EMBO/SourceData huggingface dataset:
Figure 3Images of GFP-tagged Rad54, Rdh54NRad54, Rdh54 and Rad54NRdh54 (1, 3, 10 and 30 nM each) bound to Rad51-ssDNA molecules.Images of GFP-tagged Rad54, Rdh54NRad54, Rdh54 and Rad54NRdh54 (1, 3, 10 and 30 nM each) bound to Rad51-ssDNA molecules in the presence of 30 nM Hed1.Quantification of the integrated GFP signal intensity for GFP-tagged Rad54 (N = 75), Rdh54NRad54 (N = 46), Rdh54 (N = 50) and Rad54NRdh54 (N = 35; 1, 3, 10 and 30 nM each) bound to Rad51-ssDNA molecules. The data was fit by non-linear regression and error bars represent s.d. for individual DNA molecules.Comparison of fold binding inhibition for GFP-tagged Rad54, Rdh54NRad54, Rdh54 and Rad54NRdh54 in reactions conducted in the presence of 30 nM Hed1. The dot represents the mean and the error bars represent the range for two independent experiments.
Figure 4Widefield images of Atto565-labeled PSCs (magenta) bound to dsDNA curtains stained with YOYO1 (green). The PSCs were prepared with the indicated combinations of Rad51, Rdh54 and GFP-Rad54.   Number of Atto565-labeled PSC binding events per dsDNA molecule for PCS prepared with Rad54 and Rdh54 only (minus Rad51 ; N = 372), Rad51 plus Rad54 only (N= 117), Rad51 plus Rdh54 only (N = 258), Rad51 plus Rdh54 and Rad54 (N = 303). The error bars represent the s.d. for three independent experiments.
Figure 5Kymographs of Rad51-tailed duplex PSCs (magenta) containing the indicated combinations of Rad54 and Rdh54 proteins.Translocation velocities for tailed duplex PSCs prepared with Rad54 only (lane 1; N = 86), Rad54 plus Rdh54 (lane 2; N = 144), Rad54-K341R plus Rdh54 (lane 3; N = 47), Rad54 plus Rdh54-K318R (lane 4; N = 112), or Rad54-K341R plus Rdh54-K318R (lane 5; N = 64). The error bars represent s.d.   Translocation distances for tailed duplex PSCs prepared with Rad54 only (lane 1; N = 86), Rad54 plus Rdh54 (lane 2; N = 144), Rad54-K341R plus Rdh54 (lane 3; N = 47), Rad54 plus Rdh54-K318R (lane 4; N = 112), or Rad54-K341R plus Rdh54-K318R (lane 5; N = 64). Error bars were generated by bootstrapping.   First passage recognition efficiency for tailed duplex PSCs prepared with Rad54 only (lane 1; N = 145), Rad54 plus Rdh54 (lane 2; N = 115), Rad54-K341R plus Rdh54 (lane 3; N = 45), Rad54 plus Rdh54-K318R (lane 4; N = 39), or Rad54-K341R plus Rdh54-K318R (lane 5; no translocation observed). Error bars (s.d.) were generated from three independent experiments.   PSC binding distributions following 10 min reactions for PSCs prepared with Rad54 plus Rdh54 (N= 181); Rad54 plus Rdh54-K318R (N= 115); Rad54-K341R plus Rdh54 (N = 90); or Rad54-K341R plus Rdh54­-K318R (N = 54), as indicated. Error bars were generated by bootstrapping. The asterisks denote the targeted region of homology on the donor dsDNA molecules.
Figure 6D-loop stimulation by Rad54 or Rdh54NRad54 in reactions with Rad51 (300 nM), tailed duplex substrate (15 nM) and a pUC19 plasmid dsDNA (0.3 nM).   Quantification of D-loop formation for Rad54 and Rdh54NRad54. Error bars represent s.d. of three independent experiments.   D-loop stimulation by Rdh54 and Rad54NRdh54.   Quantification of D-loop formation for Rad54 and Rdh54NRad54. Error bars represent s.d. of three independent experiments.   ATPase stimulation for GFP-tagged Rad54, Rdh54NRad54, Rdh54 or Rad54NRdh54 in the presence of Rad51-ssDNA. Error bars represent s.d. of three independent experiments.Quantification of Rad51 Atto565-tailed duplex PSCs bound per dsDNA molecule in dsDNA curtain homology search assays with Rad54 (N = 117), Rdh54NRad54 (N = 150) or Rad54NRdh54 (N=175). The error bars represent the s.d. for three independent experiments.Fraction translocating Rad51 Atto565-tailed duplex PSCs in reactions with Rad54 (N = 117), Rdh54NRad54 (N = 150), Rad54NRdh54 (N = 0) and Rdh54 (N = 0).Translocation velocities of Rad51 Atto565-tailed duplex PSCs in reactions with Rad54 (N = 86) and Rdh54NRad54 (N = 18). The error bars represent the s.d. of the data.
