Analysis of shScramble ( gray , n = 2 , 400 cells ) ‐ , shIP3R ( 1 , 3 ) A ( blue , n = 1 , 611 cells ) ‐ , and shIP3R ( 1 , 3 ) C ( green , n = 1 , 454 cells ) ‐ expressing DCs migrating in the device described in Supplementary Fig S3I , stained with Hoechst and imaged every 2 min during 24 h at 10 × magnification . Values were obtained from three independent experiments . Cell trajectories were obtained by tracking their nucleus . C Representation of individual DC trajectories .

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Analysis of shScramble (gray, n = 2,400 cells)‐, shIP3R(1,3)A (blue, n = 1,611 cells)‐, and shIP3R(1,3)C (green, n = 1,454 cells)‐expressing DCs migrating in the device described in Supplementary Fig S3I, stained with Hoechst and imaged every 2 min during 24 h at 10× magnification. Values were obtained from three independent experiments. Cell trajectories were obtained by tracking their nucleus.C Representation of individual DC trajectories.


Analysis of shScramble ( gray , n = 2 , 400 cells ) ‐ , shIP3R ( 1 , 3 ) A ( blue , n = 1 , 611 cells ) ‐ , and shIP3R ( 1 , 3 ) C ( green , n = 1 , 454 cells ) ‐ expressing DCs migrating in the device described in Supplementary Fig S3I , stained with Hoechst and imaged every 2 min during 24 h at 10 × magnification . Values were obtained from three independent experiments . Cell trajectories were obtained by tracking their nucleus . D Path persistence ( calculated as the ratio between the effective length and the total length of the cell trajectory ) of migrating DCs . P ‐ values were determined with a t ‐ test analysis .

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Analysis of shScramble (gray, n = 2,400 cells)‐, shIP3R(1,3)A (blue, n = 1,611 cells)‐, and shIP3R(1,3)C (green, n = 1,454 cells)‐expressing DCs migrating in the device described in Supplementary Fig S3I, stained with Hoechst and imaged every 2 min during 24 h at 10× magnification. Values were obtained from three independent experiments. Cell trajectories were obtained by tracking their nucleus.D Path persistence (calculated as the ratio between the effective length and the total length of the cell trajectory) of migrating DCs. P‐values were determined with a t‐test analysis.


Analysis of shScramble ( gray , n = 2 , 400 cells ) ‐ , shIP3R ( 1 , 3 ) A ( blue , n = 1 , 611 cells ) ‐ , and shIP3R ( 1 , 3 ) C ( green , n = 1 , 454 cells ) ‐ expressing DCs migrating in the device described in Supplementary Fig S3I , stained with Hoechst and imaged every 2 min during 24 h at 10 × magnification . Values were obtained from three independent experiments . Cell trajectories were obtained by tracking their nucleus . E Bars showing the proportion of the effective length displacement ( L ) of DCs . A Fisher ' s exact test ( P ‐ value 2 . 2 × 10 − 16 ) was used for statistics .

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Analysis of shScramble (gray, n = 2,400 cells)‐, shIP3R(1,3)A (blue, n = 1,611 cells)‐, and shIP3R(1,3)C (green, n = 1,454 cells)‐expressing DCs migrating in the device described in Supplementary Fig S3I, stained with Hoechst and imaged every 2 min during 24 h at 10× magnification. Values were obtained from three independent experiments. Cell trajectories were obtained by tracking their nucleus.E Bars showing the proportion of the effective length displacement (L) of DCs. A Fisher's exact test (P‐value 2.2 × 10−16) was used for statistics.


A Immunoblot showing the intracellular levels of phosphorylated MLC in DCs transduced with the shScramble or shRNAs for IP3Rs ( shIP3R ( 1 , 3 ) A ′ , shIP3R ( 2 , 3 ) B ′ , and shIP3R ( 1 , 3 ) C ′ ) . Actin was used as a control .

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A Immunoblot showing the intracellular levels of phosphorylated MLC in DCs transduced with the shScramble or shRNAs for IP3Rs (shIP3R(1,3)A′, shIP3R(2,3)B′, and shIP3R(1,3)C′). Actin was used as a control.


B Inverted sequential epifluorescence images ( 20 × ) showing myosin IIA ‐ GFP localization of a representative shScramble ( left ) ‐ and shIP3R ( 1 , 3 ) C ‐ expressing DC ( right ) migrating in micro ‐ channels . Myosin IIA ‐ GFP knock ‐ in DCs were imaged every 1 min ( 1 out of 2 images is shown ) . Asterisks highlight changes of direction during locomotion .

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B Inverted sequential epifluorescence images (20×) showing myosin IIA‐GFP localization of a representative shScramble (left)‐ and shIP3R(1,3)C‐expressing DC (right) migrating in micro‐channels. Myosin IIA‐GFP knock‐in DCs were imaged every 1 min (1 out of 2 images is shown). Asterisks highlight changes of direction during locomotion.


C , D Myosin IIA ‐ GFP localization ( red ) versus velocity fluctuations ( blue , ΔV / V0 ) in a representative shScramble ‐ transduced DC ( C ) or shIP3R ( 1 , 3 ) C ‐ silenced ( D ) migrating in micro ‐ channels . DCs were recorded as in ( B ) . E Quantification of the interval between curves ( highlighted in D ) obtained for myosin IIA positioning and velocity from shScramble ‐ and shIP3R ( 1 , 3 ) C ‐ expressing DCs . Cells were recorded as in ( B ) ( n = 20 cells from two experiments ) . The Mann - Whitney test was applied for statistical analysis .

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C, D Myosin IIA‐GFP localization (red) versus velocity fluctuations (blue, ΔV/V0) in a representative shScramble‐transduced DC (C) or shIP3R(1,3)C‐silenced (D) migrating in micro‐channels. DCs were recorded as in (B).E Quantification of the interval between curves (highlighted in D) obtained for myosin IIA positioning and velocity from shScramble‐ and shIP3R(1,3)C‐expressing DCs. Cells were recorded as in (B) (n = 20 cells from two experiments). The Mann-Whitney test was applied for statistical analysis.


A Immunoblot showing the intracellular levels of phosphorylated MLC in DCs treated overnight with increasing amounts of the MLCK inhibitor , ML7 .

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A Immunoblot showing the intracellular levels of phosphorylated MLC in DCs treated overnight with increasing amounts of the MLCK inhibitor, ML7.


B , C Analysis of immature DCs migrating in micro ‐ channels in the presence of ML7 or DMSO as a control ( n > 170 cells from two independent experiments ) . Velocity fluctuations ( ΔV / V0 ) of immature DCs migrating in micro ‐ channels ( B ) . Whiskers show 10 - 90 percentiles plus the median . P ‐ values were calculated using a Kruskal - Wallis test . Frequency of changes ( C ) in direction of DCs migrating in micro ‐ channels . Mean plus SD are shown . A Kruskal - Wallis test was applied for statistical analysis .

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B, C Analysis of immature DCs migrating in micro‐channels in the presence of ML7 or DMSO as a control (n > 170 cells from two independent experiments). Velocity fluctuations (ΔV/V0) of immature DCs migrating in micro‐channels (B). Whiskers show 10-90 percentiles plus the median. P‐values were calculated using a Kruskal-Wallis test. Frequency of changes (C) in direction of DCs migrating in micro‐channels. Mean plus SD are shown. A Kruskal-Wallis test was applied for statistical analysis.


D , E Analysis of shScramble ( gray ) ‐ and shIP3R ( 1 , 3 ) C ( green ) ‐ expressing DCs migrating in micro ‐ channels in the presence of 20 μM ML7 ( n > 70 cells per condition from 3 independent experiments ) . Velocity fluctuations ( ΔV / V0 ) of immature DCs migrating in micro ‐ channels ( D ) . Boxes illustrate 10 - 90 percentiles of values , and whiskers represent the range of values . P ‐ values were calculated using a Kruskal - Wallis test . Frequency of changes ( E ) in direction observed in DCs migrating in micro ‐ channels . Means plus SD are shown . A Kruskal - Wallis test was applied for statistical analysis .

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D, E Analysis of shScramble (gray)‐ and shIP3R(1,3)C (green)‐expressing DCs migrating in micro‐channels in the presence of 20 μM ML7 (n > 70 cells per condition from 3 independent experiments). Velocity fluctuations (ΔV/V0) of immature DCs migrating in micro‐channels (D). Boxes illustrate 10-90 percentiles of values, and whiskers represent the range of values. P‐values were calculated using a Kruskal-Wallis test. Frequency of changes (E) in direction observed in DCs migrating in micro‐channels. Means plus SD are shown. A Kruskal-Wallis test was applied for statistical analysis.


Quantification by western - blot analysis of EZH2 and H3K27me3 levels at each stage of de novo transformation . UT : untransformed , PN : pre - neoplastic , TR : transformed . Histone H3 is used as a loading control . The graph on the right displays the H3 normalized densitometric values of western - blot band from untreated and DMSO treated cells . Values represent mean ± SEM from three biological replicates . One asterisk indicates p - value < 0 . 05 ( one - tailed unpaired Student ' s t - test ) . ns : non - significant .

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Quantification by western-blot analysis of EZH2 and H3K27me3 levels at each stage of de novo transformation. UT: untransformed, PN: pre-neoplastic, TR: transformed. Histone H3 is used as a loading control. The graph on the right displays the H3 normalized densitometric values of western-blot band from untreated and DMSO treated cells. Values represent mean ± SEM from three biological replicates. One asterisk indicates p-value < 0.05 (one-tailed unpaired Student's t-test). ns: non-significant.


Volcano plot showing the relative enrichment of EZH2 binding at all sites detected in either untransformed or transformed cells . Numbers within the volcano plot indicate differential binding sites with a p - value ≤ 1e - 20 and a fold - change in normalised tag count ≥ 1 . 5 . Enrichment significance calculated based on a Poisson distribution using the ' getDifferentialPeaks ' function in homer ( see : methods ) . P - values of > 1e - 100 were set to 1e - 100 for display reasons . Tracks on the right show representative high - and low - magnitude EZH2 differential peaks . ChIP - seq signal normalised to sequencing depth is shown . Tracks are scaled to be of the same height to make samples comparable . Blue bars represent regions called as an EZH2 binding site . UT : untransformed , TR : transformed .

