Figure 2A , B : Western blot showing concentration dependent increase in γH2AX following 8 - hour exposure to etoposide in BT12 and G401 RT cells ( A ) . Red bar indicates the measured IC50 ( Figure 1C ) . Despite induction of DNA damage , 15 μM etoposide does not lead to apoptosis as indicated by live cell imaging in the presence of cleaved caspase 3 , 7 ( CC3 , 7 ) reagent that fluoresces with caspase activation ( B ) . Scale bar ( lower left ) : 150μm . C , D : Western blot showing concentration dependent increase in γH2AX following 8 - hour exposure to mithramycin ( MMA ) in BT12 and G401 RT cells ( C ) . Red bar indicates the measured IC50 ( Figure 1C ) . 100 nM Mithramycin induces apoptosis at 8 - hours as measured by CC3 , 7 fluorescence without the presence of DNA damage ( D ) . Scale bar ( lower left ) : 150μm . E : BT12 cells treated with 20nM MMA show a different cellular phenotype than with 100nM . Beyond 24h of exposure , there is evidence of mesenchymal differentiation and the appearance of maturing adipocytes . Scale bar ( lower left ) : 150μm . F , G : Western blot showing concentration dependent increase in γH2AX following 8 - hour exposure to EC8042 in BT12 and G401 RT cells . Red bar indicates the measured IC50 ( Figure 1C ) . In contrast to apoptotic induction , 75nM EC8042 exhibits evidence of mesenchymal differentiation , similar to 20nM MMA ( E , G ) . Please note , solvent control for ( E ) and ( G ) is the same although different fields are shown . Scale bar ( lower left ) : 150μm . H : Mesenchymal differentiation confirmed with oil red O staining of lipid deposits following 20nM MMA or 75nM EC8042 treatment for 24 and 48 - hours . Please note , solvent control for 20nM MMA and 75nM EC8042 is the same . Scale bar ( lower left ) : 150μm . I : PPARγ mRNA expression is induced following 20nM mithramycin treatment as measured by qPCR fold - change relative to GAPDH ( 2ddCT ) . * * , P = 0 . 002 , * * * * , P = 0 . 0001 . Data represents mean with standard deviation derived from three independent experiments . P - values were determined by one - way ANOVA using Dunnet test for multiple comparisons .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A, B: Western blot showing concentration dependent increase in γH2AX following 8-hour exposure to etoposide in BT12 and G401 RT cells (A). Red bar indicates the measured IC50 (Figure 1C). Despite induction of DNA damage, 15 μM etoposide does not lead to apoptosis as indicated by live cell imaging in the presence of cleaved caspase 3,7 (CC3,7) reagent that fluoresces with caspase activation (B). Scale bar (lower left): 150μm.  C, D: Western blot showing concentration dependent increase in γH2AX following 8-hour exposure to mithramycin (MMA) in BT12 and G401 RT cells (C). Red bar indicates the measured IC50 (Figure 1C). 100 nM Mithramycin induces apoptosis at 8-hours as measured by CC3,7 fluorescence without the presence of DNA damage (D). Scale bar (lower left): 150μm.  E: BT12 cells treated with 20nM MMA show a different cellular phenotype than with 100nM. Beyond 24h of exposure, there is evidence of mesenchymal differentiation and the appearance of maturing adipocytes. Scale bar (lower left): 150μm.  F, G: Western blot showing concentration dependent increase in γH2AX following 8-hour exposure to EC8042 in BT12 and G401 RT cells. Red bar indicates the measured IC50 (Figure 1C). In contrast to apoptotic induction, 75nM EC8042 exhibits evidence of mesenchymal differentiation, similar to 20nM MMA (E,G). Please note, solvent control for (E) and (G) is the same although different fields are shown. Scale bar (lower left): 150μm.  H: Mesenchymal differentiation confirmed with oil red O staining of lipid deposits following 20nM MMA or 75nM EC8042 treatment for 24 and 48-hours. Please note, solvent control for 20nM MMA and 75nM EC8042 is the same. Scale bar (lower left): 150μm.  I: PPARγ mRNA expression is induced following 20nM mithramycin treatment as measured by qPCR fold-change relative to GAPDH (2ddCT). **, P = 0.002, ****, P = 0.0001. Data represents mean with standard deviation derived from three independent experiments. P-values were determined by one-way ANOVA using Dunnet test for multiple comparisons.


Figure 3 A , B : Mithramycin displaces SMARCC1 and SMARCE1 SWI / SNF subunits from chromatin in a time - dependent manner in BT12 rhabdoid tumor ( A ) but not U20S osteosarcoma ( B ) cells . Western blot analysis showing whole cell lysate ( Total ) , cytoplasmic soluble ( CS ) , nuclear soluble ( NS ) , and chromatin bound ( Chr ) fractions collected after exposure to solvent ( S ) or 100nM mithramycin for 8 or 18h and probed for the SWI / SNF subunits ( BRD9 , SMARCC1 or SMARCE1 ) or H3 ( chromatin fraction control ) and GAPDH ( soluble fraction control ) . C : SMARCC1 and SMARCE1 mRNA expression does not change following 100nM mithramycin treatment as measured by qPCR fold - change relative to GAPDH ( 2ddCT ) . Data represents mean with standard deviation derived from three independent experiments . D : Addition of the proteasome inhibitor ( bortezomib ) rescues the loss of protein expression following 8 or 18 - hour mithramycin treatment . BT12 cells were treated for 8 or 18 - hours with solvent ( S ) , mithramycin ( M , 100nM ) , bortezomib ( PI , 2 . 5μM ) , or combination of 100nM mithramycin and 2 . 5μM bortezomib ( C ) . E : Loss of SWI / SNF occupancy at defined loci in the genome as measure by chromatin immunoprecipitation qPCR ( ChIP - qPCR ) at previously described SWI / SNF target genes , MYT1 ( 8 - hour , P = 0 . 0001 ; 18 - hour P = 0 . 0001 ) and CCND1 ( 8 - hour , P = 0 . 0001 ; 18 - hour P = 0 . 0001 ) . ChIP quantitation is percent input ( ng of immunoprecipitated DNA / input DNA * 100 ) determined by absolute quantitation of sheared chromatin relative to a standard curve . Data represents mean with standard deviation derived from three independent experiments . P - values were determined by one - way ANOVA using Dunnet test for multiple comparisons . F : Chromatin immunoprecipitation qPCR ( ChIP - qPCR ) of H3K27me3 at MYT1 ( 8 - hour , P = 0 . 0001 ; 18 - hour P = 0 . 0001 ) and CCND1 ( 8 - hour , P = 0 . 02 ; 18 - hour P = 0 . 0001 ) . H3K27me3 occupancy is increased in a time - dependent manner . ChIP quantitation is percent input ( ng of immunoprecipitated DNA / input DNA * 100 ) determined by absolute quantitation of sheared chromatin relative to a standard curve . Data represents mean with standard deviation derived from three independent experiments . P - values were determined by one - way ANOVA using Dunnet test for multiple comparisons . Western blot showing concentration - dependent increase in H3K27me3 following exposure to 100nM , 50nM , 25nM mithramycin for 18h in BT12 cells relative to loading control ( H3 ) . Western blot showing concentration - dependent increase in H3K27me3 following exposure to 100nM , 50nM , 25nM mithramycin for 18h in G401 cells relative to loading control ( H3 ) . Re - expression of SMARCB1 following doxycycline treatment inhibits the mithramycin - dependent effects on H3K27me3 amplification

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3 A, B: Mithramycin displaces SMARCC1 and SMARCE1 SWI/SNF subunits from chromatin in a time-dependent manner in BT12 rhabdoid tumor (A) but not U20S osteosarcoma (B) cells. Western blot analysis showing whole cell lysate (Total), cytoplasmic soluble (CS), nuclear soluble (NS), and chromatin bound (Chr) fractions collected after exposure to solvent (S) or 100nM mithramycin for 8 or 18h and probed for the SWI/SNF subunits (BRD9, SMARCC1 or SMARCE1) or H3 (chromatin fraction control) and GAPDH (soluble fraction control). C: SMARCC1 and SMARCE1 mRNA expression does not change following 100nM mithramycin treatment as measured by qPCR fold-change relative to GAPDH (2ddCT). Data represents mean with standard deviation derived from three independent experiments. D: Addition of the proteasome inhibitor (bortezomib) rescues the loss of protein expression following 8 or 18-hour mithramycin treatment. BT12 cells were treated for 8 or 18-hours with solvent (S), mithramycin (M, 100nM), bortezomib (PI, 2.5μM), or combination of 100nM mithramycin and 2.5μM bortezomib (C).  E: Loss of SWI/SNF occupancy at defined loci in the genome as measure by chromatin immunoprecipitation qPCR (ChIP-qPCR) at previously described SWI/SNF target genes, MYT1 (8-hour, P=0.0001; 18-hour P=0.0001) and CCND1 (8-hour, P=0.0001; 18-hour P=0.0001). ChIP quantitation is percent input (ng of immunoprecipitated DNA/input DNA *100) determined by absolute quantitation of sheared chromatin relative to a standard curve. Data represents mean with standard deviation derived from three independent experiments. P-values were determined by one-way ANOVA using Dunnet test for multiple comparisons.  F: Chromatin immunoprecipitation qPCR (ChIP-qPCR) of H3K27me3 at MYT1 (8-hour, P= 0.0001; 18-hour P=0.0001) and CCND1 (8-hour, P=0.02; 18-hour P=0.0001). H3K27me3 occupancy is increased in a time-dependent manner. ChIP quantitation is percent input (ng of immunoprecipitated DNA/input DNA *100) determined by absolute quantitation of sheared chromatin relative to a standard curve. Data represents mean with standard deviation derived from three independent experiments. P-values were determined by one-way ANOVA using Dunnet test for multiple comparisons. Western blot showing concentration-dependent increase in H3K27me3 following exposure to 100nM, 50nM, 25nM mithramycin for 18h in BT12 cells relative to loading control (H3). Western blot showing concentration-dependent increase in H3K27me3 following exposure to 100nM, 50nM, 25nM mithramycin for 18h in G401 cells relative to loading control (H3). Re-expression of SMARCB1 following doxycycline treatment inhibits the mithramycin-dependent effects on H3K27me3 amplification


