Figure 4 ( B ) Internally tagged HA - Mcp3 is functional . mdm10 ∆ cells were transformed with the empty plasmid pYX142 ( ) or pYX142 encoding Mdm10 , Mcp3 , or an internally HA - tagged Mcp3 ( MDM10 , MCP3 , HA - MCP3 ) . Cells were grown to an OD600 of 1 . 0 and spotted on YPG plates in a 1 : 5 dilution series . Plates were incubated for growth at 37 ° C for 4 days . ( C ) HA - Mcp3 is processed in vivo . Mitochondria isolated from mdm10 ∆ ( ∆ ) cells containing the empty plasmid or expressing HA - Mcp3 were analysed by SDS - PAGE and immunodecoration with antibodies against the indicated proteins . Two different bands are detectable in case of HA - Mcp3 ( p , precursor ; m , mature protein ) . The MOM protein Tom40 serves as loading control . ( D ) Mcp3 is a mitochondrial protein . Whole cell lysate ( WCL ) and fractions corresponding to cytosol ( Cyt . ) , light microsomal fraction ( ER ) and mitochondria ( Mito . ) of mcp3 ∆ cells either containing the empty plasmid ( ) or expressing HA - Mcp3 were analysed by SDS - PAGE and immunodecoration with antibodies against the HA - tag , the mitochondrial protein Tom70 , a marker protein for the cytosol ( Bmh1 ) and an ER marker protein ( Erv2 ) . m , mature protein . ( E ) Mcp3 co - localizes with the mitochondrial marker protein AAC . Cells over - expressing HA - Mcp3 were fixed and analysed by fluorescence microscopy using antibodies against the HA - epitope ( αHA ) and the mitochondrial ADP / ATP carrier ( αAAC ) . ( scale bar = 5 µm ) . ( F ) C - terminally tagged Mcp3 - HA is located to mitochondria . A mitochondria enriched fraction ( Mito . ) and the post mitochondrial supernatant ( PMS ) of wild - type cells carrying the empty plasmid ( ) or over - expressing the C - terminally HA - tagged Mcp3 ( Mcp3 - HA ) were analysed by SDS - PAGE and immunodecortion with antibodies against the HA - Tag , the mitochondrial protein Tom40 and a marker protein for the cytosol ( Hxk1 ) . m , mature protein . ( G ) Mcp3 is membrane - embedded . Mitochondria isolated from mcp3Δ cells expressing HA - Mcp3 were left untreated ( I , input ) or subjected to alkaline extraction . The supernatant ( SN ) and pellet ( P ) fractions were analysed by SDS - PAGE and immunodecoration with antibodies against the indicated proteins . Tom20 an integral MOM proteins ; Aco1 , soluble matrix protein . m , mature protein . ( H ) Mcp3 is a MOM protein . Mitochondria isolated from cells expressing HA - Mcp3 were treated with proteinase K ( PK , 100 µg / ml ) in the absence or presence of the detergent Triton X - 100 ( TX ) . Samples were precipitated with TCA and analysed by SDS - PAGE and immunodecoration with antibodies against the HA - tag or the indicated mitochondrial proteins . Tom20 , a MOM protein exposed to the cytosol ; Dld1 , a protein located in the IMS . ( I ) Mcp3 behaves like an outer membrane protein in density gradient centrifugation . Mitochondrial vesicles isolated from cells over - expressing HA - Mcp3 were subjected to sucrose density gradient centrifugation . Fractions of the gradient were collected and analysed by SDS - PAGE and immunodecoration with antibodies against the indicated proteins . The processed form of HA - Mcp3 is shown . Right panel : The intensities of the various bands were quantified and depicted . The sum of all intensities for each protein was set to 100 % . ( J ) Mcp3 lacking either transmembrane domain fails to rescue loss of Mdm10 . Wild - type cells carrying the empty plasmid ( wt + ) or mdm10 ∆ cells transformed with the empty plasmid ( ) or plasmid encoding Mdm10 , Mcp3 , or Mcp3 lacking TMD1 or TMD2 ( MDM10 , MCP3 , ΔTMD1 , ΔTMD2 ) were grown to an OD600 of 1 . 0 and spotted on YPG plates in a 1 : 5 dilution series . Plates were incubated for growth at 37 ° C for 4 days . ( K ) Mcp3 lacking either of the two transmembrane domains is unstable . Crude mitochondria were obtained from wild - type cells containing an empty plasmid ( Ø ) or mcp3Δ cells ( Δ ) transformed with plasmid encoding Mcp3 or Mcp3 lacking TMD1 or TMD2 ( MCP3 , ΔTMD1 , ΔTMD2 ) . Samples were analysed by SDS - PAGE and immunodecoration with antibodies against Mcp3 and Tom20 as loading control .

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 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 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 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 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 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 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 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 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 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

Figure 4(B) Internally tagged HA-Mcp3 is functional. mdm10∆ cells were transformed with the empty plasmid pYX142 () or pYX142 encoding Mdm10, Mcp3, or an internally HA-tagged Mcp3 (MDM10, MCP3, HA-MCP3). Cells were grown to an OD600 of 1.0 and spotted on YPG plates in a 1:5 dilution series. Plates were incubated for growth at 37°C for 4 days.(C) HA-Mcp3 is processed in vivo. Mitochondria isolated from mdm10∆ (∆) cells containing the empty plasmid or expressing HA-Mcp3 were analysed by SDS-PAGE and immunodecoration with antibodies against the indicated proteins. Two different bands are detectable in case of HA-Mcp3 (p, precursor; m, mature protein). The MOM protein Tom40 serves as loading control.(D) Mcp3 is a mitochondrial protein. Whole cell lysate (WCL) and fractions corresponding to cytosol (Cyt.), light microsomal fraction (ER) and mitochondria (Mito.) of mcp3∆ cells either containing the empty plasmid () or expressing HA-Mcp3 were analysed by SDS-PAGE and immunodecoration with antibodies against the HA-tag, the mitochondrial protein Tom70, a marker protein for the cytosol (Bmh1) and an ER marker protein (Erv2). m, mature protein.(E) Mcp3 co-localizes with the mitochondrial marker protein AAC. Cells over-expressing HA-Mcp3 were fixed and analysed by fluorescence microscopy using antibodies against the HA-epitope (αHA) and the mitochondrial ADP/ATP carrier (αAAC). (scale bar = 5 µm).(F) C-terminally tagged Mcp3-HA is located to mitochondria. A mitochondria enriched fraction (Mito.) and the post mitochondrial supernatant (PMS) of wild-type cells carrying the empty plasmid () or over-expressing the C-terminally HA-tagged Mcp3 (Mcp3-HA) were analysed by SDS-PAGE and immunodecortion with antibodies against the HA-Tag, the mitochondrial protein Tom40 and a marker protein for the cytosol (Hxk1). m, mature protein.(G) Mcp3 is membrane-embedded. Mitochondria isolated from mcp3Δ cells expressing HA-Mcp3 were left untreated (I, input) or subjected to alkaline extraction. The supernatant (SN) and pellet (P) fractions were analysed by SDS-PAGE and immunodecoration with antibodies against the indicated proteins. Tom20 an integral MOM proteins; Aco1, soluble matrix protein. m, mature protein.(H) Mcp3 is a MOM protein. Mitochondria isolated from cells expressing HA-Mcp3 were treated with proteinase K (PK, 100 µg/ml) in the absence or presence of the detergent Triton X-100 (TX). Samples were precipitated with TCA and analysed by SDS-PAGE and immunodecoration with antibodies against the HA-tag or the indicated mitochondrial proteins. Tom20, a MOM protein exposed to the cytosol; Dld1, a protein located in the IMS.(I) Mcp3 behaves like an outer membrane protein in density gradient centrifugation. Mitochondrial vesicles isolated from cells over-expressing HA-Mcp3 were subjected to sucrose density gradient centrifugation. Fractions of the gradient were collected and analysed by SDS-PAGE and immunodecoration with antibodies against the indicated proteins. The processed form of HA-Mcp3 is shown. Right panel: The intensities of the various bands were quantified and depicted. The sum of all intensities for each protein was set to 100%.(J) Mcp3 lacking either transmembrane domain fails to rescue loss of Mdm10. Wild-type cells carrying the empty plasmid (wt+) or mdm10∆ cells transformed with the empty plasmid () or plasmid encoding Mdm10, Mcp3, or Mcp3 lacking TMD1 or TMD2 (MDM10, MCP3, ΔTMD1, ΔTMD2) were grown to an OD600 of 1.0 and spotted on YPG plates in a 1:5 dilution series. Plates were incubated for growth at 37°C for 4 days.(K) Mcp3 lacking either of the two transmembrane domains is unstable. Crude mitochondria were obtained from wild-type cells containing an empty plasmid (Ø) or mcp3Δ cells (Δ) transformed with plasmid encoding Mcp3 or Mcp3 lacking TMD1 or TMD2 (MCP3, ΔTMD1, ΔTMD2). Samples were analysed by SDS-PAGE and immunodecoration with antibodies against Mcp3 and Tom20 as loading control.


Figure 5 ( A ) Isolated mitochondria were incubated with radiolabelled Mcp3 precursor protein for the indicated time periods . After import mitochondria were reisolated and analysed by SDS - PAGE and autoradiography . The arrowhead marks an additional band of the size of the cleaved N - terminus of Mcp3 . p and m , precursor and mature forms , respectively . ( B ) Import of Mcp3 is dependent on the receptor Tom70 . Mitochondria isolated from wild - type or tom70 / 71Δ cells were incubated with radiolabelled Mcp3 or pSu9 - DHFR ( as control ) for the indicated time periods . After import mitochondria were reisolated and analysed by SDS - PAGE and autoradiography . p and m , precursor and mature forms , respectively . I , 20 % of radiolabelled precursor protein used in each import reaction . Bands corresponding to the mature ( m ) form were quantified . Import into wild - type mitochondria after 15 min was set to 100 % . The mean with standard deviations is depicted ( n = 3 ; SD ) . ( C ) Reduced steady state levels of HA - Mcp3 in cells lacking Tom70 . Crude mitochondria isolated from the indicated cells were analysed by SDS - PAGE and immunodecoration with antibodies against the HA - tag , AAC as control substrate of Tom70 , and Fis1 as a loading control . HA - Mcp3 levels were quantified in relation to Fis1 levels and the levels in WT cells were set to 100 % . The bar diagram shows the mean with standard deviation ( n = 3 ; SD ; * , p < 0 . 05 ) . ( D ) Import of Mcp3 is dependent on Tom40 . Mitochondria isolated from wild - type and tom4025 cells were incubated with radiolabelled Mcp3 for the indicated time periods . Next , mitochondria were reisolated and analysed by SDS - PAGE and autoradiography as described in ( B ) . As a control Ugo1 was imported into the same mitochondria . Organelles were reisolated and analysed by BN - PAGE and autoradiography . The band corresponding to the Ugo1 dimer at around 150 kDa was quantified . The mean with standard deviation is shown ( n = 3 ; SD ) . ( E ) Steady state levels of HA - Mcp3 are lower in cells harbouring a mutant TOM40 allele . Crude mitochondria from wild - type and tom40 - 25 cells were obtained and analysed as described in ( C ) . Immunodecoration was performed with antibodies against the HA - tag , Tom40 , and Fis1 as loading control .

