Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Tumor cell-derived SPON2 promotes M2-polarized tumor-associated macrophage infiltration and cancer progression by activating PYK2 in CRC

doi: 10.1186/s13046-021-02108-0

Figure Lengend Snippet: SPON2 promotes monocyte transendothelial migration and tumor growth by activating integrinβ1/PYK2 axis. a GSEA of the focal adhesion signature and integrin pathway signatures in the SPON2-high group compared to the SPON2-low group. NES, normalized enrichment score. b Q-RT-PCR showed the mRNA expression of PYK2 and FAK in CRC cell lines (SW480 and SW620) and a mononuclear cell line (THP-1). c Western blot analysis showed the protein expression of PYK2 and FAK in CRC cell lines (SW480 and SW620) and a mononuclear cell line (THP-1). d THP-1 cells were treated with conditioned medium from stable cell lines as indicated, and the expression of representative molecules in the focal adhesion pathway was analyzed by western blotting. e Expression and colocalization of PYK2 and zyxin in THP-1 cells with different treatments by immunofluorescence (IF) staining. Scale bars, 10 μm. f Transendothelial migration of THP-1 toward conditioned medium from stable cell lines with different treatments. Scale bar, 100 µm. g Gross images of different treatment subcutaneous tumor groups, including MC38/Scramble, MC38/shSpon2#1, MC38/shSpon2#2, anti-integrin β1 and defactinib. h Growth curve in the different treatment groups. Two-way ANOVA test, ** p < 0.01, *** p < 0.001. i Treatment time diagram and fold change of tumor volume in each case (final volume / initiate volume). Data represent mean ± SD, Student’s t test, * p < 0.05, ** p < 0.01, **** p < 0.0001

Article Snippet: Western blotting analysis was performed as previously described [ ] using anti-SPON2 (A12077, ABclonal), anti-PYK2 (bs-3357R, Bioss), anti-p-PYK2 (bs-3400R, Bioss), anti-FAK (bs-1340R, Bioss), anti-Zyxin (60,254–1-Ig, Proteintech), anti-RhoA (HPA062346, Sigma-Aldrich), anti-cortactin (11,381–1-AP, Proteintech), anti-IL10 (20,850–1-AP, Proteintech), anti-CCL2 (66,272–1-Ig, Proteintech), and anti-CSF1 (14,779–1-AP, Proteintech) antibodies.

Techniques: Migration, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Stable Transfection, Immunofluorescence, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Tumor cell-derived SPON2 promotes M2-polarized tumor-associated macrophage infiltration and cancer progression by activating PYK2 in CRC

doi: 10.1186/s13046-021-02108-0

Figure Lengend Snippet: Proposed model. a Model of SPON2 biological function. SPON2 promotes transendothelial migration of monocytes and induces polarization of TAMs toward the M2 phenotype by stimulating gene expression of IL10, CCL2 and and CSF1 in CRC cells to maintain the infiltration of M2 tumor-associated macrophages. b Schematic representations of the role of the SPON2/integrin β1/PYK2 axis in the recruitment of TAMs. SPON2 activates PYK2 in macrophages and monocytes by binding integrin β1. Activated PYK2 recruits more zyxin to promote the formation of focal adhesion complexes that are involved in stress fiber maintenance of migrating cells

Article Snippet: Western blotting analysis was performed as previously described [ ] using anti-SPON2 (A12077, ABclonal), anti-PYK2 (bs-3357R, Bioss), anti-p-PYK2 (bs-3400R, Bioss), anti-FAK (bs-1340R, Bioss), anti-Zyxin (60,254–1-Ig, Proteintech), anti-RhoA (HPA062346, Sigma-Aldrich), anti-cortactin (11,381–1-AP, Proteintech), anti-IL10 (20,850–1-AP, Proteintech), anti-CCL2 (66,272–1-Ig, Proteintech), and anti-CSF1 (14,779–1-AP, Proteintech) antibodies.

