Shannon diversity indices of the three patients at the genus level from meta-sequencing data.

<p>Shannon diversity indices of the three patients at the genus level from meta-sequencing data.</p

Patient characteristics.

<p>Patient characteristics.</p

Taxonomic distribution of microorganisms in catheterized patients.

<p><b>(</b>A) Phyla as a percentage of taxonomic assignment in urinary catheter microbiota of the three patients across probiotic treatment and post-treatment time points based on meta-community sequence analysis. (B) Genera as a percentage of taxonomic assignment for the three patients across probiotic treatment and post-treatment time points based on meta-community sequence analysis. Only genera with an average representation of 1% or greater are shown. Error bars show standard deviations. OTUs that were not resolved at the genus level are referred to by their lowest identified phylogenetic classification and OTU number. Only phyla with a representation of at least 1% in a single individual’s catheter microbiome are shown.</p

The microbial community composition from Patient 2 during and after probiotic treatment.

<p>(A) Samples and time line for Patient 2. MDS plot of the microbial flora from Patient 2 during and after probiotic treatment based on T-RFLP (B) and Illumina sequencing data (C). The green triangle represents the sample collected in November 2011 at Month 6 of probiotic treatment. All other samples were collected post-treatment between September 2013 and February 2014. The plot also shows time points prior to and after UTI occurrence and antibiotic treatment between September and October 2013. D. Microbial community composition at the genus level for Patient 2 during and after probiotic treatment, and prior to and after UTI occurrence based on Illumina sequencing data. The sample on the left was collected in November 2011 corresponding to Month 6 of probiotic treatment. The other samples were collected at six time points in the post-treatment period, between September 2013 and February 2014. The September 2013 sample was at a time point prior to UTI. Only OTUs representing at least 1% of the total community are shown. OTUs that were not resolved at the genus level are referred to by their lowest identified phylogenetic classification.</p

MDS plot showing inter-patient differences in microbial communities of the three patients at time points of probiotic treatment and post-treatment.

<p>Data are shown for T-RFLP analysis (A) and metacommunity sequence analysis (B). The microbial flora for Patient 1 (Blue triangles), Patient 2 (Red circles) and Patient 3 (Grey squares) were plotted for each time point collected. The red arrow indicates the pre-UTI sample for patient 2. The contours group samples with a given percentage of similarity (20%, 40% and 60%) based on composition and relative abundances of taxa present.</p

The microbial community composition from Patient 3 during and after probiotic treatment.

<p>(A) Samples and time line for Patient 3. MDS plot of the microbial flora from Patient 2 during and after probiotic treatment based on T-RFLP (B) and Illumina sequencing data (C). The four green triangles represent the samples collected during the probiotic interventional period between November 2012 and April 2013, with November being the baseline time point examined. All other samples were collected at five post-treatment time points between May and October 2013. D. Microbial flora composition at the genus level for Patient 3 during and after probiotic treatment based on Illumina sequencing data. The samples collected from November 2012 to April 2013 correspond to Month 0 (Pre-treatment), and Months 3, 5 and 6 of probiotic intervention. The post-treatment samples were collected at five time points between May 2013 and October 2013. Only OTUs representing at least 1% of the total community are shown. OTUs that were not resolved at the genus level are referred to by their lowest identified phylogenetic classification.</p

α-TEA induced increased CHOP and DR5 (L/S) protein levels are JNK dependent.

<p>In turn, CHOP regulates pJNK2/1 protein levels. A. MCF-7 cells were transiently transfected with siRNA to JNK using non-specific siRNA as negative control followed by treatment with 40 µM α-TEA for 18 hrs. Western blot analyses were performed to evaluate PARP cleavage, pJNK2/1, total JNK2/1, CHOP, and DR5 (L/S) protein levels using GAPDH as loading control. B. MCF-7 cells were transiently transfected with siRNA to CHOP using non-specific siRNA as negative control followed by treatment with 40 µM α-TEA for 18 hrs. Western blot analyses were performed to evaluate pJNK2/1 protein levels using GAPDH as loading control. Data from A and B are representative of two or more independent experiments.</p

ER stress is involved in α-TEA induced apoptosis and contributes to JNK/CHOP/DR5 upregulation.