Figure 4 A , B : Mithramycin displaces BRD9 and SMARCE1 SWI / SNF subunits from chromatin in a time - dependent manner in G401 ( A ) but only BRD9 with SMARCB1 re - expression in G401 ( B ) cells . Western blot analysis showing whole cell lysate ( Total ) , cytoplasmic soluble ( CS ) , nuclear soluble ( NS ) , and chromatin bound ( Chr ) fractions collected after exposure to solvent ( S ) or 100nM mithramycin for 8 or 18h and probed for the SWI / SNF subunits ( BRD9 , SMARCB1 or SMARCE1 ) or H3 ( chromatin fraction control ) and GAPDH ( soluble fraction control ) . C : BT12 cells show a threshold of exposure that leads to irreversible growth inhibition . The cells were exposed to 100nM MMA for the indicated times followed by replacement with drug - free medium . Beyond 8h ( red ) of mithramycin exposure , the cells do not recover proliferative potential and exhibit a phenotype consistent with cell death . Data represents mean with standard deviation derived from three independent experiments . D : Mithramycin leads to H3K27me3 amplification in a time - dependent manner that precedes the induction of apoptosis as measured by the cleavage of PARP . Western blot lysates collected at 2h , 4h , 6h , 8h , 12h , 18h , 24h of continuous 100nM mithramycin treatment . E : Suppression of EZH2 expression and activity antagonizes mithramycin activity in BT12 rhabdoid tumor cells . Data represents dose response curves of mithramycin in BT12 cells following a 48h exposure in the presence of siRNA silencing of the PRC2 subunit EZH2 or treatment with the EZH2 inhibitor EPZ - 6438 relative to mithramycin alone ( media ) or a non - targeting siRNA ( siNeg ) . Data represents mean with standard deviation derived from three independent experiments . F : Suppression of SMARCB1 sensitizes U2OS osteosarcoma cells ( wild type SWI / SNF ) to mithramycin . Data represents dose response curves of mithramycin in BT12 cells following a 48h exposure in the presence of siRNA silencing of the SWI / SNF subunit SMARCB1 relative to a non - targeting siRNA ( siNeg ) . Data represents mean with standard deviation derived from three independent experiments . G . Western blot showing concentration - dependent increase in H3K27me3 following exposure to 100nM , 50nM , 25nM mithramycin for 18h in SMARCB1 - silenced U2OS cells relative to loading control ( H3 ) . Knockdown of SMARCB1 following siRNA suppression triggers mithramycin - dependent H3K27me3 amplification , while siNegative ( control ) does not have an effect on H3K27me3 following mithramycin exposure .

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

Figure 4 A, B: Mithramycin displaces BRD9 and SMARCE1 SWI/SNF subunits from chromatin in a time-dependent manner in G401 (A) but only BRD9 with SMARCB1 re-expression in G401 (B) cells. Western blot analysis showing whole cell lysate (Total), cytoplasmic soluble (CS), nuclear soluble (NS), and chromatin bound (Chr) fractions collected after exposure to solvent (S) or 100nM mithramycin for 8 or 18h and probed for the SWI/SNF subunits (BRD9, SMARCB1 or SMARCE1) or H3 (chromatin fraction control) and GAPDH (soluble fraction control).  C: BT12 cells show a threshold of exposure that leads to irreversible growth inhibition. The cells were exposed to 100nM MMA for the indicated times followed by replacement with drug-free medium. Beyond 8h (red) of mithramycin exposure, the cells do not recover proliferative potential and exhibit a phenotype consistent with cell death. Data represents mean with standard deviation derived from three independent experiments.  D: Mithramycin leads to H3K27me3 amplification in a time-dependent manner that precedes the induction of apoptosis as measured by the cleavage of PARP. Western blot lysates collected at 2h, 4h, 6h, 8h, 12h, 18h, 24h of continuous 100nM mithramycin treatment.  E: Suppression of EZH2 expression and activity antagonizes mithramycin activity in BT12 rhabdoid tumor cells. Data represents dose response curves of mithramycin in BT12 cells following a 48h exposure in the presence of siRNA silencing of the PRC2 subunit EZH2 or treatment with the EZH2 inhibitor EPZ-6438 relative to mithramycin alone (media) or a non-targeting siRNA (siNeg). Data represents mean with standard deviation derived from three independent experiments.  F: Suppression of SMARCB1 sensitizes U2OS osteosarcoma cells (wild type SWI/SNF) to mithramycin. Data represents dose response curves of mithramycin in BT12 cells following a 48h exposure in the presence of siRNA silencing of the SWI/SNF subunit SMARCB1 relative to a non-targeting siRNA (siNeg). Data represents mean with standard deviation derived from three independent experiments.  G. Western blot showing concentration-dependent increase in H3K27me3 following exposure to 100nM, 50nM, 25nM mithramycin for 18h in SMARCB1- silenced U2OS cells relative to loading control (H3). Knockdown of SMARCB1 following siRNA suppression triggers mithramycin-dependent H3K27me3 amplification, while siNegative (control) does not have an effect on H3K27me3 following mithramycin exposure.


Figure 5A - C : Volcano plot showing gene expression trends at 8h MMA treatment . Dashed lines represent a 2 log2FC ( drug over control ) ( A ) or 1 . 5 log2FC ( drug over control ) ( B ) and 10e - 7 q - value threshold . Quantification of induced and repressed genes in ( C ) . Labels indicate names of genes that meet the logFC and q - value threshold . D - F : Volcano plot showing gene expression trends at 18h MMA treatment . Dashed lines represent a 2 log2FC ( drug over control ) ( D ) or 1 . 5 log2FC ( drug over control ) ( E ) and 10e - 7 q - value threshold . Quantification of induced and repressed genes in ( F ) . Labels indicate names of genes that meet the logFC and q - value threshold . G : SP1 protein expression is reduced following mithramycin exposure . Western blot showing suppression of SP1 expression compared to loading control ( GAPDH ) after 100nM mithramycin exposure for 1h , 4h , 8h , 12h , 18h . The loss of SP1 expression is associated with a decrease in SWI / SNF occupancy of the SP1 promoter and an increase in H3K27me3 . Data represents ChIP - qPCR analysis following 8 - hours or 18 - hours of 100nM mithramycin exposure and immunoprecipitation of SMARCC1 ( 8 - hour , P = 0 . 0006 ; 18 - hour P = 0 . 0001 ) . ChIP quantitation is percent input ( ng of immunoprecipitated DNA / input DNA * 100 ) determined by absolute quantitation of sheared chromatin relative to a standard curve . Data represents mean with standard deviation derived from three independent experiments . P - values were determined by one - way ANOVA using Dunnet test for multiple comparisons . I : The loss of SP1 expression is associated with a decrease in SWI / SNF occupancy of the SP1 promoter and an increase in H3K27me3 . Data represents ChIP - qPCR analysis following 8 - hours or 18 - hours of 100nM mithramycin exposure and immunoprecipitation of H3K27me3 ( 8 - hour , P = 0 . 009 ; 18 - hour P = 0 . 0001 ) ChIP quantitation is percent input ( ng of immunoprecipitated DNA / input DNA * 100 ) determined by absolute quantitation of sheared chromatin relative to a standard curve . Data represents mean with standard deviation derived from three independent experiments . P - values were determined by one - way ANOVA using Dunnet test for multiple comparisons .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A-C: Volcano plot showing gene expression trends at 8h MMA treatment. Dashed lines represent a 2 log2FC (drug over control) (A) or 1.5 log2FC (drug over control) (B) and 10e-7 q-value threshold. Quantification of induced and repressed genes in (C). Labels indicate names of genes that meet the logFC and q-value threshold.D-F: Volcano plot showing gene expression trends at 18h MMA treatment. Dashed lines represent a 2 log2FC (drug over control) (D) or 1.5 log2FC (drug over control) (E) and 10e-7 q-value threshold. Quantification of induced and repressed genes in (F). Labels indicate names of genes that meet the logFC and q-value threshold. G: SP1 protein expression is reduced following mithramycin exposure. Western blot showing suppression of SP1 expression compared to loading control (GAPDH) after 100nM mithramycin exposure for 1h, 4h, 8h, 12h, 18h.  The loss of SP1 expression is associated with a decrease in SWI/SNF occupancy of the SP1 promoter and an increase in H3K27me3. Data represents ChIP-qPCR analysis following 8-hours or 18-hours of 100nM mithramycin exposure and immunoprecipitation of SMARCC1 (8-hour, P= 0.0006; 18-hour P=0.0001) . ChIP quantitation is percent input (ng of immunoprecipitated DNA/input DNA *100) determined by absolute quantitation of sheared chromatin relative to a standard curve. Data represents mean with standard deviation derived from three independent experiments. P-values were determined by one-way ANOVA using Dunnet test for multiple comparisons.  I: The loss of SP1 expression is associated with a decrease in SWI/SNF occupancy of the SP1 promoter and an increase in H3K27me3. Data represents ChIP-qPCR analysis following 8-hours or 18-hours of 100nM mithramycin exposure and immunoprecipitation of H3K27me3 (8-hour, P= 0.009; 18-hour P=0.0001) ChIP quantitation is percent input (ng of immunoprecipitated DNA/input DNA *100) determined by absolute quantitation of sheared chromatin relative to a standard curve. Data represents mean with standard deviation derived from three independent experiments. P-values were determined by one-way ANOVA using Dunnet test for multiple comparisons.


