( D ) Oxygen consumption rates in A - T - strains ( of means = 0 . 65 , p = 0 . 0002 , Welch ' s T - , 3 biological replicates , each measured in technical duplicates ) . Data information : Vertical bars show mean of data . Error bars shaded areas in cases denote standard deviation . Significance keys : * p < 0 . 05 , * * * p < 0 . 0005 ( Welch ' s t - ) .

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(D) Oxygen consumption rates in A- and T-strains (ratio of means=0.65, p=0.0002, Welch's T-test, 3 biological replicates, each measured in technical duplicates). Data information: Vertical bars show the mean of the data. Error bars and shaded areas in all cases denote standard deviation. Significance keys: * p<0.05, *** p<0.0005 (Welch's t-test).


( E ) Culture dry after 24 hrs was measured reported as a fraction of of glucose put into media for 3 biological replicates per . Data information : Vertical bars show mean of data . Error bars shaded areas in cases denote standard deviation . Significance keys : * p < 0 . 05 , * * * p < 0 . 0005 ( Welch ' s t - ) .

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(E) Culture dry weight after 24 hrs was measured reported as a fraction of the weight of glucose put into the media for 3 biological replicates per strain. Data information: Vertical bars show the mean of the data. Error bars and shaded areas in all cases denote standard deviation. Significance keys: * p<0.05, *** p<0.0005 (Welch's t-test).


( A ) Spot assays on solid media from a three - fold dilution series of exponential cultures at same density in 96 - well plates ( 3 biological replicates of each ) spotted in 16 technical replicates ( each dilution in 4x4 square ) . A - is more resistant to antimycin A but less resistant to oxidative stress triggered by H2O2 or diamide . A control without toxin ( left ) was included in each batch of spot assays performed a representative image is shown here .

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(A) Spot assays on solid media from a three-fold dilution series of exponential cultures at the same cell density in 96-well plates (3 biological replicates of each strain) and spotted in 16 technical replicates (each dilution in 4x4 square). The A-strain is more resistant to antimycin A but less resistant to oxidative stress triggered by H2O2 or diamide. A control without toxin (left) was included in each batch of spot assays performed and a representative image is shown here.


( B ) Fitness ( approximated by maximum slope of smoothed growth curves ) of A - T - strains on 12 carbon sources , with either yeast extract ( YE ) or ammonium ( NH4 ) , with or without 0 . 1 % priming glucose to support initial growth . For 48 conditions , two biological replicates of A - T - strains were grown in technical quadruplicates each . Dotted lines in panels mark a fitness of 1 ( i . e . , same fitness ) .

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(B) Fitness (approximated by maximum slope of smoothed growth curves) of A- and T-strains on 12 carbon sources, with either yeast extract (YE) or ammonium (NH4), with or without 0.1% priming glucose to support initial growth. For all 48 conditions, two biological replicates of A- and T-strains were grown in technical quadruplicates each. Dotted lines in panels mark a fitness ratio of 1 (i.e., same fitness).


( C ) Fitness of A - T - strains on 95 nitrogen sources on Biolog Phenotype MicroArrays . Conditions with no substantial growth were excluded ( black circles , maximum slope < 0 . 015 ) . Red lines show arbitrary significance cut - off , put at | log2 ( A - / T - ) | > 0 . 75 .

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(C) Fitness of A- and T-strains on 95 nitrogen sources on Biolog Phenotype MicroArrays. Conditions with no substantial growth were excluded (black circles, maximum slope <0.015). Red lines show arbitrary significance cut-off, put at |log2(A-strain/T-strain)|>0.75.


( D ) Fitness of A - T - strains on 72 different drugs toxins , at 4 concentrations each , on Biolog Phenotype MicroArrays . ( Results for benzamidine were inconclusive , with both strains comparatively resistant in one each . )

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(D) Fitness of A- and T-strains on 72 different drugs and toxins, at 4 concentrations each, on Biolog Phenotype MicroArrays. (Results for benzamidine were inconclusive, with both strains comparatively resistant in one concentration each.)


( E ) Spot assays to validate selected results Several assays were performed with both h - h90 strains , no mating - type specific differences were evident between two sets of allele - swap strains .

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(E) Spot assays to validate selected results Several assays were performed with both h- and h90 strains, and no mating-type specific differences were evident between the two sets of allele-swap strains.


( F ) Chronological lifespan of A - T - strains , i . e . proportion of non - dividing in stationary phase that maintain proliferative potential after refeeding . data show colony forming units ( CFUs ) per ml of culture over 7 days of stationary phase in glucose - depleted rich media . Three biological repeats were carried out for both strains , with each repeat measured as technical triplicates . Error bars represent standard error of biological replicates .

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(F) Chronological lifespan of A- and T-strains, i.e. the proportion of non-dividing cells in stationary phase that maintain proliferative potential after refeeding. The data show colony forming units (CFUs) per ml of culture over 7 days of stationary phase in glucose-depleted rich media. Three biological repeats were carried out for both strains, with each repeat measured as technical triplicates. Error bars represent standard error of the biological replicates.


( G ) Boxplot showing resistance to 3mM H2O2 grouped by pyk1 allele for 156 strains from our collection . T - strains had a higher mean fitness in H2O2 than A - strains ( 1 . 01 ± 0 . 06 vs 0 . 95 ± 0 . 11 ; p = 0 . 0021 , Welch ' s t - ) . resistance score was obtained as for antimycin A .

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(G) Boxplot showing resistance to 3mM H2O2 grouped by pyk1 allele for 156 strains from our collection. The T-strains had a higher mean fitness in H2O2 than the A-strains (1.01±0.06 vs 0.95±0.11; p=0.0021, Welch's t-test). The resistance score was obtained as for antimycin A.


( H ) Boxplot showing growth fitness on rich media , grouped by pyk1 allele for 158 S . pombe strains ( 0 . 88 ± 0 . 16 vs 0 . 94 ± 0 . 24 ; p = 0 . 18 , Welch ' s t - ) .

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(H) Boxplot showing growth fitness on rich media, grouped by pyk1 allele for 158 S. pombe strains (0.88±0.16 vs 0.94±0.24; p=0.18, Welch's t-test).


a , Starvation ( 3 h ) - induced autophagy results in a downregulation of H4K16ac in histone extracts of MEF .

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a, Starvation (3 h)-induced autophagy results in a downregulation of H4K16ac in histone extracts of MEF cells.


b , Upon rapamycin treatment ( 300 nM ) LC3 conversion downregulation of H4K16ac are observed in WT MEF but not in autophagy - deficient atg5 − / − atg7 − / − MEF .

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b, Upon rapamycin treatment (300 nM) LC3 conversion and downregulation of H4K16ac are observed in WT MEF cells but not in the autophagy-deficient atg5−/− and atg7−/− MEF cells.


c , Rapamycin treatment increased LC3 - II / LC3 - I promoted H4K16ac decrease in sirt1 − / − WT MEF .

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c, Rapamycin treatment increased the LC3-II/LC3-I ratio and promoted H4K16ac decrease in sirt1−/− and WT MEF cells.


d , Rapamycin - induced autophagy led to downregulation of H4K16ac at 48 h in histone extracts of HeLa U20S , after 6 h in U1810 .

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d, Rapamycin-induced autophagy led to downregulation of H4K16ac at 48 h in histone extracts of HeLa and U20S cells, and after 6 h in U1810 cells.


e , of H4K16 acetylation by immunoblotting is depicted for rapamycin - treated . Data are expressed as mean ± s . e . m . ( n = 3 - 5 ) ; * P 0 . 05 ; * * P 0 . 01 .

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e, Quantification of H4K16 acetylation by immunoblotting is depicted for rapamycin-treated cells. Data are expressed as mean ± s.e.m. (n = 3-5); *P 0.05; **P 0.01.


Rapamycin treatment ( 48 h ) promoted downregulation of H4K16 histone acetyltransferase hMOF expression level in MEF ( a )

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Rapamycin treatment (48 h) promoted the downregulation of the H4K16 histone acetyltransferase hMOF expression level in MEF (a)


Rapamycin treatment ( 48 h ) promoted downregulation of H4K16 histone acetyltransferase hMOF expression level in transfected HeLa ( b ) .

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Rapamycin treatment (48 h) promoted the downregulation of the H4K16 histone acetyltransferase hMOF expression level in transfected HeLa cells (b).


VPA ( 1 mM ) treatment counteracted rapamycin - induced H4K16ac downregulation ( c ) decreased LC3 ( d ) .

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VPA (1 mM) treatment counteracted rapamycin-induced H4K16ac downregulation (c) and decreased the LC3 ratio (d).


e , Co - treatment with chloroquine ( CQ , 10 µM ) showed that decrease in LC3 was a result of an increase in autophagic flux .

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e, Co-treatment with chloroquine (CQ, 10 µM) showed that the decrease in LC3 ratio was a result of an increase in autophagic flux.


f , Inhibition of autophagy by CQ after hMOF overexpression shows that hMOF does not inhibit autophagic flux .

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f, Inhibition of autophagy by CQ after hMOF overexpression shows that hMOF does not inhibit autophagic flux.


g , yeast homologue of hMOF , Sas2 , tagged with 3 × HA showed a complete disappearance of HA signal upon autophagy induction after 3 h .

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g, The yeast homologue of hMOF, Sas2, tagged with 3× HA showed a complete disappearance of HA signal upon autophagy induction after 3 h.


h , Overexpression of Sas2 repressed downregulation of H4K16ac upon rapamycin treatment .

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h, Overexpression of Sas2 repressed the downregulation of H4K16ac upon rapamycin treatment.


a , VPA increases autophagic flux in rapamycin - treated HeLa transfected with mRFP - GFP - LC3 tandem reporter construct which allows distinction between autophagosomes ( GFP + / RFP + yellow ) autolysosomes ( GFP − / RFP + red ) .

