Journal: bioRxiv

Article Title: A novel DNA repair protein, N-Myc downstream regulated gene 1 (NDRG1), links stromal tumour microenvironment to chemoresistance

doi: 10.1101/2025.01.22.634323

Figure Lengend Snippet: a, Reverse Phase Protein Array (RPPA) was performed on three pancreatic cancer cell lines (HPAC, SW1990, BxPC-3), probing for 304 phospho- and total proteins. The cells were treated with 1 µM gemcitabine (GEM) for 72h in NC or CAF-CM. The data are shown as CAF-CM media normalized over NC media. Only significantly (p<0.05) changed proteins are shown. Yellow indicates proteins that were most significantly up-regulated in the CAF-CM and blue indicates down-regulated proteins (heat-map: log2 fold change). b , Western blot validation of the RPPA. Cells were treated with NC media or CAF-CM +/-1 µM GEM for 72h, media were changed daily, and cells were harvested 1h after the last media change. Gels were run at least thrice with similar results and probed for p-NDRG1 (T346), total NDRG1, EGFR, p-FAK (Y397), and actin. c , Purified ECM proteins (Laminin-5, Fibronectin, or Collagen 1) were added to serum starved HPAC or SW1990 cells for 3h, NC and CAF-CM media were used as controls. Cells were lysed and analysed for p-NDRG1, total NDRG1, and tubulin. d , General scheme of ECM-triggered signalling events, and pharmacological inhibition approaches used in experiments e-k . e-g , SW1990 and HPAC cells were treated with Integrin β1 or Integrin β4 function blocking antibodies or control Mouse IgG1 in CAF-CM. NC media were used as a control. g, Cells were immuno-stained for p-NDRG1 T346 (white). Scale bar: 100 µm. e-f, Mean fluorescence intensities of p-NDRG1 signal were quantified in Fiji, plotted, and analysed using Mann-Whitney test. >150 cells were analysed per condition. Magenta lines represent medians. h-i , Src-family inhibitor Saracatinib (2 µM) or FAK inhibitor Defactinib (2 µM) were added to serum starved cells (SW1990, HPAC) 1h before addition of CAF-CM, the cells were lysed 3h later and immunoblotted for p-NDRG1 T346, NDRG1, p-FAK Y397, p-Src Y418, and tubulin. j-k , SW1990 and HPAC cells were treated with vehicle control (DMSO) or inhibitors targeting AKT (MK-2206 250 nM) or SGK1 (BLU6340 250 nM) and CAF-conditioned media was added for indicated times. Cells were lysed and probed for p-NDRG1, NDRG1, p-AKT S473 and vinculin.

Article Snippet: The membranes were incubated with the following antibodies: Collagen 1 (Abcam #ab34710), Fibronectin 1 (Abcam #ab6328, Invitrogen #MA5-11981), pan-Laminin (Dako #Z-0097), EGFR (#sc-03G, Santa Cruz), GAPDH (#5174S, CST), phospho-NDRG1 T346 (#5482S, CST), NDRG1 (#9485S, CST), phospho-FAK Y397 (#3283S, CST), phospho-Src Y418 (#44-660G, Invitrogen), phospho-AKT S473 (9271, CST), V5-tag (R96025, Invitrogen), PCNA (#2586S, CST), Histone H3 (#4499S, CST), pH2AX S129 (yH2AX) (#9718S, CST and 05-636 Millipore Sigma), α-tubulin (B-5-1-2) (T5168, Sigma-Aldrich), β-actin (A5316, Sigma-Aldrich), vinculin (#13901, CST), integrin ⍺5 (#4705, CST), integrin ⍺V (#4711, CST), integrin β4 (#14803, CST, Abcam #ab110167), integrin ⍺4 (#8440, CST), integrin β1 (#9699, CST, BD clone 9EG7 #553715), integrin β1 active (Millipore clone Huts-4 #MAB2079Z), integrin β3 (#13166, CST), integrin β5 (#3629, CST), integrin ⍺6 (Millipore #MAB1378).

Techniques: Protein Array, Western Blot, Purification, Inhibition, Blocking Assay, Control, Staining, Fluorescence, MANN-WHITNEY

