Fig 2: Genotoxic signaling in H460 cells. Cells were treated with FA for 3 h except for panel D. LMP7-i (0.5 μM) was present during and after FA treatments (p-CHK2, p-CHK1, p-KAP1, p-p53: phosphorylated forms). (A) Genotoxic signaling at 0 h or (B) 4 h recovery after FA exposure. (C) Westerns for markers of p53 activation at 4 h recovery post-FA. (D) Phosphorylation of CHK2 and p53 in cells treated with FA for 1 or 2 h. (E) Percentage of cells containing γ–H2AX foci (γ–H2AX+) and both γ–H2AX foci and EdU staining (γ–H2AX+/EdU+) (means ± SD, n = 3). (F) As panel E except that cells were fixed at 3 h post-FA exposure (means ± SD, n = 3).

Fig 3: Native gel electrophoresis of proteasomes. H460 and IMR90 cells were treated with 200 μM FA for 2 h. (A) Distribution of standard 20S proteasomes between free and 19S-capped (26 S) forms. (B) I-proteasome distribution between free (20S) and 19S-capped (26S) forms. (C) LMP7 levels in control and FA-treated cells. (D) Distribution of 19 S proteasome between its free and 26S-bound forms. (E) Proteasome binding of small activators PA28αβ (left panel) and PA28γ (right panel).

Fig 4: Cytotoxicity of FA and replication stressors in H460 cells. (A) Westerns for LMP2 and LMP7 in cells treated with FA and ONX-0914 (LMP7-i) for 3 h. (B) Westerns for cells treated with LMP7-i and MG132 (5 μM) for 4 h. (C) FACS profiles of EdU incorporation in cells treated with LMP7-i for 6 h. EdU was added during the last hour. (D) Clonogenic survival of cells treated with LMP7-i for 24 h. (E) Clonogenic survival of cells cotreated with FA and 0.5 μM LMP7-i for 3 h and then incubated for 24 h with 0.5 μM LMP7-i (means ± SD for three experiments in triplicates, *p < 0.05, **p < 0.01, ***p < 0.001). (F) Clonogenic survival of cells cotreated for 24 h with 0.5 μM LMP7-i and hydroxyurea or (F) camptothecin (means ± SD for two experiments in triplicates).

Fig 5: Genotoxic and cell cycle responses in normal human cells. IMR90 cells were treated with FA for 3 h. LMP7-i was present during FA treatments and the subsequent recovery times. EdU was added during the last 1 h of recovery incubations. (A) Dose-dependent effects of LMP7-i in cells immediately after FA treatments (p-p53: Ser15-phosphorylated p53). (B) Percentage of γ-H2AX-containing cells collected immediately or after 3 h recovery post-FA treatments (n = 3). (C) FACS profiles of EdU-labeled cells at 4 h or (D) 24 h recovery after FA removal. (E) FA-induced changes in G1 and G2 phases. Cells were treated with 100 μM FA for 3 h and collected for FACS after 24 h recovery. Statistics: **p < 0.01, n = 4.