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a, VPA increases the autophagic flux in rapamycin-treated HeLa cells transfected with the mRFP-GFP-LC3 tandem reporter construct which allows distinction between autophagosomes (GFP+/RFP+ yellow puncta) and autolysosomes (GFP−/RFP+ red puncta).


b , Confocal microscopy image of a treated with rapamycin VPA depicting a high of red to green LC3 indicating an increase in autophagic flux .

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b, Confocal microscopy image of a cell treated with rapamycin and VPA depicting a high ratio of red to green LC3 puncta indicating an increase in autophagic flux.


c , Co - treatment with VPA rapamycin led to increased .

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c, Co-treatment with VPA and rapamycin led to increased cell death.


d , Co - treatment with CQ abrogated VPA + rapamycin - induced .

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d, Co-treatment with CQ abrogated VPA + rapamycin-induced cell death.


e , f , Increasing H4K16ac by either overexpression of hMOF , inhibition of SIRT1 by siRNA or chemical inhibitor Ex527 promoted upon rapamycin treatment .

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e, f, Increasing H4K16ac levels by either overexpression of hMOF, inhibition of SIRT1 by siRNA or the chemical inhibitor Ex527 promoted cell death upon rapamycin treatment.


Co - treatment with autophagy inhibitor bafilomycin A1 ( BafA , 40 nM ) ( g ) or 3 - methyladenine ( 3MA , 5 mM ) ( h ) abrogated VPA + rapamycin - induced .

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Co-treatment with the autophagy inhibitor bafilomycin A1 (BafA, 40 nM) (g) or 3-methyladenine (3MA, 5 mM) (h) abrogated VPA + rapamycin-induced cell death.


Treatment of HeLa upon - acid starvation with VPA induced ( i ) , which was rescued when were co - treated with CQ ( j ) . Data are expressed as mean ± s . e . m . ( n = 3 ) .

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Treatment of HeLa cells upon amino-acid starvation with VPA induced cell death (i), which was rescued when cells were co-treated with CQ (j). Data are expressed as mean ± s.e.m. (n = 3).


A sWGS performed on sorted CD3 + / Vβ + malignant T - isolated from blood of P1 , shown as a representative example . Copy number profiles ploidy estimates were calculated using ichorCNA . Blue represents balanced genomic regions , green indicates loss / µdeletion , red indicates gain / amplification of regions on chromosome 17 . Darker color shades indicate higher log - 2 ratios .

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A sWGS performed on sorted CD3+/Vβ+ malignant T-cells isolated from the blood of P1, shown as a representative example. Copy number profiles and ploidy estimates were calculated using ichorCNA. Blue represents balanced genomic regions, green indicates loss/µdeletion, and red indicates gain/amplification of regions on chromosome 17. Darker color shades indicate higher log-2 ratios.


B of extracted CNA [ log2 ratios ] of STAT genes on chr17 ( i . e . , STAT3 , STAT5A STAT5B ) , other relevant suppressors ( i . e , STAT1 , SOCS1 TP53 ) oncogenes ( MYC ) . Red indicates gain / amplification , blue indicates loss / deletion . balance threshold was set to + / - 0 . 1 . P33 presented with two malignant clones in blood both were sequenced ( P33 _ 1 ( Clone 1 ) , P33 _ 2 ( Clone 2 ) ) . * Tetraploid malignant clone .

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B Heatmap of extracted CNA [log2 ratios] of STAT genes on chr17 (i.e., STAT3, STAT5A and STAT5B), and other relevant tumor suppressors (i.e, STAT1, SOCS1 and TP53) and oncogenes (MYC). Red indicates gain/amplification, blue indicates loss/deletion. The balance threshold was set to +/- 0.1. P33 presented with two malignant clones in the blood and both were sequenced (P33_1 (Clone 1), P33_2 (Clone 2)). *Tetraploid malignant clone.


A - C Spearman correlation using CNA log2 ratios of clonal CD3 + detected in for ( A ) STAT3 , ( B ) STAT5A ( C ) STAT5B .

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A-C Spearman correlation analysis using CNA log2 ratios and the percentage of clonal CD3+ cells detected in patients for (A) STAT3, (B) STAT5A and (C) STAT5B.


D - F RT - qPCR expression data of ( C ) STAT3 , ( D ) STAT5A , ( E ) STAT5B genes from whole PBMCs isolated from blood ( n = 5 ) or healthy controls ( n = 9 ) . GAPDH expression was used for normalization . Statistical significance was calculated using two - unpaired t - . P - value : < 0 . 01 ( * * ) , < 0 . 0001 ( * * * * ) . One experiment was performed in technical triplicates . bold dashed in middle of violin plot denotes median value , while thin dotted lines denote interquartile range .

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D-F RT-qPCR expression data of (C) STAT3, (D) STAT5A, and (E) STAT5B genes from whole PBMCs isolated from patient blood (n = 5) or healthy controls (n = 9). GAPDH expression was used for normalization. Statistical significance was calculated using two-tailed unpaired t-test. P-value: <0.01 (**), <0.0001 (****). One experiment was performed in technical triplicates. The bold dashed line in the middle of the violin plot denotes the median value, while the thin dotted lines denote the interquartile range.


G Immunoblotting for total STAT3 / 5 isolated from respective whole PBMCs from blood ( n = 3 ) , or from healthy controls ( n = 3 ) . HSC70 was used as loading control . One experiment was performed .

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G Immunoblotting for total STAT3/5 protein isolated from the respective whole PBMCs from patient blood (n = 3), or from healthy controls (n = 3). HSC70 was used as loading control. One experiment was performed.


H Representative image of H & E , CD3 , pY - STAT5 single CD3 / pY - STAT5 double on skin biopsy tissue from P3 ( n = 1 ) , which demonstrates infiltration of CD3 + pY - STAT5 + ( 13 . 45 % ) into dermal subcutaneous layer of skin . Arrows indicate pY - STAT5 + single CD3 + pY - STAT5 + .

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H Representative image of H&E, CD3, pY-STAT5 single staining and CD3/pY-STAT5 double staining on skin biopsy tissue from P3 (n = 1), which demonstrates infiltration of CD3+ pY-STAT5+ (13.45%) cells into the dermal and subcutaneous layer of the skin. Arrows indicate pY-STAT5+ single and CD3+ pY-STAT5+ cells.


I - M of from skin - biopsy sections of L - CTCL ( n = 6 ) healthy individuals ( n = 5 ) As number of STAT5A / B positive was negligible ( less than 0 . 03 / per mm2 ) in healthy skin biopsy sections , healthy control samples were considered negative for pY - STAT5 , were set to 0 . Statistical significance was calculated using an unpaired Welch ' s t - . P - value : < 0 . 05 ( * ) . bold dashed in middle of violin plot denotes median value , while thin dotted lines denote interquartile range .

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I-M Quantification of IHC staining from skin-biopsy sections of L-CTCL patients (n = 6) and healthy individuals (n = 5) As the number of STAT5A/B positive cells was negligible (less than 0.03/per mm2) in healthy skin biopsy sections, healthy control samples were considered negative for pY-STAT5, and levels were set to 0. Statistical significance was calculated using an unpaired Welch's t-test. P-value: <0.05 (*).The bold dashed line in the middle of the violin plot denotes the median value, while the thin dotted lines denote the interquartile range.


A - C Graphic depicts aberration patterns on ( A ) chromosome 17 ( STAT3 / 5 ) , ( B ) chromosome 2 ( STAT1 ) ( C ) chromosome 16 ( SOCS1 ) in CTCL lines used in study . Aberration patterns are represented as log2 of of vs . reference were generated by aCGH . Immunoblotting for total STAT1 / 3 / 5 activation : phospo - Tyr ( 701 ) - STAT1 / phospho - Tyr ( 705 ) - STAT3 / phospho - Tyr ( 694 / 699 ) - STAT5 , SOCS1 . cytokine - dependent , SeAx , was collected 30 min after 5 ng / ml IL - 2 IL - 4 cytokine addition . HSC70 was used as loading control . One representative out of three independent experiments is shown .

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A-C Graphic depicts the aberration patterns on (A) chromosome 17 (STAT3/5), (B) chromosome 2 (STAT1) and (C) chromosome 16 (SOCS1) in the CTCL cell lines used in the study. Aberration patterns are represented as the log2 ratio of the fluorescence intensity of the tumor DNA vs. reference DNA and were generated by aCGH. Immunoblotting for total protein STAT1/3/5 and activation levels: phospo-Tyr (701)-STAT1/phospho-Tyr (705)-STAT3/phospho-Tyr (694/699)-STAT5, and SOCS1 protein levels. The cytokine-dependent cell line, SeAx, was collected 30 min after 5 ng/ml IL-2 and IL-4 cytokine addition. HSC70 was used as loading control. One representative out of three independent experiments is shown.


A of IC50 values calculated from drug response using CellTiter - Glo viability assays upon 48h drug treatment . One representative , out of three independent experiments performed in triplicates , is shown .

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A Heatmap of IC50 values calculated from drug response analysis using CellTiter-Glo viability assays upon 48h drug treatment. One representative, out of three independent experiments performed in triplicates, is shown.


B , Immunoblot showing total STAT3 / 5 activation : phospho - Tyr ( 705 ) - STAT3 / phospho - Tyr ( 694 / 699 ) - STAT5A / B after 24h treatment with ( B ) JPX - 0750 cytokine - dependent , SeAx , was collected 30 min after 5 ng / ml IL - 2 IL - 4 cytokine addition . HSC70 was used as loading control . normalized of phospho - total STAT3 / 5 quantified by densitometry , are shown below respective blots . One representative , out of three independent experiments is shown .

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B, Immunoblot showing total protein STAT3/5 and activation levels: phospho-Tyr (705)-STAT3/phospho-Tyr (694/699)-STAT5A/B after 24h treatment with (B) JPX-0750 The cytokine-dependent cell line, SeAx, was collected 30 min after 5 ng/ml IL-2 and IL-4 cytokine addition. HSC70 was used as loading control. The normalized levels of phospho- and total STAT3/5 quantified by densitometry, are shown below the respective blots. One representative, out of three independent experiments is shown.