Figure 6A : Heatmaps depicting ATAC - seq global chromatin structure following 8 - hours ( middle ) and 18 - hours ( right ) 100nM MMA treatment . A 2kb window is centered on the TSS . Chromatin accessibility clusters at the TSS and does not change globally relative to solvent . Quantification of genes that gain accessibility or reduce accessibility on the right . E : IGV tracks of rhabdoid tumor genes previously identified to be occupied by non - canonical SWI / SNF ( Michel et al . , 2018 ) . ID3 and JUND3 decrease in H3K27ac occupancy and chromatin accessibility following exposure to mithramycin for 8 - hours or 18 - hours . F : IGV tracks of rhabdoid tumor genes previously identified to gain H3K27me3 upon SWI / SNF loss ( Erkek et al . , 2019 ) . CDK6 and CDK2 decrease in H3K27ac occupancy and chromatin accessibility following exposure to 8 - hours and 18 - hours mithramycin treatment . G : IGV tracks of genes identified to have an increase in accessibility , H3K27ac and gene expression following mithramycin exposure . BMP1 and ADIPOR1 play crucial roles in bone and adipogenic differentiation , respectively .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A: Heatmaps depicting ATAC-seq global chromatin structure following 8-hours (middle) and 18-hours (right) 100nM MMA treatment. A 2kb window is centered on the TSS. Chromatin accessibility clusters at the TSS and does not change globally relative to solvent. Quantification of genes that gain accessibility or reduce accessibility on the right. E: IGV tracks of rhabdoid tumor genes previously identified to be occupied by non-canonical SWI/SNF (Michel et al., 2018). ID3 and JUND3 decrease in H3K27ac occupancy and chromatin accessibility following exposure to mithramycin for 8-hours or 18-hours.  F: IGV tracks of rhabdoid tumor genes previously identified to gain H3K27me3 upon SWI/SNF loss (Erkek et al., 2019). CDK6 and CDK2 decrease in H3K27ac occupancy and chromatin accessibility following exposure to 8-hours and 18-hours mithramycin treatment.  G: IGV tracks of genes identified to have an increase in accessibility, H3K27ac and gene expression following mithramycin exposure. BMP1 and ADIPOR1 play crucial roles in bone and adipogenic differentiation, respectively.


Figure 7 A : Immunohistochemistry analysis recapitulates the biochemistry described in vitro for mithramycin . G401 tumor sections at 20X magnification stained with H & E , cleaved caspase 3 ( CC3 ; apoptosis ) or H3K27me3 . A marked increase in CC3 that correlates with H3K27me3 staining is seen only in mice treated with the continuous infusion schedule but not vehicle . Scale bar ( lower left ) : 50μm . B : Prolonged durable response and cure of mice bearing G401 xenografts treated with 30mg / kg EC8042 administered continuously over 72 - hours . Treatment duration indicated by gray shaded box . Asterisk indicate an animal sacrificed due to unknown causes not related to tumor progression or drug toxicity ( see text ) . C : Kaplan - Meier survival curves indicating extended survival for mice bearing established G401 xenografts treated with the 3 - day continuous infusions of EC8042 in ( B ) . The shaded box indicates the duration of treatment . Asterisk indicate an animal sacrificed due to unknown causes not related to tumor progression or drug toxicity ( see text ) . D : 20X image of section of G401 treated tumors stained for H3K27me3 , cleaved caspase 3 , and γH2AX . The sections compare vehicle to treatment started on day 1 with 30 mg / kg of EC8042 administered continuously for 72 - hours ( 3 - day pump ) . H3K27me3 increases and correlates with apoptosis ( CC3 ) , however induction of CC3 is modest . γH2AX staining does not increase with treatment indicating DNA damage is not responsible for these effects . Scale bar ( lower left ) : 50μm .

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

Figure 7 A: Immunohistochemistry analysis recapitulates the biochemistry described in vitro for mithramycin. G401 tumor sections at 20X magnification stained with H&E, cleaved caspase 3 (CC3; apoptosis) or H3K27me3. A marked increase in CC3 that correlates with H3K27me3 staining is seen only in mice treated with the continuous infusion schedule but not vehicle. Scale bar (lower left): 50μm.  B: Prolonged durable response and cure of mice bearing G401 xenografts treated with 30mg/kg EC8042 administered continuously over 72-hours. Treatment duration indicated by gray shaded box. Asterisk indicate an animal sacrificed due to unknown causes not related to tumor progression or drug toxicity (see text).  C: Kaplan-Meier survival curves indicating extended survival for mice bearing established G401 xenografts treated with the 3-day continuous infusions of EC8042 in (B). The shaded box indicates the duration of treatment. Asterisk indicate an animal sacrificed due to unknown causes not related to tumor progression or drug toxicity (see text).  D: 20X image of section of G401 treated tumors stained for H3K27me3, cleaved caspase 3, and γH2AX. The sections compare vehicle to treatment started on day 1 with 30 mg/kg of EC8042 administered continuously for 72-hours (3-day pump). H3K27me3 increases and correlates with apoptosis (CC3), however induction of CC3 is modest. γH2AX staining does not increase with treatment indicating DNA damage is not responsible for these effects. Scale bar (lower left): 50μm.


Figure 8 Immunohistochemistry analysis of H & E stains from G401 xenograft tumors on 1 , 3 , and 7 - days after treatment with vehicle or 3 - day EC8042 infusion . EC8042 treated xenograft tumors exhibit evidence of mesenchymal differentiation compared to vehicle . microCT analysis of xenograft tumors on 7 - days after treatment exhibit enhanced calcification compared to vehicle . IHC scale bar ( lower left ) : 100μm .

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Figure 8 Immunohistochemistry analysis of H&E stains from G401 xenograft tumors on 1, 3, and 7-days after treatment with vehicle or 3-day EC8042 infusion. EC8042 treated xenograft tumors exhibit evidence of mesenchymal differentiation compared to vehicle. microCT analysis of xenograft tumors on 7-days after treatment exhibit enhanced calcification compared to vehicle. IHC scale bar (lower left): 100μm.


Figure 1LL - 37 ( 4 kD ) detection by Western blotting . Prior to loading , samples were centrifuged through 10 kD ultrafiltration membranes and the equal amount of ultrafiltrate ( 19μg / lane ) subject to immunoblotting . High - molecular - weight proteins ( GAPDH ) were not detected in ultrafiltrate . The CFU of PAO1 was measured by culturing in ultrafiltrate ( upper panel ) or retentate ( lower panel ) of mouse BALF samples from indicated mice . Initial additions of PAO1 were 1 × 103 CFU . Coculture duration , 6h . n = 3 . The bacterial CFU ( per gram ) in lungs of indicated mice with and without PAO1 infection ( 5 × 106 CFU ) for 6 , 24 and 48 hours . n = 3 . Error bars , S . E . M . Representative histological sections of indicated lungs with PAO1 infection ( 5 × 106 CFU ) for 6 hours . H & E staining . Scale bar , 1000 μm ( upper panel ) and 50 μm ( lower panel ) . Histopathological injury score of indicated mouse lungs with PAO1 infection ( 5 × 106 CFU ) for 6 h and 24 h based on blinded expert judgement . n = 3 . Error bars , S . E . M . Gene expression level of IL - 1β and IL - 6 of indicated mouse lung with PAO1 infection ( 5 × 106 CFU ) for 6 h and 24 h . n ≥ 3 . Error bars , S . E . M . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ; * * * * , P < 0 . 0001 .

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Figure 1LL-37(4 kD) detection by Western blotting. Prior to loading, samples were centrifuged through 10 kD ultrafiltration membranes and the equal amount of ultrafiltrate (19μg/lane) subject to immunoblotting. High-molecular-weight proteins (GAPDH) were not detected in ultrafiltrate.The CFU of PAO1 was measured by culturing in ultrafiltrate (upper panel) or retentate (lower panel) of mouse BALF samples from indicated mice. Initial additions of PAO1 were 1 × 103 CFU. Coculture duration, 6h. n=3.The bacterial CFU (per gram) in lungs of indicated mice with and without PAO1 infection (5 × 106 CFU) for 6, 24 and 48 hours. n=3. Error bars, S.E.M.Representative histological sections of indicated lungs with PAO1 infection (5 × 106 CFU) for 6 hours. H&E staining. Scale bar, 1000 μm (upper panel) and 50 μm (lower panel).Histopathological injury score of indicated mouse lungs with PAO1 infection (5 × 106 CFU) for 6 h and 24 h based on blinded expert judgement. n=3. Error bars, S.E.M.Gene expression level of IL-1β and IL-6 of indicated mouse lung with PAO1 infection (5 × 106 CFU) for 6 h and 24 h. n≥3. Error bars, S.E.M. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.


Figure 2Heatmap showing transcriptome profile correlation values of indicated lung tissue samples before and after PAO1 infection . Histogram of selected differentially expressed genes of LL - 37 + / + mouse lung versus wild type FVB mouse lung prior to infection . Blue bars indicated genes up - regulated in wild type FVB mouse lungs , red bars indicated genes up - regulated in LL - 37 + / + mouse lungs .

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Figure 2Heatmap showing transcriptome profile correlation values of indicated lung tissue samples before and after PAO1 infection.Histogram of selected differentially expressed genes of LL-37+/+ mouse lung versus wild type FVB mouse lung prior to infection. Blue bars indicated genes up-regulated in wild type FVB mouse lungs, red bars indicated genes up-regulated in LL-37+/+ mouse lungs.


Figure 3Detection of LL - 37 expression in the engineered mDASCs by immunofluorescenceDetection of LL - 37 expression in the engineered mDASCs by real - time quantitative PCRDetection of LL - 37 expression in the engineered mDASCs by Western blotAnti - Krt5 ( red ) and anti - P63 ( green ) immunostaining of WT - and LL - 37 - mDASC colonies . Scale bar , 70 μm . Stem cell colony - forming efficiency of WT - and LL - 37 - mDASCs during 5 serial passages . n = 6 . Representative 3D organoid culture of mDASCs with expression of type I alveolar cell markers ( Aqp5 and Pdpn ) . Left panels , bright field imaging of 3D organoids . Right panels , immunofluorescence of organoid sections . Scale bar , 20 μm . Co - culture of bacteria with DASC shows the antimicrobial effects in dose - dependent manner . Initial additions of PAO1 were 0 . 1 × , 0 . 5 × and 1 × 104 CFU , respectively . Coculture duration , 6h . n = 4 . Error bars , S . E . M . MOI , Multiplicity of infection . Co - culture of bacteria with DASC shows antimicrobial effects in time - dependent manner . Initial additions of PAO1 was 1 × 104 CFU . MOI = 1 . n = 3 . Error bars , S . E . M . Preincubation of cells with anti - LL - 37 antibody , but not IgG control , significantly reduced anti - PAO1 and anti - E . coli ( J ) effects of LL - 37 - mDASCs . Initial dose of bacteria was 103 CFU .