Techniques: Migration, Expressing, Binding Assay

Journal: The Journal of international medical research

Article Title: Expression and activation of proline-rich tyrosine kinase 2 (PYK2) in peripheral blood mononuclear cells from patients with systemic lupus erythematosus.

doi: 10.1177/147323000903700504

Figure Lengend Snippet: FIGURE 2: Detection of phospho- proline-rich tyrosine kinase 2 (p- PYK2) protein in peripheral blood mononuclear cells (PBMCs) from (A) healthy donors (n = 15), (B) patients with rheumatoid arthritis (n = 19) and (C) patients with systemic lupus erythematosus (SLE) (n = 36) performed by immunocytochemistry using specific anti-p-PYK2 antibodies. The PBMCs from healthy donors and RA patients were weakly positive for p-PYK2, whereas an increase in positive staining for p- PYK2 was observed in PBMCs from SLE patients (original magnification ×400) (experiments repeated at least three times with similar results)

Article Snippet: Membranes were blocked with Trisbuffered saline–Tween/1% bovine serum albumin/1% non-fat dried milk and incubated with rabbit polyclonal antibodies specific for PYK2 (SC-9019), phospho-PYK2 (p-PYK2; SC-11767-R) and β-actin (SC-1616R) (all from Santa Cruz Biotechnology) overnight at 4 °C (diluted according to the at CARLETON UNIV on May 6, 2015imr.sagepub.comDownloaded from manufacturer’s instructions).

Techniques: Immunocytochemistry, Staining

Journal: bioRxiv

Article Title: PYK2 in the dorsal striatum of Huntington’s disease R6/2 mouse model

doi: 10.1101/2024.01.18.576195

Figure Lengend Snippet: PYK2 mRNA levels were quantified by RT-qPCR. A ) PYK2 mRNA in the hippocampus, striatum and cerebral cortex of 12-week old WT male C57Bl/6 mice (6–8 animals per group). Data are expressed as % of the mean in hippocampus, Kruskal-Wallis test, p < 0.0001, Dunn’s multiple comparisons test. B ) PYK2 mRNA levels in the cerebral cortex of 12-week WT and R6/2 mice (6-7 animals per group). Mann Whitney test, not significant (ns). C ) PYK2 mRNA levels in the hippocampus of WT and R6/2 mice at the indicated ages (6-10 animals per group). Kruskal-Wallis test, p = 0.0085, Mann-Whitney test, 4 w, ns, 6 w, p = 0.036, 12 w, p = 0.02. D ) PYK2 mRNA levels in the DS of WT and R6/2 mice at the indicated ages (4-8 animals per group). Kruskal-Wallis test, p = 0.0017, Mann-Whitney test, 4 w, ns, 6 w, p = 0.0047, 12 w, p = 0.0043. ( A-D ) Means ± SEM are indicated, * p < 0.05, ** p < 0.01, detailed statistical results in Supplementary Table 1 .

Article Snippet: Protein lysates from DS (10 μg) were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, electrotransferred onto nitrocellulose membranes (GE Healthcare, LC, UK), and probed with rabbit PYK2 antibodies (diluted 1/1 000, 074M4755, Sigma).

Techniques: Quantitative RT-PCR, MANN-WHITNEY

Journal: bioRxiv

Article Title: PYK2 in the dorsal striatum of Huntington’s disease R6/2 mouse model

doi: 10.1101/2024.01.18.576195

Figure Lengend Snippet: A ) PYK2 protein levels were measured by immunoblotting in the striatum of WT and R6/2 mice at the indicated ages. β-actin was used as a loading control. B ) Data as in ( A ) were quantified by densitometry (5-9 animals per group). PYK2 levels were normalized to the loading control and data expressed as % of the mean in WT at each age. Kruskal-Wallis test, p = 0.0007. Mann-Whitney test, 4 w, ns, 6 w, p = 0.0012, 12 w, p = 0.0006. C ) PYK2 protein levels were measured by immunoblotting in the putamen of human subjects deceased with HD or with other conditions . α-tubulin was used as a loading control. Data were quantified, normalized and expressed as % of the mean in controls (6 controls and 6 HD). Mann-Whitney test, p = 0.041. ( A-B ) Means ± SEM are indicated,* p < 0.05, ** p < 0.01, *** p < 0.001, detailed statistical results in Supplementary Table 1 . Uncropped immunoblots for all samples are shown in Supplementary Figure 1.

