Western blotting to show changes in expression of key molecules in autophagic signaling pathway in GSC and SNB19 cells.

<p>Cells were treated as follows: untreated control (CTL); 2 mM SS for 24 h; 2 mM SS for 24 h + 50 nM miR-30e for 12 h; 2 mM SS for 24 h + 150 μM PAC for 24 h; and 2 mM SS for 24 h + 50 nM miR-30e for 12 h + 150 μM PAC for 24 h. Representative Western blots showed changes in expression of β-actin, Beclin-1, LC3 I and LC3 II, TLR-4, mTOR, and p62.</p

Changes in levels of the molecular components of apoptosis following transfection with miR-30e or/and treatment with PAC in the SS pre-treated GSC and SNB19 cells.

<p>Treatments: untreated control (CTL); 2 mM SS for 24 h + 50 nM miR-30e for 12 h; 2 mM SS for 24 h + 150 μM PAC for 24 h; and 2 mM SS for 24 h + 50 nM miR-30e for 12 h + 150 μM PAC for 24 h. Western blots to show changes in molecules involved in apoptosis.</p

Changes in levels of expression of Beclin-1 in GSC and SNB19 cells corresponded with the miR-30e target prediction.

<p>(A) RT-PCR and Western blotting to determine relative expression of Beclin-1 mRNA and protein, respectively, in GSC and SNB19 cells after treatment with SS, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158537#pone.0158537.g001" target="_blank">Fig 1</a> legend. (B) Quantitative analysis of Beclin-1 mRNA and protein from three independent experiments to present as bar diagrams. Significant difference between CTL and any treatment was indicated by *<i>P</i> < 0.05 and **<i>P</i> < 0.01. (C) Bioinformatics analysis showing Beclin-1 as a potential target for miR-30e.</p

MTT assay to determine the changes in residual cell viability in the 2 mM SS pre-treated GSC and SNB19 cells after transfection with 25, 50, and 100 nM miR-30e for 12 h or treatment with 50, 100, and 150 μM PAC for 24 h, or their combinations.

<p>After the treatments and incubation, alterations in cell viability were measured by the MTT assay. All experiments were conducted in triplicates and the results were analyzed for statistical significance. Difference between control (CTL, the untreated group) and a monotherapy or combination therapy was considered significant at *<i>P</i> < 0.05 or **<i>P</i> < 0.01.</p

Schematic presentation to show inhibition of SS-induced autophagy and induction of apoptosis in glioblastoma cells.

<p>miR-30e inhibited autophagy via degradation of Beclin-1 and induced apoptosis via degradation of AVEN and BIRC6. PAC exerted its anti-cancer activity through activation of caspases. Combination of miR-30e and PAC most effectively inhibited autophagy and augmented apoptosis for controlling growth of glioblastoma cells.</p

Combination index (CI) of miR-30e and PAC in GSC and SNB19 Cells.

<p>Combination index (CI) of miR-30e and PAC in GSC and SNB19 Cells.</p

The AO staining to show induction of autophagy after exposure of GSC and SNB19 cells to SS.

<p>Overnight grown GSC and SNB19 cells were exposed to 0.1, 0.5, 1.0, and 2 mM SS for 24 h. Untreated cells were considered as control (CTL). (A) Staining of cells with AO followed by fluorescence microscopy for detection of AVO in autophagic cells. (B) Flow cytometric analysis of the AO stained cells from all treatment groups for detection and determination of AVO in autophagic cells. (C) Quantitative analysis of the autophagic cell populations shown in bar diagrams on the basis of flow cytometric data. Significant difference between CTL and any treatment was indicated by *<i>P</i> < 0.05.</p

Detection and determination of apoptosis after overexpression of miR30e or/and treatment with PAC in the SS pre-treated GSC and SNB19 cells.