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 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 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 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 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

Figure 5(A) Isolated mitochondria were incubated with radiolabelled Mcp3 precursor protein for the indicated time periods. After import mitochondria were reisolated and analysed by SDS-PAGE and autoradiography. The arrowhead marks an additional band of the size of the cleaved N-terminus of Mcp3. p and m, precursor and mature forms, respectively.(B) Import of Mcp3 is dependent on the receptor Tom70. Mitochondria isolated from wild-type or tom70/71Δ cells were incubated with radiolabelled Mcp3 or pSu9-DHFR (as control) for the indicated time periods. After import mitochondria were reisolated and analysed by SDS-PAGE and autoradiography. p and m, precursor and mature forms, respectively. I, 20% of radiolabelled precursor protein used in each import reaction. Bands corresponding to the mature (m) form were quantified. Import into wild-type mitochondria after 15 min was set to 100%. The mean with standard deviations is depicted (n=3; SD).(C) Reduced steady state levels of HA-Mcp3 in cells lacking Tom70. Crude mitochondria isolated from the indicated cells were analysed by SDS-PAGE and immunodecoration with antibodies against the HA-tag, AAC as control substrate of Tom70, and Fis1 as a loading control. HA-Mcp3 levels were quantified in relation to Fis1 levels and the levels in WT cells were set to 100%. The bar diagram shows the mean with standard deviation (n=3; SD; *, p < 0.05).(D) Import of Mcp3 is dependent on Tom40. Mitochondria isolated from wild-type and tom4025 cells were incubated with radiolabelled Mcp3 for the indicated time periods. Next, mitochondria were reisolated and analysed by SDS-PAGE and autoradiography as described in (B). As a control Ugo1 was imported into the same mitochondria. Organelles were reisolated and analysed by BN-PAGE and autoradiography. The band corresponding to the Ugo1 dimer at around 150 kDa was quantified. The mean with standard deviation is shown (n=3; SD).(E) Steady state levels of HA-Mcp3 are lower in cells harbouring a mutant TOM40 allele. Crude mitochondria from wild-type and tom40-25 cells were obtained and analysed as described in (C). Immunodecoration was performed with antibodies against the HA-tag, Tom40, and Fis1 as loading control.


Figure 6 ( A ) Mcp3 precursor binds in vitro to cytosolic domains of TOM receptors . Radiolabelled Mcp3 was incubated with recombinant GST alone or GST fused to the cytosolic domains of Tom70 , Tom20 or Tom22 prebound to glutathione beads or with unloaded beads . After washing bound material was eluted with sample buffer . Proteins were analysed by SDS - PAGE , blotting onto a membrane and autoradiography . To demonstrate equal amounts of GST fusion proteins the membrane was stained with Ponceau S . I , input 5 % of precursor used . ( B ) Mcp3 precursor binds to Tom22 in organello . Radiolabelled Mcp3 was incubated with mitochondria isolated from WT His - Tom22 containing cells . Mitochondria were pre - incubated with CCCP ( 40 µM ) to halt import . After lysis with β - dodecyl maltoside samples were incubated with Ni - NTA beads . After washing bound proteins were eluted with sample buffer and analysed by SDS - PAGE and autoradiography . I , 5 % of 35S - Mcp3 used ; pel . , 2 % insoluble material after clarifying spin ; sup . , 2 % of material incubated with Ni - NTA ; n . b . 2 % of non - bound material after binding to Ni - NTA ; eluate , 100 % of bound material . ( C ) Radiolabelled Mcp3 was incubated with organelles described in ( B ) . After import mitochondria were solubilized in digitonin , the lysate was cleared by centrifugation and incubated with or without an antibody against the His - tag ( α - His ) . Samples were analysed by BN - PAGE , autoradiography and afterwards immunodecoration with an antibody against Tom40 . Bands shifted by the anti - His antibody are indicated by arrowhead .

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 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 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

Figure 6(A) Mcp3 precursor binds in vitro to cytosolic domains of TOM receptors. Radiolabelled Mcp3 was incubated with recombinant GST alone or GST fused to the cytosolic domains of Tom70, Tom20 or Tom22 prebound to glutathione beads or with unloaded beads. After washing bound material was eluted with sample buffer. Proteins were analysed by SDS-PAGE, blotting onto a membrane and autoradiography. To demonstrate equal amounts of GST fusion proteins the membrane was stained with Ponceau S. I, input 5% of precursor used.(B) Mcp3 precursor binds to Tom22 in organello. Radiolabelled Mcp3 was incubated with mitochondria isolated from WT His-Tom22 containing cells. Mitochondria were pre-incubated with CCCP (40 µM) to halt import. After lysis with β-dodecyl maltoside samples were incubated with Ni-NTA beads. After washing bound proteins were eluted with sample buffer and analysed by SDS-PAGE and autoradiography. I, 5% of 35S-Mcp3 used; pel., 2% insoluble material after clarifying spin; sup., 2% of material incubated with Ni-NTA; n.b. 2% of non-bound material after binding to Ni-NTA; eluate, 100% of bound material.(C) Radiolabelled Mcp3 was incubated with organelles described in (B). After import mitochondria were solubilized in digitonin, the lysate was cleared by centrifugation and incubated with or without an antibody against the His-tag (α-His). Samples were analysed by BN-PAGE, autoradiography and afterwards immunodecoration with an antibody against Tom40. Bands shifted by the anti-His antibody are indicated by arrowhead.


Figure 7 ( A ) Import of Mcp3 is dependent on the TIM23 complex . Mitochondria isolated from wild - type and tim23ts cells were incubated with radiolabelled Mcp3 for the indicated time periods . Further treatment and analysis ( n = 3 ; SD ) was as described for Fig . 5D . ( B ) Steady state levels of HA - Mcp3 are lower in cells harbouring a temperature sensitive TIM23 allele . Crude mitochondria were obtained at the non - permissive temperature from WT or tim23ts cells containing a plasmid expressing HA - Mcp3 . Samples were analysed by SDS - PAGE and immunodecoration with antibodies against the HA - tag , the matrix proteins Mge1 and Yah1 as typical TIM23 substrates , Ugo1 and Fis1 as TIM23 - independent substrates . HA - Mcp3 levels were quantified in relation to Fis1 levels . Levels in WT cells were set to 100 % . The bar diagram shows the mean with standard deviation of six independent experiments ( n = 6 ; SD ; * * , p < 0 . 01 ) . ( C ) Import and processing of Mcp3 is dependent on the mitochondrialmembrane potential . Isolated mitochondria were incubated with radiolabelled Mcp3 or pSu9 - DHFR as control for the indicated time periods or for 15 minutes in the presence of either CCCP or o - phenantroline and EDTA ( o - Phen . / EDTA ) . After import mitochondria were reisolated and analysed by SDS - PAGE and autoradiography . L + MPP : radiolabelled proteins were incubated with recombinant MPP . ( D ) Mcp3 is processed by Imp1 / 2 . Single deletion strains of known mitochondrial proteases and peptidases were transformed with a plasmid expressing HA - Mcp3 . Crude mitochondria were isolated and analysed by SDS - PAGE and immunodecoration with antibodies against the indicated proteins and the HA - epitope . Cox2 , a known substrate of IMP ; Tom70 , a mitochondrial outer membrane protein . ( E ) C - terminally tagged Mcp3 - HA is processed by Imp1 . WT and imp1Δ cells were transformed with a plasmid expressing Mcp3 - HA and further analysis was performed as described in ( D ) . ( F ) Mcp3 is processed by Imp1 / 2 after in vitro import into mitochondria . Mitochondria isolated from WT , imp1Δ or imp2Δ strains were incubated with radiolabelled precursor of Mcp3 or pSu9 - DHFR as control . Further treatment and analysis was as described in Fig . 5A . ( G ) Full - length Mcp3 precursor accumulates during import if IMP processing is abolished . Radiolabelled internally HA - tagged Mcp3 without ( HA - Mcp3 ) or with mutation in the Imp1 cleavage site ( D70G + HA ) were incubated with mitochondria as in ( F ) . After import samples were incubated with or without PK ( 20 µg / ml ) . Samples were analysed by SDS - PAGE and autoradiography . ( H ) Mitochondrial membrane potential is required for efficient import of Mcp3 precursor into mitochondria . Isolated mitochondria were incubated with radiolabelled Mcp3 in the presence or absence of CCCP . After import mitochondria were incubated with PK as in ( G ) and analysed by SDS - PAGE and autoradiography . I , 20 % of radiolabelled precursor protein used in each import reaction ; The arrowhead marks an additional band of the size of the cleaved N - terminus of Mcp3 .