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Figure 3Detection of LL-37 expression in the engineered mDASCs by immunofluorescenceDetection of LL-37 expression in the engineered mDASCs by real-time quantitative PCRDetection of LL-37 expression in the engineered mDASCs by Western blotAnti-Krt5 (red) and anti-P63 (green) immunostaining of WT- and LL-37-mDASC colonies. Scale bar, 70 μm.Stem cell colony-forming efficiency of WT- and LL-37-mDASCs during 5 serial passages. n=6.Representative 3D organoid culture of mDASCs with expression of type I alveolar cell markers (Aqp5 and Pdpn). Left panels, bright field imaging of 3D organoids. Right panels, immunofluorescence of organoid sections. Scale bar, 20 μm.Co-culture of bacteria with DASC shows the antimicrobial effects in dose-dependent manner. Initial additions of PAO1 were 0.1×, 0.5× and 1 × 104 CFU, respectively. Coculture duration, 6h. n=4. Error bars, S.E.M. MOI, Multiplicity of infection.Co-culture of bacteria with DASC shows antimicrobial effects in time-dependent manner. Initial additions of PAO1 was 1 × 104 CFU. MOI=1. n=3. Error bars, S.E.M.Preincubation of cells with anti-LL-37 antibody, but not IgG control, significantly reduced anti-PAO1 and anti-E.coli (J) effects of LL-37-mDASCs. Initial dose of bacteria was 103 CFU.


Figure 4Bright field and direct fluorescence images of mouse lungs following transplantation of 1 × 106 GFP - labeled WT - mDASCs ( WT - Lung ) or LL - 37 - mDASCs ( LL - 37 - Lung ) on indicated days . Scale bar , 1000 μmEngraftment ratio of transplanted cells in mouse lungs on indicated days . n = 3 . Error bars , S . E . M . * P = 0 . 0474 . Morphology of engrafted GFP - labeled cells in lung parenchyma by direct fluorescence . Blue color indicates nucleus DAPI staining . Scale bar , 200 μm . Anti - Ki67 immunofluorescence of engrafted GFP - labeled WT - and LL - 37 - mDASCs in lung parenchyma 21 days after transplantation . Scale bar , 50 μm .

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Figure 4Bright field and direct fluorescence images of mouse lungs following transplantation of 1 × 106 GFP-labeled WT-mDASCs (WT-Lung) or LL-37-mDASCs (LL-37-Lung) on indicated days. Scale bar, 1000 μmEngraftment ratio of transplanted cells in mouse lungs on indicated days. n=3. Error bars, S.E.M. *P=0.0474.Morphology of engrafted GFP-labeled cells in lung parenchyma by direct fluorescence. Blue color indicates nucleus DAPI staining. Scale bar, 200 μm.Anti-Ki67 immunofluorescence of engrafted GFP-labeled WT- and LL-37-mDASCs in lung parenchyma 21 days after transplantation. Scale bar, 50 μm.


Figure 5Distribution of engrafted GFP - labeled cells in lung parenchyma by immunofluorescence 7 days after transplantation . WT - Lung : WT - mDASCs engrafted ; LL - 37 - Lung : LL - 37 - mDASCs engrafted . Scale bar , 200 μm . Arrows showed the representative cells with overlapping fluorescence of GFP and LL - 37 . Intratracheal instillation of equal amount of PAO1 ( 5 × 106 CFU per mouse ) into WT - Lung ( WT - mDASCs engrafted ) and LL - 37 - Lung ( LL - 37 - mDASCs engrafted ) followed by bacterial CFU analysis in whole lung homogenates 6 , 24 and 48 hours after infection . n = 3 . Error bars , S . E . M . Intratracheal instillation of equal amount of PAO1 into WT - Lung and LL - 37 - Lung followed by bacterial CFU analysis in BAL fluid 6 , 24 and 48 hours after infection . n = 3 . Error bars , S . E . M . Arterial blood gas analysis of mice with WT - Lung and LL - 37 - Lung following PAO1 infection . n = 6 . Error bars , S . E . M . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ; * * * * , P < 0 . 0001 .

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Figure 5Distribution of engrafted GFP-labeled cells in lung parenchyma by immunofluorescence 7 days after transplantation. WT-Lung: WT-mDASCs engrafted; LL-37-Lung: LL-37-mDASCs engrafted. Scale bar, 200 μm. Arrows showed the representative cells with overlapping fluorescence of GFP and LL-37.Intratracheal instillation of equal amount of PAO1(5 ×106 CFU per mouse) into WT-Lung (WT-mDASCs engrafted) and LL-37-Lung (LL-37-mDASCs engrafted) followed by bacterial CFU analysis in whole lung homogenates 6, 24 and 48 hours after infection. n=3. Error bars, S.E.M. Intratracheal instillation of equal amount of PAO1 into WT-Lung and LL-37-Lung followed by bacterial CFU analysis in BAL fluid 6, 24 and 48 hours after infection. n=3. Error bars, S.E.M.Arterial blood gas analysis of mice with WT-Lung and LL-37-Lung following PAO1 infection. n=6. Error bars, S.E.M. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.


Figure 6H & E staining showing histology of injured lung infected by PAO1 with WT - mDASCs or LL - 37 - mDASCs transplantation . Scale bar , 100 μm . Histopathological examination according to the lung injury scoring system based on blinded expert judgement . n = 5 mice per group . Error bars , S . E . M . CD68 immunochemistry ( brown ) in infected lung with WT - mDASCs or LL - 37 - mDASCs transplantation . Scale bar , 50 μm . Quantification of brown - stained ( CD68 + ) area by Image J software . n = 5 . Error bars , S . E . M . Gene expression level of indicated pro - inflammatory cytokines of lung infected by PAO1 with WT - mDASCs or LL - 37 - mDASCs transplantation . n = 3 . Error bars , S . E . M . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ; * * * * , P < 0 . 0001 .

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Figure 6H&E staining showing histology of injured lung infected by PAO1 with WT-mDASCs or LL-37-mDASCs transplantation. Scale bar, 100 μm. Histopathological examination according to the lung injury scoring system based on blinded expert judgement. n=5 mice per group. Error bars, S.E.M.CD68 immunochemistry (brown) in infected lung with WT-mDASCs or LL-37-mDASCs transplantation. Scale bar, 50 μm. Quantification of brown-stained (CD68+) area by Image J software. n=5. Error bars, S.E.M.Gene expression level of indicated pro-inflammatory cytokines of lung infected by PAO1 with WT-mDASCs or LL-37-mDASCs transplantation. n=3. Error bars, S.E.M. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.


Figure 7Anti - Krt5 ( red ) and anti - P63 ( green ) immunofluorescence of isolated hDASCs . Scale bar , 90 μm . The mRNA expression levels of LL - 37 was measured by qPCR for WT - and LL - 37 - hDASCs . n = 5 . Error bars , S . E . M . H & E staining of native rat lung tissue and decellularized scaffold showed the absence of nuclei following decellularization . Scale bar , 40 μm . Ex vivo biomimetic culture of LL - 37 - hDASCs recellularized lungs with constant media perfusion . Direct fluorescence image showing GFP - labeled LL - 37 - hDASCs before ( left ) and 7 days after ( right ) being engrafted onto the decellularized scaffold . Scale bar , 100 μm ( right panel ) and 1000 μm ( right panel ) Immunofluorescence of recellularized lung with indicated antibodies . Blue color indicates nucleus DAPI staining . Scale bar , 50 μm . The recellularized lung by LL - 37 - hDASCs displayed growth inhibitory effect on PAO1 and E . coli . Initial dose of bacteria was 2 × 104 CFU . Culture duration , 24h . n = 6 . Error bars , S . E . M . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ; * * * * , P < 0 . 0001 .

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Figure 7Anti-Krt5 (red) and anti-P63 (green) immunofluorescence of isolated hDASCs. Scale bar, 90 μm.The mRNA expression levels of LL-37 was measured by qPCR for WT- and LL-37-hDASCs. n=5. Error bars, S.E.M.H&E staining of native rat lung tissue and decellularized scaffold showed the absence of nuclei following decellularization. Scale bar, 40 μm.Ex vivo biomimetic culture of LL-37-hDASCs recellularized lungs with constant media perfusion.Direct fluorescence image showing GFP-labeled LL-37-hDASCs before (left) and 7 days after (right) being engrafted onto the decellularized scaffold. Scale bar, 100 μm (right panel) and 1000 μm (right panel)Immunofluorescence of recellularized lung with indicated antibodies. Blue color indicates nucleus DAPI staining. Scale bar, 50 μm.The recellularized lung by LL-37-hDASCs displayed growth inhibitory effect on PAO1 and E.coli. Initial dose of bacteria was 2× 104 CFU. Culture duration, 24h. n=6. Error bars, S.E.M. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.


Figure 1A , B . A missense mutation in SPOP gene ( Q165P , T > G ) was identified in a PCa patient . Exome sequencing revealed that the primary tumor contained a heterozygous Q165P mutation ( A ) whereas the liver metastasis harbored a homozygous Q165P mutation ( B ) . D , E . IHC analysis of BRD4 protein expression in SPOP wild type ( WT ) and mutated ( MUT ) PCa patient samples . The representative images of BRD4 IHC staining in both SPOP WT and MUT PCa patients are shown in ( D ) and the quantified IHC data are shown in ( E ) . Scale bars : 100 µm for 20 x fields ; 50 µm for 40 x fields . The red dot in ( E ) indicates SPOP Q165P sample . All data shown are means ± SEM . The P value was calculated by the unpaired two - tailed Student ' s t - test ; * * P < 0 . 01 . See Appendix Table S4 for the detailed comparison , P values and sample number ( n ) . F , G . IHC analysis of AR protein expression in SPOP WT and mutated PCa patient samples . The representative images of AR IHC staining in both SPOP WT and mutant PCa patients are shown in ( F ) and the quantified IHC data are shown in ( G ) . Scale bars : 100 µm for 20 x fields ; 50 µm for 40 x fields . The green dot indicates SPOP Q165P sample . All data shown are means ± SEM . The P value was calculated by the unpaired two - tailed Student ' s t - test ; * * * P < 0 . 01 . See Appendix Table S4 for the detailed comparison , P values and sample number ( n ) .