Article Snippet: Protein lysates from DS (10 μg) were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, electrotransferred onto nitrocellulose membranes (GE Healthcare, LC, UK), and probed with rabbit PYK2 antibodies (diluted 1/1 000, 074M4755, Sigma).

Techniques: Western Blot, MANN-WHITNEY

Journal: bioRxiv

Article Title: PYK2 in the dorsal striatum of Huntington’s disease R6/2 mouse model

doi: 10.1101/2024.01.18.576195

Figure Lengend Snippet: A ) The spontaneous locomotor activity of 3-6-month-old male WT and PYK2-KO littermate mice was studied by video tracking for 15 min in an open field. Unpaired 2-tailed Student’s t test, ns (9-10 mice per group). B ) Motor skill was evaluated as the time to reach the escape zone on a balance beam during 2 trials on 2 consecutive days (6-8 mice per group). Kruskal-Wallis test, p = 0.0007, Dunn multiple comparisons test, no difference between genotypes. C ) Motor skill was evaluated on a vertical pole by visually scoring (8 mice per group). Mann-Whitney test, ns. D ) Examples of hind limb position when mice were held by the tail showing the absence of clasping in WT and PYK2-KO mice. ( A-C ) Means ± SEM are indicated. Detailed statistical results in Supplementary Table 1 .

Article Snippet: Protein lysates from DS (10 μg) were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, electrotransferred onto nitrocellulose membranes (GE Healthcare, LC, UK), and probed with rabbit PYK2 antibodies (diluted 1/1 000, 074M4755, Sigma).

Techniques: Activity Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: PYK2 in the dorsal striatum of Huntington’s disease R6/2 mouse model

doi: 10.1101/2024.01.18.576195

Figure Lengend Snippet: A ) AAV- Camk2a -PYK2, expressing PYK2 and GFP, was stereotactically injected in one striatum of a WT mouse and AAV- Camk2a -GFP, expressing GFP, in the contralateral striatum. Coronal brain section were immunostained for GFP (left picture) and PYK2 (right picture). B ) R6/2 mice received a bilateral injection of either AAV- Camk2a -PYK2 or AAV-Camk2a-GFP in the DS and WT mice a bilateral injection of AAV-Camk2a-GFP. Their body weight was measured every week. Two way repeated measures ANOVA, interaction, F (16,80) = 8.79, p < 10 -4 , age effect, F (8,80) = 45.0, p < 10 -4 , mouse/AAV group effect, F (2,10) = 1.0, p = 0.39 (4-5 mice per group). Asterisks indicate significant post hoc Holm-Sidak multiple comparisons test. C ) Clasping responses were scored in the 3 groups at the indicated ages (4-7 mice per group and age). Kruskal-Wallis test, p = 0.0006. Horizontal bars are means. D-E ) The motor behavior of the 3 groups of mice was studied in a circular maze at 6 and 9 weeks (7-10 mice per group and age). General locomotor activity (number of beam breaks, D ) and the number of rearings ( E ) were measured. Kruskal-Wallis test, ( D ) 6 weeks, ns, 9 weeks p = 0.018, ( E ) 6 weeks, p = 0.038, 9 weeks p = 0.002, Dunn’s multiple comparisons test. F-G ) The motor coordination was evaluated every week from 6 to 11 weeks (w) in a rotarod test by measuring the latency to fall ( F ) and the speed at fall ( G ) (4-14 mice per group and age). Kruskal-Wallis test at each age, ( F ) 6 w, p = 0.0007, 7 w, p = 0.0006, 8 w p < 10 -4 , 9 w p < 10 - 4 , 10 w, p = 0.013, 11 w, p = 0.0091, ( G ) 6 w, p = 0.0047, 7 w, p < 10 -4 , 8 w p < 10 -4 , 9 w p < 10 -4 , 10 w, p = 0.0028, 11 w, p = 0.0011. ( B , D-G ) Means ± SEM are indicated. ( C-G ) Post hoc Dunn’s multiple comparisons test. Significant differences are indicated by asterisks, green, R6/2 AAV- SYN1 -GFP vs WT AAV- SYN1 -GFP, violet, R6/2 AAV- SYN1 -PYK2 vs WT AAV- SYN1 -GFP, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 10 -4 . Detailed statistical results in Supplementary Table 1 . Rpm, rotation per minute, a.u., arbitrary units.

