EMBO/SourceData huggingface dataset:
Figure 3C, Representative electron microscopy (EM) pictures of CTL and THEM6 KO 22rv1 cells taken at low (left) and high (right) magnification. Red arrows point towards abnormal ER structure. Scale bar represents 500 nm.I, Western blot analysis of THEM6, AMFR, SEC61b and XPO1 expression in CRPC cells following THEM6 immunoprecipitation in CRPC cells. Data information: Data reproducibility: representative image from 2 independent biological experiments.J, Western blot analysis of CALX and CALR expression in PCa cells following THEM6 silencing. Data information: Data reproducibility: representative image from 3 independent biological experiments.
Figure 4B, RT-qPCR analysis of MVD, FDPS and DHCR7 expression in CTL and THEM6 KO 22rv1 cells. CASC3 was used as a normalising control. Data information: Data are presented as mean values +/- SD. Statistical analysis: : *p-value < 0.05 using 1-way ANOVA with a Dunnett's multiple comparisons test. Data reproducibility: : n = 3 independent biological experiments.F, Labelled cholesterol fraction derived from 13C-glucose and 13C-glutamine in CTL and THEM6 KO CWR22res cells after 72 hours of incubation. G, Labelled cholesterol fraction derived from 13C-glucose and 13C-glutamine in CTL and THEM6 KO MCF-7 cells after 72 hours of incubation. Data information: Data are presented as mean values +/- SD. Statistical analysis: , F, : *p-value < 0.05 using 1-way ANOVA with a Dunnett's multiple comparisons test. G: *p-value < 0.05 using a two-tailed Student t-test. Data reproducibility: : n = 3 independent wells from the same cell culture.J, Labelled palmitic, oleic and stearic acid fractions derived from 13C-glucose and 13C-glutamine in CTL and THEM6 KO 22rv1 cells after 72 hours of incubation. Data information: Data are presented as mean values +/- SD. Statistical analysis: : *p-value < 0.05 using 1-way ANOVA with a Dunnett's multiple comparisons test. Data reproducibility: n = 3 independent wells from the same cell culture.
Figure 5B, Proteomic analysis highlighting proteins associated with the ER stress response significantly down-regulated in THEM6 KO 22rv1 cells when compared to CTL (p-value ≤ 0.05, FC = 1.3). Data information: Data are presented as mean values +/- SD. Data reproducibility: n = 3 independent biological experiments.E, Proteomic analysis highlighting ATF4 targets significantly down-regulated in THEM6 KO LNCaP AI cells when compared to CTL (p-value ≤ 0.05, FC = 1.3). Data information: Data are presented as mean values +/- SD. Data reproducibility: n = 3 independent biological experiments.J, Western blot analysis of ATF4 and CHOP expression in CTL and THEM6 KO LNCaP AI cells treated with palmitic acid (200 µM) or hexadecylglycerol (50 µM) for 48 hours. Data information: HSC70 was used as a sample loading control. Data reproducibility: representative image from 3 independent biological experiments.
Figure 6C, Kaplan-Meier progression-free survival analysis of PCa patients stratified according high and low THEM6 expression using the PRAD TCGA dataset. D, Kaplan-Meier recurrence-free survival analysis of PCa patients stratified according to median THEM6 expression using the GSE21034 dataset (n = 179). Data information: Statistical analysis: logrank test. Data reproducibility: C: n = 489 tumours. D: n = 179.E, IHC staining of THEM6 expression in treatment naïve, NHT-treated, CRPC and NEPC tumours. Scale bar represents 100 µm. Data information: Data reproducibility: : n = 132; 90; 66; 137; 44; 30 tumours for Untreated; NHT 1-3; NHT4-6; NHT>7; CRPC; NEPC respectively.I-J, Kaplan-Meier recurrence-free (I) and overall (J) survival analysis of PCa patients stratified according to median THEM6 expression. Data information: Statistical analysis: logrank test. Data reproducibility: n = 35 tumours for THEM6 low and n = 34 tumours for THEM6 high.K, Quantification of THEM6 expression in PCa tissue samples according to Ki67 expression in the patients analysed in F. L, Quantification of THEM6 expression in PCa tissue samples according to Ki67 expression in the patients analysed in I-J. Data information: Centre line corresponds to median of data, top and bottom of box correspond to 90th and 10th percentile, respectively. Whiskers extend to adjacent values (minimum and maximum data points not considered outliers). Statistical analysis: two-tailed Mann-Whitney U test. Data reproducibility: K: n = 269 tumours for Ki67 low and n = 270 tumours for Ki67 high. L: n = 35 tumours for Ki67 low and n = 29 tumours for Ki67 high.M, Volcano plot of the differentially modulated genes in PCa patients from the PRAD TCGA stratified according to THEM6 expression. Red dots represent the UPR-related genes extracted from the UPR-gene signature Data information: Data reproducibility: n = 489 tumours.
