Fig 2: Western blot analysis of PTPRZ and sPTPRZ in microsomal fractions and CSF from glioma patients reveals 2 forms. (A) CSF samples from glioma patients A and B were treated with or without chondroitinase ABC (Chase ABC) and probed with Santa Cruz or Sigma anti-PTPRZ antibodies. (B) Schematic of the structures of PTPRZ-Long, PTPRZ-Short, and their proteolytic cleaved forms, sPTPRZ-Long and sPTPRZ-Short, respectively. Regions recognized by the anti-PTPRZ1 (Sigma), anti-PTPRZ (Santa Cruz), and Cat-315 antibodies used are indicated. (C) Microsomal fractions prepared from glioma tissues and CSF samples were treated with or without chondroitinase ABC (Chase ABC) and used for western blot analysis with anti-PTPRZ (Santa Cruz), Cat-315, and anti-Aggrecan antibodies. Cadherin and TTR were probed as loading controls for microsomal fractions and CSF, respectively. A and B are from different patients. (D) Western blot analysis of CSF samples from a glioma patient treated with chondroitinase ABC (Chase ABC) or endo-β-galactosidase and probed with anti-PTPRZ (Santa Cruz).

Fig 3: Western blot analysis shows elevated levels of sPTPRZ in the CSF of glioma patients. (A) Western blot analysis of CSF samples from patients with iNPH, glioma (glioblastoma [GBM] and anaplastic oligodendroglioma [AO]), multiple sclerosis (MS), or schwannoma (SW). Blots were probed with anti-PTPRZ (Santa Cruz; upper) or with Cat-315 antibody (lower). Transthyretin (TTR) was probed as a loading control. (B) Western blot analysis of CSF samples from control subjects (C1, facial spasm; C2, unruptured cerebral aneurysm; and C3, iNPH) and from glioma patients collected from cerebral cisterns before surgery (G1) and from ventricles after surgery (G1*) for sPTPRZ and TTR. Relative signal intensities of sPTPRZ and sPTPRZ/TTR are shown.

Fig 4: Impact on efficacy of ASO drug-mediated target reduction by glucose concentrations in a culture medium in HepG2 cells(A) HepG2 cell viability measured by the MTT assay (left) and intracellular glucose concentrations (right) after the incubation with a culture medium containing glucose at 2.5, 5.5, 10.0, and 25.0 mM for 24 h. (B) Relative TTR mRNA levels (left) and relative TTR protein concentrations in a culture medium secreted by HepG2 cells (right) after a treatment with a culture medium containing glucose at 2.5, 5.5, 10.0, and 25.0 mM for 24 h, followed by treatment with inotersen at 100 nM for 48 h. (C) TTR mRNA (left) and TTR protein (right) levels after treatment with inotersen (0, 10, 100, and 1,000 nM) for 48 h following treatment with glucose at a high concentration (25 mM) and a low concentration (2.5 mM) for 24 h. (D and E) Same treatment as (B) and (C) for the reduction of ApoB-100 by mipomersen. Data are expressed as mean ± SD (n = 3). Comparisons were made with one-way ANOVA test, followed by Tukey post hoc test; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

Fig 5: Assessment of the efficacy of inotersen-mediated TTR reduction in HepG2 cells(A) HepG2 cell viability measured by an MTT assay after treatment of 0, 10, 100, and 1,000 nM of inotersen for 48 h. (B) Dose-dependent response on the TTR reduction at mRNA levels (left) and protein levels (right) in treatment with a culture medium containing inotersen at concentrations of 0, 10, 100, and 1,000 nM for 48 h in HepG2 cells. (C) Time-dependent response on the TTR reduction at mRNA levels (left) and protein levels (right) in treatment with a culture medium containing inotersen at 1,000 nM for 0, 24, 48, and 72 h in HepG2 cells. Data are expressed as mean ± SD (n = 3). Comparisons were made between each dose and 0 and each time point and 0 with one-way ANOVA test, followed by Tukey post hoc test; ∗p < 0.05; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. TTR, transthyretin.