Fig 2: Higher NOTCH1 expression is evident in proliferating chondrocyte cultures. (a) To assess NOTCH1 expression and modulation in human chondrocyte cultures, the active form of the receptor was evaluated in monolayer culture and in micromasses at 1 week maturation. GAPDH was used as a loading control. A representative example and a cumulative evaluation performed with samples from three different patients. * p < 0.05. (b) Western blot analysis of NOTCH1 expression in C28/I2 cells compared to primary chondrocytes, with β-actin as a loading control.

Fig 3: Effects of NOTCH1 silencing in the differentiation of chondrocytes in 3-D cultures. (a) NOTCH1 silencing impacts on RUNX2 expression: left, a representative Western blot indicates that RUNX2 protein is significantly reduced in NOTCH1 silenced 3-D cultures at 1 week as confirmed in the cumulative assessment shown in the lower graph (n = 5, * p < 0.05); right, immunohistochemistry imaging of the correlated reduced expression of RUNX2 and HES1 in micromass maturation across 1–3 weeks. (b) NOTCH1 silencing impacts on ECM maturation. Left, GAG deposition at 1, 2, and 3 weeks as evidenced by toluidin blue staining: GAG deposition resulted delayed in N1 KD 3-D constructs. Right: Alizarin Red staining on micromasses showed a markedly reduced calcium deposition at all time points in N1 KD micromasses. All images were obtained with a NIKON Eclipse 90i microscope equipped with 10× objective lens.

Fig 4: NOTCH1 mutations in CLL.A) Position of NOTCH1 mutations, superimposed on a cartoon showing the NOTCH1 protein. NRR, negative regulatory region; RAM, RAM domain; ANK, ankyrin repeat domain; TAD, transcriptional activation domain; PEST, degron domain. B) Representative confirmatory pyrosequencing results in CLL cases positive by deep sequencing for the NOTCH1 codon 2514 del(CT) mutation. Results for a case with wild type codon 2514 sequences is shown in the upper panel. The lower two panels are cases in which deep sequencing revealed high (54.1%) or low numbers of mutated reads (3.3%). Reverse-sequences of the wild type (WT) and mutant (MUT) del(CT) alleles are shown for reference. “E” corresponds to the addition of enzyme to the reaction chamber, while “S” corresponds to the addition of the substrate. In this sequencing by synthesis assay, nucleotides are added to the reaction chamber sequentially in the order shown. Nucleotide incorporation releases pyrophosphate, which catalyses a reaction in which the number of photons produced is proportional to the number of nucleotides incorporated. The codon 2514 del(CT) mutation causes the appearance of a new signal corresponding to the incorporation of 3 G residues.

Fig 5: Relationship of NICD1 staining, NOTCH1 genotype, and karyotype in CLL.A) Correlation between the fraction of mutated NOTCH1 exon 34 reads and NICD1 staining. B) NICD1 staining in CLLs with and without mutated NOTCH1 alleles. Filled squares correspond to CLLs with the NOTCH1 codon 2514 del(CT) mutation; black squares are tumors with a “high” fraction (>20%) of mutated reads, while gray squares are tumors with a “low” fraction (<5%) of mutated reads. The open square corresponds to a CLL with a nonsense mutation in codon 2444. C) Relationship of NICD1 staining to CLL karyotype. Open squares correspond to tumors with NOTCH1 mutations.