Figure 1(A) Atg5fl/fl Cre− and Atg5fl/fl Cre+ bone marrow‐derived macrophages (BMMs), pretreated overnight with 100 ng/ml LPS, were stimulated for 1 h with the inflammasome agonist nigericin (20 μM) with (Starvation; EBSS) or without (Full; full medium) autophagic induction. Cell culture supernatants were assayed for murine IL‐1β by ELISA. Data represent mean values±s.d. (n≥3); *P0.05.(B) LPS‐pretreated Atg5fl/fl Cre− and Atg5fl/fl Cre+ BMMs were stimulated with 20 μM nigericin for 1 h in OptiMEM and the release of active caspase‐1 and IL‐1β was determined by immunoblotting.(C) As in (A), assayed for IL‐18. Data represent mean values±s.d. (n≥3); *P0.05.(D) LPS‐pretreated BMMs were exposed to alum (250 μg/ml) for 1 h with or without autophagic induction by starvation. Secreted IL‐1β was measured as in (A). Data represent mean values±s.d. (n⩾3); *P0.05.(E) LPS‐pretreated BMMs were exposed to silica (250 μg/ml) for 1 h with or without autophagic induction by starvation. Secreted IL‐1β was measured as in (A). Data represent mean values±s.d. (n⩾3); *P0.05.(F) BMMs were transfected with scramble (Scr) control siRNA or siRNAs against ASC and NLRP3. After 48 h following transfection, cells were treated overnight with LPS and subjected to nigericin (20 μM) and starvation for 1 h. Data represent mean values±s.d. (n⩾3); *P0.05.(G) Immunoblot analysis of ASC and NLRP3 knockdowns.(H) BMMs were transfected with scramble (Scr) control siRNA or siRNAs against ASC and NLRP3. After 48 h following transfection, cells were treated overnight with LPS and subjected to silica (250 μg/ml) and starvation for 1 h. Data represent mean values±s.d. (n⩾3); *P0.05.(I) Colocalization of IL‐1β with the basal autophagic machinery factor LC3. Fluorescence: LC3 (green, Alexa488); IL‐1β (red, Alexa568). BMMs were from GFP-LC3 knock‐in mice, treated with LPS then prepared for immunofluorescence microscopy using fluorescently labelled antibodies against GFP and IL‐1β.(J, K) A line fluorescence tracing from images in (I).(L) Pearson's colocalization coefficient for IL‐1β and LC3. Pearson's coefficient was derived from three independent experiments with five fields per experiment, for a total of 15 fields contributing to the cumulative result. Figure source data can be found in Supplementary data.
Figure 2(A, B) LPS‐pretreated BMMs were treated with 20 μM nigericin (Nig) and 100 nM bafilomycin A1 (Baf) with (Starvation) or without (Full) autophagic induction for 1 h and secreted IL‐1β (A) and IL‐18 (B) were measured. Data represent mean values±s.d. (n⩾3); *P0.05.(C) LPS‐pretreated BMMs were treated with 250 μg/ml of silica and 100 nM bafilomycin A1 (Baf) with (Starvation) or without (Full) autophagic induction for 1 h and secreted IL‐1β were measured. Data represent mean values±s.d. (n⩾3); *P0.05.(D) Colocalization of cathepsin B with the basal autophagic machinery factor LC3 and IL‐1β. Fluorescence; LC3 (green, Alexa488), IL‐1β (red, Alexa568), and cathepsin B (blue, Alexa633). BMMs from GFP-LC3 knock‐in mice were treated with LPS and then analysed for immunofluorescence.(E) Colocalization line tracing analysis from images in (D).(F) LPS‐pretreated BMMs were treated with 20 μM nigericin and cathepsin B inhibitor CA‐074 Me (10 μM), with (Starvation) or without (Full) autophagic induction, for 1 h and secreted IL‐1β was measured. Data represent mean values±s.d. (n⩾3); *P0.05.(G) LPS‐pretreated Atg5fl/fl Cre− and Atg5fl/fl Cre+ BMMs were stimulated with 20 μM nigericin for 1 h in OptiMEM and release of cathepsin B was determined by immunoblotting. Figure source data can be found in Supplementary data.