Figure 7Yeast spot growth assays to monitor the ability of Rad54, Rdh54, Rad54NRdh54, and Rdh54NRad54 to complement MMS sensitivity in rad54∆ yeast strains.   Yeast spot growth assays to monitor the ability of Rad54, Rdh54, Rad54NRdh54, and Rdh54NRad54 to complement MMS sensitivity in rdh54∆ yeast strains.
Figure 1RIGER analysis to identify genes that are preferentially essential in FUS-DDIT3-expressing SCP-1 cells. EV-transduced SCP-1 cells and 20 FUS-DDIT3-negative cancer cell lines screened with the same shRNA library were used as reference set. Genes were ranked according to relative shRNA depletion, and YAP1 was identified as top FUS‑DDIT3‑specific essential gene. NES, normalized enrichment score.Competition assays with SCP-1 cells transduced with RFP-labeled NTC or YAP1 shRNAs. Flow cytometric quantification of RFP-positive cells on day 9 relative to day 3 showed that YAP1 knockdown was preferentially toxic to FUS-DDIT3‑expressing cultures.
Figure 2Expression of YAP1 in SCP‑1 cells transduced with FUS‑DDIT3 or EV and liposarcoma cell lines. One of at least two independent experiments with similar results is shown. FUS‑DDIT3‑expressing cell types are indicated in red.Expression of YAP1 in cytoplasmic (yellow) and nuclear (blue) fractions from SCP-1 cells transduced with FUS-DDIT3 or EV and liposarcoma cell lines. One of at least two independent experiments with similar results is shown. FUS‑DDIT3‑expressing cell types are indicated in red.Expression of FOXM1 and PLK1 in MLS cell lines. One of at least two independent experiments with similar results is shown.Strong nuclear expression of YAP1, FOXM1, and PLK1 in MLS patient samples (original magnification, x10 [inset, x20]).Intensity of nuclear YAP1 expression in liposarcoma patient samples. Immunoreactivity was assessed using a semi‑quantitative score (0, negative; 1, weak; 2, moderate; and 3, strong) defining the staining intensity in the positive control (hepatocellular carcinoma) as strong. Only tumors with at least moderate staining (semi‑quantitative score ≥2) and ≥30% YAP positive cells were considered positive for the purposes of the study.Proportion of cells with nuclear YAP1 expression in liposarcoma patient samples. Boxes represent mean values and lower and upper quartiles. Whiskers represent minimum and maximum values.
Figure 3Competition assays with liposarcoma cell lines transduced with RFP-labeled NTC or YAP1 shRNAs. Flow cytometric quantification of RFP-positive cells on day 17 relative to day 3 showed that YAP1 knockdown was preferentially toxic to MLS cells. Error bars represent the mean ± SD of two independent experiments.Aggregate data from competition assays shown in (A). Statistical significance was assessed using an unpaired t-test. LS, non-myxoid liposarcoma.Competition assays with MLS 1765-92 cells transduced with an RFP‑labeled NTC shRNA or an RFP-labeled shRNA against the YAP1 3' UTR following transduction with EV or the YAP1 coding sequence. Flow cytometric quantification of RFP‑positive cells on day 17 relative to day 3 showed that cell viability was rescued by expression of the shRNA‑resistant YAP1 cDNA. Error bars represent the mean ± SD of two independent experiments.Expression of total and exogenous V5‑tagged YAP1 in MLS 1765‑92 cells shown in (C). One of at least two independent experiments with similar results is shown.Cell viability and expression of FOXM1 and PLK1 in MLS cell lines following siRNA-mediated YAP1 knockdown. Error bars represent the mean ± SD of three independent experiments, unpaired t-test. The blots represent one of at least three independent experiments with similar results.Flow cytometric cell cycle analysis of MLS cell lines following shRNA‑mediated YAP1 knockdown. Error bars represent the mean ± SD of three independent experiments, two-way ANOVA; ns, not significant.Senescence‑associated β‑galactosidase (SABG) staining intensity in MLS cell lines following shRNA-mediated YAP1 knockdown. Error bars represent the mean ± SD of ten random microscopic fields, two-way ANOVA).Expression of CDKN1A, CDKN2A, total and phosphorylated RB1, and TP53 in MLS cell lines following shRNA‑mediated YAP1 knockdown. One of at least two independent experiments with similar results is shown.Expression of cleaved PARP and cleaved caspase 3/8 in MLS cell lines following shRNA-mediated YAP1 knockdown. One of at least two independent experiments with similar results is shown.
