Journal: Oncology reports

Article Title: Involvement of non‑triple helical type VI collagen α1 chain, NTH α1(VI), in the proliferation of cancer cells.

doi: 10.3892/or.2020.7734

Figure Lengend Snippet: Figure 1. NTH α1(VI) expression in S2‑VP10 cells. (A) Representative images of NTH α1(VI) expression in conditioned medium derived from S2‑VP10 cells. The conditioned medium was harvested after two days of culture. Western blotting was carried out under non‑reducing and reducing conditions, and probing was performed with anti‑COL6A1 polyclonal antibody (IB: αCOL6A1); NTH α1(VI) and COL6A1 are indicated by white arrowheads; * indicates non‑specific band. (B) A representative immunoblot image of COL6A2 expression in conditioned media derived from S2‑VP10 and MDA‑MB‑436 cells. The conditioned media were harvested after two days of culture. Western blotting was carried out under reducing conditions and probing with anti‑COL6A2 monoclonal antibody (IB: αCOL6A2) was performed; COL6A2 bands are indicated by an arrow. (C and D) COL6A1 mRNA and COL6A2 mRNA expression in S2‑VP10 (C) and MDA‑MB‑436 cells (D); cells were cultured for 72 h and then COL6A1 mRNA and COL6A2 mRNA expression was determined by RT‑qPCR. Data are expressed as the mean ± SD (n=3). ***P<0.001 vs. siCont by Student's t test. RT‑qPCR, reverse transcription‑quantitative PCR; COL6A1, collagen type VI alpha 1 chain; NTH α1(VI), non‑triple helical type VI collagen α1 chain.

Article Snippet: Rabbit anti-COL6A1 polyclonal antibody (cat. no. NBP1-59126) was purchased from Novus Biologicals.

Techniques: Expressing, Derivative Assay, Western Blot, Cell Culture

Journal: Oncology reports

Article Title: Involvement of non‑triple helical type VI collagen α1 chain, NTH α1(VI), in the proliferation of cancer cells.

doi: 10.3892/or.2020.7734

Figure Lengend Snippet: Figure 2. Effect of COL6A1 KD on the proliferation of S2‑VP10 cells. (A) Representative images of COL6A1 KD in S2‑VP10 cells. The indicated siRNA was transfected into S2‑VP10 cells and cell lysates were prepared. Western blotting was carried out under reducing conditions and sequential probing was performed with antibody #141 (IB: #141), anti‑COL6A1 polyclonal antibody (IB: αCOL6A1), and anti‑HSP90 monoclonal antibody (IB: αHSP90). (B) Typical proliferation curves of S2‑VP10 cells. Control siRNA was transfected into S2‑VP10 cells, which were cultured for 72 h. Cell proliferation at the indicated culture time determined by methylene blue staining is shown as absorbance at a wavelength of 660 nm. Data are expressed as the mean ± SD (n=4). (C) Effect of COL6A1 KD on the proliferation of S2‑VP10 cells. The indicated siRNA was transfected into S2‑VP10 cells, which were cultured for 72 h; relative cell numbers without transfection or with siCont transfection were increased by approximately three‑fold as shown in (B). Cell proliferation was assessed by methylene blue staining; data are expressed as the mean ± SD (n=3). ***P<0.001 vs. siCont by Dunnett's multiple comparison test. COL6A1, collagen type VI alpha 1 chain; KD, knockdown; IB, immunoblot; siCont, control siRNA; NT, no treatment.

Article Snippet: Rabbit anti-COL6A1 polyclonal antibody (cat. no. NBP1-59126) was purchased from Novus Biologicals.

Techniques: Transfection, Western Blot, Control, Cell Culture, Staining, Comparison, Knockdown

Journal: Oncology reports

Article Title: Involvement of non‑triple helical type VI collagen α1 chain, NTH α1(VI), in the proliferation of cancer cells.

doi: 10.3892/or.2020.7734

Figure Lengend Snippet: Figure 3. Effect of differently sized fractions from conditioned media on the proliferation of S2‑VP10 cells. (A) Effect of type VI collagen on the proliferation of S2‑VP10 cells. Transfection with COL6A1 siRNA#2 was the same as that shown in Fig. 2. After siRNA transfection, the indicated amount of type VI collagen (COLVI) was added to the cells, and cell proliferation was determined by methylene blue staining; data are expressed as the mean ± SD (n=3). (B‑E) Rescue activities of fractionated S2‑VP10 conditioned media on the proliferation of S2‑VP10 cells; after siRNA transfection, COL6A1#2 transfected S2‑VP10 cells were cultured for 72 h. Then, S2‑VP10 cell proliferations using DMEM supplemented with 10% FBS (negative control medium) (A), conditioned medium from S2‑VP10 cells (B), >10 kDa fraction (C), and <10 kDa fraction (D) were determined by methylene blue staining. The fractions were prepared using Amicon Ultra Centrifugal Filter Units. Data are expressed as the mean ± SD (n=3). COL6A1, collagen type VI alpha 1 chain; COL6A1#2, COL6A1#2 siRNA; fr., fraction; siCont, control siRNA; NT, no treatment.

Article Snippet: Rabbit anti-COL6A1 polyclonal antibody (cat. no. NBP1-59126) was purchased from Novus Biologicals.

Techniques: Transfection, Staining, Cell Culture, Negative Control, Control

Journal: Oncology reports

Article Title: Involvement of non‑triple helical type VI collagen α1 chain, NTH α1(VI), in the proliferation of cancer cells.

doi: 10.3892/or.2020.7734

Figure Lengend Snippet: Figure 4. Rescue activities of NTH α1(VI)‑derived peptides. (A‑C) Rescue activities of siRNA‑transfected conditioned media. Transfection with COL6A1 siRNA#2 was the same as that shown in Fig. 2. After siRNA transfection, the cells were cultured for 72 h, after which cell proliferation was determined. The cell proliferations of S2‑VP10 cells using DMEM supplemented with 10% FBS as negative control (A), control siRNA‑treated <0 kDa fraction (B), and COL6A1 #2 siRNA‑treated <10 kDa fraction (C) were determined by methylene blue staining. Data are expressed as the mean ± SD (n=3). (D) Representative immunoblot images of the indicated fraction; the fraction containing polypeptides >10 kDa (>10K) and polypeptides <10 kDa (<10K) were subjected to western blotting under reducing conditions, and probing was performed with antibody #141 (IB: #141) and anti‑COL6A1 (N‑term) antibody (IB: αCOL6A1 N term). COL6A1 is indicated by white arrowheads. (E) Coomassie Brilliant Blue (CBB) staining of the <10 kDa fraction. Acetone precipitated <10 kDa fraction was subjected to SDS‑PAGE and stained with CBB staining. Lane 1, molecular weight marker; lane 2, concentrated <10 kDa fraction. COL6A1, collagen type VI alpha 1 chain; NTH α1(VI), non‑triple helical type VI collagen α1 chain; COL6A1#2, COL6A1#2 siRNA; fr., fraction; IB, immunoblot; siCont, control siRNA.

Article Snippet: Rabbit anti-COL6A1 polyclonal antibody (cat. no. NBP1-59126) was purchased from Novus Biologicals.

Techniques: Transfection, Cell Culture, Negative Control, Control, Staining, Western Blot, Molecular Weight, Marker