Figure 3(A) Colocalization of Rab8a with the basal autophagic machinery factor LC3 and IL‐1β. Fluorescence; LC3 (green, Alexa488), IL‐1β (red, Alexa568), Rab8a (blue, Alexa633). BMMs from GFP-LC3 knock‐in mice were pretreated with LPS and analysed by immunofluorescence microscopy. Arrows indicate triple colocalization.(B) Line tracing analysis of fluorescence signal intensity.(C) Pearson's colocalization coefficient for IL‐1β and Rab8a. Pearson's coefficients were derived from three completely independent experiments with >5 fields per experiment, for a total of ⩾15 fields contributing to the cumulative result.(D) BMMs were transfected with siRNAs against Rab8a or scramble (Scr) control. At 24 h after the first transfection, cells were transfected again with siRNA, treated with LPS and the day after subjected to nigericin in full medium for 1 h, and IL‐1β secretion measured.(E) Immunoblot analysis of Rab8a knockdown in BMMs.(F) RAW 264.7 macrophages were transfected with GFP‐tagged Rab8a constructs (WT, wild type; S22N, dominant‐negative mutant), treated overnight with LPS and stimulated for 1 h with 20 μM nigericin along with induction of autophagy by starvation. IL‐1β secretion was measured by ELISA. Data represent mean values±s.d. (n≥3); *P0.05. Figure source data can be found in Supplementary data.
Figure 4(A) BMM cells were transfected with scramble (Scr) control siRNA or siRNA against GRASP55. After 48 h of transfection, cells were treated with LPS and the day after subjected to 20 μM nigericin in EBSS, and secreted IL‐1β was measured by ELISA. Data represent mean values±s.d. (n⩾3); *P0.05. Inset: Immunoblot analysis of GRASP55 knockdown.(B) Immunofluorescence confocal microscopy analysis of LC3 and GRASP55 distribution. LC3 (green, Alexa488), GRASP55 (red, Alexa568). BMMs were pretreated overnight with 100 ng/ml LPS and either not stimulated (Ctrl) or stimulated (Nig) for 30 min with the inflammasome agonist nigericin (20 μM) in full medium.(C) Line tracings, analysis of fluorescence signal intensity from images in (B).(D) Pearson's coefficients for LC3 and GRASP55 were quantified using SlideBook morphometric analysis software as a measure of adjacency between GRASP55 and LC3 profiles. Pearson's coefficients were derived from three independent experiments with five fields per experiment, for a total of 15 fields contributing to the cumulative result. Figure source data can be found in Supplementary data.
Figure 5(A, B) Effect of GRASP55 on autophagy induction by measuring LC3‐II. BMM cells were transfected with GRASP55 siRNAs or scramble (Scr) control. At 72 h post transfection, cells were induced for autophagy, treated or not with Bafilomycin A1 (Baf) to inhibit autophagic degradation and LC3‐II/actin ratios determined by immunoblotting (A) followed by densitometry (B). Data represent mean values±s.d. (n⩾3); *P0.05.(C, D) RAW 264.7 was transfected with GRASP55 siRNAs or scramble (Scr) siRNA control. Following 48 h of siRNA treatment, cells were transfected with RFP-GFP-LC3 plasmid (GFP is sensitive to acidification, whereas RFP is not), after 24 h induced for autophagy in EBSS for 1 h and autophagic induction and flux quantified (graph in D) by determining the number of early autophagic organelles (GFP+RFP+ puncta) and autolysosomal organelles (GFP−RFP+ puncta) per cell as illustrated in fluorescent images (yellow arrows, GFP+RFP+; red arrows, GFP−RFP+). Total, yellow+red puncta per cell. Data represent mean values±s.d. (n⩾3); *P0.05. Figure source data can be found in Supplementary data.