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 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 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 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 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 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 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 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

Figure 7(A) Import of Mcp3 is dependent on the TIM23 complex. Mitochondria isolated from wild-type and tim23ts cells were incubated with radiolabelled Mcp3 for the indicated time periods. Further treatment and analysis (n=3; SD) was as described for Fig. 5D.(B) Steady state levels of HA-Mcp3 are lower in cells harbouring a temperature sensitive TIM23 allele. Crude mitochondria were obtained at the non-permissive temperature from WT or tim23ts cells containing a plasmid expressing HA-Mcp3. Samples were analysed by SDS-PAGE and immunodecoration with antibodies against the HA-tag, the matrix proteins Mge1 and Yah1 as typical TIM23 substrates, Ugo1 and Fis1 as TIM23-independent substrates. HA-Mcp3 levels were quantified in relation to Fis1 levels. Levels in WT cells were set to 100%. The bar diagram shows the mean with standard deviation of six independent experiments (n=6; SD; **, p < 0.01).(C) Import and processing of Mcp3 is dependent on the mitochondrialmembrane potential. Isolated mitochondria were incubated with radiolabelled Mcp3 or pSu9-DHFR as control for the indicated time periods or for 15 minutes in the presence of either CCCP or o-phenantroline and EDTA (o-Phen./EDTA). After import mitochondria were reisolated and analysed by SDS-PAGE and autoradiography. L+MPP: radiolabelled proteins were incubated with recombinant MPP.(D) Mcp3 is processed by Imp1/2. Single deletion strains of known mitochondrial proteases and peptidases were transformed with a plasmid expressing HA-Mcp3. Crude mitochondria were isolated and analysed by SDS-PAGE and immunodecoration with antibodies against the indicated proteins and the HA-epitope. Cox2, a known substrate of IMP; Tom70, a mitochondrial outer membrane protein.(E) C-terminally tagged Mcp3-HA is processed by Imp1. WT and imp1Δ cells were transformed with a plasmid expressing Mcp3-HA and further analysis was performed as described in (D).(F) Mcp3 is processed by Imp1/2 after in vitro import into mitochondria. Mitochondria isolated from WT, imp1Δ or imp2Δ strains were incubated with radiolabelled precursor of Mcp3 or pSu9-DHFR as control. Further treatment and analysis was as described in Fig. 5A.(G) Full-length Mcp3 precursor accumulates during import if IMP processing is abolished. Radiolabelled internally HA-tagged Mcp3 without (HA-Mcp3) or with mutation in the Imp1 cleavage site (D70G+HA) were incubated with mitochondria as in (F). After import samples were incubated with or without PK (20 µg/ml). Samples were analysed by SDS-PAGE and autoradiography.(H) Mitochondrial membrane potential is required for efficient import of Mcp3 precursor into mitochondria. Isolated mitochondria were incubated with radiolabelled Mcp3 in the presence or absence of CCCP. After import mitochondria were incubated with PK as in (G) and analysed by SDS-PAGE and autoradiography. I, 20% of radiolabelled precursor protein used in each import reaction; The arrowhead marks an additional band of the size of the cleaved N-terminus of Mcp3.


Figure 8 ( A ) Import of Mcp3 is mediated by the MIM complex . Mitochondria isolated from WT , mim1Δ , or mim2Δ cells were incubated with radiolabelled Mcp3 for the indicated time periods . After import mitochondria were reisolated and analysed by SDS - PAGE and autoradiography . Bands corresponding to the mature ( m ) form were quantified . Import after 15 min into wild - type mitochondria was set to 100 % . The mean with standard deviations is depicted ( n = 3 ; SD ) . I , 20 % of radiolabelled precursor protein used in each import reactionB ) Steady state levels of over - expressed HA - Mcp3 are reduced in cells lacking MIM subunits . Crude mitochondria were obtained from WT cells containing an empty plasmid ( Ø ) and from wild - type , mim1Δ or mim2Δ cells containing a plasmid encoded HA - Mcp3 . Samples were analysed by SDS - PAGE and immunodecoration with antibodies against the HA - tag , Tom20 as MIM substrate and Fis1 as a loading control . HA - Mcp3 levels were quantified in relation to Fis1 levels . Levels in wild - type cells were set to 100 % . The bar diagram shows the mean with standard deviation ( n = 3 ; SD ; * , p < 0 . 05 ; * * , p < 0 . 01 ) . ( C ) Mcp3 precursor binds in vitro to Mim1 . Radiolabelled Mcp3 was incubated with amylose beads harbouring recombinant MBP or MBP fused to Mim1 . After washing proteins were eluted with sample buffer . Analysis was performed by SDS - PAGE , blotting and autoradiography . To demonstrate equal amounts of MBP fusion proteins the membrane was stained with Ponceau S . I , input 5 % of precursor used ; n . b . , 5 % of unbound material ; . b , 100 % of bound material .

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 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 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

Figure 8(A) Import of Mcp3 is mediated by the MIM complex. Mitochondria isolated from WT, mim1Δ, or mim2Δ cells were incubated with radiolabelled Mcp3 for the indicated time periods. After import mitochondria were reisolated and analysed by SDS-PAGE and autoradiography. Bands corresponding to the mature (m) form were quantified. Import after 15 min into wild-type mitochondria was set to 100%. The mean with standard deviations is depicted (n=3; SD). I, 20% of radiolabelled precursor protein used in each import reactionB) Steady state levels of over-expressed HA-Mcp3 are reduced in cells lacking MIM subunits. Crude mitochondria were obtained from WT cells containing an empty plasmid (Ø) and from wild-type, mim1Δ or mim2Δ cells containing a plasmid encoded HA-Mcp3. Samples were analysed by SDS-PAGE and immunodecoration with antibodies against the HA-tag, Tom20 as MIM substrate and Fis1 as a loading control. HA-Mcp3 levels were quantified in relation to Fis1 levels. Levels in wild-type cells were set to 100%. The bar diagram shows the mean with standard deviation (n=3; SD; *, p < 0.05; **, p < 0.01).(C) Mcp3 precursor binds in vitro to Mim1. Radiolabelled Mcp3 was incubated with amylose beads harbouring recombinant MBP or MBP fused to Mim1. After washing proteins were eluted with sample buffer. Analysis was performed by SDS-PAGE, blotting and autoradiography. To demonstrate equal amounts of MBP fusion proteins the membrane was stained with Ponceau S. I, input 5% of precursor used; n.b., 5% of unbound material;.b, 100% of bound material.


Figure 1 ( A ) Comparison of migration and invasion abilities between T - and B - lines . PC3 T - and B - line cells were incubated for migration ( 16h ) and invasion ( 24h ) assay . Data shown represent the means ± SD ( n = 3 biological replicates ) . Student ' s t - test was used for the statistical analysis ( * P < 0 . 05 ; * * P < 0 . 01 ) . ( B ) Comparison of cell proliferation rate between T - and B - lines . Cell proliferation assay was performed by MTS reagents . Data are the means ± SD ( n = 3 biological replicates ) . ( C ) Western blot analysis of the endogenous CTH in PC3 orthotopically implanted primary tumor - derived ( T2 and T3 - lines ) and bone - metastases - derived cancer cells ( B2 and B3 - lines ) . ( D ) Top : H2S catalyzing activity of cell lysates from PC3 T2 , B2 , T3 , and B3 lines . Lead acetate - soaked paper strips show a PbS brown stain as a result of reaction with H2S . Bottom : The level of H2S production was quantified by densitometry , and the histograms represent the means ± SD ( n = 3 biological replicates ) . ANOVA followed by Tukey ' s post - hoc test was used for the statistical analysis ( * P < 0 . 05 ) . ( E , F ) 1x106 PC3 - T2 , B2 , T3 , and B3 cells were orthotopically injected into mouse prostate for 15 - 20 days . Incidences of paraaortic lymph nodes metastasis ( E ) and bone metastasis ( F ) are shown ( n = 6 - 8 mice per group ) . Student ' s t - test was used for the statistical analysis ( * P < 0 . 05 ; * * P < 0 . 01 ) . ( G ) Top : expression of CTH in prostate tumors by immunocytochemistry analysis of commercial tissue arrays . Samples were sub - grouped by TNM stages . The representative images from different stages are shown . S : Stroma ; Scale bars : 250μm . Bottom : The statistical significance was determined using the χ2 testThe RNA - seq data of CTH was downloaded from the TCGA database ( H ) The mRNA expression of CTH in PC specimens and normal tissues . Data are presented as means ± SD ( n = 50 cases for adjancent non - tumor parts and 469 cases for PC tumors ) . Student ' s t - test was used for the statistical analysis ( * * P < 0 . 01 ) . ( I ) Kaplan - Meier survival analysis of PC patients with high or low CTH expression . The statistical significance was determined using the χ2 test .

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 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 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 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 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 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 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 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

Figure 1(A) Comparison of migration and invasion abilities between T- and B- lines. PC3 T- and B-line cells were incubated for migration (16h) and invasion (24h) assay. Data shown represent the means ± SD (n=3 biological replicates). Student's t-test was used for the statistical analysis (*P<0.05; **P<0.01).(B) Comparison of cell proliferation rate between T- and B- lines. Cell proliferation assay was performed by MTS reagents. Data are the means ± SD (n=3 biological replicates).(C) Western blot analysis of the endogenous CTH in PC3 orthotopically implanted primary tumor-derived (T2 and T3-lines) and bone-metastases-derived cancer cells (B2 and B3-lines).(D) Top: H2S catalyzing activity of cell lysates from PC3 T2, B2, T3, and B3 lines. Lead acetate-soaked paper strips show a PbS brown stain as a result of reaction with H2S. Bottom: The level of H2S production was quantified by densitometry, and the histograms represent the means ± SD (n=3 biological replicates). ANOVA followed by Tukey's post-hoc test was used for the statistical analysis (*P<0.05).(E, F) 1x106 PC3-T2, B2, T3, and B3 cells were orthotopically injected into mouse prostate for 15-20 days. Incidences of paraaortic lymph nodes metastasis (E) and bone metastasis (F) are shown (n=6-8 mice per group). Student's t-test was used for the statistical analysis (*P<0.05; **P<0.01).(G) Top: expression of CTH in prostate tumors by immunocytochemistry analysis of commercial tissue arrays. Samples were sub-grouped by TNM stages. The representative images from different stages are shown. S: Stroma; Scale bars: 250μm. Bottom: The statistical significance was determined using the χ2 testThe RNA-seq data of CTH was downloaded from the TCGA database (H) The mRNA expression of CTH in PC specimens and normal tissues. Data are presented as means ± SD (n=50 cases for adjancent non-tumor parts and 469 cases for PC tumors). Student's t-test was used for the statistical analysis (**P<0.01).(I) Kaplan-Meier survival analysis of PC patients with high or low CTH expression. The statistical significance was determined using the χ2 test.