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Volcano plot showing the relative enrichment of EZH2 binding at all sites detected in either untransformed or transformed cells. Numbers within the volcano plot indicate differential binding sites with a p-value ≤1e-20 and a fold-change in normalised tag count ≥1.5. Enrichment significance calculated based on a Poisson distribution using the 'getDifferentialPeaks' function in homer (see: methods). P-values of > 1e-100 were set to 1e-100 for display reasons. Tracks on the right show representative high- and low-magnitude EZH2 differential peaks. ChIP-seq signal normalised to sequencing depth is shown. Tracks are scaled to be of the same height to make samples comparable. Blue bars represent regions called as an EZH2 binding site. UT: untransformed, TR: transformed.


Western - blot analysis of H3K27me3 levels after treatment of cells with an EZH2 inhibitor ( EZH2i ) or DMSO as control for 12 days . Histone H3 is used as a loading control . Values represent H3 - normalized densitometric values of the H3K27me3 bands , and are expressed relative to the relevant DMSO control . UT : untransformed , PN : pre - neoplastic , TR : transformed .

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Western-blot analysis of H3K27me3 levels after treatment of cells with an EZH2 inhibitor (EZH2i) or DMSO as control for 12 days. Histone H3 is used as a loading control. Values represent H3-normalized densitometric values of the H3K27me3 bands, and are expressed relative to the relevant DMSO control. UT: untransformed, PN: pre-neoplastic, TR: transformed.


Quantification of differentially expressed genes ( false discovery rate ( FDR ) ≤ 0 . 01 , Log2FC ≥ 1 / ≤ - 1 and maximal transcripts per million ( maxTPM ) ≥ 1 ) detected by RNA - seq in each cellular state upon EZH2i treatment . UT : untransformed , PN : pre - neoplastic , TR : transformed .

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Quantification of differentially expressed genes (false discovery rate (FDR) ≤ 0.01, Log2FC ≥ 1/≤-1 and maximal transcripts per million (maxTPM) ≥ 1) detected by RNA-seq in each cellular state upon EZH2i treatment. UT: untransformed, PN: pre-neoplastic, TR: transformed.


Heatmap showing the relative expression of genes identified by the multi - step filtering strategy in the indicated cellular states . UT : untransformed , PN : pre - neoplastic , TR : transformed . Colours represent log2 - transformed , row - normalised TPM values .

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Heatmap showing the relative expression of genes identified by the multi-step filtering strategy in the indicated cellular states. UT: untransformed, PN: pre-neoplastic, TR: transformed. Colours represent log2-transformed, row-normalised TPM values.


ChIP - seq signal for EZH2 and H3K27me3 ( left ) at EMX2 and HOXB9 loci . ChIP - seq signal normalised to sequencing depth is shown . Tracks are scaled to be of the same height to make samples comparable . mRNA expression of EMX2 and HOXB9 ( right ) as detected by RNA - seq . Expression values represent mean ± SEM from three biological replicates . UT : untransformed , PN : pre - neoplastic , TR : transformed . TPM : transcripts per million .

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ChIP-seq signal for EZH2 and H3K27me3 (left) at EMX2 and HOXB9 loci. ChIP-seq signal normalised to sequencing depth is shown. Tracks are scaled to be of the same height to make samples comparable. mRNA expression of EMX2 and HOXB9 (right) as detected by RNA-seq. Expression values represent mean ± SEM from three biological replicates. UT: untransformed, PN: pre-neoplastic, TR: transformed. TPM: transcripts per million.


qRT - PCR showing the expression levels of EMX2 and HOXB9 in the indicated cells treated with EZH2i or a DMSO control for 12 days . Values represent mean ± SEM from three biological replicates . Two asterisks indicate p - value < 0 . 01 ( one - tailed unpaired Student ' s t - test ) . UT : untransformed , TR : transformed .

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qRT-PCR showing the expression levels of EMX2 and HOXB9 in the indicated cells treated with EZH2i or a DMSO control for 12 days. Values represent mean ± SEM from three biological replicates. Two asterisks indicate p-value < 0.01 (one-tailed unpaired Student's t-test). UT: untransformed, TR: transformed.


Expression levels of EMX2 and HOXB9 in primary human neural cells , as detected by RNA - seq . Data sourced from Brainseq2 . Expression values represent mean ± SEM from four , twelve , one and five biological replicates of fetal astrocytes , mature astrocytes , neurons and oligodendrocytes , respectively . FPKM : fragments per kilobase of transcript per million .

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Expression levels of EMX2 and HOXB9 in primary human neural cells, as detected by RNA-seq. Data sourced from Brainseq2. Expression values represent mean ± SEM from four, twelve, one and five biological replicates of fetal astrocytes, mature astrocytes, neurons and oligodendrocytes, respectively. FPKM: fragments per kilobase of transcript per million.


Expression levels of EMX2 and HOXB9 in 62 glioma cell lines . Data sourced from CCLE . Every dot represents a cell line . RPKM : reads per kilobase of transcript per million . In the boxplot , the top , middle and bottom box delimiters represent the 75th , 50th and 25th percentiles of the data , respectively . Top and bottom whiskers show the 75th percentile + 1 . 5 * interquartile range and 25th percentile - 1 . 5 * interquartile range , respectively .

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Expression levels of EMX2 and HOXB9 in 62 glioma cell lines. Data sourced from CCLE. Every dot represents a cell line. RPKM: reads per kilobase of transcript per million. In the boxplot, the top, middle and bottom box delimiters represent the 75th, 50th and 25th percentiles of the data, respectively. Top and bottom whiskers show the 75th percentile + 1.5*interquartile range and 25th percentile - 1.5*interquartile range, respectively.


Quantification of EZH2 binding at EMX2 , HOXB9 and GAPDH ( negative control ) promoters by ChIP - qPCR in M059K GBM cells . Values from two biological replicates are shown . Three asterisks indicate p - value < 0 . 001 of EZH2 samples relative to the negative control GAPDH ( two - way ANOVA followed by pairwise comparisons using Holm - Sidak method ) . Ns : non - significant .

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Quantification of EZH2 binding at EMX2, HOXB9 and GAPDH (negative control) promoters by ChIP-qPCR in M059K GBM cells. Values from two biological replicates are shown. Three asterisks indicate p-value < 0.001 of EZH2 samples relative to the negative control GAPDH (two-way ANOVA followed by pairwise comparisons using Holm-Sidak method). Ns: non-significant.


Quantification of EMX2 levels by qRT - PCR in five different GBM cell lines upon treatment with 1μM , 3μM or 10μM of EZH2i for eight days , or a DMSO control . Values represent mean ± SEM from three technical replicates . The SEM is indicated to show reliability of the RT - PCR values , due to the low endogenous levels of EMX2 . Two asterisks indicate p - value < 0 . 01 comparing EZH2i - and DMSO - treated cells ( Two - way ANOVA ) .

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Quantification of EMX2 levels by qRT-PCR in five different GBM cell lines upon treatment with 1μM, 3μM or 10μM of EZH2i for eight days, or a DMSO control. Values represent mean ± SEM from three technical replicates. The SEM is indicated to show reliability of the RT-PCR values, due to the low endogenous levels of EMX2. Two asterisks indicate p-value <0.01 comparing EZH2i- and DMSO-treated cells (Two-way ANOVA).


Relationship between the extent of EMX2 upregulation induced by EZH2i and the DNA methylation level at the EMX2 promoter in GBM cell lines . Values for EMX2 upregulation are the fold - change in mRNA expression induced upon treatment with 10μM EZH2i ( see : panel G ) , and are expressed relative to a DMSO control . Values represent mean ± SEM from three technical replicates . The SEM is indicated to show reliability of the RT - PCR values , due to the low endogenous levels of EMX2 . Methylation values are averages of DNA methylation at CpGs across the promoter of EMX2 in GBM cell lines . All data sourced from the CCLE . The significance of the anti - correlation between mRNA levels and DNA methylation levels across cell lines is indicated ( Spearman rank correlation ) .

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Relationship between the extent of EMX2 upregulation induced by EZH2i and the DNA methylation level at the EMX2 promoter in GBM cell lines. Values for EMX2 upregulation are the fold-change in mRNA expression induced upon treatment with 10μM EZH2i (see: panel G), and are expressed relative to a DMSO control. Values represent mean ± SEM from three technical replicates. The SEM is indicated to show reliability of the RT-PCR values, due to the low endogenous levels of EMX2. Methylation values are averages of DNA methylation at CpGs across the promoter of EMX2 in GBM cell lines. All data sourced from the CCLE. The significance of the anti-correlation between mRNA levels and DNA methylation levels across cell lines is indicated (Spearman rank correlation).


Heatmap visualizing the fold change in EMX2 expression as detected by qRT - PCR after treatment of the indicated PDX - derived ( blue ) and established cancer cell lines ( black ) with EZH2i for eight days . Values are from three technical replicates .

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Heatmap visualizing the fold change in EMX2 expression as detected by qRT-PCR after treatment of the indicated PDX-derived (blue) and established cancer cell lines (black) with EZH2i for eight days. Values are from three technical replicates.


Expression of EMX2 in normal brain and GBM patient samples . Data sourced from the Repository of Molecular Brain Neoplasia Data ( REMBRANDT ) . Four asterisks indicate p - value < 0 . 0001 ( two - tailed Mann Whitney U test ) . Bars represent median ± interquartile range . Values are expressed relative to the mean of normal brain samples . N : 21 for normal brain , 214 for GBM . Note that the extent of EMX2 repression in GBM patients is likely underestimated due to the possible presence of normal adjacent tissue in the analysed samples , and due to the intrinsic background noise of microarrays , which limits detection of truly silenced genes .

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Expression of EMX2 in normal brain and GBM patient samples. Data sourced from the Repository of Molecular Brain Neoplasia Data (REMBRANDT). Four asterisks indicate p-value < 0.0001 (two-tailed Mann Whitney U test). Bars represent median ± interquartile range. Values are expressed relative to the mean of normal brain samples. N: 21 for normal brain, 214 for GBM. Note that the extent of EMX2 repression in GBM patients is likely underestimated due to the possible presence of normal adjacent tissue in the analysed samples, and due to the intrinsic background noise of microarrays, which limits detection of truly silenced genes.


