Fig 1: Utt-B activates autophagy-related proteins and modulates autophagy by regulating the AMPK-mTOR signaling axis. (A) Immunoblot analysis shows that Autophagy markers Beclin1, Atg7 and Atg5 are elevated up to 24h upon Utt-B treatment (500nM) and declines at 48h in HepG2 cells. (B) Beclin1, Atg7, Atg5 elevates up to 24h upon Utt-B treatment(500nM) and decline at 48h in Hep3B cells. (C) Utt-B treatment inhibits both p-mTOR (S2448) and p-mTOR (S2481). (D) Utt-B treatment significantly down-regulates p-70S6 Kinase and p-4EBP1, the downstream targets of mTOR. (E) Utt-B completely abolishes PMA-induced activation of Akt. (F) Utt-B phosphorylates AMPK alpha. (G, H) Utt-B significantly inhibits the growth of HepG2 xenografts in NOD-SCID mice. (I) Change in Body weight of mice in control and Utt-B treatment is non-significant throughout the period of experiment (J–M) Immunohistochemical analysis shows that Utt-B administration down-regulates the activation of p-mTOR (S2448), p-mTOR (S2481), p-70S6 Kinase, and p-4EBP1, with no change in p-Akt status and up-regulates the expression of p-AMPK-alpha and the autophagy markers, Beclin 1 and LC3. ** level of significance 2, *** level of significance 3.
Fig 2: POL induces autophagy in A549 cells.A549 cells (A) and HELF cells (B) were treated with PBS (24 h) or 23 µg/mL POL for indicated time, and the MDC fluorescent intensity was analyzed by flow cytometry. (C) A549 cells were treated with PBS (24 h) or 23 µg/mL POL for indicated time, and the acridine orange fluorescent intensity was measured by flow cytometry. (D) A549 cells were treated with 23 µg/mL POL for indicated time, followed by Western blot analysis for detection of phosphorylated-Beclin1 and LC3 levels. ß-actin was used as an equal loading control. (E) A549 cells were transfected with Beclin1, LC3 or control siRNA for 24 h, the Beclin1 and LC3 levels were examined by Western blot analysis. (F) A549 cells were transfected with Beclin1, LC3 or control siRNA for 24 h followed by stimulation with or without 23 µg/ml POL for 24 h, the cell growth inhibition ratio was measured by the MTT assay (mean±SEM, n = 3).
Fig 3: Inhibition of autophagy results in reduction of apoptosis by POL.(A) A549 cells were pretreated with 2 mM 3MA 1 h prior to the administration of 23 µg/ml POL for indicated time, and the cell inhibitory ratio was measured by MTT assay. Control group was treated with PBS. (mean±SEM, n = 3) (*p<0.05, **p<0.001 vs. Control group, ns: no significant vs. Control group, #p<0.05, ##p<0.001 vs. POL group). (B) A549 cells were transfected with control siRNA and specific Beclin1 siRNA for 24 h, the Beclin1 level was examined by Western blot analysis. (C) A549 cells were pretreated with 2 mM 3MA or transfected with specific Beclin1 siRNA prior to the administration of 23 µg/ml POL for 24 h, and the MDC fluorescent intensity (a), SubG1 phage cell ratio (b) and early as well as late apoptosis (c) were analyzed by flow cytometry. Control group was treated with PBS for 24 h. Data were represented as bar diagram. (mean±SEM, n = 3) (*p<0.05, **p<0.001 vs. Control group, ns: no significant vs. Control group, #p<0.05, ##p<0.001 vs. POL group). (D) Cleaved PARP and LC3-II expressions in A549 cells treated with 23 µg/ml POL for 24 h in the absence or presence of 3MA (2 mM) were measured. The representative figures were shown. (E) A549 cells were preferentially transfected with control siRNA or specific Beclin1 siRNA for 24 h before 23 µg/ml POL treatment for another 24 h, and cleavage of PARP as well as expression of LC3II were measured.
Fig 4: Analysis of the autophagy inhibition potency of ATG5 or BECN1 knockdown. (A) The knockdown efficacy of Atg5 and Beclin-1 using siRNA was determined. Using MDA-MB435S, BT474, SKBr3, A431, SW480, MCF7, and KMST-6, a western blot was performed 48 h after siRNA transfection. 1, MDA-MB435S; 2, BT474; 3, SKBr3; 4, A431; 5, SW480; 6, MCF7; 7, KMST-6. (B) Using the same cell lines as in (A), western blot analysis of p62 was performed following transfection with ATG5, BECN1, or negative control siRNA with and without CDK4 inhibitor. Following a 48-h siRNA transfection period, CDK4 inhibitor or DMSO at a final concentration of 100 nM was administered to the cells and they were cultured for 24 h at 37°C before collection for western blotting. (C) Using the same cell lines as in (A), western blot analysis of Atg5 and Beclin-1 was performed after treatment with CDK4 inhibitor. A final concentration of 100 nM CDK4 inhibitor or DMSO was administered to the cells, and these were cultured for 24 h at 37°C before collection for western blotting. 1, MDA-MB435S; 2, BT474; 3, SKBr3; 4, A431; 5, SW480; 6, MCF7; 7, KMST-6. (D) Using the same cell lines as in (A), western blot analysis of Atg5 and Beclin-1 was performed after treatment with CQ. A final concentration of 50 µM CQ or DMSO was administered to the cells, and these were cultured for 24 h at 37°C before collection for western blotting. Lanes: 1, MDA-MB435S; 2, BT474; 3, SKBr3; 4, A431; 5, SW480; 6, MCF7; 7, KMST-6. CDK4i, CDK4 inhibitor.
Fig 5: Cell proliferation and cell cycle analysis for the combination of CDK4 inhibitor and ATG5 or BECN1 knockdown. (A) Cell proliferation analysis under the conditions as described for Fig. 4B. Cell viability just before treatment with siRNA was 100%, and the vertical axis corresponds to the absorbance ratio. The x-axis indicates the type of treatment. The y-axis indicates the proportion of cell proliferation. Values shown are mean ± SD (n=3). (A) shows the data for the cell lines MDA-MB435S, BT474, MCF7, and KMST-6. Data for the other cell lines are shown in Table V. 1, DMSO + siControl; 2, DMSO + siATG5; 3, DMSO + siBECN1; 4, CDK4i + siControl; 5, CDK4i + siATG5; 6, CDK4i + siBECN1. (B) Cell cycle analysis was performed under the conditions as described for (A). The x-axis indicates the phase of the cell cycle. The y-axis indicates the proportion of the cell population. Values shown are mean ± SD (n=3). (B) shows the data for the cell lines MDA-MB435S, BT474, MCF7, and KMST-6. Data for the other cell lines are shown in Table VI. Differences in sub-G1 phase between DMSO + siControl and CDK4 inhibitor + siControl, between DMSO + siATG5 and CDK4 inhibitor + siATG5, and between DMSO + siBECN1 and CDK4 inhibitor + siBECN1 were analyzed using t-test. Lanes: 1, DMSO + siControl; 2, DMSO + siATG5; 3, DMSO + siBECN1; 4, CDK4i + siControl; 5, CDK4i + siATG5; 6, CDK4i + siBECN1. (C) Images of flow cytometry under the conditions as shown in (B). Lanes: 1, DMSO + siControl; 2, DMSO + siATG5; 3, DMSO + siBECN1; 4, CDK4i + siControl; 5, CDK4i + siATG5; 6, CDK4i + siBECN1. CDK4i, CDK4 inhibitor.
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