Article Figures & Data
Figures
- Figure S1. Hikeshi orthologs.
The existence of Hikeshi orthologs in the phylogenetic dendrogram is shown. The organisms with orthologs of Hikeshi are shown by dark blue-colored boxes. The Hikeshi orthologs were only found in eukaryotes. In addition, Hikeshi orthologs are widely distributed in eukaryotic organisms. Thus, Hikeshi is inferred to have evolved in the last eukaryotic common ancestor.
- Figure 1. Hikeshi is required for the nuclear import of HSP70 under both nonstressed and proteotoxic stress conditions.
(A) Hikeshi mediates the nuclear import of HSP70 under heat-shock conditions. WT or Hikeshi-KO HeLa cells were incubated for 1 h at 43°C (HS) or 37°C (normal). (B) Hikeshi mediates the nuclear import of HSP70 under treatment with the proteasome inhibitor MG132. Cells were treated with 10 μM MG132 or DMSO for 6 h. In (A, B), DNA was counterstained with DAPI. (C) Depletion of Hikeshi affects the nuclear localization of HSP70 under nonstressed conditions. In (A, B, C), cells were fixed and immunostained with anti-Hsc70 antibodies (1B5). (A, B, C) Images were captured with an Olympus BX51 microscope (A, B) or FV1200 confocal microscope (C). Scale bars show 10 μm. In (A, C), nuclear and cytoplasmic intensities were measured with ImageJ software. Nuclear intensities (left graph in C) and nuclear intensities relative to cytoplasmic intensities (A and right graph in C) of each cell were plotted. Error bars indicate means ± SD. Statistical analyses were performed using Welch’s two-sided t tests.
- Figure 2. HSF1-regulated genes are up-regulated under nonstressed conditions in Hikeshi-KO cells.
(A) Venn diagram showing the number of genes up-regulated under nonstressed conditions in Hikeshi-KO HeLa cells (clones #1 and #2) compared with WT cells (fold change > 1.5). mRNA expression levels were quantified by RNA-seq (single replicate for each cell line). (B) Gene ontology enrichment analysis of 140 genes commonly up-regulated in the two different Hikeshi-KO HeLa clones by Metascape. (C) mRNA expression levels of HSF1-regulated genes under nonstressed conditions quantified by RNA-seq (n = 1, left panel) and qPCR (n = 3 biologically independent experiments, right panel). mRNA expression levels of HSF1-regulated genes in two HeLa Hikeshi-KO cells (clones #1 and #2) relative to that of WT cells are shown. (D) mRNA expression levels of HSF1-regulated genes under nonstressed conditions in siHSF1-treated cells. HeLa WT and Hikeshi-KO cells were transfected with siRNA targeting GL2 (siGL2, control) or HSF1 (siHSF1). mRNA expression levels were quantified by qPCR (n = 3 biologically independent experiments). In each cell, mRNA expression levels in siHSF1-transfected cells relative to that in siGL2-transfected cells were shown. Error bars indicate ± SD. Statistical significance was determined using unpaired t test (NS, no significance, P > 0.05; ***P < 0.001, **P < 0.01).
- Figure S2. mRNA expression analyses in WT, Hikeshi-KO, and siHikeshi-treated HeLa cells.
(A) Venn diagram showing the number of genes down-regulated in the Hikeshi-KO HeLa cells (clones #1 and #2) under nonstressed conditions compared with that of WT cells (fold change < 0.7). (B) Gene ontology enrichment analysis of 155 genes down-regulated in Hikeshi-KO HeLa cells by Metascape. (C) Treatment with siRNA-Hikeshi induces up-regulation of 9 HSF1-regulated genes, selected in Fig 2C. HeLa cells were transfected with siRNA targeting GL2 (siGL2, control) or Hikeshi (siHikeshi). Immunoblot of Hikeshi protein after siRNA treatment (upper panel). The band intensity of Hikeshi in siHikeshi-treated cells was normalized to that in siGL2-treated cells and the ratios are shown. mRNA expression levels of HSF1-regulated genes under nonstressed conditions quantified by RNA-seq (single replicate for each cell line, lower left panel) and qPCR (n = 3 biologically independent experiments, lower right panel). mRNA expression levels in siHikeshi-treated HeLa cells relative to that siGL2-treated HeLa cells are shown. Error bars indicate ± SD. Statistical significance was determined using unpaired t test (NS, no significance, P > 0.05).
- Figure S3. mRNA expression analyses in WT and Hikeshi-KO hTERT-RPE1 cells.
