusion model.

doi: 10.1186/s12974-024-03192-7

Figure Lengend Snippet: Fig. 5 Altered morphological features and phagocytic function in microglia of Iba-1 EGFP Tg mice brain after Pyk2-Inh infusion. A The experimental schedule for brain infusion assay in Iba1-EGFP Tg 7-week-old mice (n = 3/group). B Confocal images of GFP positive microglia (green) and β-amyloid (red). Obtained brain slices (thickness ≤ 1.0 mm) were treated with RapiClear 1.52 for tissue clearing and immunohistochemical assay was conducted. C, D Morphological changes were analyzed by IMARIS software. Cell surface, dendrite volume, and dendrite length were quantified. E Spot analysis by IMARIS software. F, G Quantification of the number of β-amyloid spots per cell and number of microglia deposits. H Representative confocal image of multinucleated microglia (indicated red arrow). Brain slices were stained with anti-NeuN (white) and nuclear DNA was stained with DAPI (white). Values are mean ± SD. *: p < 0.05; **: p < 0.01; ***: p-value < 0.001; ****: p-value < 0.0001

Article Snippet: Pyk2 inhibitor (Pyk2-Inh, PF-431396, CAS no. 717906- 29-1) was purchased from TOCRIS Biosciences/Biotechne (Minneapolis, MN) and dissolved in dimethyl sulfoxide (DMSO) (Sigma, MO), then diluted to corresponding concentrations.

Techniques: Immunohistochemical staining, Software, Staining

Journal: Journal of neuroinflammation

Article Title: Enhanced phagocytosis associated with multinucleated microglia via Pyk2 inhibition in an acute β-amyloid infusion model.

doi: 10.1186/s12974-024-03192-7

Figure Lengend Snippet: Fig. 6 Pyk2 inhibition reduces LPS-induced inflammation in human microglia. A The transcriptional expression of IL1β, IL6 and TNF in control and Pyk2-Inh treated group in human microglia cells, HMC3. B The secreted protein level of IL-6 was measured by ELISA. C The time-course Immunoblotting images of phosphor-NF-kB and NF-kB in HMC3 cells after LPS stimulation and with or without Pyk2-Inh. β-actin was used for loading control. The same experiments were repeated three times. D Quantification of immunoblotting data was measured by ImageJ. All full-length uncropped original western blots are included in a Sup. Fig. 6. Values are mean ± SD. #: significance between negative control and LPS control, #: p < 0.05; ##: p < 0.01; *: significance between LPS control and Pyk2-Inh treated groups, *: p < 0.05; **: p < 0.01; ***: p < 0.001; ns: not significant

Article Snippet: Pyk2 inhibitor (Pyk2-Inh, PF-431396, CAS no. 717906- 29-1) was purchased from TOCRIS Biosciences/Biotechne (Minneapolis, MN) and dissolved in dimethyl sulfoxide (DMSO) (Sigma, MO), then diluted to corresponding concentrations.

Techniques: Inhibition, Expressing, Control, Enzyme-linked Immunosorbent Assay, Western Blot, Negative Control