Relative expression of HOX genes in normal brain and GBM patient samples as detected by microarray analysis . Data sourced from REMBRANDT . Values are expressed relative to the median of normal brain samples . In the boxplots , the top , middle and bottom box delimiters represent the 75th , 50th and 25th percentiles of the data , respectively . Top and bottom whiskers show the 75th percentile + 1 . 5 * interquartile range and 25th percentile - 1 . 5 * interquartile range , respectively . One asterisk indicates p - value < 0 . 05 ( two - tailed Mann Whitney U test corrected for multiple comparison using Holm ' s method ) . N : 21 for normal brain , 214 for GBM . Data was not available for HOXD8 .

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Relative expression of HOX genes in normal brain and GBM patient samples as detected by microarray analysis. Data sourced from REMBRANDT. Values are expressed relative to the median of normal brain samples. In the boxplots, the top, middle and bottom box delimiters represent the 75th, 50th and 25th percentiles of the data, respectively. Top and bottom whiskers show the 75th percentile + 1.5*interquartile range and 25th percentile - 1.5*interquartile range, respectively. One asterisk indicates p-value <0.05 (two-tailed Mann Whitney U test corrected for multiple comparison using Holm's method). N: 21 for normal brain, 214 for GBM. Data was not available for HOXD8.


Covariance between EMX2 and EZH2 expression levels in GBM patient samples , as detected by microarray analysis . Data sourced from REMBRANDT . P - value and correlation coefficient ( r ) of the covariance are shown ( Spearman rank correlation ) . Every dot is a patient . N : 214 .

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Covariance between EMX2 and EZH2 expression levels in GBM patient samples, as detected by microarray analysis. Data sourced from REMBRANDT. P-value and correlation coefficient (r) of the covariance are shown (Spearman rank correlation). Every dot is a patient. N: 214.


Expression levels of EMX2 ( top ) and EZH2 ( bottom ) in tumour or adjacent normal regions laser microdissected from human GBM tumours as detected by RNA - seq . Data sourced from the Ivy Glioblastoma Atlas . FPKM : fragments per kilobase of transcript per million . The significance of the differential expression in normal and tumour regions across patients is indicated ( two - way ANOVA ) . Bars represent mean ± SEM . N : 3 regions sampled for each GBM tumour , 2 , 3 , 2 , 3 , 2 , 3 , 3 and 1 regions for normal tissue of patients 1 - 8 , respectively .

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Expression levels of EMX2 (top) and EZH2 (bottom) in tumour or adjacent normal regions laser microdissected from human GBM tumours as detected by RNA-seq. Data sourced from the Ivy Glioblastoma Atlas. FPKM: fragments per kilobase of transcript per million. The significance of the differential expression in normal and tumour regions across patients is indicated (two-way ANOVA). Bars represent mean ± SEM. N: 3 regions sampled for each GBM tumour, 2, 3, 2, 3, 2, 3, 3 and 1 regions for normal tissue of patients 1-8, respectively.


Hematoxylin and eosin ( H & E ) staining of a human GBM tumour . The black square represents the approximate location of the field shown in G ( left image ) . Serial sections were used for H & E and RNA - FISH . Scale bar : 300 µm .

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Hematoxylin and eosin (H&E) staining of a human GBM tumour. The black square represents the approximate location of the field shown in G (left image). Serial sections were used for H&E and RNA-FISH. Scale bar: 300 µm.


Visualisation of EZH2 ( green ) and EMX2 mRNA ( red ) in a human GBM tumour by RNA FISH . Nuclei were counterstained with DAPI . Scale bar : 20 µm .

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Visualisation of EZH2 (green) and EMX2 mRNA (red) in a human GBM tumour by RNA FISH. Nuclei were counterstained with DAPI. Scale bar: 20 µm.


Expression of EMX2 ( left ) and EZH2 ( right ) in glioma patient samples , grouped according to tumour grade , as detected by RNA - seq . Data sourced from the Chinese Glioma Genome Atlas . Four asterisks indicate p - value < 0 . 0001 ( Kruskal - Wallis test ) . Bars represent median ± interquartile range . N : 109 for grade II - , 72 for grade III , 144 for grade IV . RPKM : Reads per kilobase of transcript per million .

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Expression of EMX2 (left) and EZH2 (right) in glioma patient samples, grouped according to tumour grade, as detected by RNA-seq. Data sourced from the Chinese Glioma Genome Atlas. Four asterisks indicate p-value < 0.0001 (Kruskal-Wallis test). Bars represent median ± interquartile range. N: 109 for grade II -, 72 for grade III, 144 for grade IV. RPKM: Reads per kilobase of transcript per million.


Proliferation assay examining the effect of EMX2 ectopic expression in U - 87 MG and DBTRG - 05MG GBM cells , RFP is used as a control . Values represent mean ± SEM from three biological replicates . The fold change in cell number after eight days of proliferation whilst expressing the exogenous protein is shown relative to an uninduced control . Two asterisks indicate p - value < 0 . 01 ( one - tailed unpaired Student ' s t - test ) . Similar results were reported in [ 103 ] using other GBM cell lines .

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Proliferation assay examining the effect of EMX2 ectopic expression in U-87 MG and DBTRG-05MG GBM cells, RFP is used as a control. Values represent mean ± SEM from three biological replicates. The fold change in cell number after eight days of proliferation whilst expressing the exogenous protein is shown relative to an uninduced control. Two asterisks indicate p-value < 0.01 (one-tailed unpaired Student's t-test). Similar results were reported in [103] using other GBM cell lines.


Transplantation assay comparing the growth kinetics of subcutaneous DBTRG - 05MG - induced tumours expressing EMX2 at levels comparable to those expressed in normal cells , or expressing RFP as a control . Values represent mean ± SEM from six tumours . Three asterisks indicate p - value < 0 . 001 ( two - tailed single sample Student ' s t - test ) . Similar results were obtained in an independent experiment using a distinct batch of transduced cells .

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Transplantation assay comparing the growth kinetics of subcutaneous DBTRG-05MG-induced tumours expressing EMX2 at levels comparable to those expressed in normal cells, or expressing RFP as a control. Values represent mean ± SEM from six tumours. Three asterisks indicate p-value < 0.001 (two-tailed single sample Student's t-test). Similar results were obtained in an independent experiment using a distinct batch of transduced cells.


MRI scans showing representative brain tumours six weeks post - injection of DBTRG - 05MG cells expressing EMX2 or RFP . Axial anatomical scan and coronal signal intensity map indicate location of tumour in RFP control ( yellow arrow ) . Contrast enhancement in post - contrast images indicates tumour blood brain barrier breakdown . Signal was not present in pre - contrast images . Images show the same MRI slice position between mice . Scale bar : 2mm . Quantification of brain tumour size in mice injected with EMX2 - or RFP - expressing DBTRG - 05MG cells . Values represent mean ± SEM from four control and three EMX2 tumours . Five mice per conditions were injected but three ( one for RFP , and two for EMX2 ) had to be excluded from the study due to complications from the procedure .

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MRI scans showing representative brain tumours six weeks post-injection of DBTRG-05MG cells expressing EMX2 or RFP. Axial anatomical scan and coronal signal intensity map indicate location of tumour in RFP control (yellow arrow). Contrast enhancement in post-contrast images indicates tumour blood brain barrier breakdown. Signal was not present in pre-contrast images. Images show the same MRI slice position between mice. Scale bar: 2mm. Quantification of brain tumour size in mice injected with EMX2- or RFP- expressing DBTRG-05MG cells. Values represent mean ± SEM from four control and three EMX2 tumours. Five mice per conditions were injected but three (one for RFP, and two for EMX2) had to be excluded from the study due to complications from the procedure.


Representative images of euploid control or ArCK cells interacting with NK cells . The NK cell - mediated killing was measured at a 2 . 5 : 1 ( NK cells : target cells ) ratio and was recorded by live cell imaging for 36 hours at a 30 - min interval . TO - PRO - 3 ( 1 μM ) was added to the medium at the same time of NK cell addition to measure cell membrane integrity . Phase contrast ( top ) and TO - PRO - 3 signal ( bottom ) from the same field were presented . Arrowheads indicate ArCK cell death . All images were acquired at the same exposure time and light intensity . Scale bar 20 μM .

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Representative images of euploid control or ArCK cells interacting with NK cells. The NK cell-mediated killing was measured at a 2.5:1 (NK cells: target cells) ratio and was recorded by live cell imaging for 36 hours at a 30-min interval. TO-PRO-3 (1 μM) was added to the medium at the same time of NK cell addition to measure cell membrane integrity. Phase contrast (top) and TO-PRO-3 signal (bottom) from the same field were presented. Arrowheads indicate ArCK cell death. All images were acquired at the same exposure time and light intensity. Scale bar 20 μM.


NK cell - mediated cytotoxicity across various NK cell to target cell ratios . Either euploid control ( Ctrl ) or ArCK cells were co - cultured with NK cells at the indicated NK cell : target cell ratios . The cumulative killing of target cells was measured . n = 3 biological replicates ; mean ± SEM . p < 0 . 0001 for all four NK cell : target cell ratios , KS test .

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NK cell-mediated cytotoxicity across various NK cell to target cell ratios. Either euploid control (Ctrl) or ArCK cells were co-cultured with NK cells at the indicated NK cell: target cell ratios. The cumulative killing of target cells was measured. n=3 biological replicates; mean± SEM. p< 0.0001 for all four NK cell: target cell ratios, KS test.


NK cell - mediated killing for doxorubicin ( Doxo ) , palbociclib ( Palbo ) , and nutlin3 ( Nutlin ) treated samples ( NK cell : target cell = 2 . 5 : 1 ) . n = 3 biological replicates ; mean ± SEM . Ctrl vs . Doxo , p < 0 . 0001 ; Ctrl vs . Palbo , p < 0 . 0001 ; Ctrl vs . Nutlin , p < 0 . 0001 ; KS test .

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NK cell-mediated killing for doxorubicin (Doxo), palbociclib (Palbo), and nutlin3 (Nutlin) treated samples (NK cell: target cell =2.5:1). n=3 biological replicates; mean± SEM. Ctrl vs. Doxo, p< 0.0001; Ctrl vs. Palbo, p< 0.0001; Ctrl vs. Nutlin, p< 0.0001; KS test.