Figure 4Expression of YAP1 and downstream effectors in SCP-1 cells transduced with FUS‑DDIT3 or EV. One of at least three independent experiments with similar results is shown.Expression of YAP1 and downstream effectors in cytoplasmic (yellow) and nuclear (blue) fractions from SCP-1 cells transduced with FUS‑DDIT3 or EV. One of at least two independent experiments with similar results is shown.YAP1-responsive luciferase activity in SCP-1 cells transduced with FUS‑DDIT3. Relative luciferase activity is displayed as fold change relative to control. Error bars represent the mean ± SD of three independent experiments, unpaired t-test.Localization of FUS-DDIT3 and YAP1 in MLS 1765-92. Nuclei were counterstained with DAPI. The original magnification was x63, and images were zoomed in four times for co-localization analysis. The correlation between red and green fluorescence was determined by Pearson coefficient analysis (square, area for signal acquisition).Co‑IP of transiently expressed FUS‑DDIT3 and YAP1 from HEK293T cells. V5-tagged YAP1 was pulled down using an anti-V5 antibody, and interacting proteins were detected by immunoblotting. One of at least two independent experiments with similar results is shown.Co-IP of endogenous FUS-DDIT3 and YAP1 from MLS 1765-92 cells. FUS-DDIT3 was pulled down using an anti-DDIT3 antibody, and interacting proteins were detected by immunoblotting. One of at least two independent experiments with similar results is shown.
Figure 5Viability and proliferation of MLS 402‑91, MLS 1765‑92, and MLS 2645-94 cells cultured in the presence of verteporfin. Error bars represent the mean ± SEM of one representative experiment performed in quintuplicate.Flow cytometric analysis of apoptosis (cleaved PARP) and mitotic fraction (phosphorylated histone H3S10) in MLS cell lines cultured in the presence of 0.25 µM Verteporfin. One of two independent experiments with similar results is shown.Expression of total YAP1 and downstream effectors (FOXM1 and PLK1) in MLS cell lines treated with 0.5 or 1 µM verteporfin for 15 hours. One of at least three independent experiments with similar results is shown.YAP1-responsive luciferase activity in MLS cell lines transfected with a constitutively active YAP1S127A mutant and treated with 1 µM verteporfin. Relative luciferase activity is displayed relative to control. Error bars represent the mean ± SD of three independent experiments, unpaired t-test.Tumor formation on chicken CAM of MLS cell lines following shRNA-mediated YAP1 knockdown. The number of tumors is given above each bar, Fisher exact test; ns, not significant.Tumor growth on chicken CAM of MLS cell lines following treatment with 1 µM verteporfin. Shown are tumor volumes and representative photographs of tumors.
Figure 1C. Cell cycle fractions inferred from Cycleflow in IFN-β-treated NSCs. n = 3 biological replicates.
Figure 2C,D. Ribo-Seq results depicting translation efficiency as the interaction of log2 fold changes (LFC) between footprints mapped to the CDS, referred to as "CDS counts", and total RNA after 2h or 14h IFN-β treatment. FDR < 10%, LR-Test in DESeq2. Genes with a p value<0.1 after FDR correction are highlighted. n = 4 biological replicates.I. Representative WB and phosphorylation levels quantification (log2FC) of the mTOR-related proteins ribosomal protein S6 kinase (S6K), ribosomal protein S6 (S6) and eIF4E-binding protein 1 (4E-BP1) from NSCs treated with IFN-β and normalized to control (t=0h). Bars represent the mean value. One-way ANOVA for the biphasic response test p-4E-BP1S65 * (p=0.021), p-S6S235/236 & p-S6S240/244 ***(p<0.001), p-S6KT389 **(p=0.004). n = biological replicates specified. p ≡ p-value.