Figure 2 ( A ) Western blot analysis of the CTH level in PC3 - T2 cells with CTH overexpression by pCMV - CTH - HA or PC3 - B2 cells with CTH knockdown by siCTH - 1 . ( B ) Top : The H2S production capacity of cell lysates from PC3 - T2 cells with CTH overexpression by pCMV - CTH - HA or PC3 - B2 cells with CTH knockdown by siCTH - 1 . Lead acetate - soaked paper strips show a PbS brown stain as a result of reaction with H2S . Bottom : The level of H2S production was quantified by densitometry , and the histograms represent the means ± SD ( n = 3 biological replicates ) . ANOVA followed by Tukey ' s post - hoc test was used for the statistical analysis ( * P < 0 . 05 ) . ( C ) PC3 - B2 cells were transfected with control or siCTH - 1 and then were subjected to migration ( 16h ) and invasion ( 24h ) assays . Data information : Data shown represent the means ± SD ( n = 3 biological replicates ) . Student ' s t - test was used for the statistical analysis ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) . ( D ) Cell proliferation rates of PC3 - B2 cells with CTH knockdown . Cell proliferation assay was performed by MTS reagent . Data information : Data shown represent the means ± SD ( n = 3 biological replicates ) . Student ' s t - test was used for the statistical analysis ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) . ( E ) Western blot analysis of the CTH levels in PC3 cells transfected with HA - CTH or pCMV - HA vector control . ( F ) PC3 cells were transfected with HA - CTH or pCMV - HA vector control and then incubated for migration ( 16h ) and invasion assay ( 24h ) . Data information : Data shown represent the means ± SD ( n = 3 biological replicates ) . Student ' s t - test was used for the statistical analysis ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) . ( G ) PC3 cells were pre - treated with 10μM PAG for 16 hrs , and incubated for migration ( 16h ) and invasion assay ( 24h ) in the presence of 10μM PAG . Data information : Data shown represent the means ± SD ( n = 3 biological replicates ) . Student ' s t - test was used for the statistical analysis ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) .

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 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 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 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 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 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

Figure 2(A) Western blot analysis of the CTH level in PC3-T2 cells with CTH overexpression by pCMV-CTH-HA or PC3-B2 cells with CTH knockdown by siCTH-1.(B) Top: The H2S production capacity of cell lysates from PC3-T2 cells with CTH overexpression by pCMV-CTH-HA or PC3-B2 cells with CTH knockdown by siCTH-1. Lead acetate-soaked paper strips show a PbS brown stain as a result of reaction with H2S. Bottom: The level of H2S production was quantified by densitometry, and the histograms represent the means ± SD (n=3 biological replicates). ANOVA followed by Tukey's post-hoc test was used for the statistical analysis (*P<0.05).(C) PC3-B2 cells were transfected with control or siCTH-1 and then were subjected to migration (16h) and invasion (24h) assays. Data information: Data shown represent the means ± SD (n=3 biological replicates). Student's t-test was used for the statistical analysis (*P<0.05; **P<0.01; ***P<0.001).(D) Cell proliferation rates of PC3-B2 cells with CTH knockdown. Cell proliferation assay was performed by MTS reagent. Data information: Data shown represent the means ± SD (n=3 biological replicates). Student's t-test was used for the statistical analysis (*P<0.05; **P<0.01; ***P<0.001).(E) Western blot analysis of the CTH levels in PC3 cells transfected with HA-CTH or pCMV-HA vector control.(F) PC3 cells were transfected with HA-CTH or pCMV-HA vector control and then incubated for migration (16h) and invasion assay (24h). Data information: Data shown represent the means ± SD (n=3 biological replicates). Student's t-test was used for the statistical analysis (*P<0.05; **P<0.01; ***P<0.001).(G) PC3 cells were pre-treated with 10μM PAG for 16 hrs, and incubated for migration (16h) and invasion assay (24h) in the presence of 10μM PAG. Data information: Data shown represent the means ± SD (n=3 biological replicates). Student's t-test was used for the statistical analysis (*P<0.05; **P<0.01; ***P<0.001).


Figure 3 ( A ) Real - Time RT - PCR analysis for IL - 1β mRNA levels in PC3 cells with control or CTH knockdown . Recombinant IL - 1β ( 10ng / ml ) was added for 24h in serum - free medium . Data information : All data shown represent the means ± SD ( n = 3 biological replicates ) . ANOVA followed by Tukey ' s post - hoc test was used for the statistical analysis . ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) . ( B ) ELISA assay for IL - 1β protein level in the culture medium of PC3 cells with control or CTH knockdown . Data information All data shown represent the means ± SD ( n = 3 biological replicates ) . Student ' s t - test ( B ) was used for the statistical analysis . ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) . ( C ) PC3 cells transfected with control or CTH knockdown were incubated for invasion ( 24h ) assay with recombinant IL - 1β ( 10ng / ml ) added to the medium of the lower chamber . Data information : All data shown represent the means ± SD ( n = 3 biological replicates ) . ANOVA followed by Tukey ' s post - hoc test was used for the statistical analysis . ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) . ( D ) PC3 cells transfected with control or CTH siRNA were exposed to IL - 1β ( 20ng / ml ) for 1hr . Subcellular localization of p65 was detected by immunocytochemistry . Nuclei were counterstained with DAPI . The representative images are shown . Scale bars : 25μm . ( E ) The percent of nuclear translocation for p65 was scored by counting the number of nuclear positive stained cells of p65 to the total number of cells in random microscopic fields . Data information : All data shown represent the means ± SD ( n = 3 biological replicates ) . ANOVA followed by Tukey ' s post - hoc test was used for the statistical analysis . ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) . Real - Time RT - PCR analysis for VEGF ( F ) mRNA level in PC3 cells with control or CTH knockdown . Recombinant IL - 1β ( 10ng / ml ) was added for 24h in serum - free medium . Data information : All data shown represent the means ± SD ( n = 3 biological replicates ) . ANOVA followed by Tukey ' s post - hoc test was used for the statistical analysis . ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) . Real - Time RT - PCR analysis for MMP - 13 ( G ) mRNA level in PC3 cells with control or CTH knockdown . Recombinant IL - 1β ( 10ng / ml ) was added for 24h in serum - free medium . Data information : All data shown represent the means ± SD ( n = 3 biological replicates ) . ANOVA followed by Tukey ' s post - hoc test was used for the statistical analysis . ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) .

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 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 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 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 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 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 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

Figure 3(A) Real-Time RT-PCR analysis for IL-1β mRNA levels in PC3 cells with control or CTH knockdown. Recombinant IL-1β (10ng/ml) was added for 24h in serum-free medium. Data information: All data shown represent the means ± SD (n=3 biological replicates). ANOVA followed by Tukey's post-hoc test was used for the statistical analysis. (*P<0.05; **P<0.01; ***P<0.001).(B) ELISA assay for IL-1β protein level in the culture medium of PC3 cells with control or CTH knockdown. Data information All data shown represent the means ± SD (n=3 biological replicates). Student's t-test (B) was used for the statistical analysis. (*P<0.05; **P<0.01; ***P<0.001).(C) PC3 cells transfected with control or CTH knockdown were incubated for invasion (24h) assay with recombinant IL-1β (10ng/ml) added to the medium of the lower chamber. Data information: All data shown represent the means ± SD (n=3 biological replicates). ANOVA followed by Tukey's post-hoc test was used for the statistical analysis. (*P<0.05; **P<0.01; ***P<0.001).(D) PC3 cells transfected with control or CTH siRNA were exposed to IL-1β (20ng/ml) for 1hr. Subcellular localization of p65 was detected by immunocytochemistry. Nuclei were counterstained with DAPI. The representative images are shown. Scale bars: 25μm.(E) The percent of nuclear translocation for p65 was scored by counting the number of nuclear positive stained cells of p65 to the total number of cells in random microscopic fields. Data information: All data shown represent the means ± SD (n=3 biological replicates). ANOVA followed by Tukey's post-hoc test was used for the statistical analysis. (*P<0.05; **P<0.01; ***P<0.001).Real-Time RT-PCR analysis for VEGF (F) mRNA level in PC3 cells with control or CTH knockdown. Recombinant IL-1β (10ng/ml) was added for 24h in serum-free medium. Data information: All data shown represent the means ± SD (n=3 biological replicates). ANOVA followed by Tukey's post-hoc test was used for the statistical analysis. (*P<0.05; **P<0.01; ***P<0.001).Real-Time RT-PCR analysis for MMP-13 (G) mRNA level in PC3 cells with control or CTH knockdown. Recombinant IL-1β (10ng/ml) was added for 24h in serum-free medium. Data information: All data shown represent the means ± SD (n=3 biological replicates). ANOVA followed by Tukey's post-hoc test was used for the statistical analysis. (*P<0.05; **P<0.01; ***P<0.001).