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Figure 1A, B. A missense mutation in SPOP gene (Q165P, T >G) was identified in a PCa patient. Exome sequencing revealed that the primary tumor contained a heterozygous Q165P mutation (A) whereas the liver metastasis harbored a homozygous Q165P mutation (B).D, E. IHC analysis of BRD4 protein expression in SPOP wild type (WT) and mutated (MUT) PCa patient samples. The representative images of BRD4 IHC staining in both SPOP WT and MUT PCa patients are shown in (D) and the quantified IHC data are shown in (E). Scale bars: 100 µm for 20 x fields; 50 µm for 40 x fields. The red dot in (E) indicates SPOP Q165P sample. All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; ** P < 0.01. See Appendix Table S4 for the detailed comparison, P values and sample number (n).F, G. IHC analysis of AR protein expression in SPOP WT and mutated PCa patient samples. The representative images of AR IHC staining in both SPOP WT and mutant PCa patients are shown in (F) and the quantified IHC data are shown in (G). Scale bars: 100 µm for 20 x fields; 50 µm for 40 x fields. The green dot indicates SPOP Q165P sample. All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; *** P < 0.01. See Appendix Table S4 for the detailed comparison, P values and sample number (n).


Figure 2LNCaP cells were transfected with the indicated plasmids for 48 h and harvested for western blot analysis with the indicated antibodies to detect the interaction between BRD4 and SPOP ( A ) LNCaP cells were transfected with the indicated plasmids for 48 h and harvested for western blot analysis with the indicated antibodies to detect the BRD4 ubiquitination ( B ) . C . C4 - 2 cells were stably infected with the lenti - virus expressing empty vector ( EV ) or SPOP mutants and harvested for western blot analysis with the indicated antibodies . D . C4 - 2 xenograft tumors expressing EV or F133V and SPOP WT and Q165P PDX tumors were harvested for western blot analysis with the indicated antibodies . T1 : tumor 1 ; T2 : tumor 2 . E . Representative IHC images of PTEN protein in SPOP WT and Q165P PDX tumors . Scale bars : 100 µm for 20 x fields ; 50 µm for 40 x fields .

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Figure 2LNCaP cells were transfected with the indicated plasmids for 48 h and harvested for western blot analysis with the indicated antibodies to detect the interaction between BRD4 and SPOP (A)LNCaP cells were transfected with the indicated plasmids for 48 h and harvested for western blot analysis with the indicated antibodies to detect the BRD4 ubiquitination (B).C. C4-2 cells were stably infected with the lenti-virus expressing empty vector (EV) or SPOP mutants and harvested for western blot analysis with the indicated antibodies.D. C4-2 xenograft tumors expressing EV or F133V and SPOP WT and Q165P PDX tumors were harvested for western blot analysis with the indicated antibodies. T1: tumor 1; T2: tumor 2.E. Representative IHC images of PTEN protein in SPOP WT and Q165P PDX tumors. Scale bars: 100 µm for 20 x fields; 50 µm for 40 x fields.


Figure 3Co - IP assay was performed to detect the efficiency of dimerization between SPOP WT and Q165P mutant ( F ) Co - IP assay was performed to detect the interaction between CULLIN3 with SPOP WT or Q165P ( G ) .

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Figure 3Co-IP assay was performed to detect the efficiency of dimerization between SPOP WT and Q165P mutant (F)Co-IP assay was performed to detect the interaction between CULLIN3 with SPOP WT or Q165P (G).


Figure 4A . DU145 PCa cells infected with virus expressing EV or SPOP Q165P were harvested for western blot analysis . B . MTS was performed to compare the growth between EV and Q165P DU145 cells after the treatment with indicated inhibitors ( 2 µM of JQ1 , 2 µM of CPI - 637 , 2 µM NEO2734 ) for 72 h . All data shown are means ± SEM . The P value was calculated by the unpaired two - tailed Student ' s t - test ; * P < 0 . 05 , * * * P < 0 . 001 . Clonogenic survival assay was performed to determine the sensitivity of NEO2734 in SPOP Q165P DU145 cells . The survival curve showed IC50 for SPOP Q165P ( 0 . 69 µM ) and EV cells ( 1 . 08 µM ) ( C ) . DU145 cells expressing lenti - EV or lenti - HA - SPOP - Q165 constructs were treated with 1 µM of NEO2734 for 4 days and cultured for another 8 days before harvest . The number of colonies with more than 50 cells was counted . Representative images of colonies are shown in ( D ) with quantitative data shown in ( E ) . All data shown are means ± SEM . The P value was calculated by the unpaired two - tailed Student ' s t - test ; * P < 0 . 05 , * * P < 0 . 01 . F . DU145 cells were treated with the indicated inhibitors and harvested for ChIP with BRD4 antibody . The enrichment of BRD4 at c - MYC enhancer and RAC1 promoter was analyzed using qPCR . All data shown are means ± SEM . The P value was calculated by the unpaired two - tailed Student ' s t - test ; n . s . , not significant , * P < 0 . 05 , * * P < 0 . 01 . G . DU145 cells were treated with the indicated inhibitors and harvested for ChIP with Ac - H3 antibody . The level of Ac - H3 at c - MYC enhancer and RAC1 promoter is analyzed using qPCR . All data shown are means ± SEM . The P value was calculated by the unpaired two - tailed Student ' s t - test ; * * P < 0 . 01 .

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Figure 4A. DU145 PCa cells infected with virus expressing EV or SPOP Q165P were harvested for western blot analysis.B. MTS was performed to compare the growth between EV and Q165P DU145 cells after the treatment with indicated inhibitors (2 µM of JQ1, 2 µM of CPI-637, 2 µM NEO2734) for 72 h. All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; * P < 0.05, *** P < 0.001.Clonogenic survival assay was performed to determine the sensitivity of NEO2734 in SPOP Q165P DU145 cells. The survival curve showed IC50 for SPOP Q165P (0.69 µM) and EV cells (1.08 µM) (C).DU145 cells expressing lenti-EV or lenti-HA-SPOP-Q165 constructs were treated with 1 µM of NEO2734 for 4 days and cultured for another 8 days before harvest. The number of colonies with more than 50 cells was counted. Representative images of colonies are shown in (D) with quantitative data shown in (E). All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; * P < 0.05, ** P < 0.01.F. DU145 cells were treated with the indicated inhibitors and harvested for ChIP with BRD4 antibody. The enrichment of BRD4 at c-MYC enhancer and RAC1 promoter was analyzed using qPCR. All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; n.s., not significant, * P < 0.05, ** P < 0.01.G. DU145 cells were treated with the indicated inhibitors and harvested for ChIP with Ac-H3 antibody. The level of Ac-H3 at c-MYC enhancer and RAC1 promoter is analyzed using qPCR. All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; ** P < 0.01.


Figure 5B , C . SCID male mice with PDX tumors were treated with vehicle ( 40 % polyethylene glycol ) , JQ1 ( 50 mg / kg ) , CPI - 637 ( 30 mg / kg ) , combination of JQ1 ( 50 mg / kg ) and CPI - 637 ( 30 mg / kg ) or NEO2734 ( 30 mg / kg ) five days a week for three consecutive weeks . Tumors isolated from mice at day 21 of drug treatment were photographed ( B ) and tumor growth are shown in ( C ) . All data shown are means ± SEM . The P value comparing the tumor volume at day 21 post - treatment was calculated by the unpaired two - tailed Student ' s t - test ; * * * P < 0 . 001 . PDX tumors in each treatment group were harvested for protein For western blot analysis , three tumors each group ( n = 3 ) were used for protein extraction and mixed with equal ration to detect the indicated antibodies in ( D ) . PDX tumors in each treatment group were harvested for RNA extraction . For RT - qPCR , three tumors each group ( n = 3 ) were extracted RNA for RT - qPCR in ( E ) . Data are shown as means ± SEM . The P value comparing vehicle with each treatment group was calculated by unpaired two - tailed Student ' s t - test and indicated in the graph .

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Figure 5B, C. SCID male mice with PDX tumors were treated with vehicle (40% polyethylene glycol), JQ1 (50 mg/kg), CPI-637 (30 mg/kg), combination of JQ1 (50 mg/kg) and CPI-637 (30 mg/kg) or NEO2734 (30 mg/kg) five days a week for three consecutive weeks. Tumors isolated from mice at day 21 of drug treatment were photographed (B) and tumor growth are shown in (C). All data shown are means ± SEM. The P value comparing the tumor volume at day 21 post- treatment was calculated by the unpaired two-tailed Student's t-test; *** P < 0.001.PDX tumors in each treatment group were harvested for protein For western blot analysis, three tumors each group (n = 3) were used for protein extraction and mixed with equal ration to detect the indicated antibodies in (D).PDX tumors in each treatment group were harvested for RNA extraction. For RT-qPCR, three tumors each group (n = 3) were extracted RNA for RT-qPCR in (E). Data are shown as means ± SEM. The P value comparing vehicle with each treatment group was calculated by unpaired two-tailed Student's t-test and indicated in the graph.


