Fig 2: Flagellin colocalizes with ß2 integrin but not with Hsp90 inside the cells. THP-1 cells were stimulated with 1 µM ATRA for 24 h, followed by incubation with 100 ng/mL flagellin for 48 h. The collected cells were then fixed with formaldehyde, permeabilized with Triton X-100 and incubated with the FITC-conjugated anti-flagellin and Alexa 633-conjugated anti-Hsp90 antibodies (a) or with the anti-flagellin and Alexa 633-conjugated anti-integrin ß2 antibodies overnight at 4 °C (b). The cellular distribution of given proteins was detected by fluorescence microscope (×400). Scale bars represent 100 µm.

Fig 3: Identification of the Hsp90 interacting partners. (a) SDS-PAGE and Coomassie blue staining of proteins obtained in co-immunoprecipitation assay with anti-Hsp90a antibodies using cell lysates prepared from ATRA-stimulated THP-1 cells. Immunoprecipitated proteins (lane 1) or total cell lysates (lane 2) or unbounded proteins (lane 3) were separated by SDS-PAGE and visualized by Coomassie blue staining. Five main bands (indicated by arrows) obtained via Co-IP were eluted separately and analyzed by LC-MS/MS. (b) Cellular localization of Hsp90 interacting proteins by bioinformatic analysis. Hsp90 interacting partners identified by LC-MS/MS were analyzed for cellular localization. The identified proteins were classified based on their subcellular localization, of which 13% were plasma membrane proteins.

Fig 4: BRCA2 depletion results in increased TNFα signaling. a KBM-7 cells harboring indicated shRNAs were treated with doxycycline for 48 h and subsequently plated and treated with indicated concentrations of infliximab for 5 days. Error bars indicate s.d. of two independent experiments. b Levels of TNFα, IL-6, IL-8 and IL-10 secretion upon BRCA2 depletion. After 0, 2 or 4 days of doxycycline treatment, medium was harvested. TNFα was measured using ELISA. IL-6, IL-8 and IL-10 were measured using bead-arrays. Error bars indicate s.d. of two or three independent experiments. c Immunoblotting of BT-549 cells harboring indicated shRNAs after 0, 2 or 4 days of doxycycline treatment. Levels of p-p38, p-JNK, γH2AX, cleaved PARP (‘cPARP’) and HSP90 were analyzed. Both JNK isoforms (p46 and p54) are indicated. Dotted lines are used to indicate different shRNAs. d BT-549 cells harboring indicated shRNAs were treated with doxycycline for the indicated time periods, and analyzed by flow cytometry for p-JNK expression. Gating was performed as shown in the top panel. Numbers indicate the percentages of living cells stained positive for p-JNK. Error bars indicate s.d. of two independent experiments. e BT-549 cells were treated as described in d. Cells were analyzed by flow cytometry for cleaved PARP. Gating was performed as shown in the top panel. Numbers indicate the percentages of living cells stained positive for cleaved PARP. Error bars indicate s.d. of two independent experiments. Throughout the figure, P values were calculated using two-tailed Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001

Fig 5: AS6 regulates DNA repair, cell cycle, and apoptosis in MCF7 cells. (A) Key DNA repair genes affected by AS6. RNA-seq data showing a markedly reduced expression of ATM, p53, and BRCA1 in AS6-treated MCF7 cells. Light grey, untreated control; light pink, 0.5 µM AS6-treated cells. n = 3; **P < 0.005; ***P < 0.0005. (B) Real-time PCR data showing the expression of ATM, p53, and BRCA1 in AS6-treated HUMEC and MCF7 cells. ACTIN was used as a reference gene for a normalization. From left to right bars, untreated control HUMEC, 0.5 µM AS6-treated HUMEC, untreated control MCF7 cells, 0.5 µM AS6-treated MCF7 cells. n = 3; error bars show s.d.; **P < 0.005; ***P < 0.0001. (C) Immunoblotting results showing a markedly reduced protein expression of ATM, p53, and BRCA1 in AS6-treated MCF7 cells. ACTIN was used as a loading control. Left, immunoblots; right, quantitative presentations of the signals, normalized with the control. AS6 concentrations in µM. Signal intensity of immunoblotting results throughout this manuscript was measured by Image J. Full-length blots were presented in Supplementary Fig. S4A. (D) Key cell cycle regulators affected by AS6. RNA-seq data showing a significantly reduced expression of CDC20, BUB1B, PLK1, CHEK1, CDK1, MCM4, and CDC25A in AS6-treated MCF7 cells but not in HUMEC. White, untreated control HUMEC; purple, 0.5 µM AS6-treated HUMEC; Light grey, untreated control MCF7 cells; light pink, 0.5 µM AS6-treated MCF7 cells. n = 3; *P < 0.01; **P < 0.001; ***P < 0.0001; ****P < 0.00001. (E) Real-time PCR results confirming the reduced mRNA expression of CDC20, BUB1B, CHEK1, CDK1, and MCM4 in AS6-treated MCF7 cells but not in HUMEC. ACTIN was used as a reference gene for a normalization. From left to right bars, untreated control HUMEC, 0.5 µM AS6-treated HUMEC, untreated control MCF7 cells, 0.5 µM AS6-treated MCF7 cells. n = 3; error bars show s.d.; *P < 0.05; **P < 0.005; ***P < 0.0001. (F) Immunoblotting results demonstrating a dramatically disrupted protein expression of critical cell cycle regulators CDC20, BUB1B, PLK1, CHEK1, MCM4, CDC25A, cyclin B1, and p21 in AS6-treated MCF7 cells. ACTIN was used as a loading control. Left, immunoblots; right, quantitative presentations of the immunoblotting signals, normalized with the loading control. AS6 concentrations in µM. Full-length blots were presented in Supplementary Fig. S4B. (G) Key apoptotic factors affected by AS6. Real-time PCR results of CASP9 showing its increased mRNA expression in MCF7 cells but not in HUMEC. ACTIN was used as a reference gene for a normalization. From left to right bars, untreated control HUMEC, 0.5 µM AS6-treated HUMEC, untreated control MCF7 cells, 0.5 µM AS6-treated MCF7 cells. n = 3; error bars show s.d.; *P < 0.05. (H) Immunoblotting showing a marked increase of HIF1a protein expression (left) and the quantification of the signal intensity (right) in AS6-treated MCF7 cells. ACTIN was used as a loading control. Full-length blots were presented in Supplementary Fig. S4C. (I) RNA-seq data of HSP70 and HSP90 mRNA expression in AS6-treated MCF7 cells. Light grey, untreated control; light pink, 0.5 µM AS6-treated cells. n = 3; **P < 0.001; ***P < 0.0001. (J) Real-time PCR results validating the increased HSP70 and HSP90 mRNA expression in AS6-treated MCF7 cells but not in HUMEC. From left to right bars, untreated control HUMEC, 0.5 µM AS6-treated HUMEC, untreated control MCF7 cells, 0.5 µM AS6-treated MCF7 cells. n = 3; error bars show s.d.; **P < 0.005; ***P < 0.0001. (K) Immunoblotting showing increased HSP70 and HSP90 protein expression (left) and the quantification of the signal intensity (right) in AS6-treated MCF7 cells. ACTIN was used as a loading control. Full-length blots were presented in Supplementary Fig. 4D.