Journal: Mediators of Inflammation

Article Title: Toll-Like Receptor 4 Mediated Oxidized Low-Density Lipoprotein-Induced Foam Cell Formation in Vascular Smooth Muscle Cells via Src and Sirt1/3 Pathway

doi: 10.1155/2021/6639252

Figure Lengend Snippet: oxLDL regulated expression of toll-like receptor family, ROS-related genes, and sirtuin family as well as activation of Src. After oxLDL stimulation, the mRNA or protein expression of genes was detected by real-time PCR or western blot in VSMCs. The phosphorylation of Src was detected using western blot. The untreated group served as control (Con), and β -actin served as an internal reference gene to normalize protein expression. All of the real-time PCR results were calculated by 2 - ΔΔ CT method; the western blot results were calculated using grayscale value. (a) The mRNA expression of the toll-like receptor family ranging from Tlr1 to Tlr13 . ( n = 3 per group, results were expressed as mean ± SD; ∗ P < 0.01, as compared with the Con group.) (b) Upper: the phosphorylation of Src (Tyr-418, Y418) was detected after 1-hour stimulation by different doses (12.5, 25, and 50 μ g) of oxLDL in VSMCs. Bottom: the protein expression of ROS generation (Nox2 and Nox4) and elimination-related (Mnsod) genes in VSMCs, after stimulating by different doses (12.5, 25, and 50 μ g) of oxLDL for 48 hours. (c) After incubation with different doses (12.5, 25, and 50 μ g) of oxLDL, the protein levels of the sirtuin family (Sirt1-7) were measured. ( n = 3 per group, results were expressed as mean ± SD; ∗∗ P < 0.01 as compared with the Con group).

Article Snippet: Additionally, primary antibodies of β -actin (Cat#3700), p-Src Y418 (Cat#6943), t-Src (Cat#2123), Mnsod (Cat#13141), Sirt1 (Cat#2314), Sirt2 (Cat#12650), Sirt3 (Cat#5490), Sirt5 (Cat#8782), Sirt6 (Cat#12486), Sirt7 (Cat#5360), and tlr4 (Cat#14358) as well as the second antibody-conjunction HRP anti-mouse/rabbit (Cat#7076/Cat#7074) were gained from CST (MA, USA).

Techniques: Expressing, Activation Assay, Real-time Polymerase Chain Reaction, Western Blot, Incubation

Journal: Mediators of Inflammation

Article Title: Toll-Like Receptor 4 Mediated Oxidized Low-Density Lipoprotein-Induced Foam Cell Formation in Vascular Smooth Muscle Cells via Src and Sirt1/3 Pathway

doi: 10.1155/2021/6639252

Figure Lengend Snippet: Knockdown or inactivation of Src regulated lipid accumulation and cellular phenotype in VSMCs. (a, b) Tlr4(-) or NC were treated with or without oxLDL (50 μ g/mL) for 1 hour; the activation of Src (Tyr-418, Y418) had been detected by western blot. ( n = 3 per group, results were expressed as mean ± SD; ∗∗ P < 0.01, as compared with oxLDL-untreated NC group; ## P < 0.01, as compared with oxLDL-treated NC group.) Src was knockdown by siRNA transfection within VSMCs. The Src-specific antagonist, PP2, was used to block Src activation, with PP3 serving as the negative control. The VSMCs were treated with and without 50 μ g/mL oxLDL for 48 hours. (c, d) The Src-specific [Src (-)] and negative control (NC) siRNA were, respectively, transfected into VSMCs for 72 hours, and the knockdown efficiency was detected. ( n = 3 per group, results were expressed as mean ± SD; ∗∗ P < 0.01, as compared with the NC group.) (e) Nile Red (orange) was used to label the lipid, and the concentration of intracellular lipid was measured. ( n = 5 per group, results were expressed as mean ± SD; ∗∗ P < 0.01, as compared with the untreated group; @@ P < 0.01, the oxLDL-treated Src siRNA group compared with the oxLDL-treated NC group; ## P < 0.01, the oxLDL-treated PP2 group compared with the oxLDL-treated PP3 group.) (f, g) Western blot was conducted to measure the VSMC contractile phenotype markers (Myh11 and α Sma) and foam cell markers (Mac2 and Cd68). All the western blot results were calculated using grayscale value, and β -actin served as an internal reference gene to normalize protein expression. ( n = 3 per group, results were expressed as mean ± SD; ∗∗ P < 0.01, as compared with the control group (untreated group); @@ P < 0.01, the oxLDL-treated Src siRNA group compared with the oxLDL-treated NC group; ## P < 0.01, the oxLDL-treated PP2 group compared with oxLDL-treated PP3 group).

Article Snippet: Additionally, primary antibodies of β -actin (Cat#3700), p-Src Y418 (Cat#6943), t-Src (Cat#2123), Mnsod (Cat#13141), Sirt1 (Cat#2314), Sirt2 (Cat#12650), Sirt3 (Cat#5490), Sirt5 (Cat#8782), Sirt6 (Cat#12486), Sirt7 (Cat#5360), and tlr4 (Cat#14358) as well as the second antibody-conjunction HRP anti-mouse/rabbit (Cat#7076/Cat#7074) were gained from CST (MA, USA).

Techniques: Activation Assay, Western Blot, Transfection, Blocking Assay, Negative Control, Concentration Assay, Expressing

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

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