Figure 6Six organoid lines including four SPOP WT ( BM1 , BM5 , ST1 and 313HR ) and two MUT ( 573R and ASC1 ) were harvested for western blot analysis with the indicated antibodies ( A ) Asterisk , SRC3 at expected molecular mass . Six organoid lines including four SPOP WT ( BM1 , BM5 , ST1 and 313HR ) and two MUT ( 573R and ASC1 ) were harvested for IFC with p - AKT - S473 antibody ( B ) . E - cadherin antibody was used to indicate the cell membrane ( red ) and DAPI for nucleus ( blue ) . Scale bars : 25 µm . Six organoid lines including four SPOP WT ( BM1 , BM5 , ST1 and 313HR ) and two MUT ( 573R and ASC1 ) were cultured for five days , followed by the treatment with JQ1 ( 2 µM ) , CPI - 637 ( 2 µM ) , JQ1 ( 2 µM ) + CPI - 637 ( 2 µM ) or NEO2734 ( 2 µM ) for five more days . The representative images of organoids after the treatment are shown in ( C ) Six organoid lines including four SPOP WT ( BM1 , BM5 , ST1 and 313HR ) and two MUT ( 573R and ASC1 ) were cultured for five days , followed by the treatment with JQ1 ( 2 µM ) , CPI - 637 ( 2 µM ) , JQ1 ( 2 µM ) + CPI - 637 ( 2 µM ) or NEO2734 ( 2 µM ) for five more days . the quantified data of the organoid diameter are shown in ( D ) . Scale bars : 25 µm . All data shown are means ± SEM . The P value was calculated by the unpaired two - tailed Student ' s t - test ; n . s . , not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . E , F . 313HR ( SPOP WT ) and 573R ( Q165P ) organoids were cultured for 5 days , followed by the treatment with the indicated inhibitors for 5 days . The organoids were stained with Caspase - 3 antibody ( green ) to detect apoptotic cells . E - cadherin antibody staining was used to define the cell membrane ( red ) and the nucleus was counterstained with DAPI ( blue ) . The representative images of cleaved Caspase - 3 IFC staining are shown in ( E ) . Scale bars : 25 µm . All data shown are means ± SEM . The P value was calculated by the unpaired two - tailed Student ' s t - test ; * P < 0 . 05 , * * * P < 0 . 001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Six organoid lines including four SPOP WT (BM1, BM5, ST1 and 313HR) and two MUT (573R and ASC1) were harvested for western blot analysis with the indicated antibodies (A) Asterisk, SRC3 at expected molecular mass.Six organoid lines including four SPOP WT (BM1, BM5, ST1 and 313HR) and two MUT (573R and ASC1) were harvested for IFC with p-AKT-S473 antibody (B). E-cadherin antibody was used to indicate the cell membrane (red) and DAPI for nucleus (blue). Scale bars: 25 µm.Six organoid lines including four SPOP WT (BM1, BM5, ST1 and 313HR) and two MUT (573R and ASC1) were cultured for five days, followed by the treatment with JQ1 (2 µM), CPI-637 (2 µM), JQ1 (2 µM) + CPI-637 (2 µM) or NEO2734 (2 µM) for five more days. The representative images of organoids after the treatment are shown in (C)Six organoid lines including four SPOP WT (BM1, BM5, ST1 and 313HR) and two MUT (573R and ASC1) were cultured for five days, followed by the treatment with JQ1 (2 µM), CPI-637 (2 µM), JQ1 (2 µM) + CPI-637 (2 µM) or NEO2734 (2 µM) for five more days. the quantified data of the organoid diameter are shown in (D). Scale bars: 25 µm. All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; n.s., not significant, * P < 0.05, ** P < 0.01, *** P < 0.001.E, F. 313HR (SPOP WT) and 573R (Q165P) organoids were cultured for 5 days, followed by the treatment with the indicated inhibitors for 5 days. The organoids were stained with Caspase-3 antibody (green) to detect apoptotic cells. E-cadherin antibody staining was used to define the cell membrane (red) and the nucleus was counterstained with DAPI (blue). The representative images of cleaved Caspase-3 IFC staining are shown in (E). Scale bars: 25 µm. All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; * P < 0.05, *** P < 0.001.


Figure 7A . C4 - 2 PCa cells infected with virus expressing EV , SPOP Q165P or F133V were harvested for western blot analysis . B MTS was performed to compare the cell growth between EV , Q165P , F133V C4 - 2 groups after the treatment with indicated inhibitors ( 2 µM of JQ1 , 2 µM of CPI - 637 , 2 µM NEO2734 ) for 72 h . All data shown are means ± SEM . The P value was calculated by the unpaired two - tailed Student ' s t - test ; n . s . , not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . C . Clonogenic survival assay was performed to determine the sensitivity of NEO2734 in SPOP mutant C4 - 2 cells . The survival curve showed IC50 for EV ( 1 . 01 µM ) , SPOP Q165P ( 0 . 621 µM ) and SPOP F133V cells ( 0 . 395 µM ) . D , E . C4 - 2 cells expressing EV , Q165 or F133V constructs were treated with 1 µM of NEO2734 for 4 days and cultured for another 8 days before harvesting . The number of colonies with more than 50 cells was counted . Representative images of colonies are shown in ( D ) with quantification data in ( E ) . All data shown are means ± SEM . The P value was calculated by the unpaired two - tailed Student ' s t - test ; * P < 0 . 05 , * * * P < 0 . 001 . F - H . A schematic depicts the procedure of the establishment of SPOP mutant xenograft models and inhibitor administration ( F ) . When the tumor reached 100 mm3 , mice were treated with vehicle ( 40 % polyethylene glycol ) , JQ1 ( 50 mg / kg ) or NEO2734 ( 30 mg / kg ) five days a week for three consecutive weeks . Tumors isolated from mice at day 21 of treatment were photographed ( G ) and tumor growth are shown in ( H ) . All data shown are means ± SEM . The P value comparing the tumor volume at day 21 post - treatment was calculated by the unpaired two - tailed Student ' s t - test ; * P < 0 . 05 , * * P < 0 . 01 ; * * * P < 0 . 001 .

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Figure 7A. C4-2 PCa cells infected with virus expressing EV, SPOP Q165P or F133V were harvested for western blot analysis.B MTS was performed to compare the cell growth between EV, Q165P, F133V C4-2 groups after the treatment with indicated inhibitors (2 µM of JQ1, 2 µM of CPI-637, 2 µM NEO2734) for 72 h. All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; n.s., not significant, * P < 0.05, ** P < 0.01, *** P < 0.001.C. Clonogenic survival assay was performed to determine the sensitivity of NEO2734 in SPOP mutant C4-2 cells. The survival curve showed IC50 for EV (1.01 µM), SPOP Q165P (0.621 µM) and SPOP F133V cells (0.395 µM).D, E. C4-2 cells expressing EV, Q165 or F133V constructs were treated with 1 µM of NEO2734 for 4 days and cultured for another 8 days before harvesting. The number of colonies with more than 50 cells was counted. Representative images of colonies are shown in (D) with quantification data in (E). All data shown are means ± SEM. The P value was calculated by the unpaired two-tailed Student's t-test; * P < 0.05, *** P < 0.001.F - H. A schematic depicts the procedure of the establishment of SPOP mutant xenograft models and inhibitor administration (F). When the tumor reached 100 mm3, mice were treated with vehicle (40% polyethylene glycol), JQ1 (50 mg/kg) or NEO2734 (30 mg/kg) five days a week for three consecutive weeks. Tumors isolated from mice at day 21 of treatment were photographed (G) and tumor growth are shown in (H). All data shown are means ± SEM. The P value comparing the tumor volume at day 21 post- treatment was calculated by the unpaired two-tailed Student's t-test; * P < 0.05, ** P < 0.01; *** P < 0.001.


Figure 1Expression of YAP / TAZ and a smaller protein ( asterisk ) in different cell lines , protein expression was determined by immunoblotting ( IB ) . The shRNA targeting TAZ , but not YAP , knocked down the expression of the smaller protein ( asterisk ) . Antibodies targeting C - terminus YAP / TAZ , such as TAZ ( CST ) and YAP / TAZ , effectively pulled down the smaller protein ( asterisk , dubbed as cTAZ hereafter ) in RKO cells in an immunoprecipitation ( IP ) assay . cTAZ was not recognized by TAZ ( SA ) , an antibody targeting N - terminus of TAZ . Exogenous TAZ / YAP was not processed proteolytically to cTAZ . RKO cells were transfected with the indicated plasmids expressing C - terminal HA - tagged TAZ or YAP . RKO , but not HEK293A cells , expressed both full - length TAZ and cTAZ transcripts . RT PCR primers targeting different regionsAlignment of TAZ and cTAZ protein sequences . cTAZ protein sequence was derived from the Sanger sequencing results of the short PCR product ( RKO , F2 : R1 ) Knock out of cTAZ in RKO cells using CRISPR / Cas9 technology . Monoclones ( # 3 and # 14 ) were selected .

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Figure 1Expression of YAP/TAZ and a smaller protein (asterisk) in different cell lines, protein expression was determined by immunoblotting (IB).The shRNA targeting TAZ, but not YAP, knocked down the expression of the smaller protein (asterisk).Antibodies targeting C-terminus YAP/TAZ, such as TAZ (CST) and YAP/TAZ, effectively pulled down the smaller protein (asterisk, dubbed as cTAZ hereafter) in RKO cells in an immunoprecipitation (IP) assay. cTAZ was not recognized by TAZ (SA), an antibody targeting N-terminus of TAZ.Exogenous TAZ/YAP was not processed proteolytically to cTAZ. RKO cells were transfected with the indicated plasmids expressing C-terminal HA-tagged TAZ or YAP.RKO, but not HEK293A cells, expressed both full-length TAZ and cTAZ transcripts. RT PCR primers targeting different regionsAlignment of TAZ and cTAZ protein sequences. cTAZ protein sequence was derived from the Sanger sequencing results of the short PCR product (RKO, F2:R1)Knock out of cTAZ in RKO cells using CRISPR/Cas9 technology. Monoclones (#3 and #14) were selected.