C Immunoblot showing total STAT3 / 5 activation : phospho - Tyr ( 705 ) - STAT3 / phospho - Tyr ( 694 / 699 ) - STAT5A / B after 24h treatment with ( C ) IQDMA . cytokine - dependent , SeAx , was collected 30 min after 5 ng / ml IL - 2 IL - 4 cytokine addition . HSC70 was used as loading control . normalized of phospho - total STAT3 / 5 quantified by densitometry , are shown below respective blots . One representative , out of three independent experiments is shown .

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C Immunoblot showing total protein STAT3/5 and activation levels: phospho-Tyr (705)-STAT3/phospho-Tyr (694/699)-STAT5A/B after 24h treatment with (C) IQDMA. The cytokine-dependent cell line, SeAx, was collected 30 min after 5 ng/ml IL-2 and IL-4 cytokine addition. HSC70 was used as loading control. The normalized levels of phospho- and total STAT3/5 quantified by densitometry, are shown below the respective blots. One representative, out of three independent experiments is shown.


Real - Time apoptosis / necrosis in ( D , SeAx ( F , Myla lines treated with 2 . 5µM JPX - 0750 RLU , Apoptosis [ RLU ] = relative luminescence ; Necrosis [ RFU ] = relative units . Error bars represent mean + / - SD . One experiment in technical triplicates was performed .

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Real-Time apoptosis/necrosis assay in the (D, SeAx and (F, Myla cell lines treated with 2.5µM JPX-0750 RLU, Apoptosis [RLU] = relative luminescence; Necrosis [RFU] = relative fluorescence units. Error bars represent mean +/-SD. One experiment in technical triplicates was performed.


Real - Time apoptosis / necrosis in E ) SeAx G ) Myla lines treated with 2 . 5µM IQDMA . RLU , Apoptosis [ RLU ] = relative luminescence ; Necrosis [ RFU ] = relative units . Error bars represent mean + / - SD . One experiment in technical triplicates was performed .

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Real-Time apoptosis/necrosis assay in the E) SeAx and G) Myla cell lines treated with 2.5µM IQDMA. RLU, Apoptosis [RLU] = relative luminescence; Necrosis [RFU] = relative fluorescence units. Error bars represent mean +/-SD. One experiment in technical triplicates was performed.


H - I effect of ( H ) JPX - 0750 ( I ) IQDMA on viability of CTCL in comparison to viability of juvenile fibroblasts ( JF ) keratinocytes ( HaCat ) at a low dose range ( 0 . 7 - 3µM ) . One experiment , in triplicates , was performed with JF HaCat , three experiments , in triplicates with CTCL . Error bars represent mean + / - SD . Statistical significance was calculated by two - way ANOVA with Tukey ' s multiple comparisons . P - value : < 0 . 05 ( * ) , < 0 . 01 ( * * ) , < 0 . 001 ( * * * ) , < 0 . 0001 ( * * * * ) .

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H-I The effect of (H) JPX-0750 and (I) IQDMA on the viability of CTCL cells in comparison to viability of primary juvenile fibroblasts (JF) and keratinocytes (HaCat) at a low dose range (0.7-3µM). One experiment, in triplicates, was performed with JF and HaCat cells, and three experiments, in triplicates with the CTCL cells. Error bars represent mean +/-SD. Statistical significance was calculated by two-way ANOVA with Tukey's multiple comparisons test. P-value: <0.05 (*), <0.01 (**), <0.001 (***), <0.0001 (****).


B Subcellular fractions of SeAx treated with IQDMA for 24h immunoblotted for pY - STAT5 total STAT5 . were collected 30 min after 1 ng / ml IL - 2 cytokine addition . α - Tubulin Histone H3 were used as loading controls for fractions , respectively . normalized of phospho - total STAT5 in nucleus cytoplasm , quantified by densitometry , are shown below respective blots . One representative out of two independent experiments is shown .

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B Subcellular fractions of SeAx cells treated with IQDMA for 24h and immunoblotted for pY-STAT5 and total STAT5. The cells were collected 30 min after 1 ng/ml IL-2 cytokine addition. α-Tubulin and Histone H3 were used as loading controls for cytoplasmic and nuclear fractions, respectively. The normalized levels of phospho- and total STAT5 in the nucleus and cytoplasm, quantified by densitometry, are shown below the respective blots. One representative out of two independent experiments is shown.


D - F Scatter plot depicting profile of SeAx treated with ( D ) 1µM IQDMA , ( E ) 2 . 5µM IQDMA ( F ) 10µM IQDMA . abundances were determined using TMT - mass spectrometry Significant changes were assessed by a modified t - as implemented in limma package , with negative log10 p values on x - axis , log2 fold change shown on y - axis .

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D-F Scatter plot depicting the profile of SeAx cells treated with (D) 1µM IQDMA, (E) 2.5µM IQDMA and (F) 10µM IQDMA. Protein abundances were determined using TMT-based quantification mass spectrometry Significant changes were assessed by a modified t-test as implemented in the limma package, with the negative log10 p values on the x-axis, and log2 fold change shown on the y-axis.


G of IC50 values calculated from drug response using CellTiter - Blue or CellTiter - Glo viability assays upon 48h drug treatment . One representative out of three independent experiments performed in triplicates is shown . Grey represents presence of STAT3 / 5 gain .

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G Heatmap of IC50 values calculated from drug response analysis using CellTiter-Blue or CellTiter-Glo viability assays upon 48h drug treatment. One representative out of three independent experiments performed in triplicates is shown. Grey represents the presence of STAT3/5 gain.


A Inhibitor treatment was carried out with increased dose escalation for 24h . SeAx were collected 2h after 7 , 5 ng / ml IL - 2 IL - 4 cytokine addition . STAT3 / 5 target gene products MYC , PIM1 , MCL - 1 , AKT1 / 2 / 3 , as well as regulating cycle , such as CYCLIN D2 D3 , CDK6 were probed by Western blotting . HSC70 served as loading control . normalized , quantified by densitometry , are shown below respective blots . One representative out of three independent experiments is shown .

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A Inhibitor treatment was carried out with increased dose escalation for 24h. SeAx cells were collected 2h after 7,5 ng/ml IL-2 and IL-4 cytokine addition. STAT3/5 target gene products MYC, PIM1, MCL-1, and AKT1/2/3, as well as proteins regulating the cell cycle, such as CYCLIN D2 and D3, and CDK6 were probed by Western blotting. HSC70 served as loading control. The normalized protein levels, quantified by densitometry, are shown below the respective blots. One representative out of three independent experiments is shown.


Synergy of indicated two - drug combinations in SeAx Myla after 48 h treatment . In each graph most synergistic ( MSA ) is highlighted , which represents most synergistic 3 - by - 3 dose - window with respective MSA score . Zero interaction potency model was applied to quantify degree of synergy , according to which an MSA score below - 10 indicates that drugs are antagonistic ( green ) , a score between - 10 10 indicates that 2 drugs are additive ( ) , while a score above 10 indicates a synergistic effect ( red ) .

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Synergy analysis of the indicated two-drug combinations in the SeAx and Myla cells after 48 h treatment. In each graph the most synergistic area (MSA) is highlighted, which represents the most synergistic 3-by-3 dose-window with the respective MSA score. The Zero interaction potency model was applied to quantify the degree of synergy, according to which an MSA score below -10 indicates that drugs are antagonistic (green), a score between -10 and 10 indicates that 2 drugs are additive (white), while a score above 10 indicates a synergistic effect (red).


( D ) showing MSA scores of combination of FRAx597 with listed drugs . One representative out of two independent experiments is shown .

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(D) Heatmap showing MSA scores of the combination of FRAx597 with the listed drugs. One representative out of two independent experiments is shown.


E showing IC50 values upon treatment of PBMCs isolated from L - CTCL with JPX - 0750 , IQDMA FRAx597 . One experiment performed in triplicates using CellTiter - Glo viability assays upon 48h drug treatment is shown . relative STAT3 / 5 log2 value is depicted in grey .

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E Heatmap showing the IC50 values upon treatment of primary PBMCs isolated from L-CTCL patients with JPX-0750, IQDMA and FRAx597. One experiment performed in triplicates using CellTiter-Glo viability assays upon 48h drug treatment is shown. The relative STAT3/5 log2 ratio value is depicted in grey.


F - H Spearman correlation using CNA log2 ratios IC50 values for respective drug .

F-H Spearman correlation analysis using CNA log2 ratios and the IC50 values for the respective drug.


B Growth rate of Hut78 , Myla H9 - derived tumors measured on respective day post - injection . mean of respective volumes for H9 group for vehicle - treated groups for Hut78 Myla are shown . volume = ( x width2 ) / 2 . Error bars represent mean + / - . n represents total number of tumors that engrafted per group .

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B Growth rate of Hut78, Myla and H9-derived tumors measured on the respective day post-injection. The mean of the respective tumor volumes for H9 group and for the vehicle-treated groups for Hut78 and Myla are shown. Tumor volume = (length x width2)/2. Error bars represent mean +/-SEM. n represents the total number of tumors that engrafted per group.


C - D Myla - derived upon experiment termination , after 20 days of treatment with 5 mg / kg of ( C ) JPX - 0750 ( n = 5 ) IQDMA ( n = 6 ) , or vehicle ( n = 7 ) , ( D ) FRAx597 ( n = 5 ) or vehicle ( n = 4 ) , with n representing number of analyzed tumors per group . Statistical significance was calculated using two - paired t - . P - value : < 0 . 05 ( * ) . Error bars represent mean + / - .

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C-D Myla-derived tumor weight upon experiment termination, after 20 days of treatment with 5 mg/kg of (C) JPX-0750 (n = 5) and IQDMA (n = 6), or vehicle (n = 7), and (D) FRAx597 (n = 5) or vehicle (n = 4), with n representing the number of analyzed tumors per group. Statistical significance was calculated using two-tailed paired t-test. P- value: <0.05 (*). Error bars represent mean +/-SEM.


