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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 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 .

O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

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 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 .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 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 .

O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 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 ( * * * * ) .

O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 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 .

O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O

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 analysis using CNA log2 ratios and the IC50 values for the 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 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 .

O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


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 .

O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


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 .

O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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 ) .

O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O

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 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 .

O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O

(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 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 .

O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O

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 ) and Rph1 ( two lower rows ) upon hyperosmotic stress in wild type , igo1 ∆ igo2 ∆ and Cdc55 - depleted cells ( right - hand side ) .

O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O

(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 ) 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 .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 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 .

O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O

(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 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 ) .

O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O

(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 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 .

O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 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 .

O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 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 .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 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 .

O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O

(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 ) 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 .

O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O

(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 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 .

O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O

(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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O

(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 , 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 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 .

O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 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 .

O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O

(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 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 .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O

(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 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 .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O

(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 proteins identified by TAP - MS . The number of peptides and coverage of each protein are indicated .

O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O

(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 and eIF4G2 or PABP1 in C4 - 2 cells .

O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O

(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 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 .

O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O

(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 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 .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 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 .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 ) 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 ,

O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O

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


( 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 .

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 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 .

O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 ) 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 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 .

O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

(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 ) 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 .

O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O

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


( 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 .

O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 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 .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 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 ) .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O

(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 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 .

O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 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 .

O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 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 .

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 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 .

O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O

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 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 ) .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O

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 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 .

O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

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 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 ) .

O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O

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 . 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O

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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O

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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O

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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O

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 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 )

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O

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 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 )

O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O

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 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 ) .

O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

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 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 .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 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 .

O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O

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 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 ) .

O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O

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 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

O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 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

O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


Media from soluble TREM - Fc family member transfected HEK293T cells incubated with Aβ42 fibrils . Top panel shows original media sample and the presence of TREM family members associated with Aβ42 pellet . Bottom panel shows original media and Aβ42 pellet probed with

O O O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O

Media from soluble TREM-Fc family member transfected HEK293T cells incubated with Aβ42 fibrils. Top panel shows original media sample and the presence of TREM family members associated with Aβ42 pellet. Bottom panel shows original media and Aβ42 pellet probed with 6E10


Media from soluble TREM - Fc family members and APOE3 co transfected HEK293T cells incubated with agarose beads anti - Human IgG Fc . Top panel shows original media sample and the presence of soluble TREM family members associated with APOE3 . Bottom panel shows original media and purified soluble TREM - Fc family members probed with anti -

O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O

Media from soluble TREM-Fc family members and APOE3 co transfected HEK293T cells incubated with agarose beads anti-Human IgG Fc. Top panel shows original media sample and the presence of soluble TREM family members associated with APOE3. Bottom panel shows original media and purified soluble TREM-Fc family members probed with anti-V5


Schematic representation of Aβ42 oligomers ( right panel ) and APOE4 ( left panel ) binding with soluble TREM2 - Fc at various concentrations . Curves were fit by setting buffer control ( no ligand ) to y =

O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O

Schematic representation of Aβ42 oligomers (right panel) and APOE4 (left panel) binding with soluble TREM2-Fc at various concentrations. Curves were fit by setting buffer control (no ligand) to y=0


BioLayer Interferometry experiments were performed to evaluate the binding capacity of APOE3 on soluble TREM2 - Fc pre incubated with Aβ42 oligomers . Schematic representations of two sequential associations of ligand on soluble TREM2 - Fc are represented by I and II . Soluble TREM2 - Fc loaded on a sensor is first incubated with APOE3 ( association I ) followed by a second incubation with Aβ42 oligomers ( association II ) or the reverse - incubated with Aβ42 oligomers ( association I ) followed by a second incubation with APOE3 ( association II ) . All experiments were replicated 3 independent times

O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O

BioLayer Interferometry experiments were performed to evaluate the binding capacity of APOE3 on soluble TREM2-Fc pre incubated with Aβ42 oligomers. Schematic representations of two sequential associations of ligand on soluble TREM2-Fc are represented by I and II. Soluble TREM2-Fc loaded on a sensor is first incubated with APOE3 (association I) followed by a second incubation with Aβ42 oligomers (association II) or the reverse- incubated with Aβ42 oligomers (association I) followed by a second incubation with APOE3 (association II). All experiments were replicated 3 independent times