Figure 6(A) Atg5fl/fl Cre− and Atg5fl/fl Cre+ bone marrow‐derived macrophages (BMMs), pretreated overnight with 100 ng/ml LPS, were stimulated for 1 h with the inflammasome agonist nigericin (20 μM) with (Starvation; EBSS) or without (Full; full medium) autophagic induction. Cell culture supernatants were assayed for murine IL‐1β by ELISA. Data represent mean values±s.d. (n⩾3); *P0.05.(B) LPS‐pretreated Atg5fl/fl Cre− and Atg5fl/fl Cre+ BMMs were stimulated with 20 μM nigericin for 1 h in OptiMEM and the release of HMGB1 was determined by immunoblotting. Figure source data can be found in Supplementary data.
Figure 1A Coolness stimulates intracellular cyclic guanosine monophosphate (cGMP) accumulation in HEK‐293T cells expressing GC‐G. Two days after transfection of empty vector or GC‐G‐encoding plasmid, cells were exposed to ambient temperatures (37 or 15°C) for 20 min and cellular cGMP concentration was measured.B Temperature dependence of GC‐G activity. Membrane preparations from HEK‐293T cells transfected with empty vector or GC‐G‐encoding plasmid were used to determine cyclase activity at the indicated temperatures for 20 min (asterisks denote values significantly elevated compared to 37°C).C GC‐G is the only murine transmembrane GC that can be stimulated by cool temperatures. Expression plasmids encoding FLAG‐tagged GC subtypes GC‐A to GC‐G were transfected into HEK‐293T cells. Two days after transfection, membrane protein fractions were prepared and used for cyclase activity assays at 37 or 15°C. Protein expression of each receptor GC was confirmed by Western blot (WB) analysis with anti‐FLAG antibody.D The H+CYC domain is critical for stimulation by coolness. The domain structure of full‐length (FL) GC‐G and its mutant variants is shown in the upper panel. A FLAG tag was added at the N‐terminus of each protein. The ΔECD+KLD mutant protein lacks amino acids 73-455 and 556-833; the ΔCYC variant lacks amino acids 834-1,003. The H+CYC mutant protein contains amino acids 806-1,100. ECD, extracellular domain; KLD, kinase‐like domain; SP, signal peptide; TM, transmembrane region. HEK‐293T cells expressing the indicated GC‐G constructs were incubated at 37 or 15°C for 20 min, then intracellular cGMP concentration was measured (lower panel).
Figure 2In the chimeric GC‐A/G protein, the H+CYC domain of GC‐A was replaced by that of GC‐G (upper panel). HEK‐293T cells expressing the indicated proteins were exposed to ambient temperature of 37 or 15°C for 20 min; then, intracellular cGMP concentration was measured. Data are mean ± SD from three experiments in triplicate. **P 0.01.
Figure 3B, C Protein expression and coolness‐stimulated cGMP synthesis of the above shown divergent C‐terminal fragments of GC‐G with intact or ablated hinge (H) region. The expression plasmids encoding the H+CYC domain or a deletion construct (Δ1-Δ2) tagged with a FLAG epitope were transfected into HEK‐293T cells. Two days after transfection, cells were exposed to the ambient temperatures (37 or 15°C) for 20 min and cellular cGMP concentration was measured (C). Protein expression of each construct was confirmed by Western blot (WB) analysis with anti‐FLAG antibody (B).D A recombinant protein containing the H+CYC domain of GC‐G acquires coolness‐evoked activity. Diagram of recombinant GST.GC‐G‐CYC protein containing the H+CYC domain of GC‐G (amino acids 849-1,078) compared to the domain structure of full‐length GC‐G (upper panel). The purified GST.GC‐G‐CYC protein was exposed to the indicated temperatures for 20 min, and GST.GC‐G‐CYC cyclase activity was measured (lower panel; asterisks denote values significantly elevated compared to 37°C).
