Fig 1.
Effect of rs1561570 in osteoclast differentiation.
(A) The osteoclast differentiation rate (displayed as a ratio between multinucleated osteoclasts (Multi OCL) and TRAP positive (+) cells) was higher in patients with PDB carrying at least one T allele (Patients CT/TT vs Controls CC p-value < 0.01; Patients CT/TT vs Patients CC p-value < 0.05). (B) Osteoclasts from patients carrying at least one T allele contained significantly more nuclei than osteoclasts from healthy controls (Patients CT/TT vs Controls CC p-value < 0.001; Patients CT/TT vs Patients CC p-value < 0.01). (C) The images are representative of the distribution of nuclei number observed in osteoclasts generated from PBMCs derived from healthy controls and PDB patients that were stained for TRAP and counterstained with haematoxylin. (ANOVA, * represents a p-value < 0.05, ** represents a p-value < 0.01, *** represents a p-value < 0.001, **** represents a p-value < 0.0001; non-mutated controls with CC genotype (n = 3), non-mutated patients with CC genotype (n = 5) and patients carrying at least one T allele (n = 3).
Fig 2.
Effect of rs1561570 in bone resorption.
(A) Representative images of in vitro bone resorption assays (left) with (B) quantification of the results (right) by using ImageJ. The bone resorption area was higher in patients with PDB carrying at least one T allele (Patients CT/TT vs Controls CC p-value < 0.001; Patients CT/TT vs Patients CC p-value < 0.01). This result was even more evident in one PDB patient carrying one T allele (CT genotype) plus the SQSTM1/P392L mutation (p-value < 0.001). At least three different wells per patient were analysed. (ANOVA, * represents a p-value < 0.05, ** represents a p-value < 0.01, *** represents a p-value < 0.001, **** represents a p-value < 0.0001; non-mutated controls with CC genotype (n = 3), non-mutated patients with CC genotype (n = 5) and patients carrying at least one T allele (n = 3)).
Fig 3.
Rs1561570 effect in methylation and OPTN expression.
(A) In silico prediction using Methprimer tool showing a methylation site only in the presence of the allele C (not the allele T). (B) Sequencing results showing that rs1561570 C allele after bisulfite treatment remains a C (arrow head), while other C nucleotides that are not methylated change to T (asterisk). (C) Analysis of OPTN gene expression in lymphocytes from several healthy controls (CC genotype n = 10, CT/TT genotypes n = 6) and PDB patients (CC genotype n = 10, CT/TT genotypes n = 22) with the three genotypes. The levels of OPTN gene expression were measured by qPCR normalized to PPIB gene expression levels. Values are the mean of two replicates in each experiment and the experiment was repeated at least three independent times. (D) Quantification and (E) representative blots of OPTN protein expression in lymphocytes from non-mutated controls with CC genotype (n = 5), and patients carrying at least one T allele (n = 19). (F) Quantification and (G) representative blots of OPTN protein expression in osteoclasts from non-mutated controls with CC genotype (n = 3), non-mutated patients with CC genotype (n = 5) and patients carrying at least one T allele (n = 3). The levels of OPTN protein expression were measured by western blot analysis and related to levels of α-Tubulin (t-test and ANOVA, * represents a p-value < 0.05). The figures are representative of all the western blot analyses performed (n = 3).
Fig 4.
Effect of rs1561570 in NF-κB localization and in the expression of NF-κB target genes in osteoclasts of patients with PDB.
(A) NF-κB localization in osteoclasts derived from healthy controls and patients PBCMs showing an increase of the translocation of NF-κB into the nucleus, in patients with genotype CT and TT. This was also confirmed by co-localization ratio between NF-κB and DAPI–the nuclear staining. Five different fields of view were analysed per sample and at least three different samples were analysed per patient. Co-localization coefficient was calculated by Volocity software as the ratio of NF-κB staining related to DAPI. (B) Analysis of NFATc1, IL6, TRAP and ELK1 gene expression in several healthy controls (n = 16) and PDB patients (CC n = 10, CT/TT n = 22) with all genotypes. The levels of NFATc1, IL6, TRAP and ELK1 expression were measured by qPCR related to levels of PPIB. Values are the mean of at least three independent replicates. (t-test, * represents a p-value < 0.05, ** represents a p-value < 0.01).
Fig 5.
In vitro effect of OPTN demethylation in NF-κB localization and expression of NF-κB target genes.
(A) NF-κB localization in U937 cells (CC genotype). (B) NF-κB localization in T47D cells (CT genotype), following 5-Azacitidine (5-Aza) treatment. After OPTN demethylation, NF-κB was translocated exclusively to the nucleus. (C) Analysis of NF-κB target genes expression. The levels of OPTN, NF-κB and NF-κB target genes (IL-6, ELK1, NFATc1) expression were measured by qPCR related to levels of GAPDH gene. Values are the mean of at least three independent replicates. (t-test, * represents a p-value < 0.05, ** represents a p-value < 0.01 *** represents a p-value < 0.001, **** represents a p-value < 0.0001).