Figure 4 ( A ) PC3 cells were incubated for migration ( 16h ) and invasion ( 24h ) assay . DMEM / 10 % FBS , together with various concentration of NaHS ( 10nM - 1mM ) acting as a chemoattractant Data information Data shown represent the means ± SD ( n = 3 biological replicates ) ANOVA followed by Tukey ' s post - hoc tes was used for the statistical analysis ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) ( B ) PC3 cells transfected with control or CTH siRNA were incubated for invasion ( 24h ) assay . DMEM / 10 % FBS , with or without NaHS ( 1μM ) , acting as a chemoattractant Data information Data shown represent the means ± SD ( n = 3 biological replicates ) ANOVA followed by Tukey ' s post - hoc tes was used for the statistical analysis ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) ( C ) Top : PC3 cells treated with 100 μM NaHS for 30 min at 37 ° C . Cells were lyzed and treated with or without DTT for 10min at 4 ° C before subjecting to the modified biotin switch assay with the antibody against p65 to detect S - sulfhydration . Down : quantitative analysis of SSH - p65 protein level , and normalized with total p65 level Data information Data shown represent the means ± SD ( n = 3 biological replicates ) ANOVA followed by Tukey ' s post - hoc tes was used for the statistical analysis ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) ( D ) PC3 cells were incubate for invasion assay for 24 hrs . DMEM / 10 % FBS , together with 1μM NaHS and an NF - κB inhibitor ( 50μg / ml SN50 , or 100nM QNZ ) , acting as a chemoattractant Data information Data shown represent the means ± SD ( n = 3 biological replicates ) ANOVA followed by Tukey ' s post - hoc tes was used for the statistical analysis ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) ( E ) Real - Time RT - PCR analysis for IL - 1β , VEGF , and MMP - 13 mRNA levels in PC3 cells treated with 1μM NaHS for 24h Data information Data shown represent the means ± SD ( n = 3 biological replicates ) Student ' s t - tes was used for the statistical analysis ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) ( F ) ELISA assay for IL - 1β protein level in the culture medium of PC3 cells with various concentrations of NaHS ( 10nM - 10μM ) added for 24h Data information Data shown represent the means ± SD ( n = 3 biological replicates ) ANOVA followed by Tukey ' s post - hoc tes was used for the statistical analysis ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) ( G ) Western blot analysis of the p65 expression in PC3 cells with p65 knock - out and then transfected with p65 wild - type or C38S mutant Data information Data shown represent the means ± SD ( n = 3 biological replicates ) ( H ) PC3 cells with p65 knock - out with or without overexpression of p65 wild - type or C38S mutant were incubated for invasion assay for 24 hrs . DMEM / 10 % FBS was used as a chemoattractant Data information Data shown represent the means ± SD ( n = 3 biological replicates ) ANOVA followed by Tukey ' s post - hoc tes was used for the statistical analysis ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) 1x106 PC3 cells with p65 knock - out were transiently re - expressed p65 wild - type or C38S mutant and inoculated into the bloodstream via tail - vein injection . After 7 days of inoculation , mouse lung was collected to examine lung metastasis . ( I ) Images show H & E staining of the lungs from mice injected with p65 knockout PC3 cells re - expressing p65 wild - type or C38S mutant . Red arrows indicate metastatic nodules . Scale bar : 1mm1x106 PC3 cells with p65 knock - out were transiently re - expressed p65 wild - type or C38S mutant and inoculated into the bloodstream via tail - vein injection . After 7 days of inoculation , mouse lung was collected to examine lung metastasis The total metastatic nodules per lung ( J were quantified . Data are presented as means ± SEM ( n = 7 mice per group ) . Student ' s t - test was used for the statistical analysis ( * P < 0 . 05 ) 1x106 PC3 cells with p65 knock - out were transiently re - expressed p65 wild - type or C38S mutant and inoculated into the bloodstream via tail - vein injection . After 7 days of inoculation , mouse lung was collected to examine lung metastasis percentage of the metastatic area per lung ( K ) were quantified . Data are presented as means ± SEM ( n = 7 mice per group ) . Student ' s t - test was used for the statistical analysis ( * P < 0 . 05 )

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

Figure 4 (A) PC3 cells were incubated for migration (16h) and invasion (24h) assay. DMEM/10% FBS, together with various concentration of NaHS (10nM-1mM) acting as a chemoattractant Data information Data shown represent the means ± SD (n=3 biological replicates) ANOVA followed by Tukey's post-hoc tes was used for the statistical analysis (*P<0.05; **P<0.01; ***P<0.001) (B) PC3 cells transfected with control or CTH siRNA were incubated for invasion (24h) assay. DMEM/10% FBS, with or without NaHS (1μM), acting as a chemoattractant Data information Data shown represent the means ± SD (n=3 biological replicates) ANOVA followed by Tukey's post-hoc tes was used for the statistical analysis (*P<0.05; **P<0.01; ***P<0.001) (C) Top: PC3 cells treated with 100 μM NaHS for 30 min at 37°C. Cells were lyzed and treated with or without DTT for 10min at 4°C before subjecting to the modified biotin switch assay with the antibody against p65 to detect S-sulfhydration. Down: quantitative analysis of SSH-p65 protein level, and normalized with total p65 level Data information Data shown represent the means ± SD (n=3 biological replicates) ANOVA followed by Tukey's post-hoc tes was used for the statistical analysis (*P<0.05; **P<0.01; ***P<0.001) (D) PC3 cells were incubate for invasion assay for 24 hrs. DMEM/10% FBS, together with 1μM NaHS and an NF-κB inhibitor (50μg/ml SN50, or 100nM QNZ), acting as a chemoattractant Data information Data shown represent the means ± SD (n=3 biological replicates) ANOVA followed by Tukey's post-hoc tes was used for the statistical analysis (*P<0.05; **P<0.01; ***P<0.001) (E) Real-Time RT-PCR analysis for IL-1β, VEGF, and MMP-13 mRNA levels in PC3 cells treated with 1μM NaHS for 24h Data information Data shown represent the means ± SD (n=3 biological replicates) Student's t-tes was used for the statistical analysis (*P<0.05; **P<0.01; ***P<0.001) (F) ELISA assay for IL-1β protein level in the culture medium of PC3 cells with various concentrations of NaHS (10nM-10μM) added for 24h Data information Data shown represent the means ± SD (n=3 biological replicates) ANOVA followed by Tukey's post-hoc tes was used for the statistical analysis (*P<0.05; **P<0.01; ***P<0.001) (G) Western blot analysis of the p65 expression in PC3 cells with p65 knock-out and then transfected with p65 wild-type or C38S mutant Data information Data shown represent the means ± SD (n=3 biological replicates) (H) PC3 cells with p65 knock-out with or without overexpression of p65 wild-type or C38S mutant were incubated for invasion assay for 24 hrs. DMEM/10% FBS was used as a chemoattractant Data information Data shown represent the means ± SD (n=3 biological replicates) ANOVA followed by Tukey's post-hoc tes was used for the statistical analysis (*P<0.05; **P<0.01; ***P<0.001)1x106 PC3 cells with p65 knock-out were transiently re-expressed p65 wild-type or C38S mutant and inoculated into the bloodstream via tail-vein injection. After 7 days of inoculation, mouse lung was collected to examine lung metastasis. (I) Images show H&E staining of the lungs from mice injected with p65 knockout PC3 cells re-expressing p65 wild-type or C38S mutant. Red arrows indicate metastatic nodules. Scale bar: 1mm1x106 PC3 cells with p65 knock-out were transiently re-expressed p65 wild-type or C38S mutant and inoculated into the bloodstream via tail-vein injection. After 7 days of inoculation, mouse lung was collected to examine lung metastasis The total metastatic nodules per lung (J were quantified. Data are presented as means ± SEM (n=7 mice per group). Student's t-test was used for the statistical analysis (*P<0.05)1x106 PC3 cells with p65 knock-out were transiently re-expressed p65 wild-type or C38S mutant and inoculated into the bloodstream via tail-vein injection. After 7 days of inoculation, mouse lung was collected to examine lung metastasis percentage of the metastatic area per lung (K) were quantified. Data are presented as means ± SEM (n=7 mice per group). Student's t-test was used for the statistical analysis (*P<0.05)


Figure 5 ( A ) Western blot analysis of the CTH expression in PC3 cells stably expressing control or CTH shRNA . 1x106 PC3 cells with control or CTH knockdown were orthotopically injected into the mouse prostate for 60 days . The tumor weight ( B ) , and tumor volume ( C ) were compared . Data are presented as means ± SEM ( n = 10 mice per group ) . Data information : Student ' s t - test was used for the statistical analysis ( * P < 0 . 05 ) . 1x106 PC3 cells with control or CTH knockdown were orthotopically injected into the mouse prostate for 60 days . Incidences of paraaortic lymph nodes metastasis ( D ) and bone metastasis ( E ) are shown . Data are presented as means ± SEM ( n = 10 mice per group ) . Data information : Student ' s t - test was used for the statistical analysis ( * P < 0 . 05 ) . ( F ) Real - Time RT - PCR analysis for CTH mRNA levels in PC3 orthotopic tumors from shCon or shCTH group . Data shown represent normalized means ± SEM ( n = 10 mice per group ) . Data information : Student ' s t - test was used for the statistical analysis ( * P < 0 . 05 ) . Immunohistochemical analysis of paraffin - embedded xenograft prostate tumor using CD31 ( G ) antibody . The total vessels number per mm2 was quantified . Scale bars : 0 . 1 mm . Data are presented as means ± SEM ( n = 4 - 5 mice per group ) . Data information : Student ' s t - test was used for the statistical analysis ( * P < 0 . 05 ) . Immunohistochemical analysis of paraffin - embedded xenograft prostate tumor using LYVE - 1 ( H ) antibody . The total vessels number per mm2 was quantified . Scale bars : 0 . 1 mm . Data are presented as means ± SEM ( n = 4 - 5 mice per group ) . Data information : Student ' s t - test was used for the statistical analysis ( * P < 0 . 05 ) .

O O B-PANEL_START 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 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 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 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

Figure 5(A) Western blot analysis of the CTH expression in PC3 cells stably expressing control or CTH shRNA.1x106 PC3 cells with control or CTH knockdown were orthotopically injected into the mouse prostate for 60 days. The tumor weight (B), and tumor volume (C) were compared. Data are presented as means ± SEM (n = 10 mice per group). Data information: Student's t-test was used for the statistical analysis (*P<0.05).1x106 PC3 cells with control or CTH knockdown were orthotopically injected into the mouse prostate for 60 days. Incidences of paraaortic lymph nodes metastasis (D) and bone metastasis (E) are shown. Data are presented as means ± SEM (n = 10 mice per group). Data information: Student's t-test was used for the statistical analysis (*P<0.05).(F) Real-Time RT-PCR analysis for CTH mRNA levels in PC3 orthotopic tumors from shCon or shCTH group. Data shown represent normalized means ± SEM (n=10 mice per group). Data information: Student's t-test was used for the statistical analysis (*P<0.05).Immunohistochemical analysis of paraffin-embedded xenograft prostate tumor using CD31 (G) antibody. The total vessels number per mm2 was quantified. Scale bars: 0.1 mm. Data are presented as means ± SEM (n=4-5 mice per group). Data information: Student's t-test was used for the statistical analysis (*P<0.05).Immunohistochemical analysis of paraffin-embedded xenograft prostate tumor using LYVE-1 (H) antibody. The total vessels number per mm2 was quantified. Scale bars: 0.1 mm. Data are presented as means ± SEM (n=4-5 mice per group). Data information: Student's t-test was used for the statistical analysis (*P<0.05).