NK cell - mediated cytotoxicity towards torin1 - treated cells . Torin1 treated ( Torin ) cells were generated as described in ( F ) and the NK cell killing assay was performed as described in Figure 1 . n = 3 biological replicates ; mean ± SEM . Ctrl vs . Torin , p = 0 . 79 , not significant ( n . s . ) ; KS test .

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NK cell-mediated cytotoxicity towards torin1-treated cells. Torin1 treated (Torin) cells were generated as described in (F) and the NK cell killing assay was performed as described in Figure 1. n=3 biological replicates; mean± SEM. Ctrl vs. Torin, p= 0.79, not significant (n.s.); KS test.


γ - H2AX foci were analyzed in ArCK and G1 arrested cells ( generated as described in Figure 2 ) . At least 50 cells were analyzed per condition per replicate . n = 2 biological replicates ; individual values and mean are shown . The distribution of the foci number in each treated condition was compared to that of control using Kolmogorov - Smirnov test . Ctrl vs . Doxo , p < 0 . 0001 ; Ctrl vs . Palbo , p = 0 . 07 , n . s . ; Ctrl vs . Nutlin , p = 0 . 11 , n . s . ; Ctrl vs . ArCK , p = 0 . 0007 .

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γ-H2AX foci were analyzed in ArCK and G1 arrested cells (generated as described in Figure 2). At least 50 cells were analyzed per condition per replicate. n=2 biological replicates; individual values and mean are shown. The distribution of the foci number in each treated condition was compared to that of control using Kolmogorov-Smirnov test. Ctrl vs. Doxo, p< 0.0001; Ctrl vs. Palbo, p= 0.07, n.s.; Ctrl vs. Nutlin, p= 0.11, n.s.; Ctrl vs. ArCK, p= 0.0007.


Secreted cytokine and interferon levels were determined in cell supernatants . Media were collected after 36 hours of incubation with cells grown as described in Figure 1 and 2 . Cytokine and interferon levels were shown as fold change normalized to euploid control cells . Individual values and mean are shown ; n = 2 biological replicates .

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Secreted cytokine and interferon levels were determined in cell supernatants. Media were collected after 36 hours of incubation with cells grown as described in Figure 1 and 2. Cytokine and interferon levels were shown as fold change normalized to euploid control cells. Individual values and mean are shown; n=2 biological replicates.


NK cell medium was incubated with either euploid control or ArCK cells for 12 hours . At the time of NK cell addition , media were switched between ArCK and euploid control cells ( Ctrl ) . NK cell killing was measured as described in Figure 1C . For reference , NK cell killing of ArCK and euploid control cells ( Ctrl ) without medium switch were performed side by side and plotted on the graph . Black , euploid control cells without medium switch ; red , ArCK cells without medium switch ; blue , euploid control cells in ArCK cell condition medium ; green , ArCK cells in euploid control cell condition medium . n = 3 biological replicates ; mean ± SEM . Ctrl vs . ArCK , p < 0 . 0001 ; Ctrl vs . Ctrl in ArCK med , p < 0 . 0001 ; Ctrl vs . ArCK in Ctrl med , p < 0 . 0001 ; ArCK vs . ArCK in Ctrl med , p = 0 . 0002 ; KS test .

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NK cell medium was incubated with either euploid control or ArCK cells for 12 hours. At the time of NK cell addition, media were switched between ArCK and euploid control cells (Ctrl). NK cell killing was measured as described in Figure 1C. For reference, NK cell killing of ArCK and euploid control cells (Ctrl) without medium switch were performed side by side and plotted on the graph. Black, euploid control cells without medium switch; red, ArCK cells without medium switch; blue, euploid control cells in ArCK cell condition medium; green, ArCK cells in euploid control cell condition medium. n=3 biological replicates; mean± SEM. Ctrl vs. ArCK, p< 0.0001; Ctrl vs. Ctrl in ArCK med, p< 0.0001; Ctrl vs. ArCK in Ctrl med, p< 0.0001; ArCK vs. ArCK in Ctrl med, p= 0.0002; KS test.


RT - qPCR quantifying NF - κB downstream target gene expression in ArCK and euploid control ( Ctrl ) cells . n = 6 biological replicates ; mean ± SEM . IL - 1b , * p = 0 . 029 ; IL - 6 , * p = 0 . 032 ; IL - 8 , * * p = 0 . 008 ; unpaired t - test .

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RT-qPCR quantifying NF-κB downstream target gene expression in ArCK and euploid control (Ctrl) cells. n=6 biological replicates; mean± SEM. IL-1b, *p= 0.029; IL-6, *p= 0.032; IL-8, **p= 0.008; unpaired t-test.


ArCK or euploid proliferating control cells were treated with either DMSO or the NF - κB inhibitor BMS - 345541 ( 5 μM ) for 48 hours before assessing NK cell - mediated cytotoxicity . The drug was washed out during the NK cell co - culture assay . n = 3 biological replicates ; mean ± SEM . ArCK vs ArCK NF - κB inhibitor , p < 0 . 0001 ; KS test . ( H , I ) The effect of inactivating both RELA and RELB on NK cell - mediated cytotoxicity in 7 - day doxorubicin ( H ) and nutlin3 - treated ( I ) cells . n2 biological replicates ; Doxo - c1 vs . Doxo RELA RELB - c1 , p = 0 . 02 . Nutlin - c1 vs . Nutlin RELA RELB - c1 , p = 0 . 44 , n . s . ; KS test .

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ArCK or euploid proliferating control cells were treated with either DMSO or the NF-κB inhibitor BMS-345541 (5 μM) for 48 hours before assessing NK cell-mediated cytotoxicity. The drug was washed out during the NK cell co-culture assay. n=3 biological replicates; mean± SEM. ArCK vs ArCK NF-κB inhibitor, p< 0.0001; KS test. (H, I) The effect of inactivating both RELA and RELB on NK cell-mediated cytotoxicity in 7-day doxorubicin (H) and nutlin3-treated (I) cells. n2 biological replicates; Doxo-c1 vs. Doxo RELA RELB-c1, p = 0.02. Nutlin-c1 vs. Nutlin RELA RELB-c1, p = 0.44, n.s.; KS test.


HCT116 ( left ) or DLD1 cells ( right ) were treated with either DMSO or the Mps1 inhibitor reversine ( 500 nM ) for 48 hours before assessing NK cell - mediated cytotoxicity . The drug was washed out during the NK cell co - culture assay . n = 3 biological replicates ; mean ± SEM . HCT116 vs . HCT116 Mps1 inhibitor , p = 0 . 44 , n . s . ; DLD1 vs . DLD1 Mps1 inhibitor , p = 0 . 44 , n . s . ; KS test .

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HCT116 (left) or DLD1 cells (right) were treated with either DMSO or the Mps1 inhibitor reversine (500 nM) for 48 hours before assessing NK cell-mediated cytotoxicity. The drug was washed out during the NK cell co-culture assay. n=3 biological replicates; mean±SEM. HCT116 vs. HCT116 Mps1 inhibitor, p= 0.44, n.s.; DLD1 vs. DLD1 Mps1 inhibitor, p= 0.44, n.s.; KS test.


A Representative siliques from wild - type ( WT ; Col - 0 ) , herk1 , anj and herk1 anj plants prior to dehiscence . Siliques were placed on double - sided sticky tape and carpel walls separated from the replum to expose the developing seeds . Scale bar = 5 mm .

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A Representative siliques from wild-type (WT; Col-0), herk1, anj and herk1 anj plants prior to dehiscence. Siliques were placed on double-sided sticky tape and carpel walls separated from the replum to expose the developing seeds. Scale bar = 5 mm.


B Developing seeds per silique in wild - type , herk1 , anj and herk1 anj plants . Fully expanded siliques were dissected and photographed under a stereomicroscope . n = 15 ( four independent experiments with at least three plants per line and five siliques per plant ) . Data presented are means ± SEM . * * * p < 0 . 001 ( Student ' s t - test ) .

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B Developing seeds per silique in wild-type, herk1, anj and herk1 anj plants. Fully expanded siliques were dissected and photographed under a stereomicroscope. n = 15 (four independent experiments with at least three plants per line and five siliques per plant). Data presented are means ± SEM. *** p<0.001 (Student's t-test).


C Percentage of pollen tubes with normal reception at the female gametophyte ( black bars ; representative image middle centre of figure ) and with overgrowth ( grey bars ; representative image lower centre ) as assessed by aniline blue staining . 15 self - pollinated stage 16 flowers from wild - type , herk1 , anj and herk1 anj were analysed . Legend scale bars = 50 µm . * * * p < 0 . 001 ( χ - square tests ) .

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C Percentage of pollen tubes with normal reception at the female gametophyte (black bars; representative image middle centre of figure) and with overgrowth (grey bars; representative image lower centre) as assessed by aniline blue staining. 15 self-pollinated stage 16 flowers from wild-type, herk1, anj and herk1 anj were analysed. Legend scale bars = 50 µm. *** p<0.001 (χ-square tests).


D Aniline blue staining of pollen tube reception in reciprocal crosses between wild - type and herk1 anj plants with at least two siliques per cross . Legend as per ( C ) . * * * p < 0 . 001 ( χ - square tests ) .

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D Aniline blue staining of pollen tube reception in reciprocal crosses between wild-type and herk1 anj plants with at least two siliques per cross. Legend as per (C). *** p<0.001 (χ-square tests).


A , B Expression of pHERK1 : : H2B - TdTomato in mature ovules . White dotted lines delineate the egg cell and synergid cells . Data information : Scale bars = 50 µm . M , micropyle . CC , central cell . EC , egg cell . SC , synergid cell .

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A,B Expression of pHERK1::H2B-TdTomato in mature ovules. White dotted lines delineate the egg cell and synergid cells. Data information : Scale bars = 50 µm. M, micropyle. CC, central cell. EC, egg cell. SC, synergid cell.


C , D Expression of pANJ : : H2B - TdTomato in mature ovules . White dotted lines delineate the egg cell and synergid cells . Data information : Scale bars = 50 µm . M , micropyle . CC , central cell . EC , egg cell . SC , synergid cell .

C,D Expression of pANJ::H2B-TdTomato in mature ovules. White dotted lines delineate the egg cell and synergid cells. Data information : Scale bars = 50 µm. M, micropyle. CC, central cell. EC, egg cell. SC, synergid cell.


