anti- β1- integrin clone 6s6  (Millipore)

Journal: Cancers

Article Title: Integrin α2 and β1 Cross-Communication with mTOR/AKT and the CDK-Cyclin Axis in Hepatocellular Carcinoma Cells

doi: 10.3390/cancers14102430

Figure Lengend Snippet: Integrin α and β expression profile on HepG2, Huh7, and Hep3B cells. Cell number (Counts) versus fluorescence intensity is depicted (one representative of three separate experiments). Black line = specific fluorescence, grey line = isotype control.

Article Snippet: Adhesion to immobilized collagen and chemotaxis was also evaluated when tumor cells were serum starved for 12 h and stimulated with IGF-1 and simultaneously blocked with anti-integrin α2 (clone P1E6) or anti-integrin β1 (clone 6S6), both mouse mAb (all obtained from Merck Millipore, Burlington, MA, USA).

Techniques: Expressing, Fluorescence

Journal: Cancers

Article Title: Integrin α2 and β1 Cross-Communication with mTOR/AKT and the CDK-Cyclin Axis in Hepatocellular Carcinoma Cells

doi: 10.3390/cancers14102430

Figure Lengend Snippet: ( A ) Integrin α2, αV, and β1 expression level evaluated 72h after exposing HCC cells to collagen (1 µg/mL, 100 µg/mL) or Matrigel (10 µg/mL). All fluorescence values (MFU) are related to untreated controls set to 100%. Means ± SD of n = 4. * significant difference to the controls set to 100%; ( B ) Gene expression of integrin α2, αV, and β1, depicted as fold regulation relative to the control (control = 1). * indicates significant difference.

Article Snippet: Adhesion to immobilized collagen and chemotaxis was also evaluated when tumor cells were serum starved for 12 h and stimulated with IGF-1 and simultaneously blocked with anti-integrin α2 (clone P1E6) or anti-integrin β1 (clone 6S6), both mouse mAb (all obtained from Merck Millipore, Burlington, MA, USA).

Techniques: Expressing, Fluorescence

Journal: Cancers

Article Title: Integrin α2 and β1 Cross-Communication with mTOR/AKT and the CDK-Cyclin Axis in Hepatocellular Carcinoma Cells

doi: 10.3390/cancers14102430

Figure Lengend Snippet: ( A ) HepG2 cell growth in response to α2, β1 or αV knockdown with small interfering RNA (siRNA) (Western blot at right); ( B ) HepG2 cell number in response to integrin blocking antibodies targeted against integrin α2, β1, αV, α2β1 (BTT), or αVβ3 (Cyclo). The respective MTT assay was completed after 24, 48, and 72 h. Cell number of non-blocked controls was set to 100%. * ( A ) indicates significant difference as marked. * ( B ) indicates significant difference to untreated controls ( n = 3); ( C ) HepG2 cell growth in the presence of Matrigel (10 µg/mL) or collagen (1 µg/mL) and subsequent to Matrigel or collagen plus the blockade of integrin α2β1 with BTT or αV with Cyclo. Cell number was evaluated after 72 h by the MTT assay and related to untreated controls set to 100%. ( n = 3); ( D ) Cell cycle analysis of HepG2 cells under integrin α2β1 (BTT) or αV (Cyclo) blockade, or under stimulation with Matrigel (10 µg/mL) and blockade with integrin α2β1 (BTT + Matrigel) or αV (Cyclo + Matrigel). Percentage of cells in the G0/G1-, S-, and G2/M-phase is indicated (all related to respective controls set to 100%) ( n = 3); ( E ) Clonogenic growth in the presence of Matrigel (10 µg/mL), in the presence of Matrigel plus integrin α2β1 blockade (Matrigel + BTT) or in the presence of Matrigel plus integrin αV blockade (Matrigel + Cyclo). Control cells remained untreated. # significant up-regulation, * significant down-regulation to untreated controls ( n = 4).

Article Snippet: Adhesion to immobilized collagen and chemotaxis was also evaluated when tumor cells were serum starved for 12 h and stimulated with IGF-1 and simultaneously blocked with anti-integrin α2 (clone P1E6) or anti-integrin β1 (clone 6S6), both mouse mAb (all obtained from Merck Millipore, Burlington, MA, USA).

Techniques: Small Interfering RNA, Western Blot, Blocking Assay, MTT Assay, Cell Cycle Assay

Journal: Cancers

Article Title: Integrin α2 and β1 Cross-Communication with mTOR/AKT and the CDK-Cyclin Axis in Hepatocellular Carcinoma Cells

doi: 10.3390/cancers14102430

Figure Lengend Snippet: ( A ) Protein profile of cell cycle regulating proteins (AKT, mTOR, Rictor, Raptor, all total and phosphorylated, “p”), CDK1 and 2 (total and phosphorylated, “p”), Cyclin A and B, FAK, pFAK and ILK. HepG2 cells were either stimulated with IGF or stimulated with IGF and treated with an integrin α2 or β1 specific siRNA (scr = scrambled siRNA). Following siRNA transfection protein data of integrin α2 and β1 are shown on the right side of A. Controls ( C ) received cell culture medium alone. One representative experiment of three is shown; ( B ) Quantification of the protein intensity in IGF1 treated versus non-treated HepG2 cells by the pixel density analysis, all related to the unstimulated 100% controls. * significant difference to controls; ( C , D ): Pixel density analysis of the protein level in HepG2 cells following α2 ( C ) or β1 ( D ) siRNA knock-down. Values are given in percentage, related to the 100% control. * significant difference to controls.

