Figure 3 ( A ) HEK293 cells stably expressing TAP - vector , TAP - CSAG1 , or TAP - CSAG2 were subjected to pull - down followed by SDS - PAGE and LC - MS / MS . Spectral counts ( SC ) of the identified proteins in TAP - CSAG1 and TAP - CSAG2 pulldowns , but absence in TAP - vector control , are shown . ( B ) HEK293FT cells stably expressing HA - SIRT1 were transfected with Myc - CSAG1 or Myc - CSAG2 for 48 h before IP with anti - HA followed by SDS - PAGE and immunoblotting for indicated proteins . ( C ) Recombinant GST - SIRT1 , but not GST , binds in vitro translated Myc - CSAG2 . Myc - tagged CSAG2 was in vitro translated and GST - pulldown experiments were performed with recombinant GST or GST - SIRT1 followed by SDS - PAGE and immunoblotting for anti - Myc . Expression of CSAG2 decreased p53 ac - K382 levels and downregulated the expression of downstream p53 target genes in H460 cells . Cells were treated by either 1 μM doxorubicin ( D - E ) with 0 . 4 μM TSA for the indicated times . Cells were then harvested and blotted for the indicated proteins . Quantitation of expression levels is shown ( E Ac - p53 K382 levels normalized to total p53 , whereas all other proteins were normalized to GAPDH . Expression of CSAG2 decreased p53 ac - K382 levels and downregulated the expression of downstream p53 target genes in H460 cells . Cells were treated by 20 μM etoposide with 0 . 4 μM TSA for the indicated times . Ac - p53 K382 levels normalized to total p53 , whereas all other proteins were normalized to GAPDH . Expression of CSAG2 decreased p53 ac - K382 levels and downregulated the expression of downstream p53 target genes in H460 cells . Cells were treated by 20 μM etoposide with 0 . 4 μM TSA for the indicated times . Cells were then harvested and blotted for the indicated proteins . Quantitation of expression levels is shown G ) as the mean ± SD , n = 3 biological replicates . Ac - p53 K382 levels normalized to total p53 , whereas all other proteins were normalized to GAPDH . Knockdown of endogenous CSAG2 in HCT116 ( H - I ) cells increased p53 ac - K382 levels . CSAG2 - knockdown stable cells were treated by 1 μM doxorubicin / 0 . 4 μM TSA for 6 hr . Cells were then harvested and blotted for the indicated proteins . Quantitation of ac - p53 K382 levels relative to total p53 is shown ( I as the mean ± SD , n = 3 biological replicates . Knockdown of endogenous CSAG2 in A375 cells increased p53 ac - K382 levels . CSAG2 - knockdown stable cells were treated by 1 μM doxorubicin / 0 . 4 μM TSA for 6 hr . Cells were then harvested and blotted for the indicated proteins . Knockdown of endogenous CSAG2 in A375 cells increased p53 ac - K382 levels . CSAG2 - knockdown stable cells were treated by 1 μM doxorubicin / 0 . 4 μM TSA for 6 hr . Cells were then harvested and blotted for the indicated proteins . Quantitation of ac - p53 K382 levels relative to total p53 is shown K ) as the mean ± SD , n = 3 biological replicates . ( L - M ) Re - expression of CSAG2 rescued p53 ac - K382 levels in CSAG2 knockdown stable cell lines . Myc - CSAG2 was transfected in HCT116 ( L ) or A375 ( M ) CSAG2 knockdown stable cell lines . Cells were treated with 1 μM doxorubicin / 0 . 4 μM TSA for 6 hr and then blotted for the indicated proteins . Quantitation of ac - p53 K382 levels relative to total p53 is shown as the mean ± SD , n = 3 biological replicates . ( N ) CSAG2 does not enhance anchorage - independent growth of HCT116 p53 - null cells . CSAG2 was stably expressed in isogenic HCT116 p53 - wild - type or p53 - null cells . Anchorage - independent growth in soft agar colony formation assays was determined after 14 days . The number of colonies was quantified , normalized to each vector control cell line , and is shown as the mean ± SD , n = 3 biological replicates .

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

Figure 3(A) HEK293 cells stably expressing TAP-vector, TAP-CSAG1, or TAP-CSAG2 were subjected to pull-down followed by SDS-PAGE and LC-MS/MS. Spectral counts (SC) of the identified proteins in TAP-CSAG1 and TAP-CSAG2 pulldowns, but absence in TAP-vector control, are shown.(B) HEK293FT cells stably expressing HA-SIRT1 were transfected with Myc-CSAG1 or Myc-CSAG2 for 48 h before IP with anti-HA followed by SDS-PAGE and immunoblotting for indicated proteins.(C) Recombinant GST-SIRT1, but not GST, binds in vitro translated Myc-CSAG2. Myc-tagged CSAG2 was in vitro translated and GST-pulldown experiments were performed with recombinant GST or GST-SIRT1 followed by SDS-PAGE and immunoblotting for anti-Myc.Expression of CSAG2 decreased p53 ac-K382 levels and downregulated the expression of downstream p53 target genes in H460 cells. Cells were treated by either 1 μM doxorubicin (D-E) with 0.4 μM TSA for the indicated times. Cells were then harvested and blotted for the indicated proteins. Quantitation of expression levels is shown (E Ac-p53 K382 levels normalized to total p53, whereas all other proteins were normalized to GAPDH.Expression of CSAG2 decreased p53 ac-K382 levels and downregulated the expression of downstream p53 target genes in H460 cells. Cells were treated by 20 μM etoposide with 0.4 μM TSA for the indicated times. Ac-p53 K382 levels normalized to total p53, whereas all other proteins were normalized to GAPDH.Expression of CSAG2 decreased p53 ac-K382 levels and downregulated the expression of downstream p53 target genes in H460 cells. Cells were treated by 20 μM etoposide with 0.4 μM TSA for the indicated times. Cells were then harvested and blotted for the indicated proteins. Quantitation of expression levels is shown G) as the mean ± SD, n=3 biological replicates. Ac-p53 K382 levels normalized to total p53, whereas all other proteins were normalized to GAPDH.Knockdown of endogenous CSAG2 in HCT116 (H-I) cells increased p53 ac-K382 levels. CSAG2-knockdown stable cells were treated by 1 μM doxorubicin/ 0.4 μM TSA for 6 hr. Cells were then harvested and blotted for the indicated proteins. Quantitation of ac-p53 K382 levels relative to total p53 is shown (I as the mean ± SD, n=3 biological replicates.Knockdown of endogenous CSAG2 in A375 cells increased p53 ac-K382 levels. CSAG2-knockdown stable cells were treated by 1 μM doxorubicin/ 0.4 μM TSA for 6 hr. Cells were then harvested and blotted for the indicated proteins.Knockdown of endogenous CSAG2 in A375 cells increased p53 ac-K382 levels. CSAG2-knockdown stable cells were treated by 1 μM doxorubicin/ 0.4 μM TSA for 6 hr. Cells were then harvested and blotted for the indicated proteins. Quantitation of ac-p53 K382 levels relative to total p53 is shown K) as the mean ± SD, n=3 biological replicates.(L-M) Re-expression of CSAG2 rescued p53 ac-K382 levels in CSAG2 knockdown stable cell lines. Myc-CSAG2 was transfected in HCT116 (L) or A375 (M) CSAG2 knockdown stable cell lines. Cells were treated with 1 μM doxorubicin/ 0.4 μM TSA for 6 hr and then blotted for the indicated proteins. Quantitation of ac-p53 K382 levels relative to total p53 is shown as the mean ± SD, n=3 biological replicates.(N) CSAG2 does not enhance anchorage-independent growth of HCT116 p53-null cells. CSAG2 was stably expressed in isogenic HCT116 p53-wild-type or p53-null cells. Anchorage-independent growth in soft agar colony formation assays was determined after 14 days. The number of colonies was quantified, normalized to each vector control cell line, and is shown as the mean ± SD, n=3 biological replicates.


