Journal: Frontiers in Oncology

Article Title: CAR Co-Operates With Integrins to Promote Lung Cancer Cell Adhesion and Invasion

doi: 10.3389/fonc.2022.829313

Figure Lengend Snippet: CAR forms a complex with focal adhesion proteins. (A) Representative western blot of immunoprecipitation of β1 integrins from specified cell lines probed for β1 integrin and GFP. Representative of 5 independent experiments. (B) Western blots of immunoprecipitation of GFP or GFP-CAR from lysates from specified cell lines probed for indicated focal adhesion proteins and GFP. Representative of 5 independent experiments. (C) Western blots of GST pulldowns using GST only or GST-CAR cytoplasmic tail (WT, AA, DD) using from lysates from CMT parental cells probed for indicated focal adhesion proteins and GST. (D) Quantification of blots as in C from 5 independent experiments. (E, F) Western blot of lysates from CMT parental cells treated with Src inhibitor [PP2, 1μM, (E) ] or FAK inhibitor [PF228, 1μM, (F) ] probed for indicated proteins. (G) Quantification of active β1 integrins (9EG7) from images of specified CMT cell lines treated with DMSO, PP2 or PF228. Data is from at least 10 fields of view (5-10 cells per field) per condition. Representative of 3 independent experiments. (H) Quantification of recovery curves of fluorescence intensity for FRAP data from WTCAR-GFP CMT cells +/-PP2. Data is from at least 15 different ROIs per cell line, shown as mean +/-SEM, representative of 3 independent experiments. (I) Representative Western blots of lysates from Rap1 activity pulldown assays from specified CMT cell lines, probed for Rap1 and GST. (J) Quantification of data as in (I) , pooled from 5 independent experiments. (K) Quantification of CMT parental and WTCAR-GFP cell adhesion to Matrigel after 2h, following pre-treatment with DMSO or Rap1 inhibitor (GGTI298, 5μM) normalised to control. Data is from 3 wells per cell line, representative of 3 independent experiments. All values are mean ± SEM. P values *p < 0.05, **p < 0.01, ***p < 0.0005, ****p < 0.0001.

Article Snippet: Anti-integrin β1 (12G10, Santa Cruz), anti-active β1 integrin (9EG7, Merck Millipore), anti β-catenin [Santa Cruz (IF) and BD Bioscience (WB)], anti-CAR antibody (Santa-Cruz), anti E-Cadherin (Abcam), anti-FAK (Cell signalling), anti-green fluorescent protein (GFP) , anti- Heat Shock Cognate 70kDa (HSC70) (Sigma-Aldrich), anti-paxillin (BD Bioscience), anti phospho-FAK (Y397, Cell signalling), anti-phospho-paxillin (Y118, Cell signalling), anti-phospho-src (Y418, Millipore), anti-rap1 A/B (R&D Systems) and anti-src (Millipore) antibodies were used for western blot.

Techniques: Western Blot, Immunoprecipitation, Fluorescence, Activity Assay, Control

Journal: Frontiers in Oncology

Article Title: CAR Co-Operates With Integrins to Promote Lung Cancer Cell Adhesion and Invasion

doi: 10.3389/fonc.2022.829313

Figure Lengend Snippet: Working model for CAR-dependent regulation of lung cancer cell adhesion. Phosphorylated CAR promotes β1 integrin and Rap1 activation. Similarly to JAM-A, dimerised CAR may promote interaction of scaffold protein MAGI-1 and the Rap1 GEF PDZ-GEF2, resulting in Rap1 activation. CAR-mediated Rap1 activation promotes cell-ECM adhesion and may enhanceβ1 integrin activity. The latter may in turn be involved in CAR-mediated promotion of cell invasion. Phosphorylated CAR may recruit β1 integrins and/or focal adhesion components Src, FAK and Paxillin to the membrane to regulate cell-cell adhesion and to distally control focal adhesion activity. Figure created with BioRender.com .

Article Snippet: Anti-integrin β1 (12G10, Santa Cruz), anti-active β1 integrin (9EG7, Merck Millipore), anti β-catenin [Santa Cruz (IF) and BD Bioscience (WB)], anti-CAR antibody (Santa-Cruz), anti E-Cadherin (Abcam), anti-FAK (Cell signalling), anti-green fluorescent protein (GFP) , anti- Heat Shock Cognate 70kDa (HSC70) (Sigma-Aldrich), anti-paxillin (BD Bioscience), anti phospho-FAK (Y397, Cell signalling), anti-phospho-paxillin (Y118, Cell signalling), anti-phospho-src (Y418, Millipore), anti-rap1 A/B (R&D Systems) and anti-src (Millipore) antibodies were used for western blot.