Article Snippet: Protein lysates from DS (10 μg) were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, electrotransferred onto nitrocellulose membranes (GE Healthcare, LC, UK), and probed with rabbit PYK2 antibodies (diluted 1/1 000, 074M4755, Sigma).

Techniques: Expressing, Injection, Activity Assay

Journal: bioRxiv

Article Title: PYK2 in the dorsal striatum of Huntington’s disease R6/2 mouse model

doi: 10.1101/2024.01.18.576195

Figure Lengend Snippet: A ) AAV- SYN1 -PYK2, expressing PYK2 and GFP, was stereotactically injected in one striatum of a WT mouse and AAV- SYN1 -GFP, expressing GFP only, in the contralateral striatum. Coronal brain sections were immunolabeled for PYK2. B ) Four-week-old WT and R6/2 mice were injected as in A and DARPP-32 detected by immunofluorescence in coronal brain sections. Left panel, sample picture of a WT mouse. Right panel, immunofluorescence was compared in the right and left striatum of WT and R6/2 mice and the ratio quantified (DARPP-32 IF AAV- SYN1 -PYK2-injected side / AAV- SYN1 -GFP-injected side). Mann-Whitney test, p = 0.029 (n = 4). C ) R6/2 mice received a bilateral injection of either AAV- SYN1 -PYK2 or AAV- SYN1 -GFP in the DS and WT mice received a bilateral injection of AAV- SYN1 -GFP. Their body weight was measured every week (6 mice per group). Two-way repeated measures ANOVA, interaction, F (16,120) = 9.09, p < 10 -4 , age effect, F (8,120) = 18.07, p < 10 -4 , mouse/AAV group effect, F (2,15) = 3.64, p = 0.051. Asterisks indicate significant differences with Holm-Sidak’s multiple comparisons test. D ) Clasping responses were scored in the 3 groups at the indicated ages (6 mice per group). Kruskal-Wallis test, p < 10 -4 . Horizontal bars are means. E-F ) The motor behavior of the 3 groups of mice (6 mice per group) was studied in a circular maze at the indicated ages. Locomotion was measured as the number of ¼ turns ( E ) and the number of rearings counted ( F ). Kruskal-Wallis test, ( E ), 6 w, ns, 9 w, ns, 12 w, p = 0.033, ( F ), 6 w, ns, 9 w, p = 0.034, 12 w, ns. G-H ) The motor coordination was evaluated every week from 6 to 12 weeks in a rotarod test by measuring the latency to fall ( G ) and the speed at fall ( H ). Two-way repeated measures ANOVA (6 mice per group at every age), ( G ) interaction, F (12,90) = 1.94, p = 0.04, age effect, F (6,90) = 2.93, p = 0.01, mouse/AAV group, F (2,15) = 19.8, p < 10 -4 , ( H ) interaction, F (12,90) = 4.74, p < 10 -4 , age, F (6,90) = 7.84, p < 10 -4 , mouse group, F (2,15) = 14.56, p = 0.0003. Asterisks indicate significant post hoc Holm-Sidak’s multiple comparisons test. ( C , E-H ) Means ± SEM are indicated. ( D-H ) Post hoc Dunn’s multiple comparisons test. Significant differences are indicated by asterisks, green R6/2 AAV- SYN1 -GFP vs WT AAV- SYN1 -GFP, violet, R6/2 AAV- SYN1 -PYK2 vs WT AAV- SYN1 -GFP, black, R6/2 AAV- SYN1 -PYK2 vs R6/2 AAV- SYN1 -GFP, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 10 -4 . Detailed statistical results in Supplementary Table 1 . Rpm, rotation per minute, a.u., arbitrary units.

Article Snippet: Protein lysates from DS (10 μg) were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, electrotransferred onto nitrocellulose membranes (GE Healthcare, LC, UK), and probed with rabbit PYK2 antibodies (diluted 1/1 000, 074M4755, Sigma).

Techniques: Expressing, Injection, Immunolabeling, Immunofluorescence, MANN-WHITNEY