E , F Localisation of HERK1 - GFP in the synergid cell from the pFER : : HERK1 - GFP construct in ( F ) and corresponding differential interference contrast ( DIC ) image in ( E ) . White and red dotted lines delineate the egg cell and synergid cells , respectively . Data information : Scale bars = 50 µm . M , micropyle . Arrows , filiform apparatus .

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E,F Localisation of HERK1-GFP in the synergid cell from the pFER::HERK1-GFP construct in (F) and corresponding differential interference contrast (DIC) image in (E). White and red dotted lines delineate the egg cell and synergid cells, respectively. Data information : Scale bars = 50 µm. M, micropyle. Arrows, filiform apparatus.


G , H Localisation of ANJ - GFP in the synergid cell from the pANJ : : ANJ - GFP construct in ( H ) and corresponding DIC image in ( G ) . White and red dotted lines delineate the egg cell and synergid cells , respectively . Data information : Scale bars = 50 µm . M , micropyle . Arrows , filiform apparatus .

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G,H Localisation of ANJ-GFP in the synergid cell from the pANJ::ANJ-GFP construct in (H) and corresponding DIC image in (G). White and red dotted lines delineate the egg cell and synergid cells, respectively. Data information : Scale bars = 50 µm. M, micropyle. Arrows, filiform apparatus.


A Localisation of HERK1 , ANJ , LRE , FER and NTA in the synergid cell of wild - type ( Col - 0 ; WT ) , herk1 anj and lre - 5 in unfertilised ovules , as shown by pFER : : HERK1 - GFP , pANJ : : ANJ - GFP , pLRE : : LRE - Citrine , pFER : : FER - GFP and pMYB98 : : NTA - GFP . DIC and fluorescence images are shown , left to right , respectively . White and red dotted lines delineate the egg cell and synergid cells , respectively . Scale bars = 25 µm .

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A Localisation of HERK1, ANJ, LRE, FER and NTA in the synergid cell of wild-type (Col-0; WT), herk1 anj and lre-5 in unfertilised ovules, as shown by pFER::HERK1-GFP, pANJ::ANJ-GFP, pLRE::LRE-Citrine, pFER::FER-GFP and pMYB98::NTA-GFP. DIC and fluorescence images are shown, left to right, respectively. White and red dotted lines delineate the egg cell and synergid cells, respectively. Scale bars = 25 µm.


B Localisation of NTA in the synergid cell of wild - type and herk1 anj plants before ( upper panels ) and after ( lower panels ) pollen tube arrival . In green , NTA localisation as shown by pMYB98 : : NTA - GFP fluorescence . In magenta , callose of the filiform apparatus and pollen tube stained with SR2200 . From left to right , images shown are DIC , merged fluorescence images , and merged images of DIC and fluorescence . White and red dotted lines delineate the pollen tube and synergid cells , respectively . Scale bars = 25 µm . M , micropyle .

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B Localisation of NTA in the synergid cell of wild-type and herk1 anj plants before (upper panels) and after (lower panels) pollen tube arrival. In green, NTA localisation as shown by pMYB98::NTA-GFP fluorescence. In magenta, callose of the filiform apparatus and pollen tube stained with SR2200. From left to right, images shown are DIC, merged fluorescence images, and merged images of DIC and fluorescence. White and red dotted lines delineate the pollen tube and synergid cells, respectively. Scale bars = 25 µm. M, micropyle.


Profile of relative fluorescence intensity of NTA - GFP along the synergid cells of wild - type and herk1 anj ovules ( C ) before ( virgin ) and after ( pollinated ) pollen arrival . Data shown are means ± SEM , n = 25 . * * * p < 0 . 001 ( Student ' s t - test ) . FA , filiform apparatus .

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Profile of relative fluorescence intensity of NTA-GFP along the synergid cells of wild-type and herk1 anj ovules (C) before (virgin) and after (pollinated) pollen arrival. Data shown are means ± SEM, n = 25. *** p<0.001 (Student's t-test). FA, filiform apparatus.


D Profile of relative fluorescence intensity of NTA - GFP along the synergid cells of wild - type and lre - 5 ovules ( D ) before ( virgin ) and after ( pollinated ) pollen arrival . Data shown are means ± SEM , n = 25 . * * * p < 0 . 001 ( Student ' s t - test ) . FA , filiform apparatus .

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D Profile of relative fluorescence intensity of NTA-GFP along the synergid cells of wild-type and lre-5 ovules (D) before (virgin) and after (pollinated) pollen arrival. Data shown are means ± SEM, n = 25. *** p<0.001 (Student's t-test). FA, filiform apparatus.


A Representative images of ovules from wild - type ( Col - 0 ) , herk1 anj , lre - 5 and fer - 4 20 hours after emasculation ( HAE ) displaying the mature female gametophyte structure . Images presented here are maximum intensity projections from confocal microscopy images across several z - planes of ovules stained as per [ 70 ] . Scale bars = 50 µm .

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A Representative images of ovules from wild-type (Col-0), herk1 anj, lre-5 and fer-4 20 hours after emasculation (HAE) displaying the mature female gametophyte structure. Images presented here are maximum intensity projections from confocal microscopy images across several z-planes of ovules stained as per [70]. Scale bars = 50 µm.


B Quantification of H2DCF - DA staining of ROS in ovules from wild - type , herk1 anj , lre - 5 and fer - 4 plants at 20 HAE . Categories are listed in the legend Ovules dissected from at least five siliques per line . * * * p < 0 . 001 ( χ - square tests ) .

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B Quantification of H2DCF-DA staining of ROS in ovules from wild-type, herk1 anj, lre-5 and fer-4 plants at 20 HAE. Categories are listed in the legend Ovules dissected from at least five siliques per line. *** p<0.001 (χ-square tests).


C Percentage of pollen tubes with normal reception at the female gametophyte ( black bars ) and displaying overgrowth ( grey bars ) in wild - type , herk1 anj , lre - 5 and fer - 4 plants , manually selfed at 20 HAE . Fertilisation events counted from at least three siliques per line . * * * p < 0 . 001 ( Student ' s t - test ) .

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C Percentage of pollen tubes with normal reception at the female gametophyte (black bars) and displaying overgrowth (grey bars) in wild-type, herk1 anj, lre-5 and fer-4 plants, manually selfed at 20 HAE. Fertilisation events counted from at least three siliques per line. *** p<0.001 (Student's t-test).


A Yeast two hybrid ( Y2H ) assays of the extracellular juxtamembrane domains of HERK1 and ANJ ( HERK1exJM and ANJexJM , respectively ) with LRE ( residues 23 - 138 ; signal peptide and C - terminal domains excluded ) .

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A Yeast two hybrid (Y2H) assays of the extracellular juxtamembrane domains of HERK1 and ANJ (HERK1exJM and ANJexJM, respectively) with LRE (residues 23-138; signal peptide and C-terminal domains excluded).


B Y2H assays with the extracellular domains of HERK1 , ANJ and FER ( HERK1 - ECD , ANJ - ECD and FER - ECD , respectively ) . Ø represents negative controls where no sequence was cloned into the activating domain ( AD ) or DNA - binding domain ( BD ) constructs . - L - W - H , growth medium depleted of leucine ( - L ) , tryptophan ( - W ) and histidine ( - H ) .

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B Y2H assays with the extracellular domains of HERK1, ANJ and FER (HERK1-ECD, ANJ-ECD and FER-ECD, respectively). Ø represents negative controls where no sequence was cloned into the activating domain (AD) or DNA-binding domain (BD) constructs. -L-W-H, growth medium depleted of leucine (-L), tryptophan (-W) and histidine (-H).


C Co - immunoprecipitation of HA - LRE with HERK1 - GFP or ANJ - GFP following 2 days of transient expression in N . benthamiana leaves .

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C Co-immunoprecipitation of HA-LRE with HERK1-GFP or ANJ-GFP following 2 days of transient expression in N. benthamiana leaves.


D Co - immunoprecipitation of FER with HERK1 - GFP in Arabidopsis seedlings expressing pFER : : HERK1 - GFP . Numbers indicate molecular weight marker sizes in kDa . Assays were performed twice with similar results . CBB refers to Coomassie Brilliant Blue staining of total proteins .

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D Co-immunoprecipitation of FER with HERK1-GFP in Arabidopsis seedlings expressing pFER::HERK1-GFP. Numbers indicate molecular weight marker sizes in kDa. Assays were performed twice with similar results. CBB refers to Coomassie Brilliant Blue staining of total proteins.


( A ) The T cell clone 20 - 4 / A4 recognizes an epitope derived from the NeoR protein in the context of HLA ‐ DP3 . MHC class II expression on RCC1 . 24 ‐ NeoR cells following IFN ‐ γ treatment confers recognition by 20 - 4 / A4 . This recognition is almost completely blocked by the HLA ‐ DP ‐ specific antibody B7 / 21 . EBV ‐ B cells transfected with pINCO ‐ NeoR are able to present endogenous NeoR on MHC class II . As exemplified by the cell lines EBV ‐ B1 . 11 ( HLA ‐ DP3 ) and EBV ‐ B1 . 25 ( HLA ‐ DP4 ) , recognition by 20 - 4 / A4 is dependent on HLA ‐ DP3 expression . Bacterially expressed NeoR protein and a synthetic peptide encompassing amino acids NeoR216 - 229 served as controls .

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(A) The T cell clone 20-4/A4 recognizes an epitope derived from the NeoR protein in the context of HLA‐DP3. MHC class II expression on RCC1.24‐NeoR cells following IFN‐γ treatment confers recognition by 20-4/A4. This recognition is almost completely blocked by the HLA‐DP‐specific antibody B7/21. EBV‐B cells transfected with pINCO‐NeoR are able to present endogenous NeoR on MHC class II. As exemplified by the cell lines EBV‐B1.11 (HLA‐DP3) and EBV‐B1.25 (HLA‐DP4), recognition by 20-4/A4 is dependent on HLA‐DP3 expression. Bacterially expressed NeoR protein and a synthetic peptide encompassing amino acids NeoR216-229 served as controls.