Figure 1A Detection of the RBPJ / L3MBTL3 interaction using the yeast two - hybrid ( Y2H ) assay . In this Y2H experiment , RBPJ is fused to the GAL4 DNA - binding ( DB ) domain and L3MBTL3 is fused to the GAL4 activation domain ( AD ) . The DB - RBPJ and AD - L3MBTL3 fusion proteins interact with each other , leading to the activation of the ADE2 and HIS3 reporter genes and allowing yeast cells to grow on selective media lacking adenine or histidine . The six Y2H controls were previously described ( Dreze et al , 2010 ) . The experiment was independently replicated thrice . B Endogenous L3MBTL3 co - purifies specifically with HA - RBPJ but not with HA - EGFP , HA - TBL1X or HA - HEY2 . Immuno - precipitation ( IP ) of HA - tagged RBPJ , EGFP , TBL1X or HEY2 in U87 - MG cells followed by Western blot analyses using HA or L3MBTL3 antibody . The experiment was independently replicated twice . C Endogenous RBPJ co - purifies specifically with HA - L3MBTL3 but not with HA - EGFP , HA - TBL1X or HA - HEY2 . IPs of HA - tagged L3MBTL3 , EGFP , TBL1X or HEY2 in U87 - MG cells followed by Western blot analyses using HA or RBPJ antibody . The experiment was independently replicated twice . EV : Empty Vector control ; WB : Western blot ; IP : immuno - precipitation .

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 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 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

Figure 1A Detection of the RBPJ/L3MBTL3 interaction using the yeast two-hybrid (Y2H) assay. In this Y2H experiment, RBPJ is fused to the GAL4 DNA-binding (DB) domain and L3MBTL3 is fused to the GAL4 activation domain (AD). The DB-RBPJ and AD-L3MBTL3 fusion proteins interact with each other, leading to the activation of the ADE2 and HIS3 reporter genes and allowing yeast cells to grow on selective media lacking adenine or histidine. The six Y2H controls were previously described (Dreze et al, 2010). The experiment was independently replicated thrice.B Endogenous L3MBTL3 co-purifies specifically with HA-RBPJ but not with HA-EGFP, HA-TBL1X or HA-HEY2. Immuno-precipitation (IP) of HA-tagged RBPJ, EGFP, TBL1X or HEY2 in U87-MG cells followed by Western blot analyses using HA or L3MBTL3 antibody. The experiment was independently replicated twice. C Endogenous RBPJ co-purifies specifically with HA-L3MBTL3 but not with HA-EGFP, HA-TBL1X or HA-HEY2. IPs of HA-tagged L3MBTL3, EGFP, TBL1X or HEY2 in U87-MG cells followed by Western blot analyses using HA or RBPJ antibody. The experiment was independently replicated twice. EV: Empty Vector control; WB: Western blot; IP: immuno-precipitation.


Figure 2B L3MBTL3 - Δ ( 1 - 64 ) does not interact with RBPJ . IP of HA - FLAG - tagged RBPJ in the presence of FLAG - tagged L3MBTL3 ( WT or deletion mutants ) in HEK293T cells followed by Western blotting using FLAG antibody . The experiment was independently replicated twice . D Deletion of the BTD domain impairs the RBPJ / L3MBTL3 interaction . IP of HA - tagged L3MBTL3 in the presence of FLAG - tagged RBPJ ( WT and deletion mutants ) in HEK293T cells followed by Western blotting using HA or FLAG antibody . The experiment was independently replicated twice . E RBPJF261R point mutant does not interact with L3MBTL3 . IP of HA - tagged L3MBTL3 in the presence of FLAG - tagged RBPJ ( WT and point mutants ) in HEK293T cells followed by Western blotting using HA or FLAG antibody . RBPJV263R and RBPJA284R also show a reduced ability to interact with L3MBTL3 . The experiment was independently replicated twice . WB : Western blot ; IP : immuno - precipitation .

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 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

Figure 2B L3MBTL3-Δ(1-64) does not interact with RBPJ. IP of HA-FLAG-tagged RBPJ in the presence of FLAG-tagged L3MBTL3 (WT or deletion mutants) in HEK293T cells followed by Western blotting using FLAG antibody. The experiment was independently replicated twice.D Deletion of the BTD domain impairs the RBPJ/L3MBTL3 interaction. IP of HA-tagged L3MBTL3 in the presence of FLAG-tagged RBPJ (WT and deletion mutants) in HEK293T cells followed by Western blotting using HA or FLAG antibody. The experiment was independently replicated twice. E RBPJF261R point mutant does not interact with L3MBTL3. IP of HA-tagged L3MBTL3 in the presence of FLAG-tagged RBPJ (WT and point mutants) in HEK293T cells followed by Western blotting using HA or FLAG antibody. RBPJV263R and RBPJA284R also show a reduced ability to interact with L3MBTL3. The experiment was independently replicated twice. WB: Western blot; IP: immuno-precipitation.


Figure 3A Thermodynamic characterization of the RBPJ / L3MBTL3 interaction . Representative thermograms ( raw heat signal and nonlinear least squares fit to the integrated data ) for L3MBTL3 - ( 31 - 70 ) binding to RBPJ - ( 53 - 474 ) . B / C NOTCH1 ICD outcompetes L3MBTL3 for binding to RBPJ in a dose - dependent manner . IPs were performed in CRISPR / Cas9 - mediated L3MBTL3 knockout ( KO ) HEK293T cells . ( B ) SBP - FLAG - RBPJ and HA - L3MBTL3 - Δ ( SAM ) in the presence of an increasing amount of HA - NOTCH1 ICD . The L3MBTL3 - Δ ( SAM ) mutant construct was used instead of the L3MBTL3 WT construct in order to allow the analysis of both NOTCH1 ICD and L3MBTL3 proteins in the same Western blot . CRISPR / Cas9 sg - L3MBTL3 - resistant plasmids were used to express HA - L3MBTL3 - Δ ( SAM ) . The experiment was independently replicated thrice . WB : Western blot ; IP : immuno - precipitation . B / C NOTCH1 ICD outcompetes L3MBTL3 for binding to RBPJ in a dose - dependent manner . IPs were performed in CRISPR / Cas9 - mediated L3MBTL3 knockout ( KO ) HEK293T cells . ( C ) SBP - FLAG - RBPJ and HA - NOTCH1 ICD in the presence of an increasing amount of HA - L3MBTL3 - Δ ( SAM ) . The L3MBTL3 - Δ ( SAM ) mutant construct was used instead of the L3MBTL3 WT construct in order to allow the analysis of both NOTCH1 ICD and L3MBTL3 proteins in the same Western blot . CRISPR / Cas9 sg - L3MBTL3 - resistant plasmids were used to express HA - L3MBTL3 - Δ ( SAM ) . The experiment was independently replicated thrice . WB : Western blot ; IP : immuno - precipitation .

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 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 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 3A Thermodynamic characterization of the RBPJ/L3MBTL3 interaction. Representative thermograms (raw heat signal and nonlinear least squares fit to the integrated data) for L3MBTL3-(31-70) binding to RBPJ-(53-474).B/C NOTCH1 ICD outcompetes L3MBTL3 for binding to RBPJ in a dose-dependent manner. IPs were performed in CRISPR/Cas9-mediated L3MBTL3 knockout (KO) HEK293T cells. (B) SBP-FLAG-RBPJ and HA-L3MBTL3-Δ(SAM) in the presence of an increasing amount of HA-NOTCH1 ICD. The L3MBTL3-Δ(SAM) mutant construct was used instead of the L3MBTL3 WT construct in order to allow the analysis of both NOTCH1 ICD and L3MBTL3 proteins in the same Western blot. CRISPR/Cas9 sg-L3MBTL3-resistant plasmids were used to express HA-L3MBTL3-Δ(SAM). The experiment was independently replicated thrice. WB: Western blot; IP: immuno-precipitation.B/C NOTCH1 ICD outcompetes L3MBTL3 for binding to RBPJ in a dose-dependent manner. IPs were performed in CRISPR/Cas9-mediated L3MBTL3 knockout (KO) HEK293T cells. (C) SBP-FLAG-RBPJ and HA-NOTCH1 ICD in the presence of an increasing amount of HA-L3MBTL3-Δ(SAM). The L3MBTL3-Δ(SAM) mutant construct was used instead of the L3MBTL3 WT construct in order to allow the analysis of both NOTCH1 ICD and L3MBTL3 proteins in the same Western blot. CRISPR/Cas9 sg-L3MBTL3-resistant plasmids were used to express HA-L3MBTL3-Δ(SAM). The experiment was independently replicated thrice. WB: Western blot; IP: immuno-precipitation.


Figure 4A De - repression of Notch target genes upon RBPJ knockdown . Shown are means ± s . d . of quadruplicate experiments . [ * ] P < 0 . 05 , [ * * ] P < 0 . 01 , NS : Not Significant ; one - way ANOVA model on log - transformed data . Inset : Western blot analysis validates the shRNA - mediated depletion of RBPJ . B De - repression of Notch target genes in L3MBTL3 KO U87 - MG cells . Shown are means ± s . d . of quadruplicate experiments . [ * * ] P < 0 . 01 , NS : Not Significant ; two - sample T - test on log - transformed data . Inset : Western blot analysis validates the CRISPR / Cas9 - mediated KO of L3MBTL3 . C RBPJ and L3MBTL3 co - localize at the proximal Notch - responsive elements of Notch target genes . Shown are means ± s . d . of triplicate ChIP experiments . In panels C , D and F : distance in base pairs ( bp ) relative to the transcriptional start site ( TSS ) is indicated below the gene names . Chrom8 was used as negative control ( NEG ) . D L3MBTL3 occupancy at the proximal Notch - responsive elements of Notch target genes decreases upon RBPJ knockdown . Shown are means ± s . d . of triplicate ChIP experiments . In panels C , D and F : distance in base pairs ( bp ) relative to the transcriptional start site ( TSS ) is indicated below the gene names . Chrom8 was used as negative control ( NEG ) . E The repressive activity of L3MBTL3 at Notch target genes is RBPJ - dependent . Expression analysis of Notch target genes upon RBPJ knockdown and / or overexpression of L3MBTL3 . Shown are means ± s . d . of triplicate experiments . P values were estimated via a one - way ANOVA model on log - transformed data where the difference of differences was tested , which is equivalent to testing the interaction in a two - way ANOVA model . Western blot analysis validates the overexpression of L3MBTL3 and the shRNA - mediated depletion of RBPJ ( Appendix Fig S3E ) . Gene expression analyses of OCT4 was performed as control ( Appendix Fig S3F ) . F L3MBTL3 occupancy at the proximal Notch - responsive elements of Notch target genes is dependent on its RBPJ interaction domain . ChIP analyses of HA - L3MBTL3 WT and HA - L3MBTL3 - Δ ( 1 - 64 ) occupancy at the proximal Notch - responsive elements of Notch target genes . Shown are means ± s . d . of duplicate experiments measured twice each . In panels C , D and F : distance in base pairs ( bp ) relative to the transcriptional start site ( TSS ) is indicated below the gene names . Chrom8 was used as negative control ( NEG ) . G The L3MBTL3 - ( 1 - 64 ) domain is required for the downregulation of HES1 and HEY2 in U87 - MG cells . Expression analysis of Notch target genes upon overexpression of L3MBTL3 WT , L3MBTL3 - Δ ( 1 - 64 ) or LacZ control ( Control ) . Shown are means ± s . d . of triplicate experiments . [ * ] P < 0 . 05 , [ * * ] P < 0 . 01 , NS : Not Significant ; one - way ANOVA model on log - transformed data .