<p>A. MDA-MB-231 and MCF-7 cells were treated with 40 µM α-TEA for 9, 15, and 24 hrs. Western blot analyses were performed to determine ER stress markers; peIF-2α, total eIF2α and GRP78 protein levels. GAPDH levels were used as lane controls. B. Splicing of XBP-1, a marker for ER stress, was determined by RT-PCR. C. Both cell lines were treated with ER-stress inhibitor salubrinal at 40 µM plus 40 µM α-TEA for 18 hrs. Apoptosis was determined by Annexin V/FACS. D. Western blotting was conducted to evaluate salubrinal effects on α-TEA induced cleavage of PARP, upregulation of pJNK/2/1, CHOP, DR5 (L/S), peIF2α, total eIF2α, and GRP78 proteins. Data from A, B and D are representative of at least 2 individual experiments. Data from C are presented as the mean ± S.D. of three independent experiments. * <i>P</i><0.05 =  ER-stress inhibitor salubrinal at 40 µM plus 40 µM α-TEA treatment is significantly different from 40 µM α-TEA determined by <i>t</i>-test.</p

ER stress-dependent JNK/CHOP/DR5 upregulation is both upstream and downstream events of caspase-8.

<p>A. MDA-MB-231 and MCF-7 cells were cultured with caspase-8 inhibitor (Z-IETD-FMK) or DMSO vehicle control plus 40 µM α-TEA for 18 hrs. Apoptosis was determined by Annexin V/FACS. B. Samples (from A) were analyzed by western blot to evaluate caspase-8 cleavage of Bid to tBid, cleaved caspase 9 and PARP, pJNK2/1, CHOP, DR5 (L/S), peIF-2α and GRP78 protein levels using GAPDH as loading control. C. MCF-7 cells were transiently transfected with siRNA to JNK, CHOP and DR5, using non-specific siRNA as negative control followed by treatment with 40 µM α-TEA for 18 hrs. Western blot analyses were performed to evaluate caspase-8 and -9 cleavages, GAPDH served as lane controls. D. MDA-MB-231 and MCF-7 cells were cultured with ER-stress inhibitor salubrinal at 40 µM plus 40 µM α-TEA for 18 hrs. Western blot was conducted to evaluate caspase-8 and -9 cleavages, GAPDH served as lane controls. Data from B, C and D are representative of two or more individual experiments. Data from A are presented as the mean ± S.D. of three independent experiments. * <i>P</i><0.05 =  caspase-8 inhibitor (Z-IETD-FMK) at 2 µM plus 40 µM α-TEA treatment is significantly different from 40 µM α-TEA determined by <i>t</i>-test.</p

α-TEA decreased Bcl-2 and c-FLIP (L) protein levels via caspase-8 dependent ER stress-mediated JNK/CHOP/DR5 pathway.

<p>A. MDA-MB-231 and MCF-7 cells were treated with 40 µM α-TEA for 9, 15, and 24 hrs. Western blot analyses were performed to evaluate Bcl-2 and c-FLIP (L) protein levels with GADPH as loading control. B. MCF-7 cells were transiently transfected with JNK, CHOP, and DR5 siRNA or control siRNA followed by treatment with 40 µM α-TEA or vehicle for 18 hrs. Bcl-2 and c-FLIP (L) protein levels were determined by western blot analyses, using GAPDH as loading control. C. MDA-MB-231 and MCF-7 cells were cultured with ER-stress inhibitor salubrinal at 40 µM or caspase-8 inhibitor (Z-IETD-FMK) at 2 µM plus 40 µM α-TEA for 18 hrs. Bcl-2 and c-FLIP (L) protein levels were determined by western blot analyses, using GAPDH as loading control. D. MCF-7 cells were transiently transfected with Itch siRNA or control siRNA followed by treatment with 40 µM α-TEA or vehicle for 18 hrs. c-FLIP (L) protein levels were determined by western blot analyses. E. MDA-MB-231 and MCF-7 cells were transiently transfected with siRNA to itch using non-specific siRNA as negative control followed by treatment with 40 µM α-TEA for 18 hrs. Apoptosis was determined by Annexin V/FACS. F. MDA-MB-231 and MCF-7 cells were treated with 40 µM α-TEA for 9, 15, and 24 hrs. mRNA levels of Bcl-2 were determined by RT-PCR G. MCF-7 cells were transiently transfected with wild-type c-FLIP plasmid or vector control followed by treatment with 40 µM α-TEA for 18 hrs. Western blot analyses were performed to evaluate over expression of c-FLIP on ability of α-TEA to cleave PARP and caspase-8, and down-regulate c-FLIP (L), pJNK2/1, CHOP, DR5 (L/S), peIF-2α and GRP78 protein expression using GAPDH as loading control. H. MCF-7 cells were transiently transfected with siRNA to Itch, using non-specific siRNA as negative control followed by treatment with 40 µM α-TEA for 18 hrs. Western blot analyses were performed to evaluate elevated levels of c-FLIP (L) on ability of α-TEA to cleave PARP and caspase-8 and to down-regulate pJNK2/1, CHOP, DR5 (L/S) and GRP78 protein levels using GAPDH as loading control. Data for A, B, C, D, F, G and H are representative of two or more individual experiments. Data from E are the mean ± S.D. of three independent experiments. * p<0.05 = significantly different from control siRNA determined by <i>t</i>-test.</p