Additional file 1: Table S1. of Phenotype plasticity rather than repopulation from CD90/CK14+ cancer stem cells leads to cisplatin resistance of urothelial carcinoma cell lines

Primer sequences for quantitative real-time-PCR. Sequences of primers (5′-3′) used for quantitative real-time-PCR including length of PCR product and annealing temperature. bp base pair; Fwd Forward; Rev Reverse. (DOC 48 kb

Additional file 2: Figure S2. of Phenotype plasticity rather than repopulation from CD90/CK14+ cancer stem cells leads to cisplatin resistance of urothelial carcinoma cell lines

Endogenous abundance of subpopulations from differentiation states and subsequent to short-term cisplatin treatment. Original flow cytometry data displaying abundance of CD90, CD44, and CD49f positive cells in 11 UCCs corresponding to the summarized data in Fig. 1d. Unstained cells were used to set gates for positively stained cells. Subsequent to short-term treatment with cisplatin (STT, 72 h) most cell lines displayed increased numbers of CD90+ cells as also illustrated in Fig. 4b; representative results from biological triplicates. STT: Short-term cisplatin treatment. (TIF 4559 kb

Additional file 4: Figure S3. of Phenotype plasticity rather than repopulation from CD90/CK14+ cancer stem cells leads to cisplatin resistance of urothelial carcinoma cell lines

Long-term cisplatin treated UCCs are not enriched for CD90+/CK14+ cells. CD90+, CD44+, and CD49f+ cells in untreated and LTT UCCs as measured by flow cytometry and collectively illustrated in Fig. 5c; representative results from biological triplicates. Unstained cells were used to set gates for positively stained cells. One measurement is shown for each cell line as a representative of biological triplicates. Untr. Ctrl.: Untreated Control; LTT: Long-term cisplatin treatment. (TIF 3017 kb

Additional file 3: Figure S1. of Phenotype plasticity rather than repopulation from CD90/CK14+ cancer stem cells leads to cisplatin resistance of urothelial carcinoma cell lines

CD90+ UCCs do not exhibit a distinct stem cell-like phenotype. Original flow cytometry data for abundance of CD90+ cells corresponding to the summarized data in Fig. 3. a) CD90+ fraction in unsorted (top), CD90 magnetically enriched (middle, indicated by arrow) and CD90 depleted (bottom) cell cultures. b) Following reculturing for 7–8 population doublings the number of CD90+ cells was determined again in the respective fractions. (TIF 6628 kb

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

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

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 ER stress in HEp2 and 7T cells.

<p>(A) HEp2 and 7T cells were pretreated for two hours with 12.5 µM salubrinal, and then treated with different concentrations of CBP. Seventy-two hours later cell survival was measured by MTT assay. Representative data of three independent experiments are presented (mean ±SD). *Significantly different from the respective control. * p<0.05. (B) HEp2 and 7T cells were treated with 80 µg/mL CBP or 10 µg/mL tunicamycin for 4 and 8 h. At indicated time points the cells were collected and the expression of ATF4, phospho-eIFα (p-eIFα), eIFα and XBP-1 was determined. (C) HEp2 and 7T cells were treated with 40 µg/mL CBP or 1 µg/mL tunicamycin during 16 and 24 h. At indicated time points the cells were collected and the expression of ATF4 was determined. ERK2 control was used as an internal loading control. Data of one of two experiments that yielded similar results are presented. Densitometric analysis data are expressed as a ratio between examined ER markers and ERK2. Non treated cells were set as 1.0.</p

Total and DNA-platination in HEp2 and 7T cells.

<p>(A) The logarithmically grown cells were treated with 40 µg/mL CBP. The adherent cell population was collected at the indicated time points and total cell platination was measured. (B) The logarithmically grown cells were treated with 40 µg/mL CBP and the adherent cell population was collected, DNA was isolated, and DNA-platination was measured. Representative data of three independent experiments are presented (mean ±SD). *p<0.05.</p