Cell surface levels of CAR, integrin heterodimers αvβ3, αvβ5 and α3β1, integrin subunits α5, αv and β1 on HEp2 and CK2 cells.

<p>Cells were detached by Versene and incubated with murine monoclonal antibodies or isotype-matched antibody as a negative control. After incubation with the secondary reagent (i.e. PE-conjugated anti-mouse antibody), labeled cells were analyzed by flow cytometry. (A). Representative histograms and (B) mean fluorescence intensities relative to HEp2 cells obtained in one of three independent experiments that provided similar results are shown.</p

Decreasing RhoB expression increases Ad5wt-mediated transgene expression.

<p>HEp2 and CK2 cells were transfected with control or RhoB siRNA. Twenty-four hours after transfection cells were replated in 96-well plates for Ad5wt-mediated transgene expression measurement and 6-well plates for Western blot. Forty-eight hours after siRNA transfection, the level of RhoB was analyzed by Western blot and cells in 96-well plates were transduced for 1 hour at 37°C with two-fold serial dilutions of Ad5wt. Twenty-four hours after infection, cells were stained for β-galactosidase expression. The transgene expression obtained with an MOI of 10⁴ pp/cell is presented. (A) Forty-eight hours post-transfection the level of RhoB protein was analyzed by Western blot, i.e. at the time of Ad5wt transduction whose results are presented in C and D. Actin was used as a loading control. A representative blot of two independent experiments is presented. (B) Densitometric analysis of Western blot presented in (A). Data are presented as relative to RhoB expression in HEp2 cells that was set as 100%. (C) Ad5wt-mediated transgene expression in HEp2 and CK2 cells transfected with control or RhoB-specific siRNAs. (D). Data shown in (C) are presented as relative to control siRNA in HEp2 and CK2 cells, respectively. The results presented in (C) and (D) are representative of three independent experiments with similar results ± standard deviation.</p

Decreasing RhoB expression by siRNA transfection does not change Ad5wt attachment or internalization.

<p>Forty-eight hours after transfection with control or RhoB-specific siRNAs, cells were incubated with Ad5wt at an MOI 1000 pp/cell for 40 minutes on ice. To measure attachment, unbound virus was removed and cells were scraped off and pelleted. To measure internalization, unbound viruses were removed and cells were incubated at 37°C for 40 minutes, trypsinized and pelleted. Total DNA (cellular plus viral) was extracted from cells and used as a sample for quantification of viral DNA by real-time PCR using a region within hexon as the target sequence. The cell-derived DNA content per PCR reaction was normalized by a second real-time PCR assay targeting the GAPDH gene. (A). Attachment of Ad5wt in HEp2 and CK2 cells transfected with control or RhoB-specific siRNAs presented as fold of HEp2. (B). Internalization of Ad5wt in HEp2 and CK2 cells transfected with control or RhoB-specific siRNAs presented as fold of HEp2. The results presented are representative of three independent experiments with similar results ± standard deviation. Asterisks indicate significant differences (*, P<0.05; **, P<0.01).</p

RhoB influences Ad5wt intracellular trafficking.

<p>Confocal microscopy of attachment and internalization of fluorescently labeled Ad5wt at MOI 4×10⁴ pp/cell in HEp2 and CK2 cells. Cells were incubated with Alexa Fluor 488-labeled virions for 60 minutes on ice. Unbound virus was removed and cells were either fixed to measure attachment or fed with fresh medium and incubated at 37°C for 30 minutes before fixation to allow virus internalization. Overlay of virus (green), nucleus stained with DRAQ5 (blue) and phase-contrast is shown. Scale bar presents 20 µm.</p

Ad5-mediated transgene expression in cisplatin-resistant CK2 cells is higher than in parental HEp2 cell line.

<p>Cells were plated in 96-well plates and, 24 hours later, infected for 1 hour at 37°C with two-fold serial dilutions of Ad5s. Twenty-four hours after infection, cells were stained for β-galactosidase expression. A similar difference was observed over a wide range of dilutions, from 2×10⁴ to 5000 pp/cell; however, only the transgene expression obtained with a MOI of 10⁴ pp/cell is presented. (A). Transgene expression in HEp2 and CK2 cells after transduction with Ad5wt, Ad5RGD4C, Ad5Δ639 and Ad5Δ639RGD4C. (B). Data shown in (A) represented as relative to HEp2 cells. The results presented are representative of three independent experiments with similar results ± standard deviation.</p

Replication defective Ad5s used in a study.

<p>Replication defective Ad5s used in a study.</p

CBP induced Grp78 and CHOP expression in HEp2 but not in 7T cells.

<p>(A) Twenty-four hours after the seeding, HEp2 and 7T cells were treated with 40 µg/mL CBP. At indicated time points the cells were collected and the expression of ER stress markers CHOP and Grp78 was determined. Expression of ERK2 was used as an internal loading control. (B) HEp2 and 7T cells were treated with 40 µg/mL CBP for 3–24 h. At indicated time points the cells were collected and the mRNA expression of ER stress markers CHOP and Grp78 was determined. Expression of GAPDH was used as an internal control.</p

mRNA expression of <i>CTR1</i>, <i>NHE1</i>, <i>ATP7A</i> and <i>MRP2</i> in HEp2 and 7T cells.

<p>The logarithmically growing cells were collected and RNA was isolated. Semi-quantitative RT-PCR and densitometry analysis of bands were performed. As an internal control for equal RNA/cDNA loading, <i>s18</i> was used. The representative of 3 independently isolated RNAs and independently performed RT-PCRs are presented. (A) influx pumps <i>CTR1</i>and <i>NHE1</i>, (B) efflux pumps <i>ATP7A</i> and <i>MRP2</i>.</p

CHOP is involved in response of HEp2 cells to CBP.

<p>(A) Parental HEp2 and CBP-resistant 7T cells were transfected with negative control siRNA (nc siRNA) or with CHOP-specific siRNA (CHOP siRNA) The expression of CHOP was determined by western blot 48 h after transfection. ERK2 was used as equal loading control. Representative blot of two independent experiments that yielded similar results is presented. (B) HEp2 and 7T cells were seeded for MTT assay 48 h after transfection with nc siRNA or CHOP siRNA and treated with CBP 24 h after. MTT assay data are representative of two independently performed experiments ±S.D. (C) HEp2 and 7T cells were transfected with nc siRNA or CHOP siRNA, plated and 24 h later treated with 40 µg/mL CBP for 48 h. Cells were stained with fluorescein diacetate and propidium iodide and examined by fluorescence microscopy. Representative data of three independent experiments are presented (mean ±SD). *p<0.05.</p

CBP induced ROS formation in HEp2 and 7T cells.

<p>(A) The logarithmically growing cell lines were treated with 40 µg/mL CBP during the indicated period of time. The cells were collected, stained with CM-H₂DCFDA and ROS was measured by flow cytometry. (B) The HEp2 and 7T cells were pretreated with 0.1 mM tempol. Two hours later different concentrations of CBP were added. The cell survival was determined 72 hours later by MTT assay. Representative data of three independent experiments are presented (mean ±SD). *p<0.05. C_ic-isotype control, C_c-untreated cells, CBP-carboplatin treated cells, H₂O₂-hydrogen peroxide treated cells.</p