Article Snippet: Adhesion to immobilized collagen and chemotaxis was also evaluated when tumor cells were serum starved for 12 h and stimulated with IGF-1 and simultaneously blocked with anti-integrin α2 (clone P1E6) or anti-integrin β1 (clone 6S6), both mouse mAb (all obtained from Merck Millipore, Burlington, MA, USA).

Techniques: Transfection, Cell Culture

Journal: Cancers

Article Title: Integrin α2 and β1 Cross-Communication with mTOR/AKT and the CDK-Cyclin Axis in Hepatocellular Carcinoma Cells

doi: 10.3390/cancers14102430

Figure Lengend Snippet: ( A ) mTOR and pmTOR protein expression. HepG2 cells were stimulated with IGF1 (IGF), remained untreated (C), or mTOR was blocked by the mTOR-inhibitor Rapamycin and then treated with IGF1. Protein analysis was accompanied by a β-actin loading control. One representative experiment of three is shown; ( B) Surface expression of the integrin subtypes α2 and β1 on HepG2 cells. Tumor cells were either exposed to IGF1 (+IGF) or stimulated with IGF1 under AKT blockade with MK-2206. Detection was completed by FACS analysis. Mean fluorescence values (MFU) are related to untreated controls set to 100%. # indicates significant upregulation, compared to cells not exposed to IGF. * indicates significant downregulation to IGF stimulated cells. § indicates significant downregulation to cells not exposed to IGF1 ( n = 3).

Article Snippet: Adhesion to immobilized collagen and chemotaxis was also evaluated when tumor cells were serum starved for 12 h and stimulated with IGF-1 and simultaneously blocked with anti-integrin α2 (clone P1E6) or anti-integrin β1 (clone 6S6), both mouse mAb (all obtained from Merck Millipore, Burlington, MA, USA).

Techniques: Expressing, Fluorescence

Journal: Cancers

Article Title: Integrin α2 and β1 Cross-Communication with mTOR/AKT and the CDK-Cyclin Axis in Hepatocellular Carcinoma Cells

doi: 10.3390/cancers14102430

Figure Lengend Snippet: Integrin β1 interacts with AKT. IP was performed wherein integrin β1 was pulled down using mouse anti Human CD29 (Clone:18/CD29). Following SDS-PAGE electrophoresis and WB, the separated proteins were probed with antibodies against β1, AKT, and pAKT. Lysate from cells treated with IGF1 (+IGF) demonstrated strong interaction of pAKT with integrin β1. Lysate from cells treated with siRNA β1 showed knockdown efficiency of β1 protein. Mouse IgG was used as a negative con-trol. A total of 20 µg of cell lysate was used as input. IP, immunoprecipitation; WB, Western blotting.

Article Snippet: Adhesion to immobilized collagen and chemotaxis was also evaluated when tumor cells were serum starved for 12 h and stimulated with IGF-1 and simultaneously blocked with anti-integrin α2 (clone P1E6) or anti-integrin β1 (clone 6S6), both mouse mAb (all obtained from Merck Millipore, Burlington, MA, USA).

Techniques: SDS Page, Electrophoresis, Immunoprecipitation, Western Blot

Journal: Scientific Reports

Article Title: Generation of a lung squamous cell carcinoma three-dimensional culture model with keratinizing structures

doi: 10.1038/s41598-021-03708-8

Figure Lengend Snippet: PLR327F-LD41 multicellular spheres generate uniform 3D cultures. ( a ) PLR327F-LD41 cells were cultured in Corning Elplasia microcavity plates for 24 h to form multicellular spheres. Representative pre- and post-culture images are shown. Scale bar = 100 µm. ( b ) Representative bright field images of COs generated by culturing PLR327F-LD41 spheres in MG, Mix-gel, or col-I for 14 days. Scale bar = 1 mm. ( c ) Growth of COs was determined by analysing the size of each CO from day 4 to day 14. Size (µm 2 ) of COs with major axis longer than 40 µm are shown in box plot. The number of COs analysed was between 35 and 52 for each sample. ( d ) Results from 3 independent experiments were analysed to determine the difference in the ratio of IVL inner COs. Thirty or 31 COs were analysed for each sample. IVL negative CO was not observed. * p < 0.05, Tukey–Kramer HSD test. ( e ) PLR327F-LD41 spheres were cultured in MG, Mix-gel, or col-I for 14 days. Anti-integrin β1 and β4 neutralising antibodies were added to the culture from Day 7 to Day 14 at 10 µg/mL for each antibody. IVL staining pattern was analysed by whole mount immunostaining. ** p < 0.01, Fisher’s exact test. Bright field images and the size analysis of COs were obtained by cellSens Dimension software (version 2.1, Olympus, https://www.olympus-lifescience.com/en/micro/ ). Whole mount immunostaining images were obtained by NIS Elements HC software (version 5.02, Nikon, https://www.microscope.healthcare.nikon.com/en_EU/ ).

Article Snippet: For blocking integrin, 10 µg/mL each of integrin β1 (clone 6S6, Azide Free, Merck) and integrin β4 (clone UM-A9, Azide free, Novus Biologicals) neutralising antibody was added to the culture from Day 7 to Day 14.

Techniques: Cell Culture, Generated, Staining, Immunostaining, Software, Microscopy