Inhibition of autophagy using ATG5 siRNA potentiates the activity of cisplatin against A549cisR cells.

<p><b>(A)</b> A549cisR cells were treated for 24 hours with 25 μM cisplatin prior to transfection with ATG5 siRNA or NT siRNA control. Following two days incubation, media was removed and cells were treated with different cisplatin concentrations for an additional 24 or 48 hours. At the end of the incubation, cell viability was measured using CellTiter-Blue^® reagent. Experiments were performed in 1ûS media. The X-axis represents the percentage of cell viability at 24 hours (T24/T1) normalized to the negative control 1ûS. Error bars represent SEM from three independent experiment. p-value corresponding to the difference between ATG5 and NT siRNA-transfected cells was obtained by 2-way ANOVA. p-value corresponding to the individual difference between ATG5 and NT siRNA-transfected cells treated with the same concentration of cisplatin was calculated by the Sidak’s post-test analysis, with * annotating a p-value <0.05. <b>(B, C)</b> A549cisR cell lysates were prepared either immediately at the end of the siRNAs (ATG5 or NT) transfections or two days following the end of the transfection (annotated as 2d). Western Blot analysis was performed for the detection of ATG5, LC3, p62 and α-tubulin proteins.</p

Chloroquine potentiates cisplatin cytotoxicity against A549cisR lung cancer cells in an LMP-mediated manner.

<p><b>(A)</b> A549cisR cells treated overnight with cisplatin and chloroquine. Next day, cytosolic (cy), total protein (T) and lysosomal (ly) fractions were separated by ultracentrifugation and lysates were loaded on SDS/PAGE gels. Upon transfer, membranes were probed with specific antibodies against cathepsin D (cath D), LAMP-1 (lysosomes) and α-tubulin (cytosolic marker). <b>(B)</b> A549cisR cells were treated with 1ûS, chloroquine 100 μM, cisplatin 100 μM, or combination with or without pretreatment with E64 (10 μM). Cell viability was measured with CellTiter-Blue^®. X-axis, represent the % cell viability at 48 hours (T48/T1) normalized to the negative control 1ûS. *p<0.05 treatment condition versus 1ûS. <b>(C)</b> A549cisR cells were treated with different concentrations of cisplatin (cisPt) and CQ and pre-treated or not with E64 (10 μM) for 2 hours. Combination index (CI) was calculated as the ratio of the actual cell viability to the expected cell viability. Graph bars show the Mean±SEM from three independent experiments. *p<0.05 cells pre-exposed to E64 versus cells not pre-exposed to E64 and treated with the same concentration of cisplatin and chloroquine.</p

Chloroquine blocks autophagy in A549cisR and A549Pt cells.

<p><b>(A)</b> A549Pt and A549cisR cells were plated in 24-well plates and grown for 24 hours, then treated overnight with different chloroquine concentrations. Next, whole cell lysates were prepared and Western blot analysis was performed for LC3-II, p62 and α-tubulin. <b>(B)</b> Protein level was quantified by Western blot densitometry. Graph bars represent Mean±SEM from three independent experiments. *p<0.05 SF versus treatment.</p

A549cisR cells exhibit defective apoptosis following exposure to cisplatin.

<p>A549Pt and A549cisR cells were treated for 48 hours with 5 μM and 5 μM or 25 μM cisplatin (cisPt), respectively. Cells cultured in 1ûS were used as negative control, and cells treated with staurosporine (STP, 0.1 μM) were used as positive control. Subsequently, cell suspensions were incubated with Annexin V-FITC/PI or FITC-FMK-specific substrates for the detection of apoptosis or active caspases 3,8 and 9, respectively. The percentage of cells with increased Annexin-V/low PI staining (A), and increased caspases activity (B-D) measured by the Cellometer platform are plotted. Graph bars represent the Mean±SEM from at least three independent experiments. *p<0.05 treatment vs. 1ûS, ^# p<0.05 A549Pt vs. A549cisR for the same treatment conditions.</p

A549cisR cells exhibit increased autophagic flux following exposure to cisplatin, an effect that is less pronounced in parental cells.