( B ) Cross - presentation experiments to exclude release of NeoR and re ‐ uptake as exogenous protein . EBV ‐ B1 . 25 cells transfected with the neoR expression plasmid pINCO ‐ NeoR , were co ‐ cultured for 24 h with the same number of untransfected EBV ‐ B1 . 11 cells , or vice versa . Then , T cells were added and co ‐ cultured with the cells for additional 24 h . To test for NeoR release by neoR ‐ transfected EBV ‐ B1 . 11 cells , total supernatant ( 200 μl ) of 1 × 105 EBV ‐ B1 . 11 cells was harvested 48 h post transfection by centrifugation , and was used as culture media for 1 × 105 untransfected EBV ‐ B1 . 11 cells . After 48 h , 1 × 105 20 - 4 / A4 T cells were added and GM ‐ CSF concentration determined after additional 24 h .

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(B) Cross-presentation experiments to exclude release of NeoR and re‐uptake as exogenous protein. EBV‐B1.25 cells transfected with the neoR expression plasmid pINCO‐NeoR, were co‐cultured for 24 h with the same number of untransfected EBV‐B1.11 cells, or vice versa. Then, T cells were added and co‐cultured with the cells for additional 24 h. To test for NeoR release by neoR‐transfected EBV‐B1.11 cells, total supernatant (200 μl) of 1×105 EBV‐B1.11 cells was harvested 48 h post transfection by centrifugation, and was used as culture media for 1×105 untransfected EBV‐B1.11 cells. After 48 h, 1×105 20-4/A4 T cells were added and GM‐CSF concentration determined after additional 24 h.


( A ) , pINCO ‐ NeoR transfected EBV ‐ B1 . 11 cells ( B ) , and as control pINCO ‐ Tyrosinase ‐ transfected EBV ‐ B1 . 11 cells ( C ) , were either left untreated or treated with 5 μM lactacystin or 50 μM AAF ‐ CMK for 20 h . Following fixation with 0 . 5 % paraformaldehyde , cells were incubated with T cells , and 24 h later GM ‐ CSF release was determined by ELISA .

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(A), pINCO‐NeoR transfected EBV‐B1.11 cells (B), and as control pINCO‐Tyrosinase‐transfected EBV‐B1.11 cells (C), were either left untreated or treated with 5 μM lactacystin or 50 μM AAF‐CMK for 20 h. Following fixation with 0.5% paraformaldehyde, cells were incubated with T cells, and 24 h later GM‐CSF release was determined by ELISA.


( D ) FACS analysis of EBV ‐ B1 . 11 cells ( open histograms ) and EBV ‐ B1 . 11 cells stably transfected with the ICP47 gene ( shaded histograms ) . Inhibition of TAP by ICP47 down ‐ regulates HLA class I but not HLA ‐ DP expression . An isotype ‐ matched antibody was used as control ( thin ‐ lined histogram ) . EBV ‐ B1 . 11 ‐ ICP47 cells were transfected with pINCO ‐ NeoR ( E ) , pINCO ‐ Tyrosinase ( F ) , or mock transfected ( pINCO ) and MHC ‐ restricted presentation monitored with T cells .

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(D) FACS analysis of EBV‐B1.11 cells (open histograms) and EBV‐B1.11 cells stably transfected with the ICP47 gene (shaded histograms). Inhibition of TAP by ICP47 down‐regulates HLA class I but not HLA‐DP expression. An isotype‐matched antibody was used as control (thin‐lined histogram). EBV‐B1.11‐ICP47 cells were transfected with pINCO‐NeoR (E), pINCO‐Tyrosinase (F), or mock transfected (pINCO) and MHC‐restricted presentation monitored with T cells.


( A ) RCC1 . 24 ‐ NeoR incubated with IFN ‐ γ were either left untreated ( w / o ) or treated with chloroquine or leupeptin for 24 h to block lysosomal processing . Subsequently cells were fixed with 0 . 5 % paraformaldehyde and tested with 20 - 4 / A4 in a GM ‐ CSF release assay . EBV ‐ B1 . 11 cells transfected with pINCO ‐ NeoR ( B ) or pINCO ‐ Tyrosinase ( C ) were tested likewise . Both substances abrogated recognition of neoR ‐ transfected cells , while HLA ‐ A2 ‐ restricted presentation of the tyrosinase peptide was not

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(A) RCC1.24‐NeoR incubated with IFN‐γ were either left untreated (w/o) or treated with chloroquine or leupeptin for 24 h to block lysosomal processing. Subsequently cells were fixed with 0.5% paraformaldehyde and tested with 20-4/A4 in a GM‐CSF release assay. EBV‐B1.11 cells transfected with pINCO‐NeoR (B) or pINCO‐Tyrosinase (C) were tested likewise. Both substances abrogated recognition of neoR‐transfected cells, while HLA‐A2‐restricted presentation of the tyrosinase peptide was not affected


( D ) ) EBV ‐ B1 . 11 cells were incubated with 200 ng / ml of recombinant NeoR protein and treated with leupeptin or chloroquine as described in ( A ) . Presentation of exogenous NeoR on HLA ‐ DP3 is blocked by both substances as well , indicating that NeoR presentation is dependent on lysosomal processing .

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(D) ) EBV‐B1.11 cells were incubated with 200 ng/ml of recombinant NeoR protein and treated with leupeptin or chloroquine as described in (A). Presentation of exogenous NeoR on HLA‐DP3 is blocked by both substances as well, indicating that NeoR presentation is dependent on lysosomal processing.


( A ) 293T cells transfected with pINCO ‐ NeoR were harvested 6 and 96 h post transfection and NeoR localization within various cell fractions determined by Western blot . In addition , supernatant of NeoR ‐ pINCO transfected 293T cells ( 96 h post transfection ) was included to test for release of NeoR into the culture supernatant . At 6 h post transfection , NeoR protein becomes detectable in the cytoplasm , while at 96 h post transfection a substantial amount of NeoR protein is also localized in the endosomal / lysosomal fraction . For further characterization of the various fractions , blots were stripped and re ‐ probed with antibodies specific for the cytoplasmic protein LDH , the nuclear protein c ‐ myc , early endosomes ( Rab ‐ 5 ) , late endosomes and lysosomes ( Lamp ‐ 1 ) and mitochondria ( cytochrome c ) , which co ‐ purify with endosomes / lysosomes .

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(A) 293T cells transfected with pINCO‐NeoR were harvested 6 and 96 h post transfection and NeoR localization within various cell fractions determined by Western blot. In addition, supernatant of NeoR‐pINCO transfected 293T cells (96 h post transfection) was included to test for release of NeoR into the culture supernatant. At 6 h post transfection, NeoR protein becomes detectable in the cytoplasm, while at 96 h post transfection a substantial amount of NeoR protein is also localized in the endosomal/lysosomal fraction. For further characterization of the various fractions, blots were stripped and re‐probed with antibodies specific for the cytoplasmic protein LDH, the nuclear protein c‐myc, early endosomes (Rab‐5), late endosomes and lysosomes (Lamp‐1) and mitochondria (cytochrome c), which co‐purify with endosomes/lysosomes.


( B ) Endosomal / lysosomal fractions were prepared from pINCO ‐ NeoR ‐ transfected cells 96 h post transfection as in ( A ) . To prevent lysosomal degradation of NeoR , half of the cells were treated with leupeptin for the last 48 h . These fractions were separated on a 27 % Percoll gradient and eight different fractions collected . Successful separation into fractions with increasing density was verified by Western blot analysis with antibodies directed against Lamp ‐ 1 , Rab ‐ 5 and cytochrome c . Inhibition of NeoR degradation by leupeptin causes accumulation of NeoR protein in the late endosomal / lysosomal fractions 7 and 8 .

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(B) Endosomal/lysosomal fractions were prepared from pINCO‐NeoR‐transfected cells 96 h post transfection as in (A). To prevent lysosomal degradation of NeoR, half of the cells were treated with leupeptin for the last 48 h. These fractions were separated on a 27% Percoll gradient and eight different fractions collected. Successful separation into fractions with increasing density was verified by Western blot analysis with antibodies directed against Lamp‐1, Rab‐5 and cytochrome c. Inhibition of NeoR degradation by leupeptin causes accumulation of NeoR protein in the late endosomal/lysosomal fractions 7 and 8.


( C ) To determine where NeoR gains access to the vacuolar system , 293T cells were transfected with pINCO ‐ NeoR , treated with leupeptin to prevent NeoR degradation , and the endosomal / lysosomal fractions prepared at various time points post transfection and separated by Percoll gradient centrifugation . Starting at 16 h post transfection , NeoR becomes detectable in the Rab ‐ 5 ‐ positive fraction 5 , and then spreads into late endosomal / lysosomal fractions . At 26 h post transfection an almost identical NeoR distribution pattern is observed in cells treated with or without BfA .

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(C) To determine where NeoR gains access to the vacuolar system, 293T cells were transfected with pINCO‐NeoR, treated with leupeptin to prevent NeoR degradation, and the endosomal/lysosomal fractions prepared at various time points post transfection and separated by Percoll gradient centrifugation. Starting at 16 h post transfection, NeoR becomes detectable in the Rab‐5‐positive fraction 5, and then spreads into late endosomal/lysosomal fractions. At 26 h post transfection an almost identical NeoR distribution pattern is observed in cells treated with or without BfA.


( A ) RCC were transfected with the NeoR ‐ GFP expression construct and subcellular localization of the fusion protein monitored by UV fluorescence . As compared to untreated cells , cells treated with 3 ‐ MA show a much brighter UV fluorescence .

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(A) RCC were transfected with the NeoR‐GFP expression construct and subcellular localization of the fusion protein monitored by UV fluorescence. As compared to untreated cells, cells treated with 3‐MA show a much brighter UV fluorescence.


( B ) Semiquantitative analysis of fluorescence intensity of cells transfected with the NeoR ‐ GFP fusion construct . Compared to untreated cells , cells treated for 24 h with 7 . 5 mM 3 ‐ MA show an about threefold increase in mean fluorescence intensity .

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(B) Semiquantitative analysis of fluorescence intensity of cells transfected with the NeoR‐GFP fusion construct. Compared to untreated cells, cells treated for 24 h with 7.5 mM 3‐MA show an about threefold increase in mean fluorescence intensity.


( C ) Western blot analysis of neoR ‐ transfected cells . Treatment of cells with 7 . 5 mM 3 ‐ MA causes a dramatic increase in NeoR protein levels in only 12 h . The ER ‐ resident protein BiP was used as gel loading control .