Figure 3A. Representative WB image and quantification (log2FC) of p-TSC2Thr1462 & p-AktThr308 from IFNβ-treated NSCs normalized to control (t=0h). Bars represent the mean value. One-way ANOVA for the biphasic response test p-TSC2T1462 ***(p<0.001), p-AktT308 **(p=0.003). n = 4 biological replicates. Data information: p ≡ p-value.C. Representative WB images and quantifications (log2FC) of p-eIF2αS51 in IFN-β-treated NSCs normalized to control (t=0h). Bars represent the mean value. Spearman's rank correlation test. p-eIF2αS51 **(p=0.003724). n = 4 biological replicates. Data information: p ≡ p-value.E. Representative WB image and quantifications (log2FC) of p-Rb1Ser780 from IFN-β-treated NSCs normalized to control (t=0h). Bars represent the mean value. Spearman's rank correlation test. p-Rb1S780 ***(p=5.37e-06). n = 3 biological replicates. Data information: p ≡ p-value.I,J. Luciferase activity controlled by the upstream 5'UTR fragment from Sox2 (WT and mutant), Rps21 (WT and mutant) and Actb. Treatments were applied for 12h. Data is normalized to vehicle and is represented as mean ± SD. n = 6 biological replicates. Two-way ANOVA with Tukey's multiple comparisons test (p-value specified). Data information: p ≡ p-value.
Figure 4B. vSVZ niche FACS quantifications of total NSCs (Total NSCs) & immunofluorescence quantification of aNSCs (Fraction of aNSCs) in WT or IFNAGR KO mice at 2, 7 and 22 months old (MO). Data from individual mice is plotted. Inferred dynamics are depicted as a blue (WT) or red (IFNAGR KO) line over time. Sample sizes: WT total at 60 days: 8 mice, WT total at 210 days: 3 mice, WT total at 660 days: 3 mice, WT fraction aNSCs at 60 days: 3 mice, WT fraction aNSCs at 660 days: 4 mice, IFNAGRKO total at 60 days: 8 mice, IFNAGRKO total at 180 days: 3 mice, IFNAGRKO total at 660 days: 3 mice, IFNAGRKO fraction aNSCs at 60 days: 3 mice, IFNAGRKO fraction aNSCs at 660 days: 3 mice.
figf1a, Impairment of two ATG-conjugation systems (Atg12 and LC3) in the Atg7-deficient brain. Brain homogenates from P28 mice were immunoblotted with antibodies against Atg7, Atg5 and LC3. Actin was used as a loading control. Data shown are representative of three separate experiments.b, Kaplan-Meier survival curves of Atg7flox/+; nestin-Cre (n = 41) and Atg7flox/flox; nestin-Cre (n = 26) mice over 28 weeks.c, Abnormal limb-clasping reflexes in Atg7flox/flox; nestin-Cre mice at P28. When lifted by the tail, Atg7flox/+; nestin-Cre mice behave normally, extending their hind limbs and bodies. In contrast, Atg7flox/flox; nestin-Cre mice bend their legs towards their trunk or tighten their back limbs to their bodies and anterior limbs.d, Movement ataxia in Atg7flox/flox; nestin-Cre mice at P28. Motor coordination was tested using a rotarod assay. Atg7flox/+; nestin-Cre (n = 5) and Atg7flox/flox; nestin-Cre (n = 5) mice were placed on a rod rotating at 20 r.p.m., and the time spent on the rod was counted. Data show mean ± s.d. *, P 0.01 (Students t-test). There was no significant sex difference in survival rate and onset-stage of abnormal mice-clasping and tremor in miceflox/flox; nestin-Cre mice.
figf2a-f, Histological analyses of Atg7flox/+; nestin-Cre (left) and Atg7flox/flox; nestin-Cre (right) cerebral cortex at P56. Cryosections were stained with HE (a-d) or immunostained for the glial marker GFAP (e, f). Boxed areas in a and b are magnified in c and d, respectively. Arrows in c point to large pyramidal neurons in the cerebral cortex.g-j, Histological analysis of Atg7flox/+; nestin-Cre (left) and Atg7flox/flox; nestin-Cre (right) cerebellum at P56. Cryosections were stained with HE (g, h) or immunostained for the Purkinje marker calbindin (i, j). Arrows in g and h indicate cerebellar Purkinje cells.k-p, TUNEL staining of the cerebral cortex (k, l) and cerebellum (n, o) at P56 in Atg7flox/+; nestin-Cre (k, n) and Atg7flox/flox; nestin-Cre (l, o) sections. TUNEL-positive cells are indicated with arrows and shown as higher magnification images beneath panels l, n and o. Histograms show the average number (± s.d.) of TUNEL-positive cells in ten sections for three animals of each genotype (m, p). *, P 0.05 (t-test). Scale bars, 1 mm (a, b), 100 µm (c-j), 250 µm (k, l, n, o). We observed no sex difference in brain morphology or neuronal loss in Atg7flox/flox; nestin-Cre mice.
