Fig 2: MPT0B291 decreases DDR gene expression and induces DNA damage.A The fold change in the expression of RAD51, CHEK1, FANCI, and FANCD2 in TCGA-GBM dataset of primary and recurrent tumor samples, and in MPT0B291-treated U87MG-R and P3-R cells. B A172, A172-R, and C P1s cells were treated with the indicated doses of MPT0B291 for 24 h, and the protein levels of RAD51, CHEK1, γH2AX, Ac-tubulin, HDAC6, and tubulin were analyzed by western blotting (upper panels). Quantitative results (normalized to tubulin) of RAD51, CHEK1, and γH2AX from three independent experiments are shown (lower panels). D U87MG cells were fixed and stained with DAPI following DMSO or MPT0B291 treatment for 24 h. The number of micronuclei (indicated by arrows) was calculated and quantified in more than 600 cells from three independent experiments. E HR assay was performed after the treatment with DMSO or indicated doses of MPT0B291. The percentage of F HR and G NHEJ efficiency was calculated as the ratio of MPT0B291 treatment to DMSO treatment and quantified after three independent experiments.

Fig 3: DARPin F10 interferes with IAV infection(A) F10 can be efficiently degraded. Immunoblotting with lysates of F10-FKBP cells treated for the indicated times with dTAG (2 µM). The leftmost lane shows the parental A549 cells. The membrane was probed with antibodies against HDAC6, a-tubulin, and F10 (HA-F10-FKBPF36V, detected with anti-HA).(B) F10 impairs IAV infection. A549 WT, F10-FKBP, or FKBP cells were infected with IAV at a MOI of 0.05 (n = 3), and culture supernatants were collected every 12 h until 72 h. Viral titer was quantified by plaque assay. Statistical analysis was done with one-way ANOVA test; p values show the significant (< 0.05) difference between cell lines expressing or not F10. Data are represented as mean ± SD.(C) Treatment with dTAG restores IAV susceptibility. The indicated cell lines (-/+ dTAG pre-treatment) were infected with IAV at an MOI of 0.05 (n = 3). Culture supernatants were analyzed as in (B); p values show the significant (< 0.05) difference between A549 WT and F10-FKBP cells. Data are represented as mean ± SD.(D) Effect of F10 on a single IAV life cycle. The indicated cell lines were infected with IAV at an MOI of 10 (n = 3), and culture supernatants were collected every 2 h up to 8 h. Viral titer was analyzed as in (C) above.(E) IAV uncoating is impaired by F10. Left panels: confocal microscopy visualization of uncoating, staining for the capsid M1 protein (green). Parental A549 or F10-FKBP cells (-/+ dTAG pre-treatment) were used for IAV infection, and M1 localization was analyzed 3.5 hpi. Bafilomycin A1 treatment was used to block endosomal acidification. Total protein was stained to visualize the cell body (red); scale bar, 20 µm. Right panel, quantification of the image analysis in the different samples (n = 3); ca. 30 cells were selected per view (six to nine views for each condition), and M1 fluorescence intensity was analyzed. Statistical analysis as in (B); p values show the significant (< 0.05) difference against untreated A549 WT cells. Data are represented as mean ± SD.

Fig 4: GCJ-490A increases histone acetylation at the IKKα promoter and promotes IKKα transcription. (A) H3K9 acetylation in the IKKα promoter region was observed by ChIP in cells treated with GCJ-490A. (B–D) Western blot for endogenous IKKα in cells with HDAC1, HDAC6, or HDAC3 knockdown. (E) Schematic diagram depicting the proposed mechanism through which GCJ-490A overcomes gefitinib resistance in NSCLC cells. All data are representative of at least 3 independent experiments and are presented as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, p65: RelA/p65, NC: negative control; #1, #2, #3 are 3 different RNAi oligonucleotides.

Fig 5: SMAR1-mediated PKM alternative splicing is HDAC6 dependent. a Expression of PKM isoforms upon shRNA-mediated knockdown of HDAC6 in MCF7 by western blot. b Expression of PKM isoforms upon SMAR1 overexpression followed by Tubacin (5 µM) treatment in MCF7 by western blot. c Confocal microscopy to check the expression of eGFP and mCherry in MCF7 cells transfected with PKM minigene along with Flag-SMAR1. Relative fluorescence intensity of eGFP and mCherry was quantified with the help of ImageJ and fold change in eGFP/mCherry ratio was calculated for control, control + Tubacin (5 µM), Flag-SMAR1 and Flag-SMAR1 + Tubacin (5 µM). Fold change in eGFP/mCherry ratio for SMAR1 overexpression and Tubacin treatment validates the role of HDAC6 in SMAR1-mediated regulation of PKM alternative splicing (n = 3). Error bars show mean values ± SD. Differences were considered statistically significant with *p < 0.05, **p < 0.01, and ***p < 0.001; ns non-significant difference (p > 0.05)