Techniques: Activation Assay, Activity Assay, Membrane, Control

Journal: Journal of translational medicine

Article Title: N-terminal domain of CTRP9 promotes cardiac fibroblast activation in myocardial infarction via Rap1/Mek/Erk pathway.

doi: 10.1186/s12967-025-06274-z

Figure Lengend Snippet: Fig. 5 nCTRP9 promotes cardiac fibrosis through upregulation of Rap1. A. Heatmap of the different genes after nCTRP9 treated immortalized cardiac fibroblasts (iCF) activated with Tgf-β. B KEGG pathway enrichment of differentially expressed genes. C. ESI-05, an inhibitor of Rap1, down-regulated the transcription of fibrosis-related genes in iCF. n = 4. D. nCTRP9 increased MEK 1/2 and ERK 1/2 phosphorylation in fibroblasts. n = 4. E. nCTRP9 increased MEK 1/2 and ERK 1/2 phosphorylation in mice heart post MI surgery. n = 4. F. Rap1 knockdown efficiency in iCF. n = 4. G. Down-regulation of Rap1 attenu ated the promoting effect of nCTRP9 on the transcription of fibrosis-related genes in iCF. n = 4. H. Rap1 knockdown attenuated α-smooth muscle actin (α-SMA) expression increased by nCTRP9 in iCF. n = 6. I. Rap1 knockdown attenuated the promoting effect of nCTRP9 on cell proliferation of iCF. n = 6. * p < 0.05, ** p < 0.01

Article Snippet: The following are the primary antibodies: CTRP9 (1:1000, customized by Genscript), nCTRP9 (1:1000, customized by Genscript), Rap1 (1:1000, 68125-1-Ig, Proteintech, Wuhan, China) GAPDH (1:5000, AC001, Abclonal, Wuhan, China), AMPK (1:1000, 2532, CST, Danvers, USA) and p-AMPKαThr172 (1:1000, 2532, CST, Danvers, USA), Phospho-p38 MAPKαThr 172 (1:1000, 4060, CST, Danvers, USA), Akt (1:1000, 9272, CST, Danvers, USA)and p-Akt Ser 473 (1:1000, 4060, CST, Danvers, USA), ERK1/2 (1:1000, 4696, CST, Danvers, USA), p-ERK1/2 Thr202/Tyr204 (1:1000, 9106, CST, Danvers, USA), MEK1/2 (1:1000, 11049-1-AP, Proteintech, Wuhan, China)and Phospho-MEK1/2(1:1000, 9154, CST, Danvers, USA).

Techniques: Phospho-proteomics, Knockdown, Expressing

Journal: Journal of translational medicine

Article Title: N-terminal domain of CTRP9 promotes cardiac fibroblast activation in myocardial infarction via Rap1/Mek/Erk pathway.

doi: 10.1186/s12967-025-06274-z

Figure Lengend Snippet: Fig. 6 RAP1 knockdown abolishes cardiac fibrosis induced by nCTRP9. (A) Fibroblast-specific knockdown of Rap1 mice was established by administrat ing adeno-associated virus (type 9) carrying the shRNA of Rap1 via tail vein injection. nCTRP9 was administered after MI surgery for 4 weeks. (B) Cardiac ejection fraction (EF) of mice post MI surgery. n = 6. (C) Infarct size of MI mice heart was detected after surgery for 4 weeks. n = 6. (D) Heart weight (HW) to tibial length (TL) ratio. n = 6. (E) Staining with wheat germ agglutinin was done to determine the mean cross-sectional area of cardiomyocytes. n = 6. F-G. Cardiac fibrosis in Sham and MI mice heart detected by Masson staining of border region and distant region. n = 6. H. Rap1 levels in MI mice heart. n = 4. I. Knockdown of Rap1 attenuated phosphorylation increase by nCTRP9 in MI mice heart. n = 4. * p < 0.05, ** p < 0.01

Article Snippet: The following are the primary antibodies: CTRP9 (1:1000, customized by Genscript), nCTRP9 (1:1000, customized by Genscript), Rap1 (1:1000, 68125-1-Ig, Proteintech, Wuhan, China) GAPDH (1:5000, AC001, Abclonal, Wuhan, China), AMPK (1:1000, 2532, CST, Danvers, USA) and p-AMPKαThr172 (1:1000, 2532, CST, Danvers, USA), Phospho-p38 MAPKαThr 172 (1:1000, 4060, CST, Danvers, USA), Akt (1:1000, 9272, CST, Danvers, USA)and p-Akt Ser 473 (1:1000, 4060, CST, Danvers, USA), ERK1/2 (1:1000, 4696, CST, Danvers, USA), p-ERK1/2 Thr202/Tyr204 (1:1000, 9106, CST, Danvers, USA), MEK1/2 (1:1000, 11049-1-AP, Proteintech, Wuhan, China)and Phospho-MEK1/2(1:1000, 9154, CST, Danvers, USA).