ELISA plates were coated with purified soluble TREM2 - Fc , and then incubated with various concentrations of Aβ42 monomer Binding of Aβ42 monomers with soluble TREM2 - Fc shows a dose dependent signal . Results were expressed as the OD450 averaged ± standard error ( n = 3 ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , Two - way ANOVA , Bonferroni multiple comparisons , see Appendix table S2 for p value )

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

ELISA plates were coated with purified soluble TREM2-Fc, and then incubated with various concentrations of Aβ42 monomer Binding of Aβ42 monomers with soluble TREM2-Fc shows a dose dependent signal. Results were expressed as the OD450 averaged ± standard error (n =3; * p<0.05, ** p<0.01, *** p<0.001, **** p < 0.0001, Two-way ANOVA, Bonferroni multiple comparisons, see Appendix table S2 for p value)


ELISA plates were coated with purified soluble TREM2 - Fc , and then incubated wit 1 µM Aβ42 oligomers Binding of 1 µM Aβ42 oligomers with soluble TREM - Fc family members . Results were expressed as the OD450 averaged ± standard error ( n = 4 , * p < 0 . 05 , * * * * p < 0 . 0001 , ANOVA , Bonferroni multiple comparisons , see Appendix table S2 for p value )

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

ELISA plates were coated with purified soluble TREM2-Fc, and then incubated wit 1 µM Aβ42 oligomers Binding of 1 µM Aβ42 oligomers with soluble TREM-Fc family members. Results were expressed as the OD450 averaged ± standard error (n = 4, * p < 0.05, **** p<0.0001, ANOVA, Bonferroni multiple comparisons, see Appendix table S2 for p value)


2B4 NFAT - GFP reporter cells transduced with TREM2 or AD variant R47H were incubated with various concentrations of Aβ42 oligomers for 12 hours . GFP expression was detected at 1 µM Aβ42 oligomers with TREM2 transduced cells . No GFP expression was detected with Aβ42 oligomers from control transduced cells . Results were expressed as the percentage ± GFP cells averaged of ± standard error ( n = 3 , * p < 0 . 05 compared with mock , * * * * p < 0 . 0001 compared with mock , # p < 0 . 05 compared with TREM2 , # # # # p < 0 . 0001 compared with TREM2 , Two - way ANOVA , Bonferroni multiple comparisons , see Appendix table S3 for p value )

O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

2B4 NFAT-GFP reporter cells transduced with TREM2 or AD variant R47H were incubated with various concentrations of Aβ42 oligomers for 12 hours. GFP expression was detected at 1 µM Aβ42 oligomers with TREM2 transduced cells. No GFP expression was detected with Aβ42 oligomers from control transduced cells. Results were expressed as the percentage ± GFP cells averaged of ± standard error (n = 3, * p < 0.05 compared with mock, **** p< 0.0001 compared with mock, # p < 0.05 compared with TREM2, #### p < 0.0001 compared with TREM2, Two-way ANOVA, Bonferroni multiple comparisons, see Appendix table S3 for p value)


B - 2B4 NFAT - GFP reporter cells transduced with TREM2 or variants ( R47H , R62H and T96K ) were incubated with 10 µM Aβ42 oligomers for 12 hours . No GFP expression was detected with Aβ42 oligomers from control transduced cells . Results were expressed as the percentage GFP cells averaged of ± standard error ( n = 3 , * p < 0 . 05 compared with TREM2 , * * p < 0 . 05 compared with TREM2 , * * * * p < 0 . 0001 compared with TREM2 , # # p < 0 . 01 compared with TREM2 T96K , # # # # p < 0 . 0001 compared with TREM2 T96K , ANOVA , Dunnett ' s multiple comparisons test , see Appendix table S3 for p value