(A) Venn diagram showing the number of genes up-regulated in the Hikeshi-KO hTERT-RPE1 cells (clones #3 and #4) under nonstressed conditions compared with that of WT cells (fold change > 1.5). (B) Gene ontology enrichment analysis of 272 genes up-regulated in Hikeshi-KO hTERT-RPE1 cells by Metascape. (C) mRNA expression levels of HSF1-regulated genes under nonstressed conditions. Relative expression levels among WT and Hikeshi-KO hTERT-RPE1 cells (clones #3 and #4) are shown. (D) mRNA expression of HSF1-regulated genes in response to heat shock (fold change versus nonstressed condition) in WT and Hikeshi-KO cells. In (C, D), mRNA expressions were quantified by RNA-seq (single replicate for each cell line, left panels) and qPCR (n = 3 biologically independent experiments, right panels). Error bars indicate ± SD. Statistical significance was determined using unpaired t test (NS, no significance, P > 0.05).
- Figure 3. Nuclear HSP70 suppresses the transcriptional activity of HSF1.
(A) Cellular localization of EGFP-NLS-Hsc70 in HeLa cells stably expressing EGFP-NLS-Hsc70 (clones #7 and #14). (B) EGFP-NLS-Hsc70 protein stably expressed in the HeLa cell lines (clones #7 and #14) and endogenous Hsc70 (HSPA8) and Hsp70 (HSPA1) were detected by immunoblotting with anti-Hsc70/Hsp70 antibodies (1H5-1). Band intensities were measured using ImageJ software. Ratios of EGFP-NLS-Hsc70 to endogenous Hsc70 and Hsp70 proteins are shown. (C) mRNA expression levels of 9 HSF1-regulated genes, selected in Fig 2C, under nonstressed conditions quantified by RNA-seq (single replicate for each cell line). mRNA expression levels of HSF1-regulated genes in HeLa cells stably expressing EGFP-NLS-Hsc70 (clones #7 and #14) relative to that in HeLa WT cells are shown. (D) Fluc reporter gene expression driven by HSEs is highly activated in Hikeshi-KO cells. pGL4.41[luc2P/HSE/Hygro] vectors were cotransfected with pNL1.1TK (Nluc/TK) vectors, which were used as transfection controls, into WT and Hikeshi-KO HeLa cells. At 1 d posttransfection, Fluc and Nluc activities were measured using a Nano-Glo Dual-Luciferase Reporter Assay (Promage). (E) NLS-Hsc70 suppresses Fluc reporter gene expression driven by the HSE promoter. Hikeshi-KO cells were cotransfected with 37 ng of pGL4.41[luc2P/HSE/Hygro] vector, and the plasmid expressing EGFP-NLS-Hsc70 at ratios shown in the figure. (F) Exogenously expressed Hikeshi in Hikeshi-KO cells suppresses Fluc reporter gene expression driven by the HSE promoter. Hikeshi-KO cells were cotransfected with 37 ng of pGL4.41[luc2P/HSE/Hygro] vector, and the plasmid expressing FLAG-Hikeshi at ratios shown in the figure. In (E, F), Fluc activities at 1 d posttransfection were measured using a luciferase assay system (Promega). Error bars indicate ± SD.
- Figure S4. Nuclear HSP70 suppresses the transcriptional activity of HSF1.
(A) mRNA expression levels of HSF1-regulated genes in HeLa cells stably expressing EGFP-NLS-Hsc70 (clones #7 and #14) relative to that in HeLa WT cells. (B) mRNA expression levels of HSF1-regulated genes in HeLa cells transiently expressing EGFP-NLS-Hsc70 relative to that in control cells. HeLa cells were transfected with plasmid expressing EGFP-NLS-Hsc70 or EGFP (control) for 2 d. In (A, B), mRNA expression levels of HSF1-regulated genes under nonstressed conditions quantified by qPCR (n = 3 biologically independent experiments). Error bars indicate ± SD. Statistical significance was determined using unpaired t test (NS, no significance, P > 0.05). (C) NLS-Hsc70 suppresses firefly luciferase (Fluc) reporter gene expression driven by the heat-shock element (HSE) promoter. Hikeshi-KO cells were cotransfected with 37 ng of pGL4.41[luc2P/HSE/Hygro] vector and the plasmid expressing EGFP-fused non-, NLS-, or nuclear export signal-tagged Hsc70 at ratios shown in the figure. Fluc activities at 1 d posttransfection were measured using a luciferase assay system (Promega) (n = 3).
- Figure S5. mRNA expression profile of HSF1-regulated genes in HeLa WT and Hikeshi-KO cells (RNA-seq analyses).
(A, B) mRNA expression levels (rpkm) of 9 HSF1-regulated genes, selected in Fig 2C, at each time point of nonstressed (non-HS) and heat shock (HS) and beyond (R1.5h, R3h) conditions in WT and Hikeshi-KO cells were quantified by RNA-seq (single replicate for each cell line). In (B), bars (in black, blue, red) show median.
- Figure 4. Nuclear HSP70 functions in the protein activity of nuclear luciferase monitoring proteins.