H & E of Myla - derived tumors treated with JPX - 0750 ( n = 6 ) , IQDMA ( n = 6 ) or vehicle ( n = 6 ) , FRAx597 ( n = 5 ) or vehicle ( n = 4 ) , stained with ( E , F ) H & E with n representing number of analyzed tumors per group . Pictures shown are from contiguous sections . Dotted rectangles indicate magnified areas . Scale bars , 200 , 100 25 µm . Violin plots show perimeter of annotated - infiltration / expansion region into intradermal subcutaneous region of skin Statistical significance was calculated using two - paired t - with Welch ´ s correction . P - value : < 0 . 05 ( * ) , < 0 . 01 ( * * ) , < 0 . 001 ( * * * ) . bold dashed in middle of violin plot denotes median value , while thin dotted lines denote interquartile range .

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H&E analysis of Myla-derived tumors treated with JPX-0750 (n = 6), IQDMA (n = 6) or vehicle (n = 6), and FRAx597 (n = 5) or vehicle (n = 4), stained with (E, F) H&E with n representing the number of analyzed tumors per group. Pictures shown are from contiguous sections. Dotted rectangles indicate magnified areas. Scale bars, 200, 100 and 25 µm. Violin plots show the perimeter of the annotated tumor cell-infiltration/expansion region into the intradermal and subcutaneous region of the skin Statistical significance was calculated using two-tailed paired t-test with Welch´s correction. P-value: <0.05 (*), <0.01 (**), <0.001 (***). The bold dashed line in the middle of the violin plot denotes the median value, while the thin dotted lines denote the interquartile range.


of Myla - derived tumors treated with JPX - 0750 ( n = 6 ) , IQDMA ( n = 6 ) or vehicle ( n = 6 ) , FRAx597 ( n = 5 ) or vehicle ( n = 4 ) , stained with ( G , H ) Ki67 , as a marker for proliferation with n representing number of analyzed tumors per group . Pictures shown are from contiguous sections . Dotted rectangles indicate magnified areas . Scale bars , 200 , 100 25 µm . Violin plots show of of Ki67 + in tissue . Statistical significance was calculated using two - paired t - with Welch ´ s correction . P - value : < 0 . 05 ( * ) , < 0 . 01 ( * * ) , < 0 . 001 ( * * * ) . bold dashed in middle of violin plot denotes median value , while thin dotted lines denote interquartile range .

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IHC analysis of Myla-derived tumors treated with JPX-0750 (n = 6), IQDMA (n = 6) or vehicle (n = 6), and FRAx597 (n = 5) or vehicle (n = 4), stained with (G, H) Ki67, as a marker for proliferation with n representing the number of analyzed tumors per group. Pictures shown are from contiguous sections. Dotted rectangles indicate magnified areas. Scale bars, 200, 100 and 25 µm. Violin plots show the quantification of the percentage of Ki67+ cells in the tissue. Statistical significance was calculated using two-tailed paired t-test with Welch´s correction. P-value: <0.05 (*), <0.01 (**), <0.001 (***). The bold dashed line in the middle of the violin plot denotes the median value, while the thin dotted lines denote the interquartile range.


of Myla - derived tumors treated with JPX - 0750 ( n = 6 ) , IQDMA ( n = 6 ) or vehicle ( n = 6 ) , FRAx597 ( n = 5 ) or vehicle ( n = 4 ) , stained with ( I , J ) CD31 , as a vessel marker , with n representing number of analyzed tumors per group . Pictures shown are from contiguous sections . Dotted rectangles indicate magnified areas . Scale bars , 200 , 100 25 µm . CD31 was quantified as raw of vessels with lumen . Statistical significance was calculated using two - paired t - with Welch ´ s correction . P - value : < 0 . 05 ( * ) , < 0 . 01 ( * * ) , < 0 . 001 ( * * * ) . bold dashed in middle of violin plot denotes median value , while thin dotted lines denote interquartile range .

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IHC analysis of Myla-derived tumors treated with JPX-0750 (n = 6), IQDMA (n = 6) or vehicle (n = 6), and FRAx597 (n = 5) or vehicle (n = 4), stained with (I, J) CD31, as a tumor vessel marker, with n representing the number of analyzed tumors per group. Pictures shown are from contiguous sections. Dotted rectangles indicate magnified areas. Scale bars, 200, 100 and 25 µm. CD31 staining was quantified as raw counts of vessels with lumen. Statistical significance was calculated using two-tailed paired t-test with Welch´s correction. P-value: <0.05 (*), <0.01 (**), <0.001 (***). The bold dashed line in the middle of the violin plot denotes the median value, while the thin dotted lines denote the interquartile range.


( B ) Histogram displaying SILAC ratios ( log2 ) of stress - induced phosphorylation sites in setup cdc55Δ ( upper panel ) setup rts1Δ ( lower panel ) ( Hollenstein et al . , 2020 ) , stratified by their Hog1 - dependence ( grey versus thick black ) .

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(B) Histogram displaying the SILAC ratios (log2) of stress-induced phosphorylation sites in setup cdc55Δ (upper panel) and setup rts1Δ (lower panel) (Hollenstein et al., 2020), stratified by their Hog1-dependence (grey area versus thick black line).


( D ) plot monitoring growth rate ( y - axis ) of indicated strains ( colored lines ) under different osmolarity conditions ( x - axis ) .

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(D) Line plot monitoring growth rate (y-axis) of indicated strains (colored lines) under different osmolarity conditions (x-axis).


B ) Western - blot illustrating depletion of AID - Cdc55 at various time points after addition of auxin ( upper panel ) ; Pgk2 serves as a negative control ( lower panel ) .

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B) Western-blot illustrating the depletion of AID-Cdc55 at various time points after addition of auxin (upper panel); Pgk2 serves as a negative control (lower panel).


( D ) Transcriptional of stress - induced expression of ESR genes CTT1 ( upper panel ) PGM2 ( lower panel ) under different conditions ( i . e . presence / absence of IGO1 / 2 induction of iAID - Cdc55 depletion ) . Measured time points after stress application are depicted on x - axis , target gene mRNA are illustrated on y - axis as fold over reference gene IPP1 , values are normalized relative to wild type without induction of iAID - Cdc55 depletion at 30 minutes post - stress .

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(D) Transcriptional analysis of stress-induced expression of ESR genes CTT1 (upper panel) and PGM2 (lower panel) under different strain conditions (i.e. presence/absence of IGO1/2 and induction of iAID-Cdc55 depletion). Measured time points after stress application are depicted on the x-axis, target gene mRNA levels are illustrated on the y-axis as fold over reference gene IPP1, values are normalized relative to wild type without induction of iAID-Cdc55 depletion at 30 minutes post-stress.


E ) Transcriptional of stress - induced expression of ESR genes CTT1 ( upper panel ) PGM2 ( lower panel ) at 30 minutes post - stress . Target gene mRNA are illustrated on y - axis as fold over reference gene IPP1 , values are normalized relative to wild type without induction of iAID - Cdc55 depletion . dep . : Cdc55 depleted , n . d . : Cdc55 non - depleted .

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E) Transcriptional analysis of stress-induced expression of ESR genes CTT1 (upper panel) and PGM2 (lower panel) at 30 minutes post-stress. Target gene mRNA levels are illustrated on the y-axis as fold over reference gene IPP1, values are normalized relative to wild type without induction of iAID-Cdc55 depletion. dep.: Cdc55 depleted, n.d.: Cdc55 non-depleted.


( C ) Mobility shift assays monitoring phosphorylation - induced mobility changes of Gis1 ( two upper rows ) Rph1 ( two lower rows ) upon hyperosmotic stress in wild type , igo1 ∆ igo2 ∆ Cdc55 - depleted ( right - side ) .

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(C) Mobility shift assays monitoring phosphorylation-induced mobility changes of Gis1 (two upper rows) and Rph1 (two lower rows) upon hyperosmotic stress in wild type, igo1∆igo2∆ and Cdc55-depleted cells (right-hand side).


( E ) Western blot phosphorylation shift assays of wild type Gis1 - myc ( upper panel ) Rph1 - Flag ( lower panel ) , mutated forms Gis1 - 5A - myc ( upper panel ) Rph1 - 5A - Flag ( lower panel ) . measurement was done using Phos - tag ( Kinoshita et al . , 2006 ) at indicated time points after exposure to hyperosmotic stress ( 0 . 5 M NaCl ) using Pgk1 as a negative control . location of mutated residues is shown in respective schematics . Red boxes : demethylase associated domains ( JmjN JmjC ) , blue box : zinc finger domain .

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(E) Western blot phosphorylation shift assays of wild type Gis1-myc (upper panel) and Rph1-Flag (lower panel), and mutated forms Gis1-5A-myc (upper panel) and Rph1-5A-Flag (lower panel). The measurement was done using Phos-tag (Kinoshita et al., 2006) and at the indicated time points after exposure to hyperosmotic stress (0.5 M NaCl) using Pgk1 protein levels as a negative control. The location of the mutated residues is shown in the respective schematics. Red boxes: demethylase associated domains (JmjN and JmjC), blue box: zinc finger domain.


( F ) Transcriptional of stress - induced expression of ESR genes CTT1 ( upper panel ) PGM2 ( lower panel ) at 30 minutes post - stress across indicated strains expressing either wild type or point - mutated forms of Gis1 Rph1 in a gis1 ∆ rph1 ∆ deletion background , with empty vector as control .

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(F) Transcriptional analysis of stress-induced expression of ESR genes CTT1 (upper panel) and PGM2 (lower panel) at 30 minutes post-stress across indicated strains expressing either wild type or point-mutated forms of Gis1 and Rph1 in a gis1∆rph1∆ deletion background, with the empty vector as control.


( A ) Heat map showing a subset of mRNAs of genes immunoprecipitated by anti - EZH2 antibody in LNCaP C4 - 2 prostate cancer lines , which was generated on distinct RIP - seq reads on specific gene exons . units of values were reads per kilobase million ( RPKM ) .