figf3a-l, The presence of ubiquitin-positive dots was examined by immunohistochemistry in several regions including cerebral cortex (a, b), cerebellum (c, d), hippocampus (e, f), hypothalamus (g, h), amygdala (i, j) and pontine nuclei (k, l) of Atg7flox/+; nestin-Cre and Atg7flox/flox; nestin-Cre mice. Note the presence of numerous ubiquitin dots in the amygdala and hypothalamus of the representative mutants. Scale bars, 50 µm.m, n, Electron micrographs of the brain of Atg7flox/flox; nestin-Cre mice. Inclusion bodies (arrows) were often observed in Atg7flox/flox; nestin-Cre hypothalamus. The boxed region in m is shown in n. Inclusion bodies were not detected in Atg7flox/+; nestin-Cre brain (data not shown). Scale bars, 5 µm (m), 1 µm (n). o, Immunoelectron micrograph of ubiquitin in a representative Atg7flox/flox; nestin-Cre hypothalamus. N, nucleus. Scale bar, 1 µm.p, q, Immunohistochemical detection of ubiquitin-positive inclusions in the cerebral cortex (p) and hypothalamus (q) at P9, P18 and P56. Brain sections of each genotype at the indicated ages were immunostained with an anti-ubiquitin antibody. Ubiquitin-positive inclusions appeared at P18 and became larger with ageing in the brain of Atg7flox/flox; nestin-Cre mice. Scale bars, 50 µm.
figf4a, Increase in ubiquitinated proteins in Atg7flox/flox; nestin-Cre mouse brain over time. Homogenates of P9, P18 and P56 brains from Atg7flox/+; nestin-Cre and Atg7flox/flox; nestin-Cre mice were immunoblotted with an anti-ubiquitin antibody. An anti-actin antibody was used as a loading control. Data shown are representative of three separate experiments. We observed no sex difference in the accumulation of ubiquitin in Atg7flox/flox; nestin-Cre mice.b, Peptide hydrolysis activity of 20S and 26Sproteasomes. Homogenates from P28Atg7flox/+; nestin-Cre (blue) and Atg7flox/flox; nestin-Cre (pink) brains were fractionated by glyceroldensity gradient centrifugation (10-40% glycerol from fraction 1 to fraction 30). Aliquots from each fraction were used for the assay of chymotryptic activity of proteasomes using Suc-LLVY-AMC as a substrate in the absence (top) or presence (bottom) of 0.05% SDS. The sedimenting positions of 20S and 26Sproteasomes are indicated with arrowheads. Note that whereas 26Sproteasomes exist in active forms in tissues, 20Sproteasomes are latent and are activated artificially by a low concentration of SDS.c, ATP-dependent degradation of [35S]-labelled ODC. Degradation of [35S]-labelled ODC was assayed using crude extracts from P28 Atg7flox/+; nestin-Cre and Atg7flox/flox; nestin-Cre brains. The experiment was repeated three times, and values represent mean ± s.d. In the above assays, there were no significant differences between Atg7flox/+; nestin-Cre and Atg7flox/flox; nestin-Cre mice.d, Immunoblot analysis of 26S proteasome components. Homogenates from P28 Atg7flox/flox, Atg7flox/+; nestin-Cre and Atg7flox/flox; nestin-Cre brains were immunoblotted with antibodies against the indicated proteins. Data shown are representative of three separate experiments. There was no change in proteasome status for the different genotypes.
