Dataset_06_08_2017

There are five spreadsheets in this excel file. The spreadsheets "Microcosm soil A”, “Microcosm soil B” and “Environmental gradient" contain the raw data used in this paper. The spreadsheets "Microcosm metadata” and “Env. gradient metadata" contain the associated metadata, where a description of all the variables and units can be found

Bortezomib and AG 490 induce immunogenic BC3 cell death.

<p>Induction of HSP90, CRT and HSP70 traslocation on BC3 cell surface induced by Bortezomib, AG 490 or both after 12 hours (a) or 24 hours (b) treatments was evaluated by flow cytometric analysis. Mean of the percentage of positive cells plus SD of three independent experiments is indicated. Plasma membrane localization of CRT after Bortezomib, AG 490 or Bortezomib plus AG 490 treatments was also analyzed by immunofluorescence on BC3 fixed with 4% paraformaldehyde in PBS for 30 min and the arrows indicate the presence of CRT on the surface of the cells (c). One representative experiment out of three is shown. The effect of Z-VAD-fmk pre-treatment (100 µM) before exposure to Bortezomib and AG 490 on HSP90, CRT and HSP70 surface expression was evaluated by flow cytometric analysis. Mean of percentage of positive cells plus SD of three independent experiments is indicated (d). Western blot analysis showing the total HSP90 and 70 expression after 24 hours treatment of BC3 with Bortezomib, AG 490 or both (e–f). Results are representative of two independent experiments.</p

Bortezomib and AG 490 induce apoptosis in BC3 PEL cell line.

<p>PEL cells were exposed to Bortezomib, AG 490 or both for the indicated times, then resuspended in Annexin V binding buffer and stained with Annexin V (a). The effect of the drugs was also evaluated on nuclear fragmentation (sub-G₀/G₁ phase) that represent the apoptotic cells. Cells were fixed in ethanol/water (70/30, vol/vol), washed in PBS containing RNAse and then stained with Propidium Iodide (PI) (b). The analysis was performed by flow cytometry. The reduction of the percentage of Annexin V positive (c) or the sub-G₀/G₁ cells (d), obtained with Z-VAD-fmk pre-treatment at 100 µM, is also shown. The experiments were performed simultaneously with 1a and 1b at 24 hours. Mean plus SD is indicated.</p

Bortezomib induces autophagy in JNK-dependent manner.

<p>(A) BC3 and BCBL1 cells were treated with bortezomib (20 nM for 16 hrs) with or without pre-treatment with JNK inhibitor (SP600125) (20 µM). Total cell lysates were prepared and immunoblotted with the following antibodies: anti-LC3, anti-pJNK, anti-T-JNK and anti-p62. Anti-β-actin was used as loading control. The histograms indicate LC3-II/I and LC3-II/Actin ratio based on densitometric analysis (mean ± the standard deviation, n = 3 experiments). (B) BC3 cells were transfected with DN-JNK expression vector or with an control empty vector (CV) and after 16 hrs treated with bortezomib (20 nM) for an additional 16 hrs. Total cell lysates were prepared and immunoblotted with anti-LC3 and p62 antibodies. β-actin was used as internal control. The histograms indicate LC3-II/I and LC3-II/Actin ratio based on densitometric analysis (mean ± the standard deviation, n = 3 experiments). (C) BC3 and BCBL1 cells were treated with Bortezomib (20 nM) alone or in combination with JNK inhibitor (SP600125) (20 µM) or ERK inhibitor (PD98059) (10 µM) for 16 hrs. A western blotting was performed using the following antibodies: anti-pBcl2(S70) and anti-total Bcl2. β-actin was used as loading control.</p

Bortezomib treatment induces ER stress in PEL cell lines.

<p>(A) Time course assay was performed on BC3 and BCBL1 cells treated with 20 nM of bortezomib (BZ) for 0, 4, 8, 16 hrs. Total cell lysates were prepared and immunoblotted with anti-BiP, anti-CHOP, anti IRE1α, anti-phospho-JNK (p-JNK 1/2) and t-JNK antibodies. Anti-tubulin was assayed as protein loading control. (B) Electron microscopy (EM) analysis was performed on BC3 and BCBL1 cells treated with Bortezomib (20 nM) for 16 hrs (BZ). Cells showed several signs of ER stress compared to untreated control (CT). More apoptotic features such as nuclear (N) condensation were observed in BCBL1. Er (Endoplasmic reticulum), G (Golgi Apparatus), N (Nucleus), Au (Autophagosomes), Ne (Nuclear Envelope), M (Mitochondria). Bars: 1 µm. Results are representative of three independent experiments.</p

Bortezomib induces autophagy in PEL cells.