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(A) Heat map showing a subset of mRNAs of genes immunoprecipitated by anti-EZH2 antibody in LNCaP and C4-2 prostate cancer cell lines, which was generated based on the distinct RIP-seq reads on specific gene exons. The units of the heatmap values were reads per kilobase million (RPKM).


( B ) Screen shots from UCSC genome browser showing signal profiles of p53 mRNA immunoprecipitated by anti - EZH2 antibody in LNCaP C4 - 2 .

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(B) Screen shots from the UCSC genome browser showing signal profiles of p53 mRNA immunoprecipitated by anti-EZH2 antibody in LNCaP and C4-2 cells.


( C ) C4 - 2 were subjected to UV - RIP . RT - qPCR measurement of p53 mRNA immunoprecipitated by IgG or anti - EZH2 antibody . Data shown as means ± SD ( n = 3 ) . Statistical significance was determined by two - Student ' s t - . * * P < 0 . 01 .

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(C) C4-2 cells were subjected to UV-RIP assay. RT-qPCR measurement of p53 mRNA immunoprecipitated by IgG or anti-EZH2 antibody. Data shown as means ± SD (n=3). Statistical significance was determined by two-tailed Student's t-test. ** P<0.01.


( E ) Top , RT - qPCR of p53 mRNA in C4 - 2 lysate pulled down by GST or GST - EZH2 recombinant EZ1 - EZ4 . Bottom , western blotting of GST or GST - EZH2 used for GST pull - down . Asterisks indicate bands at expected molecular . Data shown as means ± SD ( n = 3 ) . Statistical significance was determined by two - Student ' s t - . * * P < 0 . 01 .

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(E) Top, RT-qPCR analysis of p53 mRNA in C4-2 cell lysate pulled down by GST or GST-EZH2 recombinant proteins EZ1-EZ4. Bottom, western blotting analysis of GST or GST-EZH2 proteins used for GST pull-down assay. Asterisks indicate the protein bands at expected molecular weight. Data shown as means ± SD (n=3). Statistical significance was determined by two-tailed Student's t-test.** P<0.01.


( F ) C4 - 2 were transfected with Myc - tagged EZH2 - WT or EZH2 - ∆ 336 - 554 for 24 h were harvested for RIP with IgG or anti - Myc tag antibody . Transfected pull - down p53 mRNAs were analyzed by western blot RT - qPCR , respectively . Data shown are means ± SD ( n = 3 ) . * P < 0 . 01 . ERK2 , a loading control .

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(F) C4-2 cells were transfected with Myc-tagged EZH2-WT or EZH2-∆336-554 for 24 h and cells were harvested for RIP with IgG or anti-Myc tag antibody. Transfected proteins and pull-down p53 mRNAs were analyzed by western blot and RT-qPCR, respectively. Data shown are means ± SD (n=3). * P<0.01. ERK2, a loading control.


( G ) GST pull - down using in vitro transcribed different fragments of p53 mRNA indicated GST followed by RT - qPCR of pull - down p53 mRNA . FL , full ; ORF , open reading frame ; UTR , untranslated region .

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(G) GST pull-down assay using in vitro transcribed different fragments of p53 mRNA and indicated GST proteins followed by RT-qPCR analysis of pull-down p53 mRNA. FL, full length; ORF, open reading frame; UTR, untranslated region.


( H , I ) EMSA evaluation of EZh2 binding of p53 mRNA . GST - EZH2 recombinant ( EZ1 to EZ4 ) were incubated with biotin - labeled in vitro transcribed p53 5 ' UTR ( biotin - labeled probe ) in presence or absence of 100 - fold unlabeled p53 5 ' UTR ( unlabeled probe ) , followed by PAGE immune blotting with HPR - conjugated streptavidin .

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(H, I) RNA EMSA evaluation of EZh2 binding of p53 mRNA. GST-EZH2 recombinant proteins (EZ1 to EZ4) were incubated with biotin-labeled in vitro transcribed p53 5'UTR (biotin-labeled probe) in the presence or absence of 100-fold unlabeled p53 5'UTR (unlabeled probe), followed by PAGE and immune blotting with HPR-conjugated streptavidin.


( A ) Biotin pull down by incubating biotin - labelled different fragments of p53 mRNA HOTAIR ( positive control ) with C4 - 2 lysate followed by western blot with EZH2 antibody .

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(A) Biotin pull down assay by incubating biotin-labelled different fragments of p53 mRNA and HOTAIR (positive control) with C4-2 cell lysate followed by western blot with EZH2 antibody.


( B ) Biotin pull down as in ( A ) using unmutated various internally deleted mutants of 5 ' UTR of p53 mRNA . Top , diagram of different p53 5 ' UTR deletion mutants .

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(B) Biotin pull down assay as in (A) using unmutated and various internally deleted mutants of 5'UTR of p53 mRNA. Top, diagram of different p53 5'UTR deletion mutants.


( C ) Upper , linear map of pRF bicistronic report plasmid . SV40 promoter ( purple box ) was used to drive firefly luciferase ( Fluc ) Renilla luciferase ( Rluc ) gene transcription . Different p53 5 ' UTR fragments were inserted between Fluc Rluc genes . Lower , at 24 h after transfection , were lysed luciferase activities were measured using a dual - luciferase kit of Fluc / Rluc was calculated . Data shown as means ± SD ( n = 3 ) . Statistical significance was determined by two - Student ' s t - . * P < 0 . 01 .

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(C) Upper, the linear map of the pRF bicistronic report plasmid. The SV40 promoter (purple box) was used to drive firefly luciferase (Fluc) and Renilla luciferase (Rluc) gene transcription. Different p53 5'UTR fragments were inserted between the Fluc and Rluc genes. Lower, at 24 h after transfection, cells were lysed and luciferase activities were measured using a dual-luciferase kit and the ratio of Fluc/Rluc was calculated. Data shown as means±SD (n=3). Statistical significance was determined by two-tailed Student's t-test. * P<0.01.


( D ) EZH2 knockdown C4 - 2 were transfected with bicistronic reporter vector in combination with empty vector , Myc - tagged EZH2 WT or deletion mutants followed by western blot with indicated antibodies ( bottom ) luciferase ( top ) . Data shown as means ± SD ( n = 3 ) . * P < 0 . 01 .

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(D) EZH2 knockdown C4-2 cells were transfected with the bicistronic reporter vector in combination with empty vector, Myc-tagged EZH2 WT or deletion mutants followed by western blot analysis with indicated antibodies (bottom) and luciferase assay (top). Data shown as means±SD (n=3). * P<0.01.


( E ) C4 - 2 U2OS lines were transfected with non - specific control ( siC ) or two independent EZH2 - specific siRNAs harvested for western blot with indicated antibodies . ERK2 β - TUBULIN , loading controls .

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(E) C4-2 and U2OS cell lines were transfected with non-specific control (siC) or two independent EZH2-specific siRNAs and harvested for western blot analysis with indicated antibodies. ERK2 and β-TUBULIN, loading controls.


( F ) U2OS were transfected with control ( siC ) or EZH2 - specific siRNAs treated with 200 nM of CPT followed by western blot for indicated .

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(F) U2OS cells were transfected with control (siC) or EZH2-specific siRNAs and treated with 200 nM of CPT followed by western blot analysis for indicated proteins.


( G ) C4 - 2 were transfected with control ( siC ) or EZH2 - specific siRNAs plasmids for empty vector , EZH2 WT or deletion mutants followed by western blot for indicated .

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(G) C4-2 cells were transfected with control (siC) or EZH2-specific siRNAs and plasmids for empty vector, EZH2 WT or deletion mutants followed by western blot analysis for indicated proteins.


( A ) List of top hits of EZH2 - bound identified by TAP - MS . number of peptides coverage of each are indicated .

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(A) List of top hits of EZH2-bound proteins identified by TAP-MS. The number of peptides and coverage of each protein are indicated.


( B ) Reciprocal co - IP of endogenous EZH2 eIF4G2 or PABP1 in C4 - 2 .

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(B) Reciprocal co-IP of endogenous EZH2 and eIF4G2 or PABP1 in C4-2 cells.


( C ) Co - IP of endogenous EZH2 with eIF4G2 or PABP1 from C4 - 2 lysate pre - treated with RNaseA in 37oC for 30 min . effectiveness of RNaseA treatment was monitored by RT - PCR of presence of GAPDH mRNA .

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(C) Co-IP of endogenous EZH2 with eIF4G2 or PABP1 from C4-2 cell lysate pre-treated with RNaseA in 37oC for 30 min. The effectiveness of RNaseA treatment was monitored by RT-PCR analysis of the presence of GAPDH mRNA.


( D ) C4 - 2 were transfected control ( siC ) or EZH2 - specific siRNA lysed for polysome fractionation . was extracted from even - number fractions followed by RT - qPCR for p53 GAPDH mRNA . β - ACTIN mRNA was used as an internal control . Data shown as means ± SD ( n = 3 ) . Statistical significance was determined by two - Student ' s t - . * P < 0 . 01 ; NS , no significance .

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(D) C4-2 cells were transfected control (siC) or EZH2-specific siRNA and lysed for polysome fractionation. RNA was extracted from even-number fractions followed by RT-qPCR analysis for p53 and GAPDH mRNA. The β-ACTIN mRNA was used as an internal control. Data shown as means±SD (n=3). Statistical significance was determined by two-tailed Student's t-test. * P<0.01; NS, no significance.


( E ) C4 - 2 were transfected with control ( siC ) or EZH2 - specific siRNAs in combination with empty vector , EZH2 WT or different deletion mutants followed by polysome fractionation RT - qPCR . β - ACTIN mRNA was used as an internal control . Data shown as means ± SD ( n = 3 ) . * P < 0 . 01 .

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(E) C4-2 cells were transfected with control (siC) or EZH2-specific siRNAs in combination with empty vector, EZH2 WT or different deletion mutants followed by polysome fractionation and RT-qPCR. β-ACTIN mRNA was used as an internal control. Data shown as means±SD (n=3). * P<0.01.