Figure 4 ( A ) Knockdown of SIRT1 abolished CSAG2 - mediated decrease in ac - p53 K382 . CSAG2 over - expressing HCT116 were transfected with SIRT1 siRNA for 72 hr before doxorubicin / TSA treatment for 6 hr . Cell lysates were blotted for the indicated proteins . ( B ) Inhibition of SIRT1 enzymatic activity blocked CSAG2 - induced decrease in p53 ac - K382 . CSAG2 over - expressing HCT116 cells were treated with 1 μM doxorubicin / 0 . 4 μM TSA with or without 1 μM EX - 527 for 6 hr . Cell lysates were blotted for the indicated proteins . Inhibition of SIRT1 enzymatic activity prevents CSAG2 - knockdown increase in p53 ac - K382 . CSAG2 - knockdown stable cells were treated by 1 μM doxorubicin / 0 . 4 μM TSA with or without 1 μM EX - 527 for 6 hr . Cells were then harvested and blotted for the indicated proteins ( C ) Inhibition of SIRT1 enzymatic activity prevents CSAG2 - knockdown increase in p53 ac - K382 . CSAG2 - knockdown stable cells were treated by 1 μM doxorubicin / 0 . 4 μM TSA with or without 1 μM EX - 527 for 6 hr . Cells were then harvested and blotted for the indicated proteins and quantitated ( D ; n = 3 biological replicates ) . Data shown as mean ± SD . ( E ) CSAG2 binds catalytically inactive SIRT1 H363Y . Cells were transfected with the indicated constructs before anti - Myc IP and blotting for the indicated proteins . ( F ) SIRT1 H363Y blocks CSAG2 regulation of p53 ac - K382 levels . HCT116 cells were transfected with the indicated constructs and blotted for the indicated proteins . ( G ) Inhibition of SIRT1 activity abolished CSAG2 - induced anchorage - independent growth . Soft agar colony formation assays were performed in control or CSAG2 over - expressing HCT116 cells with or without EX - 527 treatment . Data are represented as the mean ± SD , n = 3 biological replicates . ( H ) Summary of region on CSAG2 required for SIRT1 interaction . HEK293FT cells stably expressing HA - SIRT1 were transfected with indicated CSAG2 constructs for 48 h before IP with anti - HA followed by SDS - PAGE and immunoblotting for anti - Myc . ( I ) CSAG2 Δ37 mutant fails to interact with SIRT1 in vitro . Myc - tagged CSAG2 38 - 127 fragment was in vitro translated and followed by in vitro binding assay with recombinant GST - SIRT1 , SDS - PAGE , and immunoblotting for anti - Myc . ( J ) CSAG2 Δ37 mutant does not regulate p53 ac - K382 levels . Cells were transfected with the indicated Myc - CSAG2 constructs for 48 hr before being treated with 1 μM doxorubicin / 0 . 4 μM TSA . Cell lysates were blotted for the indicated proteins . ( K ) CSAG2 Δ37 fails to promote anchorage - independent growth of H460 cells . Soft agar colony formation assays were performed in CSAG2 - negative H460 cells stably expressing CSAG2 wild - type or CSAG2 Δ37 mutant ( n = 3 biological replicates ) . Data are represented as the mean ± SD .

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Figure 4(A) Knockdown of SIRT1 abolished CSAG2-mediated decrease in ac-p53 K382. CSAG2 over-expressing HCT116 were transfected with SIRT1 siRNA for 72 hr before doxorubicin/TSA treatment for 6 hr. Cell lysates were blotted for the indicated proteins.(B) Inhibition of SIRT1 enzymatic activity blocked CSAG2-induced decrease in p53 ac-K382. CSAG2 over-expressing HCT116 cells were treated with 1 μM doxorubicin/ 0.4 μM TSA with or without 1 μM EX-527 for 6 hr. Cell lysates were blotted for the indicated proteins.Inhibition of SIRT1 enzymatic activity prevents CSAG2-knockdown increase in p53 ac-K382. CSAG2-knockdown stable cells were treated by 1 μM doxorubicin/ 0.4 μM TSA with or without 1 μM EX-527 for 6 hr. Cells were then harvested and blotted for the indicated proteins (C)Inhibition of SIRT1 enzymatic activity prevents CSAG2-knockdown increase in p53 ac-K382. CSAG2-knockdown stable cells were treated by 1 μM doxorubicin/ 0.4 μM TSA with or without 1 μM EX-527 for 6 hr. Cells were then harvested and blotted for the indicated proteins and quantitated (D; n=3 biological replicates). Data shown as mean ± SD.(E) CSAG2 binds catalytically inactive SIRT1 H363Y. Cells were transfected with the indicated constructs before anti-Myc IP and blotting for the indicated proteins.(F) SIRT1 H363Y blocks CSAG2 regulation of p53 ac-K382 levels. HCT116 cells were transfected with the indicated constructs and blotted for the indicated proteins.(G) Inhibition of SIRT1 activity abolished CSAG2-induced anchorage-independent growth. Soft agar colony formation assays were performed in control or CSAG2 over-expressing HCT116 cells with or without EX-527 treatment. Data are represented as the mean ± SD, n=3 biological replicates.(H) Summary of region on CSAG2 required for SIRT1 interaction. HEK293FT cells stably expressing HA-SIRT1 were transfected with indicated CSAG2 constructs for 48 h before IP with anti-HA followed by SDS-PAGE and immunoblotting for anti-Myc.(I) CSAG2 Δ37 mutant fails to interact with SIRT1 in vitro. Myc-tagged CSAG2 38-127 fragment was in vitro translated and followed by in vitro binding assay with recombinant GST-SIRT1, SDS-PAGE, and immunoblotting for anti-Myc.(J) CSAG2 Δ37 mutant does not regulate p53 ac-K382 levels. Cells were transfected with the indicated Myc-CSAG2 constructs for 48 hr before being treated with 1 μM doxorubicin/ 0.4 μM TSA. Cell lysates were blotted for the indicated proteins.(K) CSAG2 Δ37 fails to promote anchorage-independent growth of H460 cells. Soft agar colony formation assays were performed in CSAG2-negative H460 cells stably expressing CSAG2 wild-type or CSAG2 Δ37 mutant (n=3 biological replicates). Data are represented as the mean ± SD.