Techniques: Knockdown, Virus, shRNA, Injection, Staining, Phospho-proteomics

Journal: Oncotarget

Article Title: Heregulin, a new regulator of telomere length in human cells

doi:

Figure Lengend Snippet: A. Retrovirally-induced HRGβ2 regulates TRF2 and RAP1 expression and telomere length in MCF-7 breast cancer cells. Left. Representative immunoblot ( n = 3 ) showing upregulation of TRF2 and RAP1 in MCF-7 cells retrovirally engineered to overexpress HRGβ 2 . PD 0 represents the first sub-passage after selection for retroviral infection. At the indicated PDs, cells were washed with cold PBS and solubilized in lysis buffer containing phosphatase and protease inhibitors. Fifty μg of protein per sample was resolved by SDS-PAGE and subjected to Western blotting for TRF2 and hRap1 as described above (see “Materials and Methods” for details). Middle panel. MCF-7/HRGβ 2 cells do not exhibit significant changes in telomerase activity as assessed by the TRAP assay. Three thousand cell equivalents were used for each reaction and a representative experiment is shown ( n = 3 ). Right panel. Telomere length changes in MCF-7 cells upon retrovirally-induced HRGβ 2 overexpression. The panel shows a representative genomic blotting analysis of telomeric restriction fragments in Hin fI/ Rsa I-digested genomic DNA from retrovirally-infected MCF-7 cells probed with a TTAGGG repeat fragment. B. HRG knockdown reduces the presence of TRF2 and RAP1 on telomeres and promotes telomere lengthening. Top left. Depletion of HRG with siRNA significantly affects TRF2 and RAP1 protein levels. A representative Western blot of MDA-MB-231 cell lysates 72 h after transfection of siRNA to HRG or siRNA control is shown ( n = 3 ). Top right. TRF length analysis in HRG knockdown MDA-MB-231 cells. The panel shows a representative genomic blotting analysis of telomeric restriction fragments in Hin fI/ Rsa I-digested genomic DNA from MDA-MB-231 cells transiently transfected with control siRNA or graded concentrations of HRG siRNA (PD 4) probed with a TTAGGG repeat fragment ( n = 3 ). Bottom. Reduced TRF2 and RAP1 telomeric signals after HRG siRNA treatment. Western blot analysis showing the significant depletion of HRGβ 2 protein with a specific siRNA. The panel shows representative immunofluorescence images of MDA-MB-231 cells 48 h after introduction of HRG siRNA: staining with anti-TRF2 ( red ) and anti-RAP1 ( green ) antibodies is shown ( n = 3 ). DAPI was used to visualize nuclear DNA ( blue ).

Article Snippet: Primary antibodies against TRF2 (IMG-124), TRF1 (IMG-283) and RAP1 (IMG-272) were purchased from Imgenex (San Diego, CA).

Techniques: Expressing, Western Blot, Selection, Infection, Lysis, SDS Page, Activity Assay, TRAP Assay, Over Expression, Transfection, Immunofluorescence, Staining

Journal: Oncotarget

Article Title: Heregulin, a new regulator of telomere length in human cells

doi:

Figure Lengend Snippet: A. Subnuclear localization of HRGβ 2 and its relationship with telomeric loci. A431 cells naturally overexpressing HRGβ 2 were permeabilized twice and sequentially stained with an antibody against HRGβ 2 ( red ) and the fluorescein-conjugated telomere-specific peptide nucleic acid (PNA) probe ( green ). DAPI was used to visualize nuclear DNA ( blue ). Merged images reveal partial co-localization of endogenous HRGβ 2 and telomeres ( yellow ). B. Representative immunofluorescent images of A431 cells co-stained for endogenous HRGβ 2 ( green ) using anti-TRF2 or anti-RAP1 ( red ) antibodies is shown ( n = 3 ). Merged images, including DAPI staining of DNA, reveal a prominent co-localization ( yellow ) of endogenous HRGβ 2 with TRF2 and RAP1.

Article Snippet: Primary antibodies against TRF2 (IMG-124), TRF1 (IMG-283) and RAP1 (IMG-272) were purchased from Imgenex (San Diego, CA).

Techniques: Staining