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 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 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 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 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 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 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

Figure 4A De-repression of Notch target genes upon RBPJ knockdown. Shown are means ±s.d. of quadruplicate experiments. [*] P<0.05, [**] P<0.01, NS: Not Significant; one-way ANOVA model on log-transformed data. Inset: Western blot analysis validates the shRNA-mediated depletion of RBPJ.B De-repression of Notch target genes in L3MBTL3 KO U87-MG cells. Shown are means ±s.d. of quadruplicate experiments. [**] P<0.01, NS: Not Significant; two-sample T-test on log-transformed data. Inset: Western blot analysis validates the CRISPR/Cas9-mediated KO of L3MBTL3.C RBPJ and L3MBTL3 co-localize at the proximal Notch-responsive elements of Notch target genes. Shown are means ±s.d. of triplicate ChIP experiments.In panels C, D and F: distance in base pairs (bp) relative to the transcriptional start site (TSS) is indicated below the gene names. Chrom8 was used as negative control (NEG).D L3MBTL3 occupancy at the proximal Notch-responsive elements of Notch target genes decreases upon RBPJ knockdown. Shown are means ±s.d. of triplicate ChIP experiments.In panels C, D and F: distance in base pairs (bp) relative to the transcriptional start site (TSS) is indicated below the gene names. Chrom8 was used as negative control (NEG).E The repressive activity of L3MBTL3 at Notch target genes is RBPJ-dependent. Expression analysis of Notch target genes upon RBPJ knockdown and/or overexpression of L3MBTL3. Shown are means ±s.d. of triplicate experiments. P values were estimated via a one-way ANOVA model on log-transformed data where the difference of differences was tested, which is equivalent to testing the interaction in a two-way ANOVA model. Western blot analysis validates the overexpression of L3MBTL3 and the shRNA-mediated depletion of RBPJ (Appendix Fig S3E). Gene expression analyses of OCT4 was performed as control (Appendix Fig S3F).F L3MBTL3 occupancy at the proximal Notch-responsive elements of Notch target genes is dependent on its RBPJ interaction domain. ChIP analyses of HA-L3MBTL3 WT and HA-L3MBTL3-Δ(1-64) occupancy at the proximal Notch-responsive elements of Notch target genes. Shown are means ±s.d. of duplicate experiments measured twice each.In panels C, D and F: distance in base pairs (bp) relative to the transcriptional start site (TSS) is indicated below the gene names. Chrom8 was used as negative control (NEG).G The L3MBTL3-(1-64) domain is required for the downregulation of HES1 and HEY2 in U87-MG cells. Expression analysis of Notch target genes upon overexpression of L3MBTL3 WT, L3MBTL3-Δ(1-64) or LacZ control (Control). Shown are means ±s.d. of triplicate experiments. [*] P<0.05, [**] P<0.01, NS: Not Significant; one-way ANOVA model on log-transformed data.


Figure 5A The RBPJ / KDM1A interaction is indirect and occurs via L3MBTL3 . IP of HA - KDM1A in the presence of overexpressed V5 - L3MBTL3 or V5 - L3MBTL3 - Δ ( 1 - 64 ) in L3MBTL3 KO U87 - MG cells . CRISPR / Cas9 sg - L3MBTL3 - resistant plasmids were used to overexpress the L3MBTL3 proteins . The experiment was independently replicated twice . B KDM1A occupancy at the proximal Notch - responsive elements of Notch target genes is L3MBTL3 - dependent . ChIP analysis of endogenous KDM1A in L3MBTL3 KO U87 - MG cells . Shown are means ± s . d . of duplicate experiments measured twice each . Panels B , C and D : distance in bp relative to the TSS is indicated below the gene names . Chrom8 was used as negative control ( NEG ) . C KDM1A occupancy at the proximal Notch - responsive elements of Notch target genes is dependent on L3MBTL3 and both its RBPJ interaction and KDM1A interaction domains . ChIP analysis of endogenous KDM1A in L3MBTL3 KO U87 - MG cells upon overexpression of L3MBTL3 , L3MBTL3 - Δ ( 1 - 64 ) or L3MBTL3 - Δ ( SAM ) . Control : empty vector . Shown are means ± s . d . of duplicate experiments measured twice each . Panels B , C and D : distance in bp relative to the TSS is indicated below the gene names . Chrom8 was used as negative control ( NEG ) . D L3MBTL3 , but neither L3MBTL3 - Δ ( 1 - 64 ) nor L3MBTL3 - Δ ( SAM ) , leads to decreasing H3K4me2 at the proximal Notch - responsive element of HES1 . ChIP analysis of H3K4me2 at the proximal Notch - responsive element of HES1 upon overexpression of LacZ control ( Control ) , L3MBTL3 , L3MBTL3 - Δ ( 1 - 64 ) or L3MBTL3 - Δ ( SAM ) in L3MBTL3 KO U87 - MG cells . Shown are means ± s . d . of duplicate experiments measured twice each . P values were estimated via a one - way ANOVA on log - transformed data . Panels B , C and D : distance in bp relative to the TSS is indicated below the gene names . Chrom8 was used as negative control ( NEG ) . E L3MBTL3 , but neither L3MBTL3 - Δ ( 1 - 64 ) nor L3MBTL3 - Δ ( SAM ) , represses HES1 . Expression analysis of HES1 upon overexpression of LacZ control ( Control ) , L3MBTL3 , L3MBTL3 - Δ ( 1 - 64 ) or L3MBTL3 - Δ ( SAM ) mutants in L3MBTL3 KO U87 - MG cells . Shown are means ± s . d . of triplicate experiments . P values were estimated via a one - way ANOVA on log - transformed data . NS : Not Significant . WB : Western blot ; IP : immuno - precipitation . We note that in the context of this experiment , i . e . , in the absence of endogenous L3MBTL3 , the overexpression of L3MBTL3 - Δ ( 1 - 64 ) does not result in the increased expression of HES1 , contrasting with the result obtained in Fig 4G , i . e . , in the presence of endogenous L3MBTL3 . Indeed , as expected , the dominant negative effect of L3MBTL3 - ( 1 - 64 ) on endogenous WT L3MBTL3 ' s ability to repress the expression of Notch target genes can only be observed when WT L3MBTL3 is expressed .

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 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 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 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 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

Figure 5A The RBPJ/KDM1A interaction is indirect and occurs via L3MBTL3. IP of HA-KDM1A in the presence of overexpressed V5-L3MBTL3 or V5-L3MBTL3-Δ(1-64) in L3MBTL3 KO U87-MG cells. CRISPR/Cas9 sg-L3MBTL3-resistant plasmids were used to overexpress the L3MBTL3 proteins. The experiment was independently replicated twice.B KDM1A occupancy at the proximal Notch-responsive elements of Notch target genes is L3MBTL3-dependent. ChIP analysis of endogenous KDM1A in L3MBTL3 KO U87-MG cells. Shown are means ±s.d. of duplicate experiments measured twice each.Panels B, C and D: distance in bp relative to the TSS is indicated below the gene names. Chrom8 was used as negative control (NEG).C KDM1A occupancy at the proximal Notch-responsive elements of Notch target genes is dependent on L3MBTL3 and both its RBPJ interaction and KDM1A interaction domains. ChIP analysis of endogenous KDM1A in L3MBTL3 KO U87-MG cells upon overexpression of L3MBTL3, L3MBTL3-Δ(1-64) or L3MBTL3-Δ(SAM). Control: empty vector. Shown are means ±s.d. of duplicate experiments measured twice each.Panels B, C and D: distance in bp relative to the TSS is indicated below the gene names. Chrom8 was used as negative control (NEG).D L3MBTL3, but neither L3MBTL3-Δ(1-64) nor L3MBTL3-Δ(SAM), leads to decreasing H3K4me2 at the proximal Notch-responsive element of HES1. ChIP analysis of H3K4me2 at the proximal Notch-responsive element of HES1 upon overexpression of LacZ control (Control), L3MBTL3, L3MBTL3-Δ(1-64) or L3MBTL3-Δ(SAM) in L3MBTL3 KO U87-MG cells. Shown are means ±s.d. of duplicate experiments measured twice each. P values were estimated via a one-way ANOVA on log-transformed data.Panels B, C and D: distance in bp relative to the TSS is indicated below the gene names. Chrom8 was used as negative control (NEG).E L3MBTL3, but neither L3MBTL3-Δ(1-64) nor L3MBTL3-Δ(SAM), represses HES1. Expression analysis of HES1 upon overexpression of LacZ control (Control), L3MBTL3, L3MBTL3-Δ(1-64) or L3MBTL3-Δ(SAM) mutants in L3MBTL3 KO U87-MG cells. Shown are means ±s.d. of triplicate experiments. P values were estimated via a one-way ANOVA on log-transformed data. NS: Not Significant. WB: Western blot; IP: immuno-precipitation. We note that in the context of this experiment, i.e., in the absence of endogenous L3MBTL3, the overexpression of L3MBTL3-Δ(1-64) does not result in the increased expression of HES1, contrasting with the result obtained in Fig 4G, i.e., in the presence of endogenous L3MBTL3. Indeed, as expected, the dominant negative effect of L3MBTL3-(1-64) on endogenous WT L3MBTL3's ability to repress the expression of Notch target genes can only be observed when WT L3MBTL3 is expressed.