Figure 5Control or CSAG2 expressing H460 cells were treated with the indicated concentrations of doxorubicin ( A ) for 24 hr before cell viability was determined by alamarBlue ( n = 6 biological replicates ) . Control or CSAG2 expressing H460 cells were treated with the indicated concentrations of H2O2 ( B ) for 24 hr before cell viability was determined by alamarBlue ( n = 6 biological replicates ) . shCTRL , shCSAG2 - 1 , or shCSAG2 - 2 stable HCT116 cells were treated with the indicated concentrations of doxorubicin ( C ) for 24 hr before cell viability was determined by alamarBlue ( n = 6 biological replicates ) . shCTRL , shCSAG2 - 1 , or shCSAG2 - 2 stable HCT116 cells were treated with the indicated concentrations of H2O2 ( D ) for 24 hr before cell viability was determined by alamarBlue ( n = 6 biological replicates ) . ( E - F ) HCT116 cells were transiently transfected with the indicated shRNAs for 48 hr before treatment with 1 μM doxorubicin / 0 . 4 μM TSA for 6 hr . Cell lysates were collected and blotted for the indicated proteins ( E ) . Quantitation ( n = 3 biological replicates ) of normalized cleaved PARP levels is shown ( F ) . ( G ) H460 cells were transfected with wild type , Δ37 , or Δ63 CSAG2 for 48 hr . Cells were then treated with the indicated concentrations of H2O2 for 24 hr before cell viability was determined by alamarBlue ( n = 6 biological replicates ) . ( H ) Inhibition of SIRT1 activity abolished CSAG2 induced cell survival under genotoxic stress . CSAG2 over - expressing H460 cells were treated with the indicated concentrations of H2O2 with or without 1 μM EX - 527 for 24 hr before cell viability was determined by alamarBlue ( n = 6 biological replicates ) . Control or Myc - CSAG2 expressing HCT116 p53 - / - cells were treated with the indicated concentrations of H2O2 ( I ) for 24 hr before cell viability was determined by alamarBlue ( n = 6 biological replicates ) . Control or Myc - CSAG2 expressing HCT116 p53 - / - cells were treated with the indicated concentrations of doxorubicin ( J ) for 24 hr before cell viability was determined by alamarBlue ( n = 6 biological replicates ) . shCTRL , shCSAG2 - 1 , or shCSAG2 - 2 stable HCT116 p53 - / - cells were treated with the indicated concentrations of H2O2 ( K ) for 24 hr before cell viability was determined by alamarBlue ( n = 6 biological replicates ) . shCTRL , shCSAG2 - 1 , or shCSAG2 - 2 stable HCT116 p53 - / - cells were treated with the indicated concentrations of doxorubicin ( L ) for 24 hr before cell viability was determined by alamarBlue ( n = 6 biological replicates ) . Control or Myc - CSAG2 expressing H1299 ( p53 mutant ) cells were treated with the indicated concentrations of H2O2 ( M ) for 24 hr before cell viability was determined by alamarBlue ( n = 6 biological replicates ) . Control or Myc - CSAG2 expressing H1299 ( p53 mutant ) cells were treated with the indicated concentrations of doxorubicin ( N ) for 24 hr before cell viability was determined by alamarBlue ( n = 6 biological replicates ) .

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Figure 5Control or CSAG2 expressing H460 cells were treated with the indicated concentrations of doxorubicin (A) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).Control or CSAG2 expressing H460 cells were treated with the indicated concentrations of H2O2 (B) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).shCTRL, shCSAG2-1, or shCSAG2-2 stable HCT116 cells were treated with the indicated concentrations of doxorubicin (C) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).shCTRL, shCSAG2-1, or shCSAG2-2 stable HCT116 cells were treated with the indicated concentrations of H2O2 (D) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).(E-F) HCT116 cells were transiently transfected with the indicated shRNAs for 48 hr before treatment with 1 μM doxorubicin/ 0.4 μM TSA for 6 hr. Cell lysates were collected and blotted for the indicated proteins (E). Quantitation (n=3 biological replicates) of normalized cleaved PARP levels is shown (F).(G) H460 cells were transfected with wild type, Δ37, or Δ63 CSAG2 for 48 hr. Cells were then treated with the indicated concentrations of H2O2 for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).(H) Inhibition of SIRT1 activity abolished CSAG2 induced cell survival under genotoxic stress. CSAG2 over-expressing H460 cells were treated with the indicated concentrations of H2O2 with or without 1 μM EX-527 for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).Control or Myc-CSAG2 expressing HCT116 p53 -/- cells were treated with the indicated concentrations of H2O2 (I) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).Control or Myc-CSAG2 expressing HCT116 p53 -/- cells were treated with the indicated concentrations of doxorubicin (J) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).shCTRL, shCSAG2-1, or shCSAG2-2 stable HCT116 p53 -/- cells were treated with the indicated concentrations of H2O2 (K) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).shCTRL, shCSAG2-1, or shCSAG2-2 stable HCT116 p53 -/- cells were treated with the indicated concentrations of doxorubicin (L) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).Control or Myc-CSAG2 expressing H1299 (p53 mutant) cells were treated with the indicated concentrations of H2O2 (M) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).Control or Myc-CSAG2 expressing H1299 (p53 mutant) cells were treated with the indicated concentrations of doxorubicin (N) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).