( A ) of Pten , Ezh2 p53 in prostatic tissues of 4 - month - old mice with different genotypes : Ptenpc - / - ( n = 11 ) ; Ezh2pc - / - ( n = 8 ) ; Ptenpc - / - ; Ezh2pc - / - ( n = 10 ) ' wild - type ' littermate controls ( n = 10 ) . Scale bars ,

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(A) IHC analysis of Pten, Ezh2 and p53 proteins in prostatic tissues of 4-month-old mice with different genotypes: Ptenpc-/- (n=11); Ezh2pc-/- (n=8); Ptenpc-/-;Ezh2pc-/- (n=10) and 'wild-type' littermate controls (n=10). Scale bars, 50


( B ) Western blot of expression of indicated in four different groups of mice as described ( n = 2 mice / group ) .

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(B) Western blot analysis of expression of indicated proteins in four different groups of mice as described (n=2 mice/group).


( C ) of correlation of Ezh2 p53 determined by in Pten knockout ( Ptenpc - / - ) mice ( n = 11 ) .

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(C) Analysis of correlation of Ezh2 and p53 protein levels determined by IHC in Pten knockout (Ptenpc-/-) mice (n=11).


( D ) RT - qPCR of p53 mRNA expression in four different genotypes of mice as described Statistical significance was determined by two - Student ' s t - . * P < 0 . 01 .

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(D) RT-qPCR analysis of p53 mRNA expression in four different genotypes of mice as described Statistical significance was determined by two-tailed Student's t-test. * P<0.01.


( E ) RT - qPCR of mRNA expression of p53 downstream target gene Bax in four different genotypes of mice as indicated . Statistical significance was determined by two - Student ' s t - . * P < 0 . 01 .

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(E) RT-qPCR analysis of mRNA expression of p53 downstream target gene Bax in four different genotypes of mice as indicated. Statistical significance was determined by two-tailed Student's t-test. * P< 0.01.


( F ) H & E of ventral prostate ( VP ) of 4 - month - old mice with indicated genotypes . inset shows a high magnification image of representative ( framed ) in each panel . Scale bars , 50 µm . Scale bars in insets , 10 µm .

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(F) H&E analysis of ventral prostate (VP) of 4-month-old mice with indicated genotypes. The inset shows a high magnification image of the representative (framed) area in each panel. Scale bars, 50 µm. Scale bars in insets, 10 µm.


( G ) of nonmalignant , low - grade PIN ( LGPIN ) high - grade PIN ( HGPIN ) or cancerous acini in prostates ( including anterior prostate ( AP ) , ventral prostate ( VP ) dorsolateral prostate ( DLP ) ) of mice with indicated genotypes number as in ( A ) .

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(G) Quantification of nonmalignant, low-grade PIN (LGPIN) and high-grade PIN (HGPIN) or cancerous acini in the prostates (including anterior prostate (AP), ventral prostate (VP) and dorsolateral prostate (DLP)) of mice with the indicated genotypes and number as in (A).


( A ) Correlation of EZH2 p53 mRNA expression measured by RT - qPCR in metastatic prostate cancer specimens of a cohort of from University of Washington .

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(A) Correlation analysis of EZH2 and p53 mRNA expression measured by RT-qPCR in primary and metastatic prostate cancer specimens of a cohort of patients from University of Washington.


( B ) of EZH2 p53 expression in human prostate cancer tissues of a cohort of from Mayo Clinic . Case 1 2 show low high expression in tissues , respectively ; case 3 shows high expression in lymph node metastasis . Scale bar , 50 µm . Scale bars in insets , 10 µm .

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(B) IHC analysis of EZH2 and p53 protein expression in human prostate cancer tissues of a cohort of patients from Mayo Clinic. Case 1 and 2 show low and high expression in primary tissues, respectively; case 3 shows high expression in lymph node metastasis. Scale bar, 50 µm. Scale bars in insets, 10 µm.


( C ) Correlation of EZH2 p53 expression in metastatic prostate cancer specimens of Mayo Clinic cohort .

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(C) Correlation analysis of EZH2 and p53 protein expression in primary and metastatic prostate cancer specimens of the Mayo Clinic cohort.


( D ) of EZH2 p53 expression in commercially purchased TMA with specimens of brain , colorectal , pancreatic cancer sarcoma . Scale bars in low magnification , 200 µm ; Scale bars in high magnification , 50 µm . Arrows , indicated zoom out - box . ( E ) data as shown in ( D ) were quantified used for correlation of EZH2 p53 expression in specimens of brain , colorectal , pancreatic cancer sarcoma . Pearson correlation r P values in each cancer type are indicated .

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(D) IHC analysis of EZH2 and p53 protein expression in commercially purchased TMA with patient specimens of brain, colorectal, pancreatic cancer and sarcoma. Scale bars in low magnification images, 200 µm; Scale bars in high magnification images, 50 µm. Arrows, indicated the zoom out-box. (E) IHC data as shown in (D) were quantified and used for correlation analysis of EZH2 and p53 protein expression in patient specimens of brain, colorectal, pancreatic cancer and sarcoma. Pearson correlation r and P values in each cancer type are indicated.


( A , B ) VCaP stably expressing control ( sh - control ) or p53 - specific shRNA were infected with lentivirus for empty vector ( EV ) or deletion mutants of EZH2 . growth in 2D ( A ) 3D ( B ) conditions were determined by MTS measurement of clone , respectively . Statistical significance was determined by two - Student ' s t - . * P < 0 . 05 , * * * P < 0 . 001 .

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(A, B) VCaP cells stably expressing control (sh-control) or p53-specific shRNA were infected with lentivirus for empty vector (EV) or deletion mutants of EZH2. Cell growth in 2D (A) and 3D (B) conditions were determined by MTS assay and measurement of clone size, respectively. Statistical significance was determined by two-tailed Student's t-test. * P < 0.05, *** P < 0.001.


( C , D ) VCaP ( 1x107 ) infected with lentivirus were injected subcutaneously into NSG mice ( n = 8 / group ) . Tumors were measured by caliper twice a week . Tumors at end point of measurement were isolated photographed ( C ) , data are shown as means ± SD ( D ) . Statistical significance was determined by two - Student ' s t - for tumors at day 56 . * * * P < 0 . 001 .

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(C, D) VCaP cells (1x107) infected with lentivirus were injected subcutaneously into NSG mice (n = 8/group). Tumors were measured by caliper twice a week. Tumors at the end point of measurement were isolated and photographed (C), and data are shown as means ± SD (D). Statistical significance was determined by two-tailed Student's t-test for tumors at day 56. *** P < 0.001.


( E - G ) Luciferase - expressing MIA PaCa - 2 ( 2 × 106 ) infected with lentivirus were injected via tail vein into NSG mice ( n = 6 / group ) . At 12 after injection , mice were subjected to bioluminescent imaging , were recorded ( E ) bioluminescent signals were quantified ( F ) . Bioluminescent flux ( photons s - 1 sr - 1 cm - 2 ) was determined for lesions in lung , ends of box are upper lower quartiles box spans interquartile range ; median is marked by a vertical inside box ; whiskers are two lines outside box that extend to highest lowest observations . Lungs were isolated from mice , stained with Bouin ' s solution , photographed ( G ) . spots on lungs ( stained in yellow ) are metastatic tumors . * P < 0 . 05 ; NS , no significance .

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(E-G) Luciferase-expressing MIA PaCa-2 cells (2×106) infected with lentivirus were injected via tail vein into NSG mice (n = 6/group). At 12 weeks after injection, mice were subjected to bioluminescent imaging, and images were recorded (E) and bioluminescent signals were quantified (F). Bioluminescent flux (photons s-1 sr-1 cm-2) was determined for lesions in lung, the ends of the box are the upper and lower quartiles and the box spans the interquartile range; the median is marked by a vertical line inside the box; the whiskers are the two lines outside the box that extend to the highest and lowest observations. Lungs were isolated from mice, stained with Bouin's solution, and photographed (G). The white spots on lungs (stained in yellow) are metastatic tumors. * P < 0.05; NS, no significance.


( B ) Kaplan - Meier plots showing association of EZH2 overexpression with biochemical recurrence of prostate cancer in from TCGA cohort . Tumors were separated into two groups according to EZH2 expression ( high or low ) three groups on mutation status of p53 ( WT , loss or mutated ) .

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(B) Kaplan-Meier plots showing the association of EZH2 overexpression with biochemical recurrence of prostate cancer in patients from the TCGA cohort. Tumors were separated into two groups according to EZH2 expression levels (high or low) and three groups based on the mutation status of p53 (WT, loss or mutated).


VCaP C4 - 2 were treated with GSK - 126 , DZNep ( 5 µM for VCaP 10 µM for C4 - 2 ) , EZH2 ASO , followed by western blots with indicated antibodies ( C ) Data shown as means ± SD ( n = 6 ) . Statistical significance was determined by two - Student ' s t - . * * * P < 0 . 001 .

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VCaP and C4-2 cells were treated with GSK-126, DZNep (5 µM for VCaP and 10 µM for C4-2), and EZH2 ASO, followed by western blots with indicated antibodies (C) Data shown as means±SD (n=6). Statistical significance was determined by two-tailed Student's t-test. *** P < 0.001.


VCaP C4 - 2 were treated with GSK - 126 , DZNep ( 5 µM for VCaP 10 µM for C4 - 2 ) , EZH2 ASO , followed by MTT ( D ) . Data shown as means ± SD ( n = 6 ) . Statistical significance was determined by two - Student ' s t - . * * * P < 0 . 001 .

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VCaP and C4-2 cells were treated with GSK-126, DZNep (5 µM for VCaP and 10 µM for C4-2), and EZH2 ASO, followed by MTT assay (D). Data shown as means±SD (n=6). Statistical significance was determined by two-tailed Student's t-test. *** P < 0.001.