Figure 4A HEK‐293T cells were co‐transfected with plasmids encoding two differential epitope‐tagged (FLAG or Myc) versions of GC‐G or GC‐A, respectively. After 2 days, transfected cells were lysed with detergent and lysates were subjected to immunoprecipitation (IP) for 2 h with anti‐Myc antibody at two different temperatures (15 or 25°C), followed by Western blotting (WB) using anti‐FLAG antibody to determine the amount of dimerized/oligomerized GC proteins (upper panel). These approaches demonstrated that in contrast to GC‐A, homomeric protein/protein association of GC‐G seems to be enhanced by cool temperatures (upper panel). Cell lysates were also precipitated and immunoblotted with the anti‐Myc antibody, demonstrating that cooling does not interfere with immunoprecipitation of Myc‐tagged GC‐G via the anti‐Myc antibody (middle panel). Likewise, precipitation and immunoblotting with the anti‐FLAG antibody yielded bands of similar intensity for 15 and 25°C, respectively (lower panel).B Relative quantification of homomeric protein/protein association of GC‐G and GC‐A was performed by densitometric scanning. Each intensity value from the upper panel was subsequently normalized to the total protein levels as determined in the middle and the lower panel of Fig A (intensity of the immunoreactive bands obtained with the anti‐Myc or anti‐FLAG antibody, respectively). Relative dimerization level at 15°C was further calculated by normalizing to values obtained at 25°C; the latter were set as 1. Results are means ± SD of three experiments, **P 0.01 (compared to 25°C).
Figure 5HEK‐293T cells expressing CNGA3 alone (upper panel) or together with GC‐G (full‐length in the middle panel and H+CYC domain in the lower panel) were loaded with the Ca2+ indicator Fura‐2 to monitor intracellular [Ca2+]i concentration. Cells co‐expressing GC‐G and CNGA3 showed a rapid increase in [Ca2+]i when the temperature was lowered from 37 to 15°C (middle and lower panel); cells only expressing CNGA3 are not responsive to cool temperatures (top panel). Removal of extracellular Ca2+ (−Ca2+; middle panel) completely suppressed the response to cooling. ΔF represents changes in the ratio of the fluorescence intensity of Fura‐2 at 340/380 nm excitation. The ratiometric Ca2+ responses are representative of 30 cells recorded from three experiments.
Figure 6A-H In situ hybridization experiments with an antisense probe for c‐Fos on coronal sections through the GG of wild‐type (WT; left panel) or GC‐G‐KO (right panel) mouse pups (P0-P4) exposed to warm (35°C; A-D) or cool (22°C; E-H) ambient temperature for 2 h. Panels (C, D, G, H) show higher magnifications of the boxed areas in (A, B, E, F). At 35°C (A-D), no c‐Fos expression was detectable in the GG of both WT (A, C) and GC‐G‐KO (B, D) individuals. Upon exposure to 22°C, strong c‐Fos expression was observed in the GG of WT pups (E, G), whereas c‐Fos expression was barely detectable in the GG of GC‐G‐KO animals (F, H). The data shown are representative of 10 independent experiments. Pups originated from six different litters for each genotype. Scale bars: 200 μm in (A, B, E, F); 50 μm in (C, D, G, H).I Quantification of c‐Fos‐positive GG cells in WT and GC‐G‐KO pups on exposure to 22°C for 2 h. All stained cells on every section along the rostrocaudal extent of the GG were counted; the results shown are based on the above‐mentioned 10 experiments. The number of c‐Fos‐positive cells in the GG of a GC‐G‐KO mouse was determined relative to that in a concomitantly processed WT pup; the latter number was set to 100%. Data are mean ± SD (n = 11 in each genotype), **P 0.0001.J-N Calcium imaging of coronal sections through the GG of olfactory marker protein‐green fluorescent protein (OMP‐GFP)/GC‐G+/+ or OMP‐GFP/GC‐G‐KO pups (P1-P4). High magnification image (J) of a tissue slice through the GG of an OMP‐GFP pup with GG neurons labeled by intrinsic GFP fluorescence (GFP fluorescence was merged with the transmitted‐light channel). Cells analyzed are circled in blue (GFP‐negative) or red (GFP‐positive). NC, nasal cavity. Scale bar: 30 μm. (K-M) Representative ratiometric Ca2+ transients induced by cooling from 37 to 15°C in GFP‐negative non‐neuronal cells (K) and in GFP‐positive GG neurons from OMP‐GFP/GC‐G+/+ (L) and OMP‐GFP/GC‐G‐KO pups (M). The numbers in the bottom right hand corners are the number of cells with Ca2+ transient similar to what is shown in the respective graph (left) and total number of measured cells (right). (N) Quantification of coolness‐induced ΔF in GG neurons from OMP‐GFP/GC‐G+/+ and OMP‐GFP/GC‐G‐KO mice. Coolness‐induced ΔF was calculated by subtracting the baseline fluorescence ratio (340/380 nm) at 37°C from the peak fluorescence ratio (340/380 nm) measured at 15°C. For OMP‐GFP/GC‐G+/+, the 29 coolness‐responsive neurons (black bar) of all 54 analyzed neurons (gray bar) from five slices (obtained from different animals) were analyzed. For OMP‐GFP/GC‐G‐KO, the 50 analyzed neurons (shaded bar) from five slices (obtained from different animals) were analyzed. Data are mean ± SD, **P 0.01.
