Fig 1.
Heavy metal induced HSR in C5 cells.
(A) C5 cells were treated with the indicated concentrations of CdSO4 in DMEM complete for 1 h. Then the cells were washed once and recovered in DMEM complete for different time points (2, 6, 24, 48 h). Y-axis shows relative luciferase activity compared to untreated control cells. (B) C5 cells were treated with CdSO4 (Cd, 50 μM), CuSO4 (Cu, 3.5 mM) or HgCl2 (Hg, 75 μM) in DMEM complete for 1 h. Then the cells were washed once and recovered in DMEM complete for 6, 24 or 48 h. Y-axis shows relative luciferase activity compared to untreated control cells. (C-E) For viability assay with flow cytometry, HEK293 cells were treated with different concentrations of CdSO4 in DMEM complete for 1 h. Samples were taken after 6 h and 24 h, then stained with AnnexinV-FITC and 7-AAD. Y-Axis shows percentage of viable cells (no 7-AAD staining) compared to total cell count (C), early apoptotic cells, positive for AnnexinV-FITC (D) and of necrotic cells, positive for 7-AAD (E). All values show means of at least three independent experiments, luciferase measurements were performed with 6 technical replicates each. Error bars indicate SEM.
Fig 2.
HSF1 target gene expression on mRNA level.
C5 cells were treated with 250 μM CdSO4 for 1 h, then washed once, and afterwards recovered for different time periods (2 h, 6 h, 24 h and 48 h), before mRNA harvest. Quantitative PCR was performed for HSPA1A/1B and DNAJB1 (A), HSPB1 and HSPA6 (B) and firefly luciferase (C). GAPDH was used for normalization. Y-axis shows x-fold mRNA levels compared to untreated control cells. All values show means of at least three independent experiments. Error bars indicate SEM.
Fig 3.
Mutations of HSEs in the HSPA1A promoter.
(A) Scheme of the luciferase reporter construct, derived from the natural HSPA1A promoter, with NanoLuc (Nluc) inserted downstream of the transcription start site. The three specific HSF1 binding sites (HSEs) are marked in different colours. Alignment shows HSEs compared to the consensus sequence, the pentameric elements are indicated by arrows, matches with the core sequences [35] of the consensus are highlighted in red. The HSE used in C5 cells is derived from the consensus HSE. (B-D) Effects of individual HSEs on HSPA1A promoter activity analysed by transient transfection experiments in HEK 293 cells; induction with 50 μM CdSO4 for 1 h, followed by 6 h recovery. All constructs are in the same plasmid background (pM Nluc PAUM; see S1 Table); (B) Isolated HSEs 3-fold multimerised; (C) combinations of the HSEs with or without spacers, compared to wildtype HSPA1A and (D) mutations of HSEs in the context of the promoter, (m) following the number of the HSE in the name indicates a mutation. P-values in (C) were calculated relative to HSPA1A. Values shown are means of at least 3 independent experiments with 6–12 replicates each. Y-axis shows relative luciferase activity compared to untreated control cells (B-D) in relation to HSPA1A (C,D). Error bars indicate SEM.
Fig 4.
Analysis of C5 cells with different heavy metals.
(A-C) C5 cells were treated with CuSO4 for 1 h. Then the cells were recovered for up to 24 h. (A) Luciferase reporter assay, Y-axis shows relative luciferase activity compared to untreated control cells for 6 h and 24 h. Viability (curve fitted red dotted line, right y-axis) was measured with trypan blue. (B,C) qPCR, Y-axis shows x-fold luciferase mRNA compared to untreated control cells relative to GADPH mRNA. (B) mRNA induction after 2 and 24 h; (C) time course of mRNA induction for 6 mM CuSO4 treatment. (D–F) Luciferase reporter assay, C5 cells were treated with different inducers for 1 h and 6 or 24 h recovery. Y-axis shows relative luciferase activity compared to untreated control cells. X-axis shows concentration of heavy metals. Viability (curve fitted, red dotted line, right y-axis) was measured with trypan blue. (D) ZnCl2 (E) HgCl2 (F) NiCl2. All values show means of at least three independent experiments. Error bars indicate SEM.
Fig 5.
Detection of heavy metals with C5 cells.
(A) C5 cells were incubated with different potential inducers for 24 h. All substances were diluted in complete medium. Viability (curve fitted, red dotted line, right y-axis) was measured with resazurin assay (A) or PI staining (B) and is shown relative to untreated cells. Red lines delimit regions above 75% viability. Left y-axis shows relative luciferase activity compared to untreated control cells. Green line (at relative luciferase activity of 2) indicates a threshold for significant induction. All values show means of at least three independent experiments with 12 technical replicates per plate. Error bars indicate SEM. (C) Pictures of C5 cells taken with fluorescence microscopy (scale bar 50 μm) after treatment with 25 μM CdSO4 for 48 h. From left to right: phase contrast, GFP, PI staining. Under the same conditions as described in (A), percentage of GFP positive cells and dead cells (PI positive) was determined with flow cytometry (B).
Fig 6.
Activation of HSEs by heavy metals stress.
Heavy metal stress acts on the heat shock pathway by activating HSF1 which interacts with the HSEs of target genes. Of key importance in this respect are affinity and position of the HSEs within the target promoter. Consensus HSEs were used to generate a high sensitivity reporter construct for detection of heavy metals.