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(C) Western blot analysis of neoR‐transfected cells. Treatment of cells with 7.5 mM 3‐MA causes a dramatic increase in NeoR protein levels in only 12 h. The ER‐resident protein BiP was used as gel loading control.


( D ) This increase in total NeoR protein in 3 ‐ MA ‐ treated cells is associated with an increase in cytoplasmic , and a decrease in endosomal / lysosomal NeoR protein levels .

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(D) This increase in total NeoR protein in 3‐MA‐treated cells is associated with an increase in cytoplasmic, and a decrease in endosomal/lysosomal NeoR protein levels.


( E ) Western blot analysis of whole cell lysates prepared from neoR ‐ transfected cells . Inhibition of protein synthesis by cycloheximide treatment leads to a rapid decrease in NeoR protein levels . Simultaneous treatment of cells with 3 ‐ MA prevents NeoR degradation . BiP was again used as a gel loading control .

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(E) Western blot analysis of whole cell lysates prepared from neoR‐transfected cells. Inhibition of protein synthesis by cycloheximide treatment leads to a rapid decrease in NeoR protein levels. Simultaneous treatment of cells with 3‐MA prevents NeoR degradation. BiP was again used as a gel loading control.


( A ) RCC1 . 24 ‐ NeoR ( endogenous ) , and RCC1 . 24 cells to which NeoR protein had been added ( exogenous ) , were treated with different concentrations of 3 ‐ MA and cell surface presentation of the NeoR ‐ derived peptide on HLA ‐ DP3 monitored with the T cell clone 20 - 4 / A4 ‐ specific T cell clone 20 - 4 / A4 . Inhibition of autophagy by 3 ‐ MA causes an almost complete inhibition of endogenous antigen presentation , while presentation of exogenous NeoR is not affected .

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(A) RCC1.24‐NeoR (endogenous), and RCC1.24 cells to which NeoR protein had been added (exogenous), were treated with different concentrations of 3‐MA and cell surface presentation of the NeoR‐derived peptide on HLA‐DP3 monitored with the T cell clone 20-4/A4‐specific T cell clone 20-4/A4. Inhibition of autophagy by 3‐MA causes an almost complete inhibition of endogenous antigen presentation, while presentation of exogenous NeoR is not affected.


( B ) EBV ‐ B1 . 11 cells transfected with pINCO ‐ NeoR ( endogenous ) , or incubated with recombinant NeoR protein ( exogenous ) , were treated with the inhibitors of autophagy , wortmannin and 3 ‐ MA , and HLA ‐ DP3 ‐ restricted presentation of the NeoR ‐ derived peptide determined . Inhibition of antigen presentation by leupeptin served as control .

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(B) EBV‐B1.11 cells transfected with pINCO‐NeoR (endogenous), or incubated with recombinant NeoR protein (exogenous), were treated with the inhibitors of autophagy, wortmannin and 3‐MA, and HLA‐DP3‐restricted presentation of the NeoR‐derived peptide determined. Inhibition of antigen presentation by leupeptin served as control.


( C ) Presentation of the HLA ‐ A2 ‐ restricted tyrosinase peptide in EBV ‐ B1 . 11 cells transfected with pINCO ‐ Tyrosinase is not affected by these inhibitors of autophagy .

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(C) Presentation of the HLA‐A2‐restricted tyrosinase peptide in EBV‐B1.11 cells transfected with pINCO‐Tyrosinase is not affected by these inhibitors of autophagy.


FACS analysis of HLA ‐ class I and HLA ‐ DP expression on RCC1 . 24 ( D ) and EBV ‐ B1 . 11 cells ( E ) As compared to untreated cells ( thick ‐ lined histogram ) , treatment of cells with 7 . 5 mM 3 ‐ MA ( shaded histogram ) does not cause down ‐ regulation of HLA ‐ class I or HLA ‐ DP . Isotype control is shown as thin ‐ lined histogram .

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FACS analysis of HLA‐class I and HLA‐DP expression on RCC1.24 (D) and EBV‐B1.11 cells (E) As compared to untreated cells (thick‐lined histogram), treatment of cells with 7.5 mM 3‐MA (shaded histogram) does not cause down‐regulation of HLA‐class I or HLA‐DP. Isotype control is shown as thin‐lined histogram.


Viability of diploid ( SC5314 ) and tetraploid ( RBY18 ) cells grown on PRE - SPO medium at 37 ° C ( n = 5 , error bars represent S . E . M . ) .

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Viability of diploid (SC5314) and tetraploid (RBY18) cells grown on PRE-SPO medium at 37°C (n=5, error bars represent S.E.M.).


A tetraploid β - tubulin ( TUB2 ) - GFP , histone H2B ( HTB ) - RFP strain was cultured on YPD or PRE - SPO medium at 30 ° C or 37 ° C , respectively , for 24 h . Cell images indicate maximum intensity Z projections for GFP and RFP channels , and merged images of the GFP / RFP and GFP / RFP / DIC channels .

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A tetraploid β-tubulin (TUB2)-GFP, histone H2B (HTB)-RFP strain was cultured on YPD or PRE-SPO medium at 30°C or 37°C, respectively, for 24 h. Cell images indicate maximum intensity Z projections for GFP and RFP channels, and merged images of the GFP/RFP and GFP/RFP/DIC channels.


Diploid strain RBY1 and tetraploid strain RBY18 were cultured on YPD or PRE - SPO medium at 30 ° C or 37 ° C , respectively , for 7 days and plated onto 2 - DOG medium to monitor for GAL1 loss .

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Diploid strain RBY1 and tetraploid strain RBY18 were cultured on YPD or PRE-SPO medium at 30°C or 37°C, respectively, for 7 days and plated onto 2-DOG medium to monitor for GAL1 loss.


2 - DOGR progeny arising from RBY18 on PRE - SPO medium were DNA stained and their ploidy determined via flow cytometry .

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2-DOGR progeny arising from RBY18 on PRE-SPO medium were DNA stained and their ploidy determined via flow cytometry.


Heatmap of RNA - seq data showing global gene expression changes in diploid ( SC5314 , 2n ) and tetraploid ( RBY18 , 4n ) cells on YPD and PRE - SPO medium over 24 h .

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Heatmap of RNA-seq data showing global gene expression changes in diploid (SC5314, 2n) and tetraploid (RBY18, 4n) cells on YPD and PRE-SPO medium over 24 h.


Heatmap of RNA - seq data for glycolysis and oxidative stress genes in diploid ( 2n ) and tetraploid ( 4n ) cells grown on YPD or PRE - SPO medium over 24 h .

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Heatmap of RNA-seq data for glycolysis and oxidative stress genes in diploid (2n) and tetraploid (4n) cells grown on YPD or PRE-SPO medium over 24 h.


TTC assay results in diploid and tetraploid cells grown on YPD or PRE - SPO medium at 30 ° C or 37 ° C , respectively . Cells were grown for 24 h at which point a TTC overlay solution was applied and color allowed to develop for 15 min .

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TTC assay results in diploid and tetraploid cells grown on YPD or PRE-SPO medium at 30°C or 37°C, respectively. Cells were grown for 24 h at which point a TTC overlay solution was applied and color allowed to develop for 15 min.


Diploid and tetraploid strains were spot inoculated onto YPD or PRE - SPO medium supplemented with the pH indicator bromocresol purple and cultured for 24 h at 30 ° C or 37 ° C , respectively .

Diploid and tetraploid strains were spot inoculated onto YPD or PRE-SPO medium supplemented with the pH indicator bromocresol purple and cultured for 24 h at 30°C or 37°C, respectively.


Analysis of metabolic flux in diploid and tetraploid cells following growth for 24 h on YPD or PRE - SPO medium ( 30 ° C or 37 ° C , respectively ) , using a Seahorse instrument . OCR values represent rates of aerobic respiration while ECAR values represent rates of fermentation .

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Analysis of metabolic flux in diploid and tetraploid cells following growth for 24 h on YPD or PRE-SPO medium (30°C or 37°C, respectively), using a Seahorse instrument. OCR values represent rates of aerobic respiration while ECAR values represent rates of fermentation.


Cell size and mean MitoTracker Green signal intensity in diploid and tetraploid cells grown on YPD medium for 24 h at 30 ° C ( n = 100 and n = 5 for cell volume and MitoTracker Green measurements , respectively , error bars represent S . E . M . ) .

Cell size and mean MitoTracker Green signal intensity in diploid and tetraploid cells grown on YPD medium for 24 h at 30°C (n=100 and n=5 for cell volume and MitoTracker Green measurements, respectively, error bars represent S.E.M.).


Diploid and tetraploid cells were grown on YPD or PRE - SPO medium at 30 ° C or 37 ° C , respectively , for 24 h and stained with CellROX Green . Cell images indicate calcofluor white staining ( cell wall ; blue ) , GFP , and a merged image of GFP / DAPI / DIC channels . Scale bar = 10 μm . Diploid and tetraploid cells were grown on YPD or PRE - SPO medium at 30 ° C or 37 ° C , respectively , for 24 h , stained with CellROX Green , and signal analyzed via flow cytometry .

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Diploid and tetraploid cells were grown on YPD or PRE-SPO medium at 30°C or 37°C, respectively, for 24 h and stained with CellROX Green. Cell images indicate calcofluor white staining (cell wall; blue), GFP, and a merged image of GFP/DAPI/DIC channels. Scale bar = 10 μm. Diploid and tetraploid cells were grown on YPD or PRE-SPO medium at 30°C or 37°C, respectively, for 24 h, stained with CellROX Green, and signal analyzed via flow cytometry.


Diploid and tetraploid cells containing a fluorescently labeled version of the transcription factor Cap1 ( Cap1 - mNeonGreen ) were grown on YPD or PRE - SPO medium at 30 ° C or 37 ° C , respectively , for 24 h . Cell images indicate calcofluor white staining ( cell wall ; blue ) , GFP , and a merged image of GFP / DAPI / DIC channels . Scale bar = 10 μm . Quantitation of the percentage of diploid and tetraploid cells with activated Cap1 on YPD or PRE - SPO medium . " n . d . " indicates that no cells with activated Cap1 were detected .