Figure 7A Reduced responsive rate of coolness‐stimulated USV in GC‐G‐KO pups. Pups were removed from their littermates and dams at postnatal (P) day 2, 4, and 6 and exposed to 15°C. USV was measured by use of the Anabat SD1 Bat detector. The number of responsive pups versus total number of pups tested for each WT and GC‐G‐KO group at different ages is indicated on the top of each bar. *P 0.05.B, C Total number of USV calls (B) and response latency (C) in GC‐G‐KO and WT pups at P2. The horizontal bars indicate the mean. *P 0.05; **P 0.01.
Figure 1Western blot for p53, phosphorylated p53 at Ser15 (p53 pS15), and vinculin (vinc) in HCT116 treated with 300 μM 5-FU for 8 h and silenced for PHD1, PHD2, or PHD3.qRT-PCR for PHD1, PHD2, and PHD3 in HCT116 silenced for PHD1 (*P = 0.0006 toward Scr), PHD2 (*P = 0.0004 toward Scr), or PHD3 (*P = 0.0075 toward Scr). A two-tailed unpaired t-test was performed with n = 3/group.Western blot of p53 pS15, p53, PHD1, and vinculin in HCT116 treated with 300 μM 5-FU for 8 h and silenced for PHD1 or a scrambled (Scr) control.qRT-PCR for PHD1 showing silencing efficacy with both siRNAs 1 (*P = 0.0017 toward Scr) and 2 (*P = 0.0023 toward Scr) compared to the scrambled control in HCT116. A two-tailed unpaired t-test was performed with n = 3/group.Western blot for p53, p53 pS15, and vinc in HCT116 silenced for PHD1 with two different constructs (constructs 1 and 2) upon exposure to 300 μM 5-FU for 8 h.Western blot for p53 pS15, p53, and vinculin in HCT116 silenced for PHD1 and treated with either 200 nM SN-38 or 20 μM oxaliplatin for 8 h.RNA levels of PHD1 in LIM1215 silenced for PHD1. (*P = 0.0099 toward Scr in a two-tailed unpaired t-test with n = 3/group.)Western blot for p53 pS15, p53, and vinc in LIM1215 upon silencing of PHD1 and treatment for 8 h with 200 μM 5-FU.
Figure 2A Western blot for p53, cleaved caspase-3 (cleaved casp3), cleaved parp, and vinculin (vinc) in p53wt/wt and p53−/− HCT116 silenced for PHD1 upon exposure to 300 μM 5-FU for 24 h, showing an increased apoptotic response to 5-FU treatment in PHD1-silenced cells compared to the scrambled control.B Confirmation of these results by FACS analysis of propidium iodide-stained PHD1-silenced p53wt/wt and p53−/− HCT116 cells exposed for 26 h to 300-μM 5-FU treatment. *P = 0.002 toward p53wt/wt HCT116 shScr 5-FU-treated (two-tailed unpaired t-test) with n = 6 for non-treated p53wt/wt HCT116 and n = 3 in all other groups.C Detection of cleaved casp3, cleaved parp, and vinc in HCT116 silenced with a second siRNA for PHD1.D, E Apoptosis as detected by Western blot for cleaved casp3 and parp in PHD1-silenced HCT116 treated for 20 h with 200 nM SN-38 (D) or for 24 h with 20 μM oxaliplatin (E).F Western blot for cleaved casp3, parp, and vinc in LIM1215 silenced for PHD1 and treated for 24 h with 200 μM 5-FU.G Similar apoptosis levels were detected by Western blot for cleaved casp3 and parp with vinc as a loading control in p53S15A→p53−/− HCT116 silenced for PHD1 or scrambled (Scr) control and treated for 20 h with 300 μM 5-FU.