Figure 6A GST pulldown showing that dL ( 3 ) mbt , the Drosophila homolog of L3MBTL3 , directly interacts with Su ( H ) , the Drosophila homolog of RBPJ . In vitro transcribed and translated dL ( 3 ) mbt or dNotch ICD ( dNotch ICD fragment containing the RAM domain and ANK repeats ) , as positive control , was incubated with bacterially purified GST - Su ( H ) or GST alone pre - bound to GSH beads . Proteins were resolved via SDS - PAGE and signals were acquired via X - ray exposure . The experiment was independently replicated four times . C Snapshot showing the co - localization of dL ( 3 ) mbt and Su ( H ) at the dNotch ( N ) locus . D In the wing imaginal disc , dL ( 3 ) mbt overexpression in the dorso - ventral ( D - V ) boundary results in the downregulation of the Notch target gene cut . Wing discs expressing UAS - GFP ( top panels ) or UAS - HA - dL ( 3 ) mbt ; UAS - GFP ( bottom panels ) under the vg - Gal4 driver at 25 ° C were stained for cut and HA . GFP marks the vg - Gal4 expression domain . Insets below each panel show a closer view of the D - V boundary with yellow arrows marking the regions where HA - dL ( 3 ) mbt is expressed and cut is downregulated . At least 20 discs for each genotype were analyzed . Representative images are shown . Scale bars : 100μm . E The vg - Gal4 - driven HA - dL ( 3 ) mbt overexpression causes a serrated wing ( wing notching ) phenotype . Flies expressing either UAS - GFP or UAS - HA - dL ( 3 ) mbt ; UAS - GFP under vg - Gal4 were reared to adulthood at 25 ° C . P values were estimated by comparing the proportions via a two - proportion Z - test . Scale bars : 200μm . F Functional interaction between lag - 1 / RBPJ and lin - 61 / L3MBTL3 during C . elegans vulva development . Proportion of animals ( n ≥ 100 ) displaying a protruding vulva ( Pvl ) phenotype after RNAi treatment for two generations . Worms were grown at 25 ° C . Shown are means ± s . d of duplicate experiments . EV : Empty Vector control .

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 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 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 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 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

Figure 6A GST pulldown showing that dL(3)mbt, the Drosophila homolog of L3MBTL3, directly interacts with Su(H), the Drosophila homolog of RBPJ. In vitro transcribed and translated dL(3)mbt or dNotch ICD (dNotch ICD fragment containing the RAM domain and ANK repeats), as positive control, was incubated with bacterially purified GST-Su(H) or GST alone pre-bound to GSH beads. Proteins were resolved via SDS-PAGE and signals were acquired via X-ray exposure. The experiment was independently replicated four times.C Snapshot showing the co-localization of dL(3)mbt and Su(H) at the dNotch (N) locus.D In the wing imaginal disc, dL(3)mbt overexpression in the dorso-ventral (D-V) boundary results in the downregulation of the Notch target gene cut. Wing discs expressing UAS-GFP (top panels) or UAS-HA-dL(3)mbt;UAS-GFP (bottom panels) under the vg-Gal4 driver at 25°C were stained for cut and HA. GFP marks the vg-Gal4 expression domain. Insets below each panel show a closer view of the D-V boundary with yellow arrows marking the regions where HA-dL(3)mbt is expressed and cut is downregulated. At least 20 discs for each genotype were analyzed. Representative images are shown. Scale bars: 100μm.E The vg-Gal4-driven HA-dL(3)mbt overexpression causes a serrated wing (wing notching) phenotype. Flies expressing either UAS-GFP or UAS-HA-dL(3)mbt;UAS-GFP under vg-Gal4 were reared to adulthood at 25°C. P values were estimated by comparing the proportions via a two-proportion Z-test. Scale bars: 200μm.F Functional interaction between lag-1/RBPJ and lin-61/L3MBTL3 during C. elegans vulva development. Proportion of animals (n ≥100) displaying a protruding vulva (Pvl) phenotype after RNAi treatment for two generations. Worms were grown at 25°C. Shown are means ±s.d of duplicate experiments. EV: Empty Vector control.


Figure 2 ( A ) Relative share of the three PIKKs in NCS - responsive phosphorylations . ( C ) The profile of a group of substrates targeted by ATM 20 min after neocarzinostatin ( NCS ) addition and by ATR 1 hr after hydroxyurea ( HU ) addition . Box plots depict 23 phosphopeptides measured in two independent biological replicates . The box indicates the range from first to third quartiles , and the central band represents the median . Upper and lower whiskers extend to the maximum and minimum values which are not farther than 1 . 5 times the interquartile range ( IQR ) . ( D ) Western blotting analysis confirming that the ATM substrate , pS824 / KAP1 is targeted by ATR in response to HU . pS345 / CHK1 - an established ATR substrate - served as a positive control for ATR activation .

O O B-PANEL_START 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 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

Figure 2(A) Relative share of the three PIKKs in NCS-responsive phosphorylations.(C) The profile of a group of substrates targeted by ATM 20 min after neocarzinostatin (NCS) addition and by ATR 1 hr after hydroxyurea (HU) addition. Box plots depict 23 phosphopeptides measured in two independent biological replicates. The box indicates the range from first to third quartiles, and the central band represents the median. Upper and lower whiskers extend to the maximum and minimum values which are not farther than 1.5 times the interquartile range (IQR).(D) Western blotting analysis confirming that the ATM substrate, pS824/KAP1 is targeted by ATR in response to HU. pS345/CHK1 - an established ATR substrate - served as a positive control for ATR activation.


Figure 3 ( A ) A cluster of 71 neocarzinostatin ( NCS ) - induced phosphorylations that were enhanced upon continuous inhibition of DNA - PK . Untreated cells are marked UT . This cluster breaks down into two distinct groups according to their ATM dependence 240 min after NCS addition . Clusters were obtained using K - means algorithm implemented in Perseus on Z - scored intensities . Box plots depict 71 phosphopeptides measured in two independent biological replicates . The box indicates the range from first to third quartiles , and the central band represents the median . Upper and lower whiskers extend from the box to the maximum and minimum values which are not farther than 1 . 5 times the interquartile range ( IQR ) . ( B ) Depicted are phosphopeptides that responded to NCS treatment in A - T cells . No significant elevation was observed in these phosphorylations in A - T cells following continuous inhibition of DNA - PK . Box plots depict 71 phosphopeptides measured in two independent biological replicates . The box indicates the range from first to third quartiles , and the central band represents the median . Upper and lower whiskers extend from the box to the maximum and minimum values which are not farther than 1 . 5 time the interquartile range ( IQR ) . ( E ) Western blotting analysis of selected phosphorylations in the above cluster showing DNA - PKi - induced elevation . ( F ) Temporal dynamics of DNA - PK - dependent attenuation of pS824 / KAP - 1 .

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 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 B-PANEL_START 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

Figure 3(A) A cluster of 71 neocarzinostatin (NCS)-induced phosphorylations that were enhanced upon continuous inhibition of DNA-PK. Untreated cells are marked UT. This cluster breaks down into two distinct groups according to their ATM dependence 240 min after NCS addition. Clusters were obtained using K-means algorithm implemented in Perseus on Z-scored intensities. Box plots depict 71 phosphopeptides measured in two independent biological replicates. The box indicates the range from first to third quartiles, and the central band represents the median. Upper and lower whiskers extend from the box to the maximum and minimum values which are not farther than 1.5 times the interquartile range (IQR).(B) Depicted are phosphopeptides that responded to NCS treatment in A-T cells. No significant elevation was observed in these phosphorylations in A-T cells following continuous inhibition of DNA-PK. Box plots depict 71 phosphopeptides measured in two independent biological replicates. The box indicates the range from first to third quartiles, and the central band represents the median. Upper and lower whiskers extend from the box to the maximum and minimum values which are not farther than 1.5 time the interquartile range (IQR).(E) Western blotting analysis of selected phosphorylations in the above cluster showing DNA-PKi-induced elevation.(F) Temporal dynamics of DNA-PK-dependent attenuation of pS824/KAP-1.


Figure 4 ( C ) The first time points at which ' compensated sites ' responded to NCS in WT and A - T cells are presented . ( D ) Temporal kinase dependencies of the ' compensated sites ' in A - T cells . Shown are the numbers of ' compensated sites ' in A - T cells , which depended on each kinase at the 20 and 240 min time points . The early ones depend mainly on DNA - PK , while the later ones - on ATR . ( F ) Western blotting analysis confirming high - throughput results for selected ATM substrates .

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Figure 4(C) The first time points at which 'compensated sites' responded to NCS in WT and A-T cells are presented.(D) Temporal kinase dependencies of the 'compensated sites' in A-T cells. Shown are the numbers of 'compensated sites' in A-T cells, which depended on each kinase at the 20 and 240 min time points. The early ones depend mainly on DNA-PK, while the later ones - on ATR.(F) Western blotting analysis confirming high-throughput results for selected ATM substrates.


Figure 5 ( A ) A group of exclusively ATM - dependent sites that were ATM - dependent one hr after NCS addition , were still phosphorylated or dephosphorylated 4 hr after treatment , but were not affected by ATMi at that time point . Box plots depict 18 phosphopeptides measured in two independent biological replicates . The box indicates the range from first to third quartiles , and the central band represents the median . Upper and lower whiskers extend from the box to the maximum and minimum values which are not farther than 1 . 5 times the interquartile range ( IQR ) . ( B ) This group was enriched for sites that were compensated in an ATR - dependent manner in A - T cells . Box plots depict 9 phosphopeptides measured in two independent biological replicates . The box indicates the range from first to third quartiles , and the central band represents the median . Upper and lower whiskers extend from the box to the maximum and minimum values which are not farther than 1 . 5 times the interquartile range ( IQR ) . ( C ) Western blotting analysis of pS343 / NBS1 , which is compensated upon ATM inhibition , and pS824 / KAP - 1 and pS395 / NUMA , which are not compensated under these conditions .

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 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 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

Figure 5(A) A group of exclusively ATM-dependent sites that were ATM-dependent one hr after NCS addition, were still phosphorylated or dephosphorylated 4 hr after treatment, but were not affected by ATMi at that time point. Box plots depict 18 phosphopeptides measured in two independent biological replicates. The box indicates the range from first to third quartiles, and the central band represents the median. Upper and lower whiskers extend from the box to the maximum and minimum values which are not farther than 1.5 times the interquartile range (IQR).(B) This group was enriched for sites that were compensated in an ATR-dependent manner in A-T cells. Box plots depict 9 phosphopeptides measured in two independent biological replicates. The box indicates the range from first to third quartiles, and the central band represents the median. Upper and lower whiskers extend from the box to the maximum and minimum values which are not farther than 1.5 times the interquartile range (IQR).(C) Western blotting analysis of pS343/NBS1, which is compensated upon ATM inhibition, and pS824/ KAP-1 and pS395/NUMA, which are not compensated under these conditions.