<p><b>(A, B)</b> A549Pt and A549cisR cells were plated in 24-well plates and grown for 24 hours, then treated overnight with different cisplatin concentrations in 1% FBS media; whenever present, E64 (10 μM) was added for 2h prior to the cisplatin treatment. Next, whole cell lysates were prepared and Western blot analysis was performed for the indicated proteins. <b>(C)</b> Whole cell lysates of cells treated with different concentration of cisplatin without E64 were extracted and western blot was performed for p62 and α-tubulin. Levels of p62 and α-tubulin were determined by densitometry. The graph on the right panel represents the ratio of p62/α-tubulin normalized to the negative control 1% FBS in A549Pt and A549cisR cells exposed to cisplatin at concentration close to their corresponding IC₅₀ (10 μM and 50 μM respectively). Quantitative data are reported as Mean±SEM from three independent experiments. *p<0.05 A549Pt versus A549cisR. (<b>D)</b> A549Pt and A549cisR cells were treated for 48 hours with CQ (50 μM), cisplatin (5 μM for A549Pt or 25 μM for A549cisR) or a drug combination. 1ûS was used as a negative control. Following incubation with Cyto-ID green detection reagent, cell suspensions were mounted on a Cellometer imager and fluorescence was measured. Y-value depicts the fluorescence value of cyto-ID that stains autophagosomes. Graph bars represent Mean±SEM from three independent experiments. *p<0.05 1ûS vs. treatment, **p<0.05 A549Pt vs. A549cisR and ^#p<0.05 chloroquine/cisplatin combination vs. cisplatin alone. <b>(E)</b> The histogram plot generated by the Cellometer in A549cisR cells following incubation with different treatment conditions.</p

Additional file 4: of Ajuba inhibits hepatocellular carcinoma cell growth via targeting of β-catenin and YAP signaling and is regulated by E3 ligase Hakai through neddylation

Figure S4. Hakai mediates Ajuba degradation via neddylation. (A) Immunoblot analysis and quantification of the half-life of Ajuba in the presence of cycloheximide (CHX, 80 μg/ml), and in the presence or absence of MLN4924 (5 μM) in BEL7402 and HepG2 cells. GAPDH was used as a loading control. (B) Ubiquitination (Ub) assay of Ajuba in 293 T cells transfected with the indicated plasmids. (C) Neddylation assay of Ajuba in 293 T cells transfected with the indicated plasmids. IB, immnoblot. IP, immunoprecipitation. WCL, Whole-cell lysates. (JPG 103 kb

Additional file 6: of Ajuba inhibits hepatocellular carcinoma cell growth via targeting of β-catenin and YAP signaling and is regulated by E3 ligase Hakai through neddylation

Figure S6. Hakai promotes BEL7402 cells invasion and growth. (A) Representative images and quantification of invasion in GFP-tagged Hakai-overexpressing BEL7402 cells by adenovirus. Scale bar = 200 μm. (B) Analysis of the ability of Hakai-overexpressing BEL7402 cells by adenovirus to form colonies. Data are presented as Mean ± SEM from three independent experiments (**p < 0.01, ***p < 0.001). (JPG 146 kb

Additional file 1: of Ajuba inhibits hepatocellular carcinoma cell growth via targeting of β-catenin and YAP signaling and is regulated by E3 ligase Hakai through neddylation

Figure S1. The regulation of β-Catenin and cell growth in HCC cells. (A, B) HCC cells were transfected with specific siRNAs to silence GSK3β protein in Ajuba-depleted HCC cell lines. The expression of GSK3β, Ajuba, CyclinD1 and GAPDH were tested by immunoblot assay (A). β-Catenin translocation were tested by confocal assay, Scale bar = 25 μm (B). (C, D) HCC cells were transfected with specific siRNAs to silence β-Catenin protein in Ajuba-depleted HCC cell lines. The expression of β-Catenin, Ajuba, CyclinD1 and GAPDH were tested by immunoblot assay (C). Cell growth was tested by colony formation (D). (E) HCC cells were transfected with specific siRNAs to silence YAP protein in Ajuba-depleted HCC cell lines. Cell growth was tested by colony formation. Data are presented as Mean ± SEM from three independent experiments (***p < 0.001). (JPG 515 kb

Additional file 2: of Ajuba inhibits hepatocellular carcinoma cell growth via targeting of β-catenin and YAP signaling and is regulated by E3 ligase Hakai through neddylation

Figure S2. Ajuba was co-localized with Hakai in HepG2 cells. (A) HepG2 cells were co-transfected with Myc-Ajuba or Myc-Vector and GFP-Hakai for 24 h. Cells were analyzed for GFP-Hakai/Myc-Ajuba co-localization, Scale bar = 25 μm. (JPG 97 kb

Additional file 5: of Ajuba inhibits hepatocellular carcinoma cell growth via targeting of β-catenin and YAP signaling and is regulated by E3 ligase Hakai through neddylation

Figure S5. Ajuba knockdown-mediated β-catenin translocation into nucleus is not dependent on Hakai. (A, B) HepG2 cells were transfected with specific siRNAs to silence Ajuba protein in Hakai-depleted HepG2 cells. β-catenin translocation were tested by confocal assay, Scale bar = 25 μm (A). The expression of Ajuba, Hakai and β-catenin were tested by immunoblot assay, GAPDH was used as a loading control (B). (C) Immunoblot analysis of Ajuba and Hakai in BEL7402 and HepG2 cell lysis. GAPDH was used as a loading control. (JPG 617 kb