Fig 2: Loss of CHK2 activity is required to recover from a DNA damage-induced arrest in G1. a G1-synchronized RPE-1 cells were irradiated with a dose of 4 Gy; at the same time, BrdU and STLC were added and trypsinized at the indicated days after IR for flow cytometry analysis of BrdU-positive cells. Irradiated cells progressively entered into S-phase up to 6 days after IR. Depicted are the means and standard deviation of five independent experiments. b G1-synchronized RPE-Fucci cells were left unirradiated (-) or irradiated with a dose of 4 Gy (+); protein was harvested at the indicated hours post-damage (hpd) and analyzed by western blot. HSP90 was used as a loading control. IR induced a robust phosphorylation of CHK2 at S516 that decreased over time. c RPE-Fucci cells were trypsinized 40 hpd and arrested (expressing a red fluorofore) vs recovered (expressing a green fluorofore) cells were isolated using Fluorescence Activated Cell Sorting for subsequent western blot analysis. FACS plot indicates the gating windows of arrested (red) and recovered (green) cells. d Western blot analysis of arrested (A) vs recovered (R) cells 40 h after irradiation; pCHK2 S516 phosphorylation is lost in cells that recovered from IR

Fig 3: Internalization and protein analysis of ADSC-EXOs. (A) Confocal images of 293T, HaCAT, HUVECs, and fibroblasts incubated with 20 µg PKH26-labeled ADSC-EXOs for 24 h; scale bar: 20 µm. (B) Confocal images of co-cultured Schwann cells and fibroblasts incubated with PKH26-labeled ADSC-EXOs at different times; scale bar: 100 µm. (C) Silver staining analysis of ADSC-EXOs protein gels. (D) Illustration of the top ten high-abundance surface proteins in ADSC-EXOs. (E) Western blot analysis of HSP90 levels in ADSCs and ADSC-EXOs.

Fig 4: PU-H71 Is Cytotoxic in a Subset of AML Cells while Leaving Normal Blood Cells Unharmed(A) Growth of cells treated for 72 hr with PU-H71 (relative to untreated cells). PBL, peripheral blood lymphocytes.(B) Cells were treated with or were not treated (control) with the Hsp90 inhibitor (Hsp90i), PU-H71. Percent annexin V+ cells relative to control after 48 hr treatment with PU-H71 is shown.(C) Immunoblots showing cleaved PARP after 24 hr treatment with PU-H71.(D) Percent increase in caspase-3, 7 activity assay after 24 hr treatment with PU-H71(E) Immunoblots for Bcl-xL after 24 hr treatment with PU-H71(F and G) Percent annexin V+ cells relative to control for primary AML cells (F) or cord blood mononuclear cells (MNCs) (G) after 48 hr treatment with PU-H71. (A, B, and D) Values denote mean ± SD (n = 4).(F and G) Values denote SEM. See also Figure S1 and Tables S1 and S2.

Fig 5: Cell death induction by merosesquiterpenes in p53-mutant CRC cells: (A) HT29 cells were incubated with increasing concentrations of SP or IQ, and 5-FU was included as positive control. Cell death induction was assessed by Annexin V-FITC/PI staining and flow cytometry. Data are depicted as mean + SEM (n = 5). Not significant (ns): p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001; (B) representative dot plots of (A); (C) HT29 cells were exposed to increasing concentrations of IQ or SP for 48 h, and 5-FU was included as positive control. Samples were separated by SDS–PAGE, followed by Western blot analysis of cleaved caspase-3, cleaved caspase-9, and Bim. Hsp90 served as loading control. Representative blots are shown including the densitometric evaluation; (D) knockdown of p53 in HT29 cells. The cells were transfected with p53 siRNA or scrambled RNA, followed by treatment with 50 µM IQ and 5 µM 5-FU. Cell death induction was analyzed as described in panel A. Data are given as mean + SEM (n = 3). Not significant (ns): p > 0.05; * p < 0.05; ** p < 0.01; (E) representative dot plots of (D).