Figure 2Cluster analysis of RNA expression of WT , cTAZ - / - , and cTAZ - / - / GFP - cTAZ put - back RKO cells . Genes down - and up - regulated more than 2 folds ( P < 0 . 05 ) in cTAZ KO cells were included in cluster analysis . Green and red indicate down - and up - regulated genes respectively . cTAZ suppressed expression of ISGs . RNA levels of selected ISGs in WT , cTAZ - / - , cTAZ - / - / GFP - cTAZ RKO cells were quantified by qPCR . Error bars indicate SD , n = 3 . * * P < 0 . 01 ; * * * P < 0 . 001 ; Student ' s t - test . cTAZ repressed 5 × ISRE - luciferase activity induced by IFN - α . HEK293A cells were transfected with 5 × ISRE - luciferase reporter along with the indicated plasmids , treated with or without IFN - α ( 50 ng / ml ) for 12 hours . The expression of ectopic genes was determined by IB . Error bars indicate SD , n = 3 . * * * P < 0 . 001 ; one - way ANOVA test was used for statistical analysis . Knockout of cTAZ promoted ISGs expression in the presence of IFN - α . WT and cTAZ - / - RKO cells were treated with or without of IFN - α ( 50 ng / ml ) for indicated time , the expression of selected genes was quantified by qPCR . WT or cTAZ - / - group was normalized by basal level , respectively . Error bars indicate SD , n = 3 . * P < 0 . 05 ; * * P < 0 . 01 ; Student ' s t - test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Cluster analysis of RNA expression of WT, cTAZ-/-, and cTAZ-/- /GFP-cTAZ put-back RKO cells. Genes down- and up-regulated more than 2 folds (P < 0.05) in cTAZ KO cells were included in cluster analysis. Green and red indicate down- and up-regulated genes respectively.cTAZ suppressed expression of ISGs. RNA levels of selected ISGs in WT, cTAZ-/-, cTAZ-/-/GFP-cTAZ RKO cells were quantified by qPCR. Error bars indicate SD, n =3. **P<0.01; ***P<0.001; Student's t-test.cTAZ repressed 5×ISRE-luciferase activity induced by IFN-α. HEK293A cells were transfected with 5×ISRE-luciferase reporter along with the indicated plasmids, treated with or without IFN-α (50 ng/ml) for 12 hours. The expression of ectopic genes was determined by IB. Error bars indicate SD, n =3. ***P<0.001; one-way ANOVA test was used for statistical analysis.Knockout of cTAZ promoted ISGs expression in the presence of IFN-α. WT and cTAZ-/- RKO cells were treated with or without of IFN-α (50 ng/ml) for indicated time, the expression of selected genes was quantified by qPCR. WT or cTAZ-/- group was normalized by basal level, respectively. Error bars indicate SD, n =3. *P<0.05; **P<0.01; Student's t-test.


Figure 3STAT1 interacted with cTAZ , and the interaction was enhanced by IFN - α . HEK293A cells were transfected with indicated plasmids , treated with or without IFN - α ( 50 ng / ml ) for 1 hour . IP were performed using Flag antibody . cTAZ attenuated heterodimerization between STAT1 and STAT2 induced by IFN - α . Following transfection , cells were treated with or without IFN - α ( 50 ng / ml ) for 1 hour and subjected to IP assays using Flag antibody . Depletion of cTAZ enhanced the interaction between STAT1 and STAT2 . RKO cell lines ( WT and # 14 ) were treated with IFN - α as in ( B ) . IP assays were performed using STAT1 antibody . cTAZ attenuated interaction between STAT1 and importin α5 ( Im - α5 ) . Following transfection , HEK293A cells were stimulated using IFN - α IP assays were performed using Flag antibody . Deletion of cTAZ promoted nuclear accumulation of STAT1 upon IFN - α stimulation . RKO ( WT and cTAZ - / - ) cells treated with or without IFN - α ( 50 ng / ml ) for 1 hour , fixed , and subjected to IF using STAT1 antibody ( green ) . DNA was labeled by DAPI ( blue ) . Scale bar , 50 μm . Quantification was performed using ImageJ software ( right ) . N > C : nuclear ; N = C : nuclear plus cytoplasmic ; N < C : cytoplasmic localization . Deletion of cTAZ promoted nuclear accumulation of STAT1 upon IFN - α stimulation . Cells were treated and subjected to subcellular fractionation . cTAZ defective in STAT1 - binding failed to block nuclear translocation of STAT1 . HEK293A stably overexpressing STAT1 were transfected with the indicated plasmids , treated with or without IFN - α ( 50 ng / ml ) for 1 hour , fixed , and subjected to IF Scale bar , 25 μm .

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Figure 3STAT1 interacted with cTAZ, and the interaction was enhanced by IFN-α. HEK293A cells were transfected with indicated plasmids, treated with or without IFN-α (50 ng/ml) for 1 hour. IP were performed using Flag antibody.cTAZ attenuated heterodimerization between STAT1 and STAT2 induced by IFN-α. Following transfection, cells were treated with or without IFN-α (50 ng/ml) for 1 hour and subjected to IP assays using Flag antibody.Depletion of cTAZ enhanced the interaction between STAT1 and STAT2. RKO cell lines (WT and #14) were treated with IFN-α as in (B). IP assays were performed using STAT1 antibody.cTAZ attenuated interaction between STAT1 and importin α5 (Im-α5). Following transfection, HEK293A cells were stimulated using IFN-α IP assays were performed using Flag antibody.Deletion of cTAZ promoted nuclear accumulation of STAT1 upon IFN-α stimulation. RKO (WT and cTAZ-/-) cells treated with or without IFN-α (50 ng/ml) for 1 hour, fixed, and subjected to IF using STAT1 antibody (green). DNA was labeled by DAPI (blue). Scale bar, 50 μm. Quantification was performed using ImageJ software (right). N>C: nuclear; N=C: nuclear plus cytoplasmic; N<C: cytoplasmic localization.Deletion of cTAZ promoted nuclear accumulation of STAT1 upon IFN-α stimulation. Cells were treated and subjected to subcellular fractionation.cTAZ defective in STAT1-binding failed to block nuclear translocation of STAT1. HEK293A stably overexpressing STAT1 were transfected with the indicated plasmids, treated with or without IFN-α (50 ng/ml) for 1 hour, fixed, and subjected to IF Scale bar, 25 μm.


Figure 4cTAZ repressed the antiviral activity of ectopic STAT1 / 2 . HEK293A were transfected with indicated plasmids , infected with gVSV ( MOI = 0 . 01 ) for 12 hours , fixed , and subjected to microscopy ( Left ) . Viral infection and replication were marked by green fluorescence . Scale bar , 100 μm . ( Right ) Quantification of GFP positive cells . About 300 cells ( from 3 different fields ) were analyzed . Error bars indicate SD . * * P < 0 . 01 ; * * * P < 0 . 001 ; Student ' s t - test . cTAZ and STAT1 / 2 expression levels of cellscTAZ effectively repressed antiviral activity of IFN - α . Transfection and infection were performed Top : GFP intensity of cell lysates was measured by a fluorometer . Bottom : viral GFP and ectopic cTAZ protein levels were detected by IB . Depletion of cTAZ repressed viral replication . RKO cell lines ( WT and two independent cTAZ - / - clones ) were infected with gVSV ( MOI = 0 . 1 ) for 12 hours . Scale bar , 100 μm . The ratio of GFP positive cells were calculated About 300 cells ( from 3 different fields ) were analyzed . Error bars indicate SD . * * * P < 0 . 001 ; Student ' s t - test . Depletion of cTAZ repressed viral replication . Cells were treated GFP intensity of cell lysates was measured . Error bars indicate SD , n = 3 . * * P < 0 . 01 ; * * * P < 0 . 001 ; Student ' s t - test . Depletion of cTAZ repressed viral replication . Cells were treated the viral genomic RNA was quantified by qPCR ( normalized to human GAPDH ) . Error bars indicate SD , n = 3 . * * * P < 0 . 001 ; Student ' s t - test . cTAZ deficiency enhanced ISGs expression . Cells were treated the expression of IFIH1 and DDX58 were assessed by qPCR . Error bars indicate SD , n = 3 . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; Student ' s t - test . Overexpression of cTAZ promotes virus infection . DOX - inducible cTAZ - overexpressing RKO cell lines were pre - treated with DOX for 48 hours ( 1 μg / ml ) . gVSV infection and GFP Fluorescence intensity quantification were performed Error bars indicate SD , n = 3 . * * P < 0 . 01 ; * * * P < 0 . 001 ; Student ' s t - test . Overexpression of cTAZ repressed ISGs expression . Cells were treated the expression of IFIH1 and DDX58 were assessed by qPCR . Error bars indicate SD , n = 3 . * P < 0 . 05 ; * * P < 0 . 01 ; Student ' s t - test .

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Figure 4cTAZ repressed the antiviral activity of ectopic STAT1/2. HEK293A were transfected with indicated plasmids, infected with gVSV (MOI = 0.01) for 12 hours, fixed, and subjected to microscopy (Left). Viral infection and replication were marked by green fluorescence. Scale bar, 100 μm. (Right) Quantification of GFP positive cells. About 300 cells (from 3 different fields) were analyzed. Error bars indicate SD. **P<0.01; ***P<0.001; Student's t-test.cTAZ and STAT1/2 expression levels of cellscTAZ effectively repressed antiviral activity of IFN-α. Transfection and infection were performed Top: GFP intensity of cell lysates was measured by a fluorometer. Bottom: viral GFP and ectopic cTAZ protein levels were detected by IB.Depletion of cTAZ repressed viral replication. RKO cell lines (WT and two independent cTAZ -/- clones) were infected with gVSV (MOI = 0.1) for 12 hours. Scale bar, 100 μm. The ratio of GFP positive cells were calculated About 300 cells (from 3 different fields) were analyzed. Error bars indicate SD. ***P<0.001; Student's t-test.Depletion of cTAZ repressed viral replication. Cells were treated GFP intensity of cell lysates was measured. Error bars indicate SD, n = 3. **P<0.01; ***P<0.001; Student's t-test.Depletion of cTAZ repressed viral replication. Cells were treated the viral genomic RNA was quantified by qPCR (normalized to human GAPDH). Error bars indicate SD, n = 3. ***P<0.001; Student's t-test.cTAZ deficiency enhanced ISGs expression. Cells were treated the expression of IFIH1 and DDX58 were assessed by qPCR. Error bars indicate SD, n = 3. *P<0.05; **P<0.01; ***P<0.001; Student's t-test.Overexpression of cTAZ promotes virus infection. DOX-inducible cTAZ-overexpressing RKO cell lines were pre-treated with DOX for 48 hours (1 μg/ml). gVSV infection and GFP Fluorescence intensity quantification were performed Error bars indicate SD, n = 3. **P<0.01; ***P<0.001; Student's t-test.Overexpression of cTAZ repressed ISGs expression. Cells were treated the expression of IFIH1 and DDX58 were assessed by qPCR. Error bars indicate SD, n = 3. *P<0.05; **P<0.01; Student's t-test.