O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O

B-2B4 NFAT-GFP reporter cells transduced with TREM2 or variants (R47H, R62H and T96K) were incubated with 10 µM Aβ42 oligomers for 12 hours. No GFP expression was detected with Aβ42 oligomers from control transduced cells. Results were expressed as the percentage GFP cells averaged of ± standard error (n = 3, * p < 0.05 compared with TREM2, ** p < 0.05 compared with TREM2, **** p< 0.0001 compared with TREM2, ## p < 0.01 compared with TREM2 T96K, #### p < 0.0001 compared with TREM2 T96K, ANOVA, Dunnett's multiple comparisons test, see Appendix table S3 for p value


HEK293T cells transfected with TREM2 or AD variant incubated with 500 nM Aβ42 monomers were lysed for Aβ42 quantifications by ELISA . Expression of TREM2 increases Aβ42 level detected compared to mock control as well the AD variant . Results were averaged ± standard error ( n = 9 , # p < 0 . 05 compared with TREM2 , # # p < 0 . 01 compared with TREM2 , * * * p < 0 . 001 compared with mock , * * * * p < 0 . 0001 compared with mock , ANOVA , Tukey ' s Multiple Comparison Test , see Appendix table S3 for p value )

O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

HEK293T cells transfected with TREM2 or AD variant incubated with 500 nM Aβ42 monomers were lysed for Aβ42 quantifications by ELISA. Expression of TREM2 increases Aβ42 level detected compared to mock control as well the AD variant. Results were averaged ± standard error (n = 9, # p < 0.05 compared with TREM2, ## p< 0.01 compared with TREM2, *** p< 0.001 compared with mock, **** p <0.0001 compared with mock, ANOVA, Tukey's Multiple Comparison Test, see Appendix table S3 for p value)


HEK293T cells transfected with TREM2 were incubated with Aβ42 oligomers for 16 hours . Conditioned media and cell lysate were analyzed by Western blot for TREM2 ( RIPA lysate ) and soluble TREM2 ( media ) detection . Experiment was replicated 3 independent

O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O

HEK293T cells transfected with TREM2 were incubated with Aβ42 oligomers for 16 hours. Conditioned media and cell lysate were analyzed by Western blot for TREM2 (RIPA lysate) and soluble TREM2 (media) detection. Experiment was replicated 3 independent times


HEK293T cells transfected with TREM2 were subjected to cell membrane biotinylation for purification . Bottom panel in D shows the presence of TREM2 in the purified product . Endogenous APP and β - tubulin were used as controls for the purification ( middle and upper panels ) . Experiment was replicated 3 independent

O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

HEK293T cells transfected with TREM2 were subjected to cell membrane biotinylation for purification. Bottom panel in D shows the presence of TREM2 in the purified product. Endogenous APP and β-tubulin were used as controls for the purification (middle and upper panels). Experiment was replicated 3 independent time


( A ) Immunofluorescence images of PDAC tissues from KPC mice treated with vehicle control of tamoxifen , scale bar 100

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O

(A) Immunofluorescence images of PDAC tissues from KPC mice treated with vehicle control of tamoxifen, scale bar 100 μm


( B Quantification of GLUT1 ( hypoxia marker Control ( n = 5 ) , 2mg ( n = 5 ) , and 5mg ( n = 4 ) . In all cases , bars represent mean ± s . e .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B Quantification of GLUT1 (hypoxia marker Control (n=5), 2mg (n=5), and 5mg (n=4). In all cases, bars represent mean ± s.e.m


( C ) Relative values of protein levels for Glut1 in PDAC tumors assessed by proteomic analysis ( 6 mice per condition and samples were analysed in duplicates )

O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

(C) Relative values of protein levels for Glut1 in PDAC tumors assessed by proteomic analysis (6 mice per condition and samples were analysed in duplicates)


, D ) Quantification o CD31 ( endothelial cell marker ) . Control ( n = 5 ) , 2mg ( n = 5 ) , and 5mg ( n = 4 ) . In all cases , bars represent mean ± s . e . m . (

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

,D) Quantification o CD31 (endothelial cell marker). Control (n=5), 2mg (n=5), and 5mg (n=4). In all cases, bars represent mean ± s.e.m. (C


( E , F ) Expression levels of DEGs relevant to response to hypoxia ( left ) and blood vessel morphogenesis ( right ) . The values were normalized by tubulin family

