Fig 1.
Endocytotic responses of HTR-8/SVneo cells treated with Malmö and Prague PM.
(A-C): Fluorescence images showing PKH67 membrane stain (green), nuclei DAPI stain (blue) after 24 hr of PM exposure. Control cells show very few PKH67-stained endosomes (A, arrowhead). Cells exposed to Malmö PM show many small endosomes IB, arrowheads), whereas larger endosomes were detected in the Prague-treated cells (C, arrowheads), often in perinuclear region. Scale bars: 20 μm. (D-E): Quantification of numbers of endocytotic vesicles per cell (D) and cell number per field (E). Histograms display mean ± SEM. Significance of statistical analyses of particle-treated cultures with respective controls are indicated by: *, P<0.05; **, p<0.001; ***p<0.0001 (2-way ANOVA and t-test with Holm-Sidak correction for multiple comparison). See Supporting information S1 Fig and S1 Movie showing uptake of endocytic vesicles.
Fig 2.
Effect of PM exposure on trophoblast protein secretion.
The level of hCGβ (A, B) and IL-6 (C, D) secretion was measured in culture supernatant following exposure for 48 hours with varying doses of Prague (A, C) or Malmö (B, D) PM or PM-conditioned media. Results are expressed as ± S.D of triplicate wells. * p < 0.05. Abbreviations: Control (CTL), PM-conditioned media (S-).
Fig 3.
Differentially expressed proteins following acute Prague PM exposure.
Heatmap showing replicate expression intensities for control (CTL) and acute exposed samples with matching table showing levels of differential protein (UniProt code and description) expression (FC, fold change) following acute PM exposure (48 hr, 50 ng/ml, single dose). Shades of red and blue indicate relative levels of up- or down-regulation respectively. The Heatmap was generated with Morpheus (https://software.broadinstitute.org/morpheus).
Fig 4.
Top networks affected in trophoblast cells acute Prague PM exposure.
IPA network diagram showing combination of the top two networks (‘Amino acid metabolism, molecular transport, small molecule biochemistry’ and ‘Cell death and survival, cellular compromise, cancer’) affected in trophoblast cells exposed to Prague PM (50 ng/ml) for 48 h. Actual up- and down-regulated proteins are shown in red and green respectively, predicted activated and inhibited proteins are shown in orange and blue respectively, orange lines indicate activation, blue lines inhibition, yellow lines indicate inconsistent findings and grey lines no effect predicted. The Networks & Functional analyses were generated through the use of Ingenuity Pathways Analysis, Qiagen.
Fig 5.
Differentially expressed proteins following chronic Prague PM exposure.
Heatmap showing replicate expression intensities for control (CTL) and chronic exposed samples with matching table showing levels of differential protein (UniProt code and description) expression (FC, fold change) following chronic PM (7 day, 50 ng/ml daily) exposure. Shades of red and blue indicate relative levels of up- or down-regulation respectively. The Heatmap was generated with Morpheus (https://software.broadinstitute.org/morpheus).
Fig 6.
Top networks affected in trophoblast cells chronic Prague PM exposure.
IPA network diagram showing combination of the top two networks (‘Cellular development, cellular growth and proliferation, connective tissue development and function’ and ‘Cell-to-cell signalling and interaction, drug metabolism, molecular transport’) affected in trophoblast cells following chronic Prague PM (7 days, 50 ng/ml daily) exposure. Actual up- and down-regulated proteins are shown in red and green respectively, predicted activated and inhibited proteins are shown in orange and blue respectively, orange lines indicate activation, blue lines inhibition, yellow lines indicate inconsistent findings, and grey lines no effect predicted. The Networks & Functional analyses were generated through the use of Ingenuity Pathways Analysis, Qiagen.