Figure 1A-C. Immunodetection of BES1-CFP from heat-treated plants. Plants pre-grown at 21°C were (A) exposed for 60 minutes to the indicated temperatures, (B) to 45°C for the indicated period of time, or (C) to 45°C and, following sampling after 30 minutes (lane 2) and 60 minutes (lane 3), returned to 21°C for 3 hours (lane 4). CBB, Coomassie Brilliant Blue stained gel.D. Subcellular localization of BES1-CFP in roots of 7-day-old plants after heat shock. 35S:BES1-CFP seedlings were grown at 21°C and either left untreated or exposed for 60 minutes to 45°C. Left: representative photos. Right: quantification of the nuclear/cytoplasmic signal. Data show the mean ±SD. n=25. The asterisks indicate significant differences to the untreated plants by Student's t-test (***P≤0.001).F. Heat stress resistance of bes1-1, bes1-2 and bes1-D as compared to wild-type. For basal resistance assays (top charts) plants were directly exposed to 45°C for the indicated time. For acquired resistance (bottom charts), plants were primed with the respective temperatures for 120 minutes before transfer to 45°C for 150 minutes. Data show the mean ±SE. n=7. The asterisks indicate significant differences compared to wild-type by Student's t-test (*P≤0.05; **P≤0.01; ***P≤0.001).
Figure 2B. Immunodetection of BES1-CFP from heat-stressed plants, treated with epiBL or BRZ. 35S:BES1-CFP plants were treated for 24 hours with DMSO (control), 1 μM 24-epiBL or 5 μM BRZ and then heat-stressed as follows: lane 1: untreated control; lane 2: 45°C for 30 minutes; lane 3: 45°C for 60 minutes; lane 4: 45°C for 60 minutes and recovery at 21°C for 180 minutes.C, D. Immunodetection of BES1-CFP from heat-treated bri1-1 or det2-1 plants. Left: phenotypes of 10-day-old plants of all lines as compared to wild-type Col-0 (wt). Right: Immunoblots from the lines grown and treated as in B.F. Immunodetection of BES1-CFP from heat-treated bin2-1 plants.
Figure 3A. Immunodetection of BES1-CFP from heat-stressed plants, treated with ABA or Fluridone (Flu). 35S:BES1-CFP plants were treated for 24 hours with DMSO (control), 5 μM ABA or 10 μM Fluridone and heat-stressed as follows: lane 1: untreated control; lane 2: 45°C for 30 minutes; lane 3: 45°C for 60 minutes. The non-phosphorylated BES1 bands were quantified with ImageJ and the obtained values are shown in blue.C. Immunodetection of native BES1 from heat-stressed and epiBL-treated abitM plants. abitM and wild-type plants were grown on ½ MS medium either containing DMSO as a control or 1 μM epiBL and either left untreated or exposed to 45°C for 60 minutes. The non-phosphorylated BES1 bands were quantified with ImageJ and the obtained values are shown in blue.
Figure 4C. ChIP to determine BES1-CFP enrichment on the promoters of HSP70.3, HSP70.4 and HSP90.1 following heat stress. 35S:BES1-CFP and wild-type plants grown at 21°C were either left untreated or treated with 45°C for 60 minutes. BES1-CFP was immuno-precipitated with α-GFP beads. Fragments were quantified in the precipitates with qPCR and the ratios of samples with antibody to without antibody were calculated. TA3 (70.3p and 90.1p) or 5s rRNA (70.4p) were used for normalization. UBQ5 served as an internal control. Data show the mean ±SE. n=3 biological replicates measured in 3 technical repeats. Significant differences as compared to wild-type at 21°C, calculated by Student's t-test, are shown (*P≤0.05; **P≤0.01; ***P≤0.001).
Figure 5A. EMSAs showing in vitro DNA interaction of recombinant BES1 and/or HSFA1 with HSE-containing fragments of the HSP70.3 and HSP90.1 promoters. Unlabeled competitor fragments with functional HSEs (C1 +HSE) or mutated versions (C2 -HSE) were used for competition studies in 100x molar access (lanes 6 and 7).B. BiFC assays showing cells co-transformed with nYFP-BES1 and cYFP-HSFA1a. The fusion proteins were co-expressed in N. benthamiana leaves and fluorescence was visualized. Two nuclei are highlighted and shown in a 6-fold magnification. Scale bar=20 μm.C. LUC assays in protoplasts of wild-type, bes1-D or the hsfA1qM plants. LUC reporter constructs, driven by 2 kb of the HSP70.3, HSP70.4 and HSP90.1 promoters, were tested either alone (0, with empty effector plasmid) or with BES1 or HSFA1a as effectors and LUC transactivation was analyzed. Data show the mean ±SD. n=5. Statistically significant difference at P≤0.05 of results is indicated with different letters and was determined by Student's t-test.