Fig 1: Versican-based growth differentiation factor 11 (GDF11) trapping is required for the proliferation-promoting effects of hyaluronan and proteoglycan link protein 1 (Hapln1). (A) Western blotting assays were conducted in the cytoplasmic and nucleus fractions to explore the effect of GDF11 manipulation on HAPLN1-induced SMAD2/3 phosphorylation in hiPSC-CMs (4 weeks, (4W)). Cells were treated with 100 ng/mL Hapln1 for 48 h, with or without GDF11 (20 ng/mL) or GDF11-neutralizing antibody (anti-GDF11, 100 ng/mL). (B, C) Western blotting assays were conducted to explore the expression of (B) the human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) maturation markers (TNNI3 and MYL2), dedifferentiation markers (NKX2.5 and α-SMA) and (C) cell-cycle related proteins (P21, Cyclin D1, and Cyclin D2) in whole-cell lysate from hiPSC-CMs, as treated in Fig. 5A. (D) Western blotting assays were conducted of the cytoplasmic and nucleus fractions, to explore the effect of VCAN manipulation on HAPLN1-induced SMAD2/3 phosphorylation in hiPSC-CMs (4W). Cells with or without lentivirus-mediated VCAN knockdown were treated with 100 ng/mL HAPLN1 for 72 h. One VCAN knockdown group received both HAPLN1 (100 ng/mL) and GDF11 (20 ng/mL) treatment. (E, F) Western blotting assays were conducted to explore the expression of (E) the hiPSC-CM maturation markers (TNNI3 and MYL2), dedifferentiation markers (NKX2.5 and α-SMA) and (F) cell-cycle related proteins (P21, Cyclin D1, and Cyclin D2) in whole-cell lysate from hiPSC-CMs, as treated in Fig. 5D. (G–L) The proliferative abilities of hiPSC-CMs as treated in Figs. 5A and D, were investigated using EdU incorporation assays (G, I–J) and immunofluorescence staining of Ki67 (H, K–L). Two-way analysis of variance (ANOVA) with Bonferroni correction for post-hoc comparisons (G–J) was conducted. Data are presented as the mean ± standard deviation (n = 3). ∗P < 0.05. GAPDH: glyceraldehyde-3-phosphate dehydrogenase; rhHapln1: recombinant human hyaluronan and proteoglycan link protein 1.
Fig 2: Effect of ADAMTS1 knock down on VCAN expression in OC lines. A After 48 h post transfection of ADAMTS1 siRNA, the level of VCAN in the conditioned medium of OVCAR4, OVCAR5 and ES2 cell lines was measured by ELISA as described in the Methods. The experiment was repeated three times in triplicate. Graphs represent mean of total ± SEM. Significance is indicated by *p < 0.05, **p < 0.01 by one-way ANOVA. The mRNA expression of B G1 domain and C V0 and D V1 isoforms was analysed at the mRNA level by qRT-PCR. P are the parental cell lines; C are cells transfected with scrambled siRNA and A-KD is a representative of a pool of all three ADAMTS1 siRNAs knock down cells. The experiment was repeated three times in triplicate. Graphs represent mean of total ± SEM. Significance is indicated by *p < 0.05, and **p < 0.01, one-way ANOVA
Fig 3: Model of ADAMTS1 knock down related functional changes in OC cell lines. (a) Normal functional status for OC cells. (b) Knock down of ADAMTS1 (A-KD) in OC cell lines induces enhanced Cdc42 GTPase activity and high intracellular VCAN production. This results in cells becoming more rounded, polarised epithelial-like cells with reduced adhesion and enhanced migration. (c) Knock down of VCAN in A-KD cells reverses the effect of increased migration and reduced adhesion imposed by A-KD in OC cells
Fig 4: Effect of VCAN knockdown on the adhesion and migration of ADAMTS1 KD cells. A Adhesion and B migration in OVCAR4 and OVCAR5 cell lines treated 48 h with VCAN and ADAMTS1 siRNAs was measured as described in the Methods. B also demonstrates significance in migration at 48 h under different experimental condition. Grey bar (C) indicates cell line transfected with scrambled siRNA; red bar (A-KD) is a representative of a pool of all three ADAMTS1 siRNAs knock down cells; light green bar (V-KD) is cell line transfected with VCAN siRNA; dark red bar (C + V-KD) is cell line transfected with control siRNA + VCAN siRNA; and dark green bar (A-KD + V-KD) is a cell line transfected with ADAMTS1 siRNA and VCAN siRNA as described in the method section. The experiments were repeated three times in triplicate. Graphs represent mean of total ± SEM. Significance is indicated by *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA
Fig 5: Effect of VCAN knock down in ADAMTS1 knocked down OC cell lines. A After 48 h post transfection of VCAN and/or ADAMTS1 siRNA the levels of VCAN in conditioned medium of OVCAR4 and OVCAR5 cell lines were measured by ELISA as described in the Methods. The experiment was repeated three times in triplicate. Graphs represent mean of total ± SEM. Significance is indicated by *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA. The mRNA expression of B G1 domain picks up all isoforms of VCAN and C V1 isoform of VCAN and D ADAMTS1 was analysed in OVCAR4 and OVCAR5 cell lines treated with VCAN and ADAMTS1 siRNAs. Grey bar (C) indicates cell line transfected with scrambled siRNA; red bar (A-KD) is a representative of a pool of all three ADAMTS1 siRNAs knock down cells; light green bar (V-KD) is cell line transfected with VCAN siRNA; dark red bar (C + V-KD) is cell line transfected with C siRNA + VCAN siRNA; and dark green bar (A-KD + V-KD) is a cell line transfected with ADAMTS1 and VCAN siRNAs as described in the method section. The experiment was repeated three times in triplicate. Graphs represent mean of total ± SEM. Significance is indicated by *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way. Red asterisks indicate pairs analysed by unpaired t-test
Supplier Page from Abcam for Human Versican ELISA Kit