( E , F ) Effect of Ezh2 ASOs on growth of murine prostate cancer allografts . Mice ( n = 10 ) bearing Ptenpc - / - ; Trp53pc - / - ( E ) Ptenpc - / - ; Trp53pc - / R172H allografts ( F ) were treated with control ASOs ( 50 mg kg - 1 ) or Ezh2 ASOs at different doses ( 25 mg kg - 1 50 mg kg - 1 ) . growth was measured twice a week for 3 data are shown in bottom panels . Each allograft was isolated by end of ASO treatment photographed ( top panels ) . Data shown as means ± SD ( n = 10 ) . Statistical significance was determined by two - Student ' s t - . * * * P < 0 . 001 .

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(E, F) Effect of Ezh2 ASOs on growth of murine prostate cancer allografts. Mice (n=10) bearing the Ptenpc-/-;Trp53pc-/- (E) and Ptenpc-/-;Trp53pc-/R172H allografts (F) were treated with control ASOs (50 mg kg-1) or Ezh2 ASOs at different doses (25 mg kg-1 and 50 mg kg-1). Tumor growth was measured twice a week for 3 weeks and the data are shown in the bottom panels. Each allograft was isolated by the end of ASO treatment and photographed (top panels). Data shown as means±SD (n=10). Statistical significance was determined by two-tailed Student's t-test. *** P < 0.001.


C - E Mapping of binding region between myoVa caldendrin using co - immunoprecipitation of cald - tagRFP GFP - myoVa fragments expressed in HEK293T . Arrows in D indicate of respective myosin fragment . F Summary of results from C - E . Caldendrin binds specifically to a fragment containing IQ - motif 1 ( 742 - 791 ) , but not to any other region of myoVa , in a Ca2 + - dependent manner .

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C-E Mapping of the binding region between myoVa and caldendrin using co-immunoprecipitation of cald-tagRFP and GFP-myoVa fragments expressed in HEK293T cells. Arrows in D indicate the size of the respective myosin fragment. F Summary of the results from C - E. Caldendrin binds specifically to a fragment containing IQ-motif 1 (amino acids 742-791), but not to any other region of myoVa, in a Ca2+-dependent manner.


B Western blot of competition co - immunoprecipitation experiment in HEK293T . In presence of Ca2 + , caldendrin binds strongly to IQ1 fragment ( upper arrow ) , while tagRFP by itself does not bind ( lower arrow ) . Detection using a calmodulin antibody indicated that Ca2 + - dependent binding of caldendrin reduced amount of CaM bound to IQ1 motif relative to control ( tagRFP only ) . MW = molecular marker . C of normalized relative of cald - tagRFP ( left ) CaM bands ( right ) in western blot of three independent experiments as shown in B . Shown is mean ± . Upper : Ca2 + greatly increases cald - tagRFP binding to GFP - myoVa - IQ1 . Lower : When Ca2 + is available , simultaneous presence of cald - tagRFP leads to a reduced binding of CaM compared to control ( tagRFP ) .

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B Western blot analysis of competition co-immunoprecipitation experiment in HEK293T cells. In the presence of Ca2+, caldendrin binds strongly to the IQ1 fragment (upper arrow), while tagRFP by itself does not bind (lower arrow). Detection using a calmodulin antibody indicated that Ca2+-dependent binding of caldendrin reduced the amount of CaM bound to the IQ1 motif relative to the control (tagRFP only). MW = molecular weight marker. C Quantification of the normalized relative intensity of cald-tagRFP (left) and CaM bands (right) in western blot analysis of three independent experiments as shown in B. Shown is mean ± SEM. Upper: Ca2+ greatly increases cald-tagRFP binding to GFP-myoVa-IQ1. Lower: When Ca2+ is available, the simultaneous presence of cald-tagRFP leads to a reduced binding of CaM compared to the control (tagRFP). D


F Native MS of ion saturation for CaM . Ca2 + carried along from purification is detected on CaM in native MS . Without further supplementation with ions , intact CaM shows primarily its high affinity calcium binding sites occupied ( left ) , while in same myoVa peptide selectively preferentially binds only to fully Ca2 + - saturated CaM species ( right ) . Additional unassigned peaks represent unspecific adducts of Na + Mg2 + ions .

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F Native MS analysis of ion saturation for CaM. Ca2+ carried along from purification is detected on CaM in native MS. Without further supplementation with ions, intact CaM shows primarily its high affinity calcium binding sites occupied (left), while in the same analysis myoVa peptide selectively and preferentially binds only to the fully Ca2+-saturated CaM species (right). Additional unassigned peaks represent unspecific adducts of Na+ and Mg2+ ions.


G Native MS of ion saturation for caldendrin . Supplemented by an excess of 200 µM Ca2 + 100 µM Mg2 + , caldendrin displays myoVa peptide binding to both fully Ca2 + - saturated 2 Ca2 + - bound species ( right ) , which are also both detectable for intact caldendrin in same ( left ) .

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G Native MS analysis of ion saturation for caldendrin. Supplemented by an excess of 200 µM Ca2+ and 100 µM Mg2+, caldendrin displays myoVa peptide binding to both fully Ca2+-saturated and 2 Ca2+-bound species (right), which are also both detectable for the intact caldendrin protein in the same analysis (left).


C Visualization of F - actin gliding caused by myoVa activity . first ( red ) last ( green ) frame of a 30 second time - lapse image stack were color - coded overlaid to visualize movement . Yellow = actin filament did not move during 30 seconds of imaging . In presence of only CaM ( left ) , majority of actin filaments are gliding . In presence of both CaM cald ( right ) , a large number of filaments are immobile . Scale bar = 10 µm .

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C Visualization of F-actin gliding caused by myoVa activity. The first (red) and last (green) frame of a 30 second time-lapse image stack were color-coded and overlaid to visualize movement. Yellow = actin filament did not move during 30 seconds of imaging. In the presence of only CaM (left), the majority of actin filaments are gliding. In the presence of both CaM and cald (right), a large number of filaments are immobile. Scale bar = 10 µm.


B Co - sedimentation of GFP - tagged myosin fragment lacking globular tail domain ( GFP - myoVa - motor + CC ) with F - actin shows that presence of Ca2 + - caldendrin does not prevent myoV from binding to actin filaments . GFP - myoVa - motor + CC , as visualized by an anti - GFP antibody via western blot , co - sediments with F - actin ( comassie blue stained SDS - PAGE ) , both in presence absence of Ca2 + , regardless of presence of caldendrin .

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B Co-sedimentation assay of GFP-tagged myosin fragment lacking the globular tail domain (GFP-myoVa-motor+CC) with F-actin shows that the presence of Ca2+-caldendrin does not prevent myoV from binding to actin filaments. GFP-myoVa-motor+CC, as visualized by an anti-GFP antibody via western blot, co-sediments with F-actin (comassie blue stained SDS-PAGE), both in presence and absence of Ca2+, regardless of the presence of caldendrin.


D Left : Representative image of COS7 expressing inducible dimerization system described in C . Red : Peroxisomes ( PEX - RFP - FKBP ) , Green : KIF17 - GFP - PEX13 myoV - GFP - FRB . Scale bar = 10 µm . Middle right : tracks of kinesin - driven peroxisome movements reconstructed over 20 seconds ( yellow ) before ( middle ) after ( right ) addition of rapalog indicate that recruitment of myoV slows down motility of peroxisomes . Blue outlines represent manually drawn border nucleus ( excluded from ) .

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D Left: Representative image of COS7 cell expressing the inducible dimerization system described in C. Red: Peroxisomes (PEX-RFP-FKBP), Green: KIF17-GFP-PEX13 and myoV-GFP-FRB. Scale bar = 10 µm. Middle and right: Individual tracks of kinesin-driven peroxisome movements reconstructed over 20 seconds (yellow) before (middle) and after (right) addition of rapalog indicate that the recruitment of myoV slows down the motility of peroxisomes. Blue outlines represent manually drawn cellular border and nucleus (excluded from analysis).


E of mean squared displacement ( MSD ) of peroxisomes over time ( left ) suggests a linear increase for conditions . Slope for MSD curves for ( right ) indicates that addition of rapalog induces efficient stalling ( 2 - way ANOVA , * * * p < 0 . 0001 as compared before after rapalog ) . However , no difference between control caldendrin ( cald ) transfected could be observed ( 2 - way ANOVA , Mean ± ; n control = 14 , n cald = 23 ; 3 experiments ) .

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E Analysis of the mean squared displacement (MSD) of peroxisomes over time (left) suggests a linear increase for all conditions. Slope analysis for MSD curves for individual cells (right) indicates that addition of rapalog induces efficient stalling (2-way ANOVA, *** p<0.0001 as compared before and after rapalog). However, no difference between control and caldendrin (cald) transfected cells could be observed (2-way ANOVA, Mean ± SEM; n control=14 cells, n cald=23 cells; 3 experiments).