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Diploid and tetraploid cells containing a fluorescently labeled version of the transcription factor Cap1 (Cap1-mNeonGreen) were grown on YPD or PRE-SPO medium at 30°C or 37°C, respectively, for 24 h. Cell images indicate calcofluor white staining (cell wall; blue), GFP, and a merged image of GFP/DAPI/DIC channels. Scale bar = 10 μm. Quantitation of the percentage of diploid and tetraploid cells with activated Cap1 on YPD or PRE-SPO medium. "n.d." indicates that no cells with activated Cap1 were detected.


Analysis of cell viability in tetraploid cells after culture on PRE - SPO medium ( 37 ° C for 24 h ) when supplemented with ascorbic acid , DTT , or glutathione at the indicated concentrations .

Analysis of cell viability in tetraploid cells after culture on PRE-SPO medium (37°C for 24 h) when supplemented with ascorbic acid, DTT, or glutathione at the indicated concentrations.


Analysis of GAL1 loss ( 2 - DOG resistant colonies ) in tetraploid strain RBY18 after growth on PRE - SPO medium with or without antioxidants for 7 days at 37 ° C .

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Analysis of GAL1 loss (2-DOG resistant colonies) in tetraploid strain RBY18 after growth on PRE-SPO medium with or without antioxidants for 7 days at 37°C.


Tetraploid cells expressing Cap1 - mNeonGreen were grown on PRE - SPO medium supplemented with 20 mM ascorbic acid , 1 mM DTT , or 1 mM glutathione for 24 h at 37 ° C . Cell images were analyzed to determine the percentage of viable cells with activated Cap1 .

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Tetraploid cells expressing Cap1-mNeonGreen were grown on PRE-SPO medium supplemented with 20 mM ascorbic acid, 1 mM DTT, or 1 mM glutathione for 24 h at 37°C. Cell images were analyzed to determine the percentage of viable cells with activated Cap1.


Assay to monitor for loss of GAL1 from tetraploid RBY18 overexpressing the indicated genes . Cells were grown for 7 d at 37 ° C on PRE - SPO medium in the presence ( gene OFF ) or absence ( gene ON ) of doxycycline ( DOX ) .

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Assay to monitor for loss of GAL1 from tetraploid RBY18 overexpressing the indicated genes. Cells were grown for 7 d at 37°C on PRE-SPO medium in the presence (gene OFF) or absence (gene ON) of doxycycline (DOX).


Diploid ( 2n ) and tetraploid ( 4n ) strains expressing a Gam - GFP reporter were cultured on YPD or PRE - SPO medium at 30 ° C or 37 ° C , respectively , in the presence ( Gam - GFP ON ) or absence ( Gam - GFP OFF ) of doxycycline ( DOX ) for 24 h . Cell images indicate calcofluor white staining ( cell wall ; blue ) , GFP , and a merged image of GFP / DAPI / DIC channels . Scale bar = 10 μm .

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Diploid (2n) and tetraploid (4n) strains expressing a Gam-GFP reporter were cultured on YPD or PRE-SPO medium at 30°C or 37°C, respectively, in the presence (Gam-GFP ON) or absence (Gam-GFP OFF) of doxycycline (DOX) for 24 h. Cell images indicate calcofluor white staining (cell wall; blue), GFP, and a merged image of GFP/DAPI/DIC channels. Scale bar = 10 μm.


Flow cytometric analysis of the tetraploid Gam - GFP reporter strain grown on YPD or PRE - SPO medium at 30 ° C or 37 ° C , respectively , in the presence or absence of doxycycline for 24 h .

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Flow cytometric analysis of the tetraploid Gam-GFP reporter strain grown on YPD or PRE-SPO medium at 30°C or 37°C, respectively, in the presence or absence of doxycycline for 24 h.


Flow cytometric analysis of the tetraploid Gam - GFP reporter strain grown on PRE - SPO medium with or without 20 mM ascorbic acid , 1 mM DTT , or 1 mM glutathione at 37 ° C for 24 h .

Flow cytometric analysis of the tetraploid Gam-GFP reporter strain grown on PRE-SPO medium with or without 20 mM ascorbic acid, 1 mM DTT, or 1 mM glutathione at 37°C for 24 h.


10 - fold dilutions of diploid and tetraploid cells were spot inoculated onto SCD medium with or without the DNA - damaging agents hydroxyurea ( HU , 20 mM ) or methyl methanesulfonate ( MMS , 0 . 01 % ) . Cells were grown for 48 h at 30 ° C or 37 ° C and the plates imaged .

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10-fold dilutions of diploid and tetraploid cells were spot inoculated onto SCD medium with or without the DNA-damaging agents hydroxyurea (HU, 20 mM) or methyl methanesulfonate (MMS, 0.01%). Cells were grown for 48 h at 30°C or 37°C and the plates imaged.


Assay to monitor for loss of GAL1 ( 2 - DOG resistant cells ) arising from tetraploid strain RBY18 after culture at 37 ° C on SCD medium alone , SCD supplemented with HU ( 20 mM ) or MMS ( 0 . 01 % ) , or SCD medium with exposure to UV light for five seconds a day for 7 days .

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Assay to monitor for loss of GAL1 (2-DOG resistant cells) arising from tetraploid strain RBY18 after culture at 37°C on SCD medium alone, SCD supplemented with HU (20 mM) or MMS (0.01%), or SCD medium with exposure to UV light for five seconds a day for 7 days.


Tetraploid cells were grown on SCD medium with or without the oxidative - stress inducing agents paraquat ( PQT ; 600 μg / mL ) , hydrogen peroxide ( H2O2 ; 2 mM ) , or piperlongumine ( PL ; 100 μg / mL ) for 24 h at 37 ° C and stained with CellROX Green . Cell images indicate calcofluor white staining ( cell wall ; blue ) , GFP , and a merged image of GFP / DAPI / DIC channels . Scale bar = 10 μm .

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Tetraploid cells were grown on SCD medium with or without the oxidative-stress inducing agents paraquat (PQT; 600 μg/mL), hydrogen peroxide (H2O2; 2 mM), or piperlongumine (PL; 100 μg/mL) for 24 h at 37°C and stained with CellROX Green. Cell images indicate calcofluor white staining (cell wall; blue), GFP, and a merged image of GFP/DAPI/DIC channels. Scale bar = 10 μm.


Tetraploid cells expressing Cap1 - mNeonGreen were grown on SCD medium with or without PQT ( 600 μg / mL ) , H2O2 ( 2 mM ) , or PL ( 100 μg / mL ) for 24 h at 37 ° C . Cell images as in ( A ) . Scale bar = 10 μm .

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Tetraploid cells expressing Cap1-mNeonGreen were grown on SCD medium with or without PQT (600 μg/mL), H2O2 (2 mM), or PL (100 μg/mL) for 24 h at 37°C. Cell images as in (A). Scale bar = 10 μm.


Tetraploid cells expressing a Gam - GFP reporter were cultured on SCD medium with or without PQT ( 600 μg / mL ) , H2O2 ( 2 mM ) , or PL ( 100 μg / mL ) for 24 h at 37 ° C and analyzed via fluorescence microscopy . Cell images as in ( A ) . Scale bar = 10 μm .

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Tetraploid cells expressing a Gam-GFP reporter were cultured on SCD medium with or without PQT (600 μg/mL), H2O2 (2 mM), or PL (100 μg/mL) for 24 h at 37°C and analyzed via fluorescence microscopy. Cell images as in (A). Scale bar = 10 μm.


Flow cytometric analysis of the tetraploid Gam - GFP reporter strain grown on SCD with or without PQT ( 600 μg / mL ) , H2O2 ( 2 mM ) , or PL ( 100 μg / mL ) for 24 h at 37 ° C .

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Flow cytometric analysis of the tetraploid Gam-GFP reporter strain grown on SCD with or without PQT (600 μg/mL), H2O2 (2 mM), or PL (100 μg/mL) for 24 h at 37°C.


2 - DOG assay to monitor for loss of GAL1 function from tetraploid strain RBY18 after culture on SCD medium with or without PQT ( 600 μg / mL ) , H2O2 ( 2 mM ) , or PL ( 100 μg / mL ) at 37 ° C for 7 d .

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2-DOG assay to monitor for loss of GAL1 function from tetraploid strain RBY18 after culture on SCD medium with or without PQT (600 μg/mL), H2O2 (2 mM), or PL (100 μg/mL) at 37°C for 7 d.


( A ) Representative images of COS7 cells expressing GFP - COP1 ( green ) co - transfected with empty vector or the indicated TRIB1 - FLAG construct ( red ) . Cells were stained with anti - FLAG ( Sigma ) and anti - mouse Alexa Fluor 568 . Nuclei were counterstained with DAPI ( blue ) . Scale bar , 10 µm . See also Appendix Fig S1B for western blots showing the expression levels of the constructs used .

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(A) Representative images of COS7 cells expressing GFP-COP1 (green) co-transfected with empty vector or the indicated TRIB1-FLAG construct (red). Cells were stained with anti-FLAG (Sigma) and anti-mouse Alexa Fluor 568. Nuclei were counterstained with DAPI (blue). Scale bar, 10 µm. See also Appendix Fig S1B for western blots showing the expression levels of the constructs used.


( B ) Quantification of the average ratio of nuclear / cytoplasmic fluorescence of GFP - COP1 in ( A ) . Mean values ± S . E . M . are shown for three independent experiments where 50 individual cells per experiment were analyzed . Significance relative to GFP - COP1 with empty vector was calculated using the Student t test ( * * * * p < 0 . 00001 ) .

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(B) Quantification of the average ratio of nuclear/cytoplasmic fluorescence of GFP-COP1 in (A). Mean values ± S.E.M. are shown for three independent experiments where 50 individual cells per experiment were analyzed. Significance relative to GFP-COP1 with empty vector was calculated using the Student t test (****p < 0.00001).


( C ) Representative images of COS7 cells showing GFP - COP1 ( green ) and TRIB1 - FLAG ( red ) colocalizing in punctate nuclear structures . Insets show enlarged images of these nuclear puncta . Cells were stained with anti - FLAG and anti - mouse Alexa Fluor 568 . Nuclei were counterstained with DAPI ( blue ) . Scale bar , 10 µm .

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(C) Representative images of COS7 cells showing GFP-COP1 (green) and TRIB1-FLAG (red) colocalizing in punctate nuclear structures. Insets show enlarged images of these nuclear puncta. Cells were stained with anti-FLAG and anti-mouse Alexa Fluor 568. Nuclei were counterstained with DAPI (blue). Scale bar, 10 µm.