Autophagy

Effects of NVP-BKM120 and KU-55933 and their combination on the DNA damage response. A, HCC1937 cells were treated for 18 hours with NVP-BKM120 at 2.5 μmol/L, KU-55933 at 10 μmol/L, or their combination, subjected to ionizing radiation(IR) with 10 Gy or mock, lysed 6 hours later, and subjected to immunoblotting with antibodies as indicated.

Cells were stimulated with TPA (10 nM) for 15 min in the presence of the indicated concentrations of U0126. Samples were collected and analyzed by Western blot to detect phosphorylated p42/p44 MAPK.

Microscope image (406) magnification of U937 cells after exposure to SM-TNs. a) Untreated U937 cells. b) U937 cells treated with 200 nM, and 35 nM ODS empty shells. c) U937 cells treated with microcrystalline (free) Taxol prepared by sonicating Taxol in 20%H3PO4/0.8% SDS solution. d) U937 cells treated with 200 nM SM-TN. A SM-TN is seen in the middle of the picture. e) U937 cells treated with 35 nM SMTN.The nanowires are too small to see at this magnification, but their effect on the cells is clearly visible. Scale bar = 10 mm.

L3.6pl cells at 6,000 cells per well were incubated in MEM with 5% FBS in triplicate in a 96-well culture plate and then treated alone with 5 umol/L BMS-777607, 10 umol/L wortmannin, or with BMS-777607 in combination with individual inhibitors. Polyploidy was examined under BK71 Olympus microscope and photographed 72 hours after treatment.

Cells were treated with brefeldin A or manumycin A, and the resulting supernatant was collected after 48 h for exosomal preparation (lanes 1 and 2), or exosomes obtained from C81 cells were trypsin-treated or freeze/thawed (F/T) and then trypsin-treated (lanes 3 and 4). Lanes 5 and 6, input exosome controls from C81 or CEM cells, respectively. Resulting exosomes were assayed for the presence of Tax by Western blotting.

A, HeLa cells were treated with DMSO, Taxol (100 nM for 16 h), or Nocodazole (Noco, 100 ng/ml for 16 h). Total cell lysates were probed with the indicated antibodies against Hippo components on Phos-tag SDS-polyacrylamide gels. O and * mark the non-phosphorylated and phosphorylated proteins, respectively.

NCI-H929 EV and miR-137 OE cells were treated with ATM activator Chloroquine Phosphate (CQ), specific ATM inhibitor KU-55933, and ATR inhibitor AZ20 for 12 hr. Immunoblotting showed the expression of p-ATM, p-Chk2, p-BRCA1, p-ATR and p-Chk1.

b EdU incorporation assays indicated PFK15 inhibited the cell proliferation of Cal27 cells. c The quantitative data of the EdU incorporation assays.

Inhibition of autophagy advances LPS-induced accumulation of G-MDSCs in vivo and in vitro. (A-E) C57BL/6 mice were treated with SBI-0206965 (5 μg/g) or vehicle for 2 h followed by LPS challenge (10 μg/g). Percentages of CD11b+Gr-1+ MDSCs (A, C), CD11b+Ly6G+Ly6Clow G-MDSCs (B, D) and CD11b+Ly6G-Ly6Chigh M-MDSCs (B, E) in spleens of these mice were analyzed with FACS at 12, 24 and 36 hours. The data are shown as the means ± SEM (n=5 replicates/group) and are representative of three independent experiments. (F, G) Bone marrow cells were pretreated with SBI-0206965 (1 μg/ml) or vehicle for 2 h and then cultured with GM-CSF (40 ng/ml) and IL-6 (40 ng/ml). Four days later, the percentages of CD11b+Gr-1+ MDSCs (F), CD11b+Ly6G+Ly6Clow G-MDSCs (G) and CD11b+Ly6G- Ly6Chigh M-MDSCs (G) were analyzed with FACS. Data are representative from one out of three biological replicates, each with three technical replicates. Error bars represent S.E.M. * p < 0.05, ** p < 0.01, *** p < 0.001, as determined by ANOVA tests; ns denotes p > 0.05.

(c) Functional bile canaliculi or lack thereof in PHHs within MPTCs (12 d of drug treatment) as visualized by transport of fluorescent (green) dye into the canaliculi between PHHs. DMSO-treated MPCC control image is shown to the far right. (d) Neutral lipid (Nile red, green) staining of PHHs within MPTCs (12 d of drug treatment). DMSO-treated MPCC control image is shown to the far right. (e) NR1I2 (PXR) gene expression in drug-treated MPTCs relative to DMSOtreated MPTC controls (12 d of treatment). (f) ABCC2 (MRP2) gene expression in drug-treated MPTCs relative to DMSO-treated MPTC controls (12 d of treatment). (g) IL-6 levels in drug-treated MPTC supernatants (6 d of treatment). In all panels, statistical significance is displayed relative to DMSO-treated MPTCs. *p r 0.05, **p r 0.01, ***p r 0.001, and ****p r 0.0001. Scale bars on images represent 80 mm.

LC3 and DAPI immunofluorescence staining were performed to detect autophagy in VSC4.1 cells treated with 25 mM HD alone, 25 mM HD + 5 μM PIK-III, 5 μM PIK-III and 0.1% DMSO alone (control group). Scale bar: 20 μm

(D) Microglial cultures treated with 300 μM H2O2 alone or 100 μM SNAP plus 300 μM H2O2 for 24 h in the absence or presence of a chemical inhibitor (20 μM VER-155008 or 20 μM SnPP) were subjected to the determination of cell viability (MTT reduction assay). ∗p < 0.05 vs. H2O2 (300 μM) alone. +p < 0.05 vs. SNAP (100 μM) plus H2O2 (300 μM).