Figure 6 ( A ) CSAG2 does not alter SIRT1 protein levels . HEK293FT cells were transfected with different amounts of Myc - CSAG2 for 48 hr before cells lysates were harvested and immunoblotted for indicated proteins . ( B ) CSAG2 does not alter SIRT1 subcellular location . U2OS cells were transfected with or without mcherry - CSAG2 for 48 hr before fixation , SIRT1 immunostaining , and imaging . Scale bar : 10 µm . ( C ) Summary of results mapping interaction region of SIRT1 recognized by CSAG2 . HEK293FT cells stably expressing HA - CSAG2 were transfected with indicated Myc - SIRT1 constructs for 48 h before IP with anti - HA followed by SDS - PAGE and immunoblotting for anti - Myc . ( D ) CSAG2 directly binds SIRT1 catalytic domain in vitro . HA - tagged SIRT1 fragments were in vitro translated . In vitro binding assays with recombinant GST or GST - CSAG2 were performed followed by SDS - PAGE and immunoblotting for anti - HA . ( E ) CSAG2 enhances SIRT1 activity in vitro . In vitro SIRT1 activity towards p53 ac - K382 peptide was determined in the presence of GST or GST - CSAG2 , n = 3 biological replicates . Nicotinamide ( 1 mM ) was added to the reaction were indicated to block SIRT1 activity . ( F ) CSAG2 Δ37 mutant that does not bind SIRT1 does not enhance SIRT1 activity in vitro . In vitro SIRT1 activity towards p53 ac - K382 peptide was determined in the presence of GST , GST - CSAG2 or GST - CSAG2 38 - 127 , n = 3 biological replicates . ( G ) CSAG2 enhances SIRT1 deacetylation of p53 ac - K382 peptide through increasing kcat , but not Km . not affecting the binding ability with its substrate p53 . SIRT1 activity assays were performed as described above , except varying amounts of p53 ac - K382 fluorometric peptide were used in the presence of saturating NAD + ( 3 mM ) , n = 3 biological replicates . ( H - I ) U2OS cells were transfected with Myc - Vector or Myc - CSAG2 for 48 hr before being treated with or without 0 . 4 μM TSA for 6 hr . Cell lysates were blotted for the indicated proteins ( H ) . Quantitation of expression levels of acetylated Histone relative to total Histone is shown as the mean ± SD , n = 3 biological replicates . White circles indicate Myc - Vector and black circles indicate Myc - CSAG2 . ( J ) HeLa , U2OS , or HCT116 cells were transfected with Myc - Vector or Myc - CSAG2 for 48 hr . Cell lysates were blotted for the indicated proteins . Quantitation of expression levels of acetylated Histone relative to total Histone is shown as the mean ± SD , n = 3 biological replicates . White circles indicate Myc - Vector and black circles indicate Myc - CSAG2 . ( K ) Knockdown of endogenous CSAG2 in A375 cells increased ac - H4K16 levels . Cell lysates were blotted for the indicated proteins . Quantitation of expression levels of ac - H4K16 relative to total H4 is shown as the mean ± SD , n = 3 biological replicates .

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 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 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 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 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 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 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

Figure 6(A) CSAG2 does not alter SIRT1 protein levels. HEK293FT cells were transfected with different amounts of Myc-CSAG2 for 48 hr before cells lysates were harvested and immunoblotted for indicated proteins.(B) CSAG2 does not alter SIRT1 subcellular location. U2OS cells were transfected with or without mcherry-CSAG2 for 48 hr before fixation, SIRT1 immunostaining, and imaging. Scale bar: 10 µm.(C) Summary of results mapping interaction region of SIRT1 recognized by CSAG2. HEK293FT cells stably expressing HA-CSAG2 were transfected with indicated Myc-SIRT1 constructs for 48 h before IP with anti-HA followed by SDS-PAGE and immunoblotting for anti-Myc.(D) CSAG2 directly binds SIRT1 catalytic domain in vitro. HA-tagged SIRT1 fragments were in vitro translated. In vitro binding assays with recombinant GST or GST-CSAG2 were performed followed by SDS-PAGE and immunoblotting for anti-HA.(E) CSAG2 enhances SIRT1 activity in vitro. In vitro SIRT1 activity towards p53 ac-K382 peptide was determined in the presence of GST or GST-CSAG2, n=3 biological replicates. Nicotinamide (1 mM) was added to the reaction were indicated to block SIRT1 activity.(F) CSAG2 Δ37 mutant that does not bind SIRT1 does not enhance SIRT1 activity in vitro. In vitro SIRT1 activity towards p53 ac-K382 peptide was determined in the presence of GST, GST-CSAG2 or GST-CSAG2 38-127, n=3 biological replicates.(G) CSAG2 enhances SIRT1 deacetylation of p53 ac-K382 peptide through increasing kcat, but not Km. not affecting the binding ability with its substrate p53. SIRT1 activity assays were performed as described above, except varying amounts of p53 ac-K382 fluorometric peptide were used in the presence of saturating NAD+ (3 mM), n=3 biological replicates.(H-I) U2OS cells were transfected with Myc-Vector or Myc-CSAG2 for 48 hr before being treated with or without 0.4 μM TSA for 6 hr. Cell lysates were blotted for the indicated proteins (H). Quantitation of expression levels of acetylated Histone relative to total Histone is shown as the mean ± SD, n=3 biological replicates. White circles indicate Myc-Vector and black circles indicate Myc-CSAG2.(J) HeLa, U2OS, or HCT116 cells were transfected with Myc-Vector or Myc-CSAG2 for 48 hr. Cell lysates were blotted for the indicated proteins. Quantitation of expression levels of acetylated Histone relative to total Histone is shown as the mean ± SD, n=3 biological replicates. White circles indicate Myc-Vector and black circles indicate Myc-CSAG2.(K) Knockdown of endogenous CSAG2 in A375 cells increased ac-H4K16 levels. Cell lysates were blotted for the indicated proteins. Quantitation of expression levels of ac-H4K16 relative to total H4 is shown as the mean ± SD, n=3 biological replicates.


Figure 1 ( D , E ) Change in pO2 of osteoclasts in the mice upon exposure to various concentration of oxygen from 21 % to 14 % pO2 . Magnified PLIM images of osteoclasts under different peripheral oxygen saturation ( SpO2 , D ) . The pO2 of each osteoclast in these mice was plotted ( E , n = 47 from three mice for each SpO2 ) . Scale bar , 20 μm . ( F ) Effect of physioxia ( 5 % pO2 ) or physiological hypoxia ( 2 % pO2 ) on osteoclastogenesis . TRAP - stained cells ( left panel ) and the number of TRAP - positive cells with more than three nuclei ( right ) . Scale bar , 100 μm .

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 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

Figure 1(D, E) Change in pO2 of osteoclasts in the mice upon exposure to various concentration of oxygen from 21% to 14% pO2. Magnified PLIM images of osteoclasts under different peripheral oxygen saturation (SpO2, D). The pO2 of each osteoclast in these mice was plotted (E, n = 47 from three mice for each SpO2). Scale bar, 20 μm.(F) Effect of physioxia (5% pO2) or physiological hypoxia (2% pO2) on osteoclastogenesis. TRAP-stained cells (left panel) and the number of TRAP-positive cells with more than three nuclei (right). Scale bar, 100 μm.


Figure 2 ( A ) μCT analysis of the femurs of 10 - week - old male mice upon exposure to low oxygen concentration ( 14 % pO2 , n = 5 ) or ambient air ( n = 7 ) , and of 10 - week - old Hif1aRank - / - ; Hif2aRank - / - male mice upon exposure to low oxygen concentration ( 14 % pO2 , n = 6 ) or ambient air ( n = 5 ) for 10 days ( axial view of the metaphyseal region ) . Scale , 0 . 5 mm . ( B ) Histological analysis of the proximal tibias of 10 - week - old male mice upon exposure to low oxygen concentration ( 14 % pO2 ) or ambient air for 10 days , and of 10 - week - old Hif1aRank - / - ; Hif2aRank - / - male mice upon exposure to low oxygen concentration ( 14 % pO2 ) or ambient air ( toluidine blue staining [ arrow , osteoclasts ] ) . Scale , 100 μm . ( C ) Parameters for osteoclasts and osteoblasts during bone morphometric analysis of 10 - week - old male mice under low oxygen concentration ( n = 3 ) or ambient air ( n = 5 ) , and 10 - week - old Hif1aRank - / - ; Hif2aRank - / - male under low oxygen concentration ( n = 3 ) or ambient air ( n = 5 ) for 10 days .

