Fig 2: Pluripotent factors Oct4 and Sox2 regulate Set7 expression in mESCs. (A) Schematic illustration of the Set7 proximal promoter region. Mouse Set7 gene is located on chromosome 3qC. Putative binding sites of three transcription factors; CTCF (R1 at -11 to -132 bp), OCT4/SOX2 (R2 at -149 to -303 bp), STAT3 (R3 at -277 to -381 bp) and negative control region (R4 at -880 to -1018 bp) are located upstream of the transcriptional start site (TSS) of the Set7 (Setd7) gene. (B) Identification of the Oct4 and Sox2 binding element necessary for Set7 transcription in mESCs. The reporter constructs (left panel) were generated and transfected into mESCs. Luciferase activity was measured using the FLUOstar Omega (BMG Labtech). Data are expressed as the relative response ratio (RRR%) that quantifies the ratio of the Luciferase firefly luminescence/Renilla luminescence of the experimental reporter activity relative to the control reporter activity. (C) Deletion of the Oct4/Sox2 binding site activates Set7 transcription. Reporter constructs (1.3, 1.0, 0.7 and 0.3) were deleted for the Oct4/Sox2 binding sites and transfected into mESCs. Luciferase activity was normalized against cells transfected with the reporter constructs with wild-type (WT) Set7 promoter. (D) Reduced binding of OCT4 and SOX2 on the Set7 gene promoter following LIF removal. Soluble chromatin was isolated from mESCs stimulated with (black) or without LIF (white) for 4 days, and immunopurified with antibodies that recognize CTCF, OCT4, SOX2, STAT3, H3K4me3 and H3K27me3. qPCR was performed to assay quantitative binding relative to the IgG-negative control. The level of enrichment is shown as the percentage of corrected input. (E) Knockdown of OCT4 reactivates Set7 expression. mESCs were transfected with siRNA targeted to the Oct4 transcript. Immunoblot analysis was performed using anti-OCT4, SOX2, SET7 and GAPDH antibodies (left panel). mRNA levels of Set7, Oct4, Sox2 and Nanog genes were quantified by qRT-PCR using Gapdh as the internal control (right panel). Gene expression was normalized against cells transfected with non-target control (NTC). (F) Overexpression of OCT4 and SOX2 down-regulates Set7 gene expression. Sca1+ cells were transfected with HA-tagged OCT4 (HA-OCT4) and SOX2 (HA-SOX2) constructs followed by protein and RNA isolation after 48 h. Immunoblot analysis was performed using anti-HA, OCT4, SOX2, SET7 and GAPDH antibodies (left panel). Set7 gene expression was assessed by qRT-PCR using Gapdh as the internal control (right panel). Gene expression was normalized against the vector control. Error bars represent the standard error of the mean from three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001.

Fig 3: Biofilm formation by Candida species. Candida bloodstream isolates were evaluated for biofilm formation with standardized methods. Candida isolates standardized (1 × 106 cells/mL) in RPMI-1640 were grown in flat-bottomed 96-well microtitre plates for 24 h at 37°C. Mature biofilms were carefully washed with phosphate-buffered saline and allowed to air dry, and biomass quantified by staining with 0.05% w/v crystal violet solution. The biofilms were washed and destained with 100% ethanol. Biomass was quantified spectrophotometrically by reading the optical density (OD) at 570 nm in a microtitre plate reader (FluoStar Omega BMG Labtech, Aylesbury, UK). Six replicates were used for each isolate, and the mean of each is represented. ***p <0.0001.

Fig 4: Biofilm formation on Candida species isolated from patients with persistent and non-persistent candidemia. Candida bloodstream strains isolated from all included patients were evaluated for biofilm formation with standardized methods. The first Candida spp. isolate was conducted for biofilm formation evaluation when multiple isolates were collected from some of these patients within the study period. Candida isolates standardized (1 × 106 cells/mL) in RPMI-1640 were grown in flat-bottomed 96-well microtiter plates for 24 h at 37°C. The biomass quantified by staining with 0.05% w/v crystal violet solution. Biomass was quantified spectrophotometrically by reading the optical density (OD) at 550 nm in a microtiter plate reader (FluoStar Omega BMG Labtech, Aylesbury, United Kingdom). Three replicates were used for each isolate. The outlier was abandoned and the average of the 2 remaining values was represented. Higher biofilm formation in C. albicans, C. tropicalis, and C. glabrata were found among isolates in the persistent candidemia group compared with those in the non-persistent candidemia group.

Fig 5: Analysis of the temporal response of the nunF promoter to rhizosphere-associated carbon sources using a nunF promoter mCherry reporter strain. A nunF promoter fusion to mCherry was constructed and used as a reporter system in P. fluorescens In5 for monitoring nunF gene expression in vitro. Pseudomonas fluorescens In5 reporter strain (pSEVA237R::PnunF::mCherry) and the control strain (pSEVA237R::mCherry) were screened in a 96-well microtiter plates with varying rhizosphere-associated carbon sources. Dashed lines represent fungal-associated carbon sources and nondashed show plant-associated carbon sources with glucose control in black. Growth and fluorescence of mCherry was measured every hour for 48 hr in FLUOstar Omega Microplate Reader (BMG LABTECH). Graph shows mCherry signal from the reporter strain (pSEVA237R::mCherry) over the control strain (empty vector; (pSEVA237R::mCherry)