Fig. 1. LNA043 induces chondrogenesis and DKK1, PRG4 and collagen type II secretion, but not angiogenesis.

a, Left, PRG4 production by 3D hMSCs. PRG4 IHC staining after 28 d of LNA043 treatment. Green, PRG4; blue, DAPI. Comparable effects of LNA043 were detected in hMSCs from four (out of four) donors. Scale bar, 100 μm. Right, PRG4 secretion, as determined by ELISA, generated from one donor in quadruplicate (one representative experiment shown out of eight). b, DKK1 secretion by 3D hMSCs. The accumulation of DKK1 between days 8 and 11, 15 and 18 and 22 and 25 was quantified by ELISA (one representative experiment shown out of three; n = 3 biological replicates). Comparable effects of LNA043 were detected in hMSCs from two (out of four) donors. c, Osteogenic and adipogenic marker expression in 3D hMSCs. ALPL (left) and LEP expression (middle) were determined by quantitative reverse transcription PCR (one representative experiment shown out of six; n = 3 biological replicates). Right, LEP secretion as determined by ELISA (one representative experiment shown out of four; n = 6 biological replicates) following 28 d of LNA043 treatment. Comparable LNA043 effects on ALPL, LEP and LEP levels were detected in hMSCs from four (out of four) donors. d, DKK1 secretion by C-28/I2 cells, as determined by ELISA, following 24 h culture with LNA043 (one representative experiment shown out of >10; n = 3 biological replicates). e, DKK1 secretion by C-28/I2 cells, as determined by ELISA, following 24 h culture in the presence of LNA043 (23 μM = ~600 μg ml⁻¹), ANGPTL3, ANGPTL2 or ANGPTL4 one representative experiment shown out of two; n = 2–3 biological replicates). Single, double and triple crosses represent the first signs of toxicity, few intact cells remaining and many particles or cell debris present, respectively. The toxicity scores are based on cell morphology (bright-field microscopy). f, PRG4 (left) and collagen type II increase (right) in the supernatants of human OA cartilage explants, as determined by ELISA (n = 15; from two donors). One half of each explant was cultured with LNA043, while the other half was cultured with vehicle for 24 h. g, LNA043 (left), unlike ANGPTL3 (right), does not induce angiogenesis. The cumulative sprout length (CSL) was measured in HUVEC spheroids incubated with LNA043, ANGPTL3, VEGF-A (25 ng ml⁻¹) or bFGF (25 ng ml⁻¹) for 24 h. The data are representative of three independent experiments. Each individual data point per experiment represents the mean CSL of ten randomly selected spheroids and is expressed as a percentage of basal control. In a–g, the values represent means ± s.d. Statistical significance was determined by one-way ANOVA (a, b, d, e and g), one-way ANOVA performed on each day separately (c) or two-tailed paired t-test (f). P values are provided in Extended Data Table 1 (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).