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

Figure 2(A) μCT analysis of the femurs of 10-week-old male mice upon exposure to low oxygen concentration (14% pO2, n = 5) or ambient air (n = 7), and of 10-week-old Hif1aRank-/-; Hif2aRank-/- male mice upon exposure to low oxygen concentration (14% pO2, n = 6) or ambient air (n = 5) for 10 days (axial view of the metaphyseal region). Scale, 0.5 mm. (B) Histological analysis of the proximal tibias of 10-week-old male mice upon exposure to low oxygen concentration (14% pO2) or ambient air for 10 days, and of 10-week-old Hif1aRank-/-; Hif2aRank-/- male mice upon exposure to low oxygen concentration (14% pO2 ) or ambient air (toluidine blue staining [arrow, osteoclasts]). Scale, 100 μm. (C) Parameters for osteoclasts and osteoblasts during bone morphometric analysis of 10-week-old male mice under low oxygen concentration (n = 3) or ambient air (n = 5), and 10-week-old Hif1aRank-/-; Hif2aRank-/- male under low oxygen concentration (n = 3) or ambient air (n = 5) for 10 days.


Figure 3 ( A ) Protein expression of HIF - 1α , HIF - 2α , Blimp1 , NFATc1 , Ctsk and TRAP in control and Hif1aRank - / - ; Hif2aRank - / - BMMs stimulated with RANKL and cultured under different conditions of oxygen concentration for 2 days . ( B ) Effect of physioxia ( 5 % pO2 ) or physiological hypoxia ( 2 % pO2 ) on the formation of osteoclast from control and Hif1aRank - / - ; Hif2aRank - / - BMMs . TRAP - stained cells ( left panel ) and the number of TRAP - positive cells with more than three nuclei ( right ) . Scale bar , 100 μm . ( C ) Expression of osteoclastogenic and canonical HIF - target genes in wild - type control and Hif1aRank - / - ; Hif2aRank - / - BMMs under conditions of 5 % or 2 % oxygen .

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

Figure 3(A) Protein expression of HIF-1α, HIF-2α, Blimp1, NFATc1, Ctsk and TRAP in control and Hif1aRank-/-; Hif2aRank-/- BMMs stimulated with RANKL and cultured under different conditions of oxygen concentration for 2 days.(B) Effect of physioxia (5% pO2) or physiological hypoxia (2% pO2) on the formation of osteoclast from control and Hif1aRank-/-; Hif2aRank-/- BMMs. TRAP-stained cells (left panel) and the number of TRAP-positive cells with more than three nuclei (right). Scale bar, 100 μm.(C) Expression of osteoclastogenic and canonical HIF-target genes in wild-type control and Hif1aRank-/-; Hif2aRank-/- BMMs under conditions of 5% or 2% oxygen.


Figure 4 ( A ) Representative intravital image of calvarial bone marrow of Rosa26GO - ATeam / + ; Tg ( Acp5 - tdTomato ) male mice showing osteoclasts ( left , tdTomato fluorescence ) and FLIM image for ATP changes ( middle and right , fluorescence lifetime of EGFP ) . The FRET efficiencies of each osteoclast were plotted from the mice upon exposure to low oxygen atmosphere ( 14 % pO2 ) or ambient air ( right , n = 11 from three mice for each SpO2 ) . ( B ) Representative intravital image of calvarial bone marrow of Tg ( Acp5 - tdTomato ) male mice showing osteoclasts ( left , tdTomato fluorescence ) and NAD ( P ) H autofluorescence lifetime images ( middle and right ) . Average of autofluorescence lifetime of each osteoclast was plotted from the mice upon exposure to low oxygen concentration ( 14 % pO2 ) or ambient air ( right , n = 64 from three mice for each SpO2 ) .

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 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

Figure 4(A) Representative intravital image of calvarial bone marrow of Rosa26GO-ATeam/+; Tg(Acp5-tdTomato) male mice showing osteoclasts (left, tdTomato fluorescence) and FLIM image for ATP changes (middle and right, fluorescence lifetime of EGFP). The FRET efficiencies of each osteoclast were plotted from the mice upon exposure to low oxygen atmosphere (14% pO2) or ambient air (right, n = 11 from three mice for each SpO2).(B) Representative intravital image of calvarial bone marrow of Tg(Acp5-tdTomato) male mice showing osteoclasts (left, tdTomato fluorescence) and NAD(P)H autofluorescence lifetime images (middle and right). Average of autofluorescence lifetime of each osteoclast was plotted from the mice upon exposure to low oxygen concentration (14% pO2) or ambient air (right, n = 64 from three mice for each SpO2).


Figure 5 ( B ) μCT analysis of the femurs of 10 - week - old control ( n = 6 ) , Tet2Rank - / - ( n = 5 ) , Tet3Rank - / - ( n = 5 ) , Tet2Rank - / - ; Tet3aRank + / - ( n = 6 ) , Tet2Rank + / - ; Tet3aRank - / - ( n = 6 ) and Tet2Rank - / - ; Tet3aRank - / - ( n = 4 ) male mice ( axial view of the metaphyseal region ) . ( C ) Histological analysis of the proximal tibias of 10 - week - old control , Tet2Rank - / - , Tet3Rank - / - , Tet2Rank - / - ; Tet3aRank + / - , Tet2Rank + / - ; Tet3aRank - / - and Tet2Rank - / - ; Tet3aRank - / - male mice ( TRAP staining [ red , osteoclasts ] ) . ( E ) Effect of Tet2 and Tet3 double deficiency on osteoclastogenesis . TRAP - stained cells ( left panel ) and the number of TRAP - positive cells with more than three nuclei ( right ) . ( n = 6 ( control ) , n = 10 ( Tet2Rank - / - ; Tet3aRank + / - ) , n = 3 ( Tet2Rank + / - ; Tet3aRank - / - ) and n = 4 ( Tet2Rank - / - ; Tet3aRank - / - ) biological

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 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

Figure 5(B) μCT analysis of the femurs of 10-week-old control (n = 6), Tet2Rank-/- (n = 5), Tet3Rank-/- (n = 5), Tet2Rank-/-; Tet3aRank+/- (n = 6), Tet2Rank+/-; Tet3aRank-/- (n = 6) and Tet2Rank-/-; Tet3aRank-/- (n = 4) male mice (axial view of the metaphyseal region). (C) Histological analysis of the proximal tibias of 10-week-old control, Tet2Rank-/-, Tet3Rank-/-, Tet2Rank-/-; Tet3aRank+/-, Tet2Rank+/-; Tet3aRank-/- and Tet2Rank-/-; Tet3aRank-/- male mice (TRAP staining [red, osteoclasts]).(E) Effect of Tet2 and Tet3 double deficiency on osteoclastogenesis. TRAP-stained cells (left panel) and the number of TRAP-positive cells with more than three nuclei (right). (n = 6 (control), n = 10 (Tet2Rank-/-; Tet3aRank+/-), n = 3 (Tet2Rank+/-; Tet3aRank-/-) and n = 4 (Tet2Rank-/-; Tet3aRank-/-) biological replicates