<p>Cells were treated as follows: untreated control (CTL); 2 mM SS for 24 h + 50 nM miR-30e for 12 h; 2 mM SS for 24 h + 150 μM PAC for 24 h; and 2 mM SS for 24 h + 50 nM miR-30e for 12 h + 150 μM PAC for 24 h. (A) <i>In situ</i> Wright staining was performed to show morphological features of apoptosis in both cell lines. (B) Determination of amounts of apoptosis based on <i>in situ</i> Wright staining. (C) Annexin V-FITC/PI binding assay followed by flow cytometry to show accumulation of apoptotic populations (A4). (D) Determination of amounts of apoptosis based on flow cytometry. All experiments were performed in triplicates. Significant difference between CTL and a treatment was indicated by *<i>P</i> < 0.05 or **<i>P</i> < 0.01.</p

(A) The role of Atg4 and the Atg12–Atg5 conjugation system in Atg8–PE formation

MKO () cells transformed with different combinations of plasmids were grown in selective SMD medium, collected at mid-log phase or 2 h after starvation, and then subjected to Western blot analysis using anti-Atg8 antiserum. 0.2 OD units of cells were loaded in each lane. Pgk1 was used as a loading control. Plasmids expressing Atg8 (pATG8(414)); Atg8, Atg4, Atg7, and Atg10 (pATG8-ATG4-ATG7-ATG10(414)); Atg5, HA-Atg12, and Atg16 (pATG5-HA-ATG12-ATG16(416)); and Atg8ΔR, Atg7, and Atg10 (pATG8ΔR-ATG7-ATG10(414)) were used as indicated. Atg8–PE was hardly detected in both growing and starvation conditions even when all the known components from the Atg8–PE and Atg12–Atg5 conjugation systems were expressed (lane 4). However, when Atg8ΔR was expressed and Atg4 was absent, a significant amount of Atg8–PE was observed (lane 5), and the amount was further increased when all the components from the Atg12–Atg5 conjugation system were also expressed (lane 6). Note that Atg8–PE migrates aberrantly during SDS-PAGE and runs lower than Atg8. (B) The role of the Atg12–Atg5 conjugation system on Atg8–PE formation. The experimental procedures were the same as in A. Plasmids expressing Atg8ΔR (pATG8ΔR(414)); Atg8ΔR, Atg4, Atg7, and Atg10 (pATG8ΔR-ATG4-ATG7-ATG10(414)); Atg8ΔR, Atg7, and Atg10; Atg5 (pATG5(416)); HA-tagged Atg12 (pHA-ATG12(416)); Atg16 (pATG16(416)); Atg5 and HA-Atg12 (pATG5-HA-ATG12(416)); and Atg5, HA-Atg12, and Atg16 were used as indicated. The strain transformed with the plasmid expressing Atg8ΔR (pATG8ΔR(414)) and the strain were used as controls (lanes 10 and 11). When Atg4 was present, an Atg8–PE band was not detected (compare lanes 3 and 4). Expression of Atg5, Atg12, or Atg16 alone did not improve Atg8–PE formation (compare lanes 5–7 to lane 4). When the Atg12–Atg5 conjugate was formed through the expression of Atg7, Atg10, Atg12, and Atg5, the efficiency of Atg8–PE formation was greatly enhanced (lane 8). Atg16 further facilitated Atg8–PE conjugation and/or enhanced the stability of the conjugate (lane 9).<p><b>Copyright information:</b></p><p>Taken from "In vivo reconstitution of autophagy in "</p><p></p><p>The Journal of Cell Biology 2008;182(4):703-713.</p><p>Published online 25 Aug 2008</p><p>PMCID:PMC2518709.</p><p></p

Localization of the cargo prApe1 in the MKO strain, and localization of the receptor Atg19 and the adaptor Atg11 in the MKO strain

The MKO () strain and the MKO () strain transformed with a plasmid expressing YFP-Atg19 (pYFPATG19(416)) or HA-tagged CFP-Atg11 (pCuHACFPATG11(414)) were grown in selective SMD medium to mid-log phase and observed by fluorescence microscopy. DIC, differential interference contrast. (B) Colocalization of prApe1, Atg19, and Atg11. The MKO () strain was transformed with a plasmid expressing YFP-Atg19, HA-tagged CFP-Atg11, or both, grown to mid-log phase and observed by fluorescence microscopy. When Atg19 was coexpressed with prApe1 in the MKO () strain, the cargo prApe1 colocalized with the receptor Atg19 (top). When Atg19 was absent, the adaptor Atg11 (arrows) did not colocalize with the cargo (middle). When prApe1, Atg19, and Atg11 were all present, the three proteins colocalized to the same structure (bottom). Bars, 2.5 μm.<p><b>Copyright information:</b></p><p>Taken from "In vivo reconstitution of autophagy in "</p><p></p><p>The Journal of Cell Biology 2008;182(4):703-713.</p><p>Published online 25 Aug 2008</p><p>PMCID:PMC2518709.</p><p></p