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. 2020 Jan 9;9:e53404. doi: 10.7554/eLife.53404

Figure 5. The effects of CTGF and CYR61 on cell size and number.

CTGF or CYR61 were neutralized by the addition of antibodies at the indicated concentration to the culture medium when cells were seeded. (A–C) In co-cultures of 6x YAP5SA: 1x nuclear mCherry expressing cells (n-mCherry), CTGF neutralization increases the n-mCherry cells’ carrying capacity (Ymax), while decreasing their rate of growth (k). (D–F) Neutralizing CYR61 in these cultures has opposing effects of larger magnitude compared to CTGF. (G–I) Treating cells expressing empty vector (WT) with 1 μg/ml of CYR61 increases Ymax and decreases k. (J–L) YAPKD cells respond to exogenous CYR61 with an increase in Ymax and a decrease in k. (M–N) Cell size is not affected by the presence of anti-Cyr61 or anti-CTGF. (O, P) Adding recombinant CYR61 at 1 μg/ml also has little effect on cell size in YAPKDs and controls. (B), (C), (E), (F), (H), (I), (K), (L) n = 5 wells; mean ± SEM. (M–P) n = 5 wells; mean ± SD).

Figure 5.

Figure 5—figure supplement 1. Validation of anti-CYR61 and anti-CTGF antibodies.

Figure 5—figure supplement 1.

(A) The primary anti-Cyr61 antibody used in our assays recognizes titrated amounts of recombinant full length CYR61 (Peprotech). (B) The anti-CTGF antibody (Peprotech) recognizes the 12 kD peptide of recombinant active form of CTGF (Peprotech). Serum-free conditioned medium from FlipinTrex cells expressing nGFP or 5SA for 6 days was concentrated using Amicon filters. Equal amounts of total protein were loaded on a gradient tris-acetate gel and transferred to a nitrocellulose membrane for further probing using the antibodies in (A and B) (C, E). No full length CYR61 or CTGF was detectable in the concentrates, just the processed forms of CYR61 (D) and CTGF (F) which appear to be post-translationally modified to various degrees as indicated by the presence of several bands of higher molecular weight. (G, H) Pure cultures of flipinTrex-293 cells treated with 10 μg/ml of anti-CYR61 have a lower Ymax compared to those treated with polycolonal rabbit IgG as a control, but k is not significantly different. n = 5, mean ± SEM. (I, J) The effect of a second anti-CYR61 antibody raised against a different epitope of CYR61 was validated to have similar effects on population growth dynamics as the antibody we use throughout the manuscript. Pure cultures of YAP5SA and nGFP FlipinTrex cells show a titratable reduction in Ymax when treated with the 2nd antibody in comparison with 10 μg/ml of the polyclonal IgG control.