Figure 6 ( B ) MeDIP - qPCR analysis to validate regions that are hypermethylated in Tet2Rank - / - ; Tet3aRank + / - BMMs stimulated with RANKL for 2 days . ( C ) Gene expression of TET target candidates in BMMs stimulated with RANKL for 2 days . ( E ) Bisulfate sequencing analysis of selected region of the second intron of the Prdm1 gene in control and Tet2Rank - / - ; Tet3aRank + / - BMMs stimulated with RANKL for 2 days . ( F ) Effect of retroviral NFATc1 expression on osteoclastogenesis of control and Tet2Rank - / - ; Tet3aRank + / - BMMs stimulated with RANKL for 3 days . TRAP - stained cells ( left panel ) and the number of TRAP - positive cells with more than three nuclei ( right ) . ( G ) Bisulfate sequencing analysis of the selected region of the second intron of the Prdm1 gene in BMMs stimulated with RANKL and culture under 5 % and 2 % oxygen for 3 days . ( H ) Effect of retroviral NFATc1 expression on osteoclastogenesis of BMMs stimulated with RANKL and culture under 5 % and 2 % oxygen for 5 days . Control and Tet2Rank - / - ; Tet3aRank + / - BMMs stimulated with RANKL for 3 days . TRAP - stained cells ( left panel ) and the number of TRAP - positive cells with more than three nuclei ( right ) .

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 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

Figure 6(B) MeDIP-qPCR analysis to validate regions that are hypermethylated in Tet2Rank-/-; Tet3aRank+/- BMMs stimulated with RANKL for 2 days. (C) Gene expression of TET target candidates in BMMs stimulated with RANKL for 2 days.(E) Bisulfate sequencing analysis of selected region of the second intron of the Prdm1 gene in control and Tet2Rank-/-; Tet3aRank+/- BMMs stimulated with RANKL for 2 days. (F) Effect of retroviral NFATc1 expression on osteoclastogenesis of control and Tet2Rank-/-; Tet3aRank+/- BMMs stimulated with RANKL for 3 days. TRAP-stained cells (left panel) and the number of TRAP-positive cells with more than three nuclei (right). (G) Bisulfate sequencing analysis of the selected region of the second intron of the Prdm1 gene in BMMs stimulated with RANKL and culture under 5% and 2% oxygen for 3 days. (H) Effect of retroviral NFATc1 expression on osteoclastogenesis of BMMs stimulated with RANKL and culture under 5% and 2% oxygen for 5 days. Control and Tet2Rank-/-; Tet3aRank+/- BMMs stimulated with RANKL for 3 days. TRAP-stained cells (left panel) and the number of TRAP-positive cells with more than three nuclei (right).


Figure 1Dual - color SMLM shows co - localization of BAX and DRP1 at the nanoscale . B ) Magnified SMLM images of GFP - BAX and DRP1 assemblies co - localizing up to 30 nm during apoptosis . Scale bar 100 nm . C ) Co - localization control of DRP1 immunostaining with the same primary and different secondary antibodies ( labelled with AF647 and CF680 ) in HeLa cells . Scale bar 100 nm . D ) Quantification of the distance from GFP - BAX to DRP1 structures ( distance measured at the center of each structure ) from dual - color SMLM images . Data are quantified from n = 4 independent experiments with a total of 720 BAX structure . E , F ) Representative confocal microscopy images of HeLa cells transfected with ddFP RA - BAX and GB - DRP1 ( E ) in untreated or apoptotic ( STS ) conditions , or with RA - BAX and GB - BAX during apoptosis ( F ) . RA - BAX / GB - DRP1 and RA - BAX / GB - BAX complexes shown in green , mitochondria labeled with mito - BFP in magenta . Scale bar 10 μm . Right panels are zoomed areas representing individual and merged channels . Scale bar 5 μm . G ) BAX / DRP1 interaction compared to BAX / BAX and BCL ‑ xL / DRP1 by ddFP . % mito - BFP transfected cells that show RA / GB signal was quantified in HeLa cells untreated ( untr . ) and 3 h after apoptosis induction ( STS ) . Box plots represent the interquartile ( box ) , median ( line ) and SD ( whiskers ) of n = 3 independent experiments ( with n = 100 cells each ) . Levels of significance were determined by paired two - tailed Student ´ s t - test ( * p < 0 . 05 , * * * p < 0 . 001 ) compared to BAX / DRP1 after apoptotic induction ( STS ) .

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 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 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

Figure 1Dual-color SMLM shows co-localization of BAX and DRP1 at the nanoscale. B) Magnified SMLM images of GFP-BAX and DRP1 assemblies co-localizing up to 30 nm during apoptosis. Scale bar 100 nm. C) Co-localization control of DRP1 immunostaining with the same primary and different secondary antibodies (labelled with AF647 and CF680) in HeLa cells. Scale bar 100 nm. D) Quantification of the distance from GFP-BAX to DRP1 structures (distance measured at the center of each structure) from dual-color SMLM images. Data are quantified from n=4 independent experiments with a total of 720 BAX structure.E,F) Representative confocal microscopy images of HeLa cells transfected with ddFP RA-BAX and GB-DRP1 (E) in untreated or apoptotic (STS) conditions, or with RA-BAX and GB-BAX during apoptosis (F). RA-BAX/GB-DRP1 and RA-BAX/GB-BAX complexes shown in green, mitochondria labeled with mito-BFP in magenta. Scale bar 10 μm. Right panels are zoomed areas representing individual and merged channels. Scale bar 5 μm.G) BAX/DRP1 interaction compared to BAX/BAX and BCL‑xL/DRP1 by ddFP. % mito-BFP transfected cells that show RA/GB signal was quantified in HeLa cells untreated (untr.) and 3 h after apoptosis induction (STS). Box plots represent the interquartile (box), median (line) and SD (whiskers) of n=3 independent experiments (with n=100 cells each). Levels of significance were determined by paired two-tailed Student´s t-test (* p < 0.05, *** p < 0.001) compared to BAX/DRP1 after apoptotic induction (STS).


Figure 2A ) Confocal microscopy time - series images of the increase in cytosolic Smac - GFP ( magenta ) in relation to the detection of RA - BAX / GB - DRP1 complexes ( green ) during apoptosis induction in MEF DRP1 KO cells . Scale bar 10 μm . Images are representative for n = 3 independent experiments . C ) Quantification of BAX / DRP1 interaction ( % cells with RA - BAX / GB - DRP1 foci normalized to the mito - BFP positive cells , n = 100 cells per experiment ) during inhibition of effector caspases ( Q - VD - OPh ) , the mitochondrial permeability transition pore ( CsA ) versus control ( DMSO ) , with or without apoptosis induction ( + / - STS ) . Box plots represent the interquartile ( box ) , median ( line ) and SD ( whiskers ) . Significance was determined from n = 3 independent experiments ( with n = 100 cells each ) by paired two - tailed Student ´ s t - test ( n . s . indicating p > 0 . 05 ) compared to DMSO control after apoptotic induction ( STS ) .