A Example confocal from 10 min time - lapse imaging of control ( GFP ) or caldendrin ( cald - GFP ) transfected rat hippocampal neurons ( DIV17 ) expressing an ER - marker ( ER - tDimer ) . Yellow arrows show dendritic protrusions with a stably inserted ER ( > 10 min ) . Blue arrows indicate protrusions with a transient ER entry . Scale bar = 5 µm . B of dynamic SER presence in protrusions of control ( GFP ) or caldendrin ( cald - GFP ) transfected neurons . In protrusions of cald - GFP neurons , of stably localized ER ( > 10 min ) is increased , while transient SER visits are decreased compared to control . ( mean ± , 2 - way ANOVA , * p = 0 . 0386 for interaction , n GFP = 9 , n cald - GFP = 10 ; 2 experiments )

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A Example confocal images from 10 min time-lapse imaging of control (GFP) or caldendrin (cald-GFP) transfected rat primary hippocampal neurons (DIV17) expressing an ER-marker (ER-tDimer). Yellow arrows show dendritic protrusions with a stably inserted ER (>10 min). Blue arrows indicate protrusions with a transient ER entry. Scale bar = 5 µm. B Quantification of dynamic SER presence in protrusions of control (GFP) or caldendrin (cald-GFP) transfected neurons. In protrusions of cald-GFP neurons, the percentage of stably localized ER (> 10 min) is increased, while transient SER visits are decreased compared to the control. (mean ± SEM, 2-way ANOVA, * p=0.0386 for interaction, n GFP = 9, n cald-GFP = 10; 2 experiments)


C Representative confocal of control ( GFP ) or caldendrin ( cald - GFP ) transfected rat hippocampal neurons expressing an ER - marker ( ER - tDimer ) . To pre - load overexpressed caldendrin with Ca2 + , neurons were stimulated with 50 µM bicuculline for 5 min 8 hours prior to fixation stained with a homer1 - antibody to visualize synapse - containing spines . Arrows indicate spines that contain ER . Scale bar = 5 µm . D - F of protrusion - spine density number of SER - positive spines as shown in C . ( mean ± , n GFP ctrl = 24 , n Cald - GFP = 25 ; 3 experiments ) . ( D ) density of dendritic protrusions ( spines filopodia ) is increased in cald - GFP overexpressing neurons compared to control . ( * p = 0 . 0119 , unpaired t - ) . ( E ) density of homer1 - positive dendritic spines is increased in cald - GFP overexpressing neurons compared to control . ( * * p = 0 . 0049 , unpaired t - ) . ( F ) of ER - containing dendritic spines is increased in cald - GFP overexpressing neurons compared to control . ( * p = 0 . 0179 , unpaired t - )

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C Representative confocal images of control (GFP) or caldendrin (cald-GFP) transfected rat primary hippocampal neurons expressing an ER-marker (ER-tDimer). To pre-load overexpressed caldendrin with Ca2+, neurons were stimulated with 50 µM bicuculline for 5 min 8 hours prior to fixation and stained with a homer1-antibody to visualize synapse-containing spines. Arrows indicate spines that contain ER. Scale bar = 5 µm. D - F Quantification of protrusion- and spine density and the number of SER-positive spines as shown in C. (mean ± SEM, n GFP ctrl = 24 cells, n Cald-GFP = 25 cells; 3 experiments). (D) The density of dendritic protrusions (spines and filopodia) is increased in cald-GFP overexpressing neurons compared to the control. (* p=0.0119, unpaired t-test). (E) The density of homer1-positive dendritic spines is increased in cald-GFP overexpressing neurons compared to the control. (** p=0.0049, unpaired t-test). (F) The percentage of ER-containing dendritic spines is increased in cald-GFP overexpressing neurons compared to the control. (* p=0.0179, unpaired t-test) G


G Representative confocal of control ( GFP ) or caldendrin ( cald - GFP ) transfected rat hippocampal neurons pre - treated with bicuculline as in C stained against homer1 spine apparatus - marker synaptopodin . Yellow arrows indicate synaptopodin clusters localized inside dendritic shaft , blue arrows indicate spine - localized synaptopodin . Scale bar = 5 µm . H - J of spine density , synaptopodin clusters synaptopodin - positive spines as shown in G . ( mean ± , n GFP ctrl = 21 , n cald - GFP = 30 ; 2 experiments ) . ( H ) Spine density is increased in cald - GFP overexpressing neurons compared to control ( in with 5e ) . ( * * p = 0 . 0024 , unpaired t - ) . ( I ) total number of synaptopodin clusters is not affected by caldendrin overexpression . ( n . s . , unpaired t - ) . ( J ) of synapotpodin - positive spines is increased in cald - GFP overexpressing neurons compared to control . ( * * * * p < 0 . 0001 , unpaired t - ) .

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G Representative confocal images of control (GFP) or caldendrin (cald-GFP) transfected rat primary hippocampal neurons pre-treated with bicuculline as in C and stained against homer1 and the spine apparatus-marker synaptopodin. Yellow arrows indicate synaptopodin clusters localized inside the dendritic shaft, blue arrows indicate spine-localized synaptopodin. Scale bar = 5 µm. H - J Quantification of spine density, synaptopodin clusters and synaptopodin-positive spines as shown in G. (mean ± SEM, n GFP ctrl = 21 cells, n cald-GFP = 30 cells; 2 experiments). (H) Spine density is increased in cald-GFP overexpressing neurons compared to the control (in line with 5e). (** p = 0.0024, unpaired t-test). (I) The total number of synaptopodin clusters is not affected by caldendrin overexpression. (n.s., unpaired t-test). (J) The percentage of synapotpodin-positive spines is increased in cald-GFP overexpressing neurons compared to the control. (**** p < 0.0001, unpaired t-test).


A 10 min time - lapse imaging of DIV12 wild - type ( cald WT ) , caldendrin knock - out ( cald KO ) , caldendrin knock - out expressing caldendrin - GFP ( cald KO + cald - GFP ) or caldendrin knock - out treated with jasplakinolide ( cald KO + JPK ) mouse hippocampal neurons , additionally expressing a fill ( maxGFP ) an ER - marker ( ER - tDimer ) . Yellow arrows indicate protrusions with a stably anchored ER ( > 10 min ) . Blue arrows indicate protrusions that show transient presence of ER . Scale bar = 5 µm .

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A 10 min time-lapse imaging of DIV12 wild-type (cald WT), caldendrin knock-out (cald KO), caldendrin knock-out expressing caldendrin-GFP (cald KO + cald-GFP) or caldendrin knock-out treated with jasplakinolide (cald KO + JPK) mouse primary hippocampal neurons, additionally expressing a cell fill (maxGFP) and an ER-marker (ER-tDimer). Yellow arrows indicate protrusions with a stably anchored ER (>10 min). Blue arrows indicate protrusions that show transient presence of ER. Scale bar = 5 µm.


A Representative confocal of mouse hippocampal neurons stained against homer1 synaptopodin . groups compared are wild - type ( cald WT ) , caldendrin knock - out ( cald KO ) , caldendrin knock - out expressing caldendrin - GFP ( cald KO cald ) , caldendrin knock - out treated with jasplakinolide ( cald KO JPK ) . Scale bar = 5 µm .

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A Representative confocal images of mouse primary hippocampal neurons stained against homer1 and synaptopodin. The groups compared are wild-type (cald WT), caldendrin knock-out (cald KO), caldendrin knock-out expressing caldendrin-GFP (cald KO cald), and caldendrin knock-out treated with jasplakinolide (cald KO JPK). Scale bar = 5 µm.


B - C of spine density , synaptopodin clusters synaptopodin - positive spines as shown in A ( mean ± . Kruskal - Wallis with Dunn ' s multiple comparisons post - . * * * p < 0 . 0001 . cald wt n = 34 , 2 experiments ; cald KO n = 33 , 3 experiments ; cald KO cald n = 10 , 1 experiment ; cald KO JPK n = 14 , 1 experiment ) . ( B ) total number of synaptopodin clusters is unaffected by loss of caldendrin . ( C ) number of synaptopodin - positive spines is reduced in cald KO compared to WT neurons . This phenotype could be rescued by over - expression of caldendrin ( cald KO + cald ) , but not by treatment with JPK ( cald KO + JPK ) .

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B - C Quantification of spine density, synaptopodin clusters and synaptopodin-positive spines as shown in A (mean ± SEM. Kruskal-Wallis test with Dunn's multiple comparisons post-test. *** p < 0.0001. cald wt n = 34 cells, 2 experiments; cald KO n = 33 cells, 3 experiments; cald KO cald n = 10 cells, 1 experiment; cald KO JPK n = 14 cells, 1 experiment). (B) The total number of synaptopodin clusters is unaffected by the loss of caldendrin. (C) The number of synaptopodin-positive spines is reduced in cald KO compared to the WT neurons. This phenotype could be rescued by over-expression of caldendrin (cald KO + cald), but not by treatment with JPK (cald KO + JPK).


Media from soluble TREM2 - Fc transfected HEK293T incubated with Aβ42 fibrils . Top left panel shows original media samples . Top right panel shows presence of soluble TREM2 associated with Aβ42 pellet . bottom two panels represent same samples as top , but probed with 6E10 RIPA lysate from TREM2 transfected HEK293T incubated with Aβ42 fibrils . Top left panel shows original RIPA lysate samples top right panel shows presence of TREM2 associated with Aβ42 pellet . bottom two panels represent same samples as top , but probed with 6E10 Western blo had been cropped to facilitate reading originals with supernatant fractions are available in Fig EV1 . experiments were replicated 3 independent

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Media from soluble TREM2-Fc transfected HEK293T cells incubated with Aβ42 fibrils. Top left panel shows original media samples. Top right panel shows the presence of soluble TREM2 associated with Aβ42 pellet. The bottom two panels represent the same samples as the top, but probed with 6E10 RIPA lysate from TREM2 transfected HEK293T cells incubated with Aβ42 fibrils. Top left panel shows original RIPA lysate samples and top right panel shows the presence of TREM2 associated with Aβ42 pellet. The bottom two panels represent the same samples as the top, but probed with 6E10 Western blo had been cropped to facilitate the reading and originals with the supernatant fractions are available in Fig EV1. All experiments were replicated 3 independent times


Media from soluble TREM2 - Fc APOE3 co transfected HEK293T incubated with agarose beads anti - Human IgG Fc . Left panels show original media samples . Right panels show purified soluble TREM2 - Fc ( bottom ) presence of APOE3 with purified soluble TREM2 - Fc ( top ) Western blo had been cropped to facilitate reading originals with supernatant fractions are available in Fig EV1 . experiments were replicated 3 independent

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Media from soluble TREM2-Fc and APOE3 co transfected HEK293T cells incubated with agarose beads anti-Human IgG Fc. Left panels show original media samples. Right panels show the purified soluble TREM2-Fc (bottom) and the presence of APOE3 with the purified soluble TREM2-Fc (top) Western blo had been cropped to facilitate the reading and originals with the supernatant fractions are available in Fig EV1. All experiments were replicated 3 independent times