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E,F) Expression levels of DEGs relevant to response to hypoxia (left) and blood vessel morphogenesis (right). The values were normalized by tubulin family genes


( G ) Immunofluorescence images of PDAC tissues from KPC mice treated with vehicle control and 5mg of tamoxifen , scale bar 100

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O

(G) Immunofluorescence images of PDAC tissues from KPC mice treated with vehicle control and 5mg of tamoxifen, scale bar 100 μm


( H ) Quantification of HIF - 1A in PDAC tissues Control ( n = 5 ) , and 5mg ( n = 4 ) . In the box - and - whisker plot , the central box represents values from the lower to upper quartile . The middle line represents the mean . The vertical line extends from the minimum to the maximum

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) Quantification of HIF-1A in PDAC tissues Control (n=5), and 5mg (n=4). In the box-and-whisker plot, the central box represents values from the lower to upper quartile. The middle line represents the mean. The vertical line extends from the minimum to the maximum value


( I ) qPCR levels of HIF - 1A in PSCs , normalized to RPLP0 and relative to

O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O

(I) qPCR levels of HIF-1A in PSCs, normalized to RPLP0 and relative to control


( J ) Western blot bands for protein expression in PSCs ( p - Tmod is post - translational modification ) . The plot shows the quantification of the sum of band intensities corresponding to isoform 1 , isoform 2 , isoform 3 , and post - transcriptionally modified HIF - 1A ( n = 8 control and n = 8 tam )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O

(J) Western blot bands for protein expression in PSCs (p-Tmod is post-translational modification). The plot shows the quantification of the sum of band intensities corresponding to isoform 1, isoform 2, isoform 3, and post-transcriptionally modified HIF-1A (n=8 control and n=8 tam)


( A ) qPCR levels of LOX - L2 in PSCs , normalized to RPLP0 and relative to

(A) qPCR levels of LOX-L2 in PSCs, normalized to RPLP0 and relative to control


( B ) Western blot levels of LOX - L2 in PSCs ( n = 3 experimental replicates , * * P < 0 . 01 )

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O

(B) Western blot levels of LOX-L2 in PSCs (n=3 experimental replicates, **P<0.01)


( C ) Expression of LOX family genes obtained from RNA - seq data in control and tamoxifen treated PSCs ( n = 3 experimental replicates ) . Expression value was normalized by tubulin family

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

(C) Expression of LOX family genes obtained from RNA-seq data in control and tamoxifen treated PSCs (n = 3 experimental replicates). Expression value was normalized by tubulin family genes


( D ) qPCR levels of LOX family in PSCs , normalized to RPLP0 and relative to

(D) qPCR levels of LOX family in PSCs, normalized to RPLP0 and relative to control


( E ) Relative values of protein levels for LOX members in PDAC tumors assessed by proteomic analysis ( 6 mice per condition and samples were analysed in

O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Relative values of protein levels for LOX members in PDAC tumors assessed by proteomic analysis (6 mice per condition and samples were analysed in duplicates


( F ) Immunofluorescence images of PDAC tissues from KPC mice treated with vehicle ( control ) , and 2mg and 5mg of tamoxifen , scale bar 50 μm . ( G ) Quantification of LOX - L2 for images in F . n ≥ 4 mice per condition , and n > 10 sections per

O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O

(F) Immunofluorescence images of PDAC tissues from KPC mice treated with vehicle (control), and 2mg and 5mg of tamoxifen, scale bar 50 μm. (G) Quantification of LOX-L2 for images in F. n ≥ 4 mice per condition, and n> 10 sections per animal


( H ) qPCR levels of LOX - L2 and HIF - 1A in PSCs , normalized to RPLP0 and relative to 1

O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O

(H) qPCR levels of LOX-L2 and HIF-1A in PSCs, normalized to RPLP0 and relative to 1 kPa


( I ) qPCR levels of LOX - L2 and HIF - 1A in PSCs , normalized to RPLP0 and relative to

O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O

(I) qPCR levels of LOX-L2 and HIF-1A in PSCs, normalized to RPLP0 and relative to control