<p>(A) Electron microscopy (EM) on BC3 and BCBL1 cells treated with 20 nM of bortezomib after 16 hrs shows pronounced cell vacuolization (A a, a₄) with autophagosome membrane-bound compartment in different stage of maturation (A a_1, a₂, a_4, a₅). Small intracellular structures consistent with aggresomes were evident in BCLB1 cells (A a₆). N (Nucleus), Au (Autophagosomes), M (Mitochondria), ag (aggresomes). Bars 1 µm. Results are representative of three independent experiments. (B) PEL cells were treated with bortezomib (20 nM), bafilomycin A (Baf) (10 nM) or combination of both. The western blot analysis was performed to detect the two isoforms of LC3 protein. The histograms indicate LC3-II/I and LC3-II/Actin ratio based on densitometric analysis (mean ± the standard deviation, n = 3 experiments). An antibody against β-actin was used as loading control.</p

Inhibition of autophagy and JNK increases the Bortezomib anti-proliferative effect in BC3 and BCBL1 PEL cells.

<p>(A) Viability assay evaluated by trypan blue exclusion was performed in BC3 (left panel) and BCBL1 (right panel) cells treated with bortezomib (20 nM) or 3-MA (5 mM) alone or in combination for 16 hrs. Mean ± the standard deviation was also indicated (n = 3 experiments). * p-value  =  0.05, ** p-value  =  0.02. Western blotting analysis was performed on BC3 cells to evaluate the expression of cleaved (cl) PARP p85 fragment (middle panel). β-actin was used as loading control. (B) BC3 and BCBL1 cells were transfected with ATG5 siRNA or scramble siRNA (siRNASc), and than a western blot was performed with the anti-ATG5 antibody. β-actin was used as loading control. Viability assay evaluated by trypan blue exclusion was performed in BC3 and BCBL1 cells ATG5 or scramble-knocked down upon bortezomib treatment (20 nM) for 16 hrs. Mean ± the standard deviation was indicated (n = 3 experiments). ♦ p-value  =  0.02, ♦♦ p-value  =  0.03. Western blotting analysis was also performed on BC3 cells to evaluate the expression of PARP p85 fragment (cl PARP) and β-actin was used as internal control. (C) Cells viability assay on BC3 and BCBL1 cells treated with bortezomib (20 nM) and SP600125 (20 µM) alone or in combination for 16 hrs. The percentage of live cells was evaluated by trypan blue exclusion assay. Mean ± the standard deviation was indicated (n = 3 experiments). p-value  =  0.01, p-value  =  0.01. Western blotting analysis was performed on BC3 cells to evaluate the expression of PARP p85 fragment (cl PARP) and β-actin was used as loading control.</p

MiR-200c-3p contrasts PD-L1 induction by combinatorial therapies and slows proliferation of epithelial ovarian cancer through downregulation of β-catenin and c-myc

Conventional/targeted chemotherapies and ionizing radiation (IR) are being used both as monotherapies and in combination for the treatment of epithelial ovarian cancer (EOC). Several studies show that these therapies might favor oncogenic signaling and impede anti-tumor responses. MiR-200c is considered a master regulator of EOC-related oncogenes. In this study, we sought to investigate if chemotherapy and IR could influence the expression of miR-200c-3p and its target genes, like the immune checkpoint PD-L1 and other oncogenes in a cohort of EOC patients’ biopsies. Indeed, PD-L1 expression was induced, while miR-200c-3p was significantly reduced in these biopsies posttherapy. The effect of miR-200c-3p target genes was assessed in miR-200c transfected SKOV3 cells untreated and treated with olaparib and IR alone. Under all experimental conditions, miR-200c-3p concomitantly reduced PD-L1, c-Myc and β-catenin expression and sensitized ovarian cancer cells to olaparib and irradiation. In silico analyses further confirmed the anti-correlation between miR-200c3p with c-Myc and β-catenin in 46 OC cell lines and showed that a higher miR-200c-3p expression associates with a less tumorigenic microenvironment. These findings provide new insights into how miR-200c-3p could be used to hold in check the adverse effects of conventional chemotherapy, targeted therapy and radiation therapy, and offer a novel therapeutic strategy for EOC

Dataset of "Unearthing the soil-borne microbiome of land plants"

We described the soil-borne microbiome of land plants</p