Figure 5gVSV induced expression of cTAZ . RKO cells were infected with gVSV ( MOI = 0 , 0 . 001 , 0 . 01 , 0 . 01 , 1 ) for 8 hours . Protein expression was determined by IB ( Left ) and quantified ( Right ) . Error bars indicate SD , n = 3 . * * * P < 0 . 001 ; two - way ANOVA test was used for statistical analysis . IFN - α induced expression of cTAZ . RKO cells were treated with different doses of IFN - α for 8 hours . Protein expression was determined Error bars indicate SD , n = 3 . * * * P < 0 . 001 ; two - way ANOVA test was used for statistical analysis . IFN - α induced mRNA level of cTAZ . RKO cells were treated with different doses of IFN - α for 8 hours . RNA levels were measured by qPCR . Error bars indicate SD , n = 3 . * * * P < 0 . 001 ; two - way ANOVA test was used for statistical analysis . STAT1 / 2 synergistically activated a cTAZ promoter . HEK293A cells were transfected with cTAZ reporter with or without STAT proteins . Promoter activity was determined using luciferase assay , and protein expression was determined by IB . Error bars indicate SD , n = 3 . * * * P < 0 . 001 ; Student ' s t - test . STAT1 / 2 binding sites on cTAZ promoter . HEK293A were transfected with cTAZ promoters ( wild type or mutants ) . Luciferase activity and responses to STAT1 / 2 expression were measured . Error bars indicate SD , n = 3 . * * P < 0 . 01 ; Student ' s t - test . STAT1 occupied cTAZ promoter in an IFN - α - sensitive manner . RKO cells were treated with or without IFN - α ( 50 ng / ml ) for 1 hour and subjected to ChIP assays . cTAZ PCR primers were designed to target STAT1 / 2 binding site . ISG15 promoter was included as a positive control .

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Figure 5gVSV induced expression of cTAZ. RKO cells were infected with gVSV (MOI = 0, 0.001, 0.01, 0.01, 1) for 8 hours. Protein expression was determined by IB (Left) and quantified (Right). Error bars indicate SD, n = 3. ***P<0.001; two-way ANOVA test was used for statistical analysis.IFN-α induced expression of cTAZ. RKO cells were treated with different doses of IFN-α for 8 hours. Protein expression was determined Error bars indicate SD, n = 3. ***P<0.001; two-way ANOVA test was used for statistical analysis.IFN-α induced mRNA level of cTAZ. RKO cells were treated with different doses of IFN-α for 8 hours. RNA levels were measured by qPCR. Error bars indicate SD, n = 3. ***P<0.001; two-way ANOVA test was used for statistical analysis.STAT1/2 synergistically activated a cTAZ promoter. HEK293A cells were transfected with cTAZ reporter with or without STAT proteins. Promoter activity was determined using luciferase assay, and protein expression was determined by IB. Error bars indicate SD, n = 3. ***P<0.001; Student's t-test.STAT1/2 binding sites on cTAZ promoter. HEK293A were transfected with cTAZ promoters (wild type or mutants). Luciferase activity and responses to STAT1/2 expression were measured. Error bars indicate SD, n = 3. **P<0.01; Student's t-test.STAT1 occupied cTAZ promoter in an IFN-α-sensitive manner. RKO cells were treated with or without IFN-α (50 ng/ml) for 1 hour and subjected to ChIP assays. cTAZ PCR primers were designed to target STAT1/2 binding site. ISG15 promoter was included as a positive control.


Figure 1A Left , time lapse confocal images of PHcrac ( red ) and LimE ( green ) in waves at the basal surface of a giant cell . Right , intensity plot across the blue dotted arrow in image " 30 sec " . Various biosensors in excitable waves . snapshots of biosensors ( top ) and intensity plots ( bottom ) across the blue dotted arrows , with black plots showing the corresponding biosensors in images above , red ( B ) showing PHcrac in the same cell , Scatter plots of biosensor peak locations relative to the peak of PHcrac ( C ) across propagating waves ( blue solid lines indicate median ) . Positive values indicate that the peak precedes that of PHcrac along the direction of wave propagation . n = 26 , 26 , 24 , 21 , 21 , 32 cells for RalGDS , RBD , PKBA , , respectively . Scale bars in images above represent 20 μm . Grey arrows in all plots represent the direction of wave propagation . Various biosensors in excitable waves . D , snapshots of biosensors ( top ) and intensity plots ( bottom ) across the blue dotted arrows , with black plots showing the corresponding biosensors in images above , green ( D ) showing LimE . E , Scatter plots of biosensor peak locations relative to the peak of or the first peak of LimE ( E ) across propagating waves ( blue solid lines indicate median ) . Positive values indicate that the peak precedes that of LimE ) along the direction of wave propagation . n = 26 , 26 , 24 , 21 , 21 , 32 cells for , RacGEF1 , PAK1 - GBD , and Coronin , respectively . Scale bars in images above represent 20 μm . Grey arrows in all plots represent the direction of wave propagation . F Left , confocal images of PHcrac ( red ) and PAK1 - GBD ( green ) in the same cells treated with LatA . Scale bars represent 10 μm . Right , temporal profiles of normalized mean cytosolic intensities of PHcrac ( red ) and PAK1 - GBD ( green ) ( mean ± s . e . m . , n = 18 cells ) in response to cAMP .

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Figure 1A Left, time lapse confocal images of PHcrac (red) and LimE (green) in waves at the basal surface of a giant cell. Right, intensity plot across the blue dotted arrow in image "30 sec".Various biosensors in excitable waves. snapshots of biosensors (top) and intensity plots (bottom) across the blue dotted arrows, with black plots showing the corresponding biosensors in images above, red (B) showing PHcrac in the same cell, Scatter plots of biosensor peak locations relative to the peak of PHcrac (C) across propagating waves (blue solid lines indicate median). Positive values indicate that the peak precedes that of PHcrac along the direction of wave propagation. n=26, 26, 24, 21, 21, 32 cells for RalGDS, RBD, PKBA, , respectively. Scale bars in images above represent 20 μm. Grey arrows in all plots represent the direction of wave propagation.Various biosensors in excitable waves. D, snapshots of biosensors (top) and intensity plots (bottom) across the blue dotted arrows, with black plots showing the corresponding biosensors in images above, green (D) showing LimE. E, Scatter plots of biosensor peak locations relative to the peak of or the first peak of LimE (E) across propagating waves (blue solid lines indicate median). Positive values indicate that the peak precedes that of LimE) along the direction of wave propagation. n=26, 26, 24, 21, 21, 32 cells for , RacGEF1, PAK1-GBD, and Coronin, respectively. Scale bars in images above represent 20 μm. Grey arrows in all plots represent the direction of wave propagation.F Left, confocal images of PHcrac (red) and PAK1-GBD (green) in the same cells treated with LatA. Scale bars represent 10 μm. Right, temporal profiles of normalized mean cytosolic intensities of PHcrac (red) and PAK1-GBD (green) (mean±s.e.m., n=18 cells) in response to cAMP.


Figure 2A Confocal image of LimE in waves ( scale bar , 20 μm ) . B Top , kymograph of LimE in a fixed region across which a piece of wave propagates , with white arrows pointing to the same puncta . Bottom , box plot of puncta ' s lifetime ( n = 100 puncta ) . H A two - dimensional snapshot of a propagating wave ( direction of propagation indicated with the dashed arrow ) . A line scan corresponding to the STEN - CEN profile is shown on the right .

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Figure 2A Confocal image of LimE in waves (scale bar, 20 μm).B Top, kymograph of LimE in a fixed region across which a piece of wave propagates, with white arrows pointing to the same puncta. Bottom, box plot of puncta's lifetime (n=100 puncta).H A two-dimensional snapshot of a propagating wave (direction of propagation indicated with the dashed arrow). A line scan corresponding to the STEN-CEN profile is shown on the right.


Figure 3A Time lapse confocal images of PHcrac ( top ) and LimE ( bottom ) in the same giant cell ( scale bar , 20 μm ) . White arrows point to wave breaking events .

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Figure 3A Time lapse confocal images of PHcrac (top) and LimE (bottom) in the same giant cell (scale bar, 20 μm). White arrows point to wave breaking events.


Figure 4E Time lapse confocal images of LimE at the basal surface of a giant cell , which recruits Inp54p to membrane induced by rapamycin at time 0 . The white arrow ( - 45 s ) points to a nascent wave before Inp54p recruitment , while the red ( 60 s ) arrow after . F Color coded overlays of LimE before and after Inp54p recruitment . White arrows point to nascent waves at time 0 . G Kymographs of LimE in regions outlined by dotted line boxes in f . J Time lapse confocal images of PHcrac at the bottom of giant cells under LatA treatment , where rapamycin is added at time 0 to induce Inp54p recruitment . White arrows point to wave patches right before rapamycin addition . K Confocal image of PI5K ( left ) and LimE ( right ) at the bottom of the same cell . All scale bars in images represent 20 μm .

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Figure 4E Time lapse confocal images of LimE at the basal surface of a giant cell, which recruits Inp54p to membrane induced by rapamycin at time 0. The white arrow (-45 s) points to a nascent wave before Inp54p recruitment, while the red (60 s) arrow after.F Color coded overlays of LimE before and after Inp54p recruitment. White arrows point to nascent waves at time 0. G Kymographs of LimE in regions outlined by dotted line boxes in f.J Time lapse confocal images of PHcrac at the bottom of giant cells under LatA treatment, where rapamycin is added at time 0 to induce Inp54p recruitment. White arrows point to wave patches right before rapamycin addition.K Confocal image of PI5K (left) and LimE (right) at the bottom of the same cell. All scale bars in images represent 20 μm.