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 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

Figure 2A) Confocal microscopy time-series images of the increase in cytosolic Smac-GFP (magenta) in relation to the detection of RA-BAX/GB-DRP1 complexes (green) during apoptosis induction in MEF DRP1 KO cells. Scale bar 10 μm. Images are representative for n=3 independent experiments.C) Quantification of BAX/DRP1 interaction (% cells with RA-BAX/GB-DRP1 foci normalized to the mito-BFP positive cells, n=100 cells per experiment) during inhibition of effector caspases (Q-VD-OPh), the mitochondrial permeability transition pore (CsA) versus control (DMSO), with or without apoptosis induction (+/- STS). Box plots represent the interquartile (box), median (line) and SD (whiskers). Significance was determined from n=3 independent experiments (with n=100 cells each) by paired two-tailed Student´s t-test (n.s. indicating p > 0.05) compared to DMSO control after apoptotic induction (STS).


Figure 3C ) Quantification of % CC between DRP1 - AF488 and BAX - AF633 in solution ( grey ) , in the membrane ( violet ) , and in the membrane ( beige ) in presence of excess unlabeled cBID . Box plots represent the interquartile ( outer box ) , mean ( inner box ) , median ( line ) and range ( whiskers ) . Levels of significance were determined by paired two - tailed Student ´ s t - test ( * p < 0 . 05 , * * p < 0 . 01 ) from n = 9 measurements in solution , n = 46 individually measured GUVs in the membrane or n = 17 GUVs in presence of cBID . E ) Effect of DRP1 on BAX - induced GUV permeabilization . Left : % GUVs permeabilized to Cytochrome c488 ( Cyt c , 12 kDa , beige ) and allophycocianin ( APC , 104 kDa , blue ) in the absence or presence of cBID , BAX and DRP1 combined as indicated . Data are presented as mean ± SD of n = 4 independent experiments . * p < 0 . 05 ( paired two - tailed Student ´ s t - test ) . Right : representative confocal microscopy images showing GUVs ( grey ) in a solution of Cyt c ( green ) and APC ( magenta ) . Scale bar 10 µm . Effect of BAX on DRP1 membrane density and DRP1 - induced shape alterations of liposomes measured by flow cytometry . G ) Membrane density of DRP1 ( corrected fluorescence units , cFU , left graph ) and DRP1 - induced membrane tethering ( indicated by a shape index > 1 , right graph ) in LUVs in the absence or presence of different concentrations of BAX . Data are presented as mean ± SD of n = 3 independent experiments . * p < 0 . 05 ( paired two - tailed Student ´ s t - test ) vs . DRP1 without BAX .

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Figure 3C) Quantification of %CC between DRP1-AF488 and BAX-AF633 in solution (grey), in the membrane (violet), and in the membrane (beige) in presence of excess unlabeled cBID. Box plots represent the interquartile (outer box), mean (inner box), median (line) and range (whiskers). Levels of significance were determined by paired two-tailed Student´s t-test (* p < 0.05, ** p < 0.01) from n=9 measurements in solution, n=46 individually measured GUVs in the membrane or n=17 GUVs in presence of cBID.E) Effect of DRP1 on BAX-induced GUV permeabilization. Left: % GUVs permeabilized to Cytochrome c488 (Cyt c, 12 kDa, beige) and allophycocianin (APC, 104 kDa, blue) in the absence or presence of cBID, BAX and DRP1 combined as indicated. Data are presented as mean ± SD of n=4 independent experiments. * p < 0.05 (paired two-tailed Student´s t-test). Right: representative confocal microscopy images showing GUVs (grey) in a solution of Cyt c (green) and APC (magenta). Scale bar 10 µm.Effect of BAX on DRP1 membrane density and DRP1-induced shape alterations of liposomes measured by flow cytometry. G) Membrane density of DRP1 (corrected fluorescence units, cFU, left graph) and DRP1-induced membrane tethering (indicated by a shape index > 1, right graph) in LUVs in the absence or presence of different concentrations of BAX. Data are presented as mean ± SD of n=3 independent experiments. * p < 0.05 (paired two-tailed Student´s t-test) vs. DRP1 without BAX.


Figure 4BAX peptide array to define interaction - site with DRP1 . D ) Quantification of the relative fluorescence intensity of DRP1 - AF488 at the rim of the GUVs for each BAX peptide of n = 3 individual experiments ( with n = 100 vesicles each ) . Black lines indicate the mean value . Interacting BAX peptides are highlighted in beige .

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Figure 4BAX peptide array to define interaction-site with DRP1. D) Quantification of the relative fluorescence intensity of DRP1-AF488 at the rim of the GUVs for each BAX peptide of n=3 individual experiments (with n=100 vesicles each). Black lines indicate the mean value. Interacting BAX peptides are highlighted in beige.


Figure 5B ) Representative confocal microscopy images of U2OS BAX / BAK DKO cells of the interactions between RA - BAX variants and GB - DRP1 , shown as green signal , 3 h after apoptosis induction . Mitochondria labeled with mito - BFP in magenta . Scale bar 10 μm . C - E ) Quantification of the interaction of DRP1 with variants of BAX based on the % cells with detectable RA - BAX / GB - DRP1 fluorescence signal ( interacting cells ) normalized to the number of mito - BFP positive cells . Box plots represent the interquartile ( box ) , median ( line ) and SD ( whiskers ) of n = 3 independent experiments ( with n = 100 cells each ) . Levels of significance were determined by paired two - tailed Student ´ s t - test ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ) compared to BAX wild type ( WT ) . G ) Representative confocal microscopy images of U2OS BAX / BAK DKO cells of the interactions between RA - BAX and GB - DRP1 variants , shown as green signal , 3 h after apoptosis induction . Mitochondria labeled with mito - BFP in magenta . Scale bar 10 μm . H ) Quantification of the interaction of DRP1 variants with BAX based on the % cells with detectable RA - BAX / GB - DRP1 fluorescence signal ( interacting cells ) normalized to the number of mito - BFP positive cells . Box plots represent the interquartile ( box ) , median ( line ) and SD ( whiskers ) of n = 3 independent experiments ( with n = 100 cells each ) . Significance was tested using paired two - tailed Student ´ s t - test ( n . s . p > 0 . 05 ) compared to DRP1 WT .