(A) Schema of experimental procedures. (B, C, D) In (B, C, D), at 1 d posttransfection, cells were incubated with or without 25 μg/ml of the protein synthesis inhibitor cycloheximide (CHX) for 2 h, and then Fluc activities were measured using a luciferase assay system (Promega). Ratios of Fluc activities of CHX-treated cells to that of untreated cells are plotted in the graph. Error bars indicate means ± SD. Statistical analyses were performed using Mann–Whitney two-tailed analysis (n.s., no significance, P > 0.05). (B) Protein stability of nuclear Fluc decreases in Hikeshi-KO cells. EGFP-NLS–tagged Fluc-WT or SM (more unstable Fluc mutant) was transiently expressed in HeLa WT or Hikeshi-KO cells. (C) Nuclear HSP70 suppresses the decrease in nuclear Fluc protein stability. HeLa WT and Hikeshi-KO cells were cotransfected with 50 or 60 ng of EGFP-NLS-Fluc-WT-expressing plasmid and NLS-Hsc70–expressing plasmid. The amount of transfected plasmid expressing NLS-Hsc70 is shown in the figure. (D) Protein stability of cytoplasmic Fluc-WT and SM. EGFP-Fluc-WT or SM was transiently expressed in HeLa WT or Hikeshi-KO cells.
- Figure 5. Hikeshi reduces nuclear polyQ-induced apoptosis.
(A) Cellular localization of NLS or nuclear export signal (NES)-tagged polyQ81-EGFP. Plasmids expressing pathologic polyQ81 (from human DRPLA) fused to NLS or NES-EGFP proteins were transfected into WT and Hikeshi-KO HeLa cells. At 1 d posttransfection, cells were fixed, and fluorescence images of polyQ81-NLS-EGFP (left panels) and polyQ81-NES-EGFP (right panels) were captured with an Olympus BX51 microscope. DNA was counterstained with DAPI. (B) Hikeshi-KO cells expressing nuclear polyQ81 were more sensitive to apoptosis than WT cells. Cells were transfected with plasmids expressing EGFP (control), polyQ81-NLS-EGFP, or polyQ81-NES-EGFP. (C, D) Hikeshi and NLS-Hsc70 expression significantly reduces cellular apoptosis induced by nuclear polyQ81 protein expression. Hikeshi-KO cells were transfected with plasmids expressing EGFP (control) or polyQ81-NLS-EGFP with or without Hikeshi (C) or NLS-Hsc70 (D). In (B, C, D), at 2 d posttransfection, the induction of apoptosis by polyQ81 was measured by using the caspase-Glo 3/7 assay system (Promega). Error bars indicate ± SD. Statistical analyses were performed using Mann–Whitney two-tailed analysis.
- Figure 6. Heat-shock response is impaired in the Hikeshi-KO cells.
(A) mRNA expression levels of 162 heat-shock–responsive genes in HeLa WT and Hikeshi-KO cells. Genes whose expression level are induced more than twofold from normal to heat stress conditions in WT cells were categorized as heat-shock–responsive genes. Horizontal lines (in black, blue, red) show median with interquartile range. (B, C) mRNA expression levels of 9 HSF1-regulated genes, selected in Fig 2C, in HeLa WT and Hikeshi-KO cells. Bars (in black, blue, red; in left panels) and horizontal lines (in blue, red; in light panels) show median. In (A, B, C), mRNA expression levels relative to that under nonstressed (non-HS) conditions in each cell (left panels) or that in WT cells (right panels) were plotted. HeLa WT and Hikeshi-KO cells under non-HS conditions were exposed to heat shock (HS) at 43°C for 1 h and returned to nonstressed conditions at 37°C for 1.5 h (R1.5h), 3 h (R3h), and 4.5 h (R4.5h, in C). Gene expression at each time point was analyzed with RNA-seq (single replicate for each cell line, A, B) or qPCR (n = 3 biologically independent experiments, C).
- Figure S6. mRNA expression profile of HSF1-regulated genes in HeLa WT and Hikeshi-KO cells (qPCR analyses).
(A, B) Relative mRNA expression levels of 9 HSF1-regulated genes at each time point of nonstressed (non-HS) and heat shock (HS) and beyond (R1.5h, R3h, and R4.5h) conditions in WT and Hikeshi-KO cells were quantified by qPCR (n = 3 biologically independent experiments). Data were normalized to non-HS in WT cell. In (B), bars (in black, blue, red) show median.
- Figure S7. Relative mRNA expression profiles of heat-shock proteins (HSPs) in HeLa Hikeshi-KO cells to that in WT cell.
mRNA expressions of HSPs (HSPAs, HSPHs, DNAJAs, and DNAJBs) at each time point of nonstressed (non-HS) and heat shock (HS) and beyond (R1.5h, R3h) conditions were quantified by RNA-seq (single replicate for each cell line). In left table, red and blue shows mRNA expression levels relative to that in WT cells (fold change > 1.2 and < 0.8, respectively). In right panel, horizontal lines (in blue, red) show median.
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