Fig 1: Fortilin and TGF-ßs specifically interact with each other.IP, immunoprecipitation; IB, immunoblot; a-Flag, anti-FLAG antibody; a-TGF-ß1, anti-TGF-ß1 antibody; a-His6, anti-hexa-histidine antibody; ELISA, enzyme-linked immunosorbent assay; TMB, 3,3’,5,5’-tetramethylbenzidine; HRP, horse-radish peroxidase; Capture, capturing antibody; Detection, detecting antibody; SeraTGF-ß1+, fortilin+, sera generated from platelet-rich plasma; SeraTGF-ß1-, fortilin-, SeraTGF-ß1+, fortilin+ immunodepleted of both fortilin and TGF-ß1; BSA, bovine serum albumin; Ab, antibody; ABS450, absorbance at 450 nm; Data points, means ± SD; statistical analyses performed using ANOVA with Fisher’s multiple comparison; ****P < 0.001. a Fortilin interacts with TGF-ß1. Recombinant Flag-tagged fortilin, TGF-ß1, Flag-tagged p53, and His6-tagged NQO2 were incubated in binding buffer. a-fortilin antibody was used to immunoprecipitate fortilin, and immune complexes were resolved in SDS-PAGE and blotted onto a nitrocellulose membrane. Immunoblot analyses using a-TGF-ß1, a-Flag, and a-His6 antibodies showed that fortilin co-immunoprecipitated TGF-ß1 and p53 but not NQO2. Fortilin is known to interact with p53 but not with NQO2. INPUT represented 10% of the total reaction mixture used for IP. b Fortilin interacts with TGF-ß2 and -ß3. Recombinant Flag-tagged fortilin, TGF-ß2 or TGF-ß3, and His6-tagged NQO2 were incubated in binding buffer. a-fortilin antibody was used to immunoprecipitate fortilin, and immune complexes were resolved in SDS-PAGE and blotted onto a nitrocellulose membrane. Immunoblot analyses using a-Flag, a-TGF-ß, and a-His6 antibodies showed that fortilin co-immunoprecipitated TGF-ß2 and -ß3, but not NQO2. INPUT represented 10% of the total reaction mixture used for IP. c Detection of TGF-ß1 in normal human serum. The TGF-ß1 ELISA that used a-TGF-ß1 capturing and detecting Abs showed high TGF-ß1 levels in SeraTGF-ß1+, fortilin+ (column 1) compared with those in SeraTGF-ß1-, fortilin- (column 2). N = 3. d Detection of fortilin in normal human serum. The in-house fortilin ELISA that used a-fortilin capturing and detecting Abs showed high fortilin levels in SeraTGF-ß1+, fortilin+ (column 1) compared with those in SeraTGF-ß1-, fortilin- (column 2). N = 3. e Detection of the fortilin-TGF-ß1 interaction in normal human serum. The modified ELISA system that used a-TGF-ß1 capturing and a-fortilin detecting Abs yielded a robust signal in SeraTGF-ß1+, fortilin+ (column 1) compared with those in SeraTGF-ß1-, fortilin- (column 2). N = 3.
Fig 2: Rigosertib induces ROS production and activates the JNK signaling by targeting the ERO1A and NQO2(A) Human recombinant NQO2 protein was incubated with DMSO, rigosertib (2, 10, 20, or 100 μM) and imatinib (1 or 100 μM) for 10 min. The enzymatic reactions were initiated by adding 300 μM menadione as a substrate along with 500 μM NMEH as a cosubstrate. The enzymatic activity of NQO2 was determined by measuring the absorbance at 595 nm of the reduced MTT. Data are represented as a mean with SD.(B) Quantification of the NQO2 enzymatic activity immediately after incubation with rigosertib or imatinib. Shown is the result of 3 experiments and ordinary one-way ANOVA, Dunnett’s multiple comparisons test was used to determine significant differences.Data are represented as a median; ns: not significant p > 0.05, (∗) p ≤ 0.05, (∗∗) p ≤ 0.01, (∗∗∗) p ≤ 0.001, (∗∗∗∗) p ≤ 0.0001.(C) Quantification from 3 independent experiments of the live cell imaging of the effect of single ERO1A or NQO2 knock-down on ROS generation in H358 cells treated with rigosertib 2 μM. Significance was determined using two-way ANOVA test.Data are represented as a mean with SD; ns: not significant p > 0.05, (∗) p ≤ 0.05, (∗∗) p ≤ 0.01, (∗∗∗) p ≤ 0.001, (∗∗∗∗) p ≤ 0.0001.(D) Western blot of the lysates that were used for (C) to confirm the ERO1A and NQO2 knockdown.(E) Western blot analysis of the effect of rigosertib on the phosphorylated JNK levels in H358-shERO1A or -shNQO2 knockdown cells from 2 to 24 h.(F) Western blot analysis of the effect of ERO1A and NQO2 single or double knockdown in H358 cells treated with rigosertib 2 μM.(G) Comparison of the levels of phosphorylated Thr183/Tyr185 residues of SAPK/JNK between double and single knockdown of ERO1A and NQO2 after 24 h rigosertib treatment from 3 independent experiments. Two-way ANOVA, Tukey’s multiple comparisons test was used to determine significant differences.Data are represented as a median; ns: not significant p > 0.05, (∗) p ≤ 0.05, (∗∗) p ≤ 0.01, (∗∗∗) p ≤ 0.001, (∗∗∗∗) p ≤ 0.0001.
Supplier Page from Abcam for Recombinant Human NQO2 protein (His tag N-Terminus)