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Figure 5B) Representative confocal microscopy images of U2OS BAX/BAK DKO cells of the interactions between RA-BAX variants and GB-DRP1, shown as green signal, 3 h after apoptosis induction. Mitochondria labeled with mito-BFP in magenta. Scale bar 10 μm.C-E) Quantification of the interaction of DRP1 with variants of BAX based on the % cells with detectable RA-BAX/GB-DRP1 fluorescence signal (interacting cells) normalized to the number of mito-BFP positive cells. Box plots represent the interquartile (box), median (line) and SD (whiskers) of n=3 independent experiments (with n=100 cells each). Levels of significance were determined by paired two-tailed Student´s t-test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001) compared to BAX wild type (WT).G) Representative confocal microscopy images of U2OS BAX/BAK DKO cells of the interactions between RA-BAX and GB-DRP1 variants, shown as green signal, 3 h after apoptosis induction. Mitochondria labeled with mito-BFP in magenta. Scale bar 10 μm.H) Quantification of the interaction of DRP1 variants with BAX based on the % cells with detectable RA-BAX/GB-DRP1 fluorescence signal (interacting cells) normalized to the number of mito-BFP positive cells. Box plots represent the interquartile (box), median (line) and SD (whiskers) of n=3 independent experiments (with n=100 cells each). Significance was tested using paired two-tailed Student´s t-test (n.s. p > 0.05) compared to DRP1 WT.


Figure 6C ) Forced dimerization of BAX and DRP1 in healthy cells induces their translocation to mitochondria and foci formation . Confocal microscopy images of U2OS BAX / BAK DKO cells transfected with FKBP - mCherry - DRP1 ( red ) and FRB - EGFP - BAX ( green ) and stained with MitoTracker Deep Red FM ( cyan ) were acquired before ( 0 min ) and after induction of BAX / DRP1 dimerization ( 5 - 60 min ) . From top to down , the individual rows correspond to fluorescence signal of BAX and DRP1 and the merge of both together with the MitoTracker signal , respectively . Scale bar 20 µm . E ) Induced dimerization of BAX and DRP1 causes mitochondrial depolarization . U2OS BAX / BAK DKO cells were transfected as described in C ) and stained with the mitochondrial membrane potential - sensitive dye MitoSpy NIR ( cyan ) . Confocal microscopy images were acquired before ( 0 min ) and after induced dimerization of BAX and DRP1 ( 10 min ) . Scale Bar 20 µm . F ) Quantification of mitochondrial depolarization normalized to the number of transfected cells before ( Ctrl ) and after induced dimerization of BAX and DRP1 ( Dim ) . Significance was tested using paired two - tailed Student ´ s t - test ( * p < 0 . 05 ) compared to non - induced control . GG - K ) Induced dimerization of DRP1 with itself ( G ) , BAX with itself ( H ) , BAX with TOM20 ( I ) in U2OS BAX / BAK DKO cells , or BAX with DRP1 in HCT OctaKO cells ( J , K ) , transected with FKBP - and FRB - mCherry DRP1 ( G , red ) , FKBP - and FRB - EGFP - BAX ( H , green ) , TOM20 - mCherry - FKBP ( red ) and FRB - EGFP - BAX ( green , I ) , respectively . Mitochondria were stained using MitoTracker Deep Red FM or MitoSpy NIR ( cyan ) as indicated . Scale bar 20 µm ( G - I ) and 10 µm ( J , K ) . All images are representative of n = 3 independent experiments .

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Figure 6C) Forced dimerization of BAX and DRP1 in healthy cells induces their translocation to mitochondria and foci formation. Confocal microscopy images of U2OS BAX/BAK DKO cells transfected with FKBP-mCherry-DRP1 (red) and FRB-EGFP-BAX (green) and stained with MitoTracker Deep Red FM (cyan) were acquired before (0 min) and after induction of BAX/DRP1 dimerization (5-60 min). From top to down, the individual rows correspond to fluorescence signal of BAX and DRP1 and the merge of both together with the MitoTracker signal, respectively. Scale bar 20 µm.E) Induced dimerization of BAX and DRP1 causes mitochondrial depolarization. U2OS BAX/BAK DKO cells were transfected as described in C) and stained with the mitochondrial membrane potential-sensitive dye MitoSpy NIR (cyan). Confocal microscopy images were acquired before (0 min) and after induced dimerization of BAX and DRP1 (10 min). Scale Bar 20 µm. F) Quantification of mitochondrial depolarization normalized to the number of transfected cells before (Ctrl) and after induced dimerization of BAX and DRP1 (Dim). Significance was tested using paired two-tailed Student´s t-test (* p < 0.05) compared to non-induced control. GG-K) Induced dimerization of DRP1 with itself (G), BAX with itself (H), BAX with TOM20 (I) in U2OS BAX/BAK DKO cells, or BAX with DRP1 in HCT OctaKO cells (J,K), transected with FKBP- and FRB-mCherry DRP1 (G, red), FKBP- and FRB-EGFP-BAX (H, green), TOM20-mCherry-FKBP (red) and FRB-EGFP-BAX (green, I), respectively. Mitochondria were stained using MitoTracker Deep Red FM or MitoSpy NIR (cyan) as indicated. Scale bar 20 µm (G-I) and 10 µm (J,K ). All images are representative of n=3 independent experiments.


Figure 1Confocal micrographs show CK7 ( green ) and vimentin ( red ) in patient - derived HGSC cells cultured in 3D collagen for 7 d . Scale bars : 50 μm . Confocal micrographs of EphA2 ( red ) in HGSC cells cultured in 3D collagen for 7 d and treated for 72 h with 10 μM cisplatin ( 5 μM for OCKI _ p01 ) . The intensity of EphA2 is comparable only between mock and treatment conditions for each patient . Scale bars : 20 μm . Chart illustrates EphA2 fold change after treatment . Mock is set to one . N = 3 .

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Figure 1Confocal micrographs show CK7 (green) and vimentin (red) in patient-derived HGSC cells cultured in 3D collagen for 7 d. Scale bars: 50 μm.Confocal micrographs of EphA2 (red) in HGSC cells cultured in 3D collagen for 7 d and treated for 72 h with 10 μM cisplatin (5 μM for OCKI_p01). The intensity of EphA2 is comparable only between mock and treatment conditions for each patient. Scale bars: 20 μm.Chart illustrates EphA2 fold change after treatment. Mock is set to one. N = 3.


Figure 2EphA2 ( total and phosphorylated at S897 or Y588 ) in OVCAR3 , OVCAR4 and OVCAR8 after treatment with 0 - 20 μM cisplatin for 72 h was assessed by immunoblotting ( A ) and quantified for pS897 / pY588 ratio ( B ) . N = 4 . EphA2 ( total and phosphorylated ) in corresponding untreated cells . Cytotoxicity assay results after cell treatment with 0 - 20 μM cisplatin for 72 h . N = 6 . Quantitative assessment of EphA2 ( total and phosphorylated ) and pS897 / pY588 ratio in early passage patient - derived HGSC cultures treated with 0 - 20 μM cisplatin for 72 h ( see immunoblots in Appendix Fig S2A ) . N = 6 patients , pooled . Corresponding EphA2 pS897 / pY588 ratios for individual patient cells . EphA2 ( total and phosphorylated ) in OVCAR4 and OVCAR8 after treatment with 0 - 80 μM carboplatin for 72 h ( G ) along with pS897 / pY588 quantification ( H ) . N = 3 .

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Figure 2EphA2 (total and phosphