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. 2023 Sep 15;133(18):e159181. doi: 10.1172/JCI159181

Figure 3. ColXVIII promotes BC cell proliferation through its N-terminal domain.

Figure 3

(A and B) Representative immunoblots of ColXVIII in human BC and mammary epithelial cell lines. (A) In cell lysates, the size of the major ColXVIII band of approximately 180 kDa corresponds to the core polypeptide of the short isoform (60). (B) JIMT-1 and MDA-MB-231 cells secreted high amounts of glycosylated ColXVIII, which appears as a broad smear over 250 kDa. In A and B, biological replicates: lysates, n ≥5; culture media, n = 3. (C) Relative expression of the short, medium, and long COL18A1 mRNA transcripts normalized to GAPDH in human BC cell lines (n = 3). The primer pairs are listed in Supplemental Table 5. (D) qRT-PCR analysis of total COL18A1 mRNA after its KD in BC cell lines (n = 6). (E and F) Examples of immunoblots of ColXVIII protein levels in various KD cell lines and corresponding scrambled controls (S). n ≥3 biological replicates for each cell line. In A, E, and F, the loading control was β-tubulin. (G) Confluence of ColXVIII-KD versus scrambled cell lines (percentage), measured by an IncuCyte live-cell analysis system for 96 hours (n = 9). (H–J) Confluence of KD cell lines after administration of recombinant NC ColXVIII fragments (500 ng/mL) to the KD cells (n = 3). Untreated scrambled cell lines are shown as controls. TSP-1, TSP-1 domain; NC11, full-length N-terminal NC. Data in C, D, G, and H–J are presented as the mean ± SD. **P < 0.01, ***P < 0.001, and ****P < 0.0001, by 2-tailed Student’s t test (C, D, and G) and 2-way ANOVA with Dunnett’s multiple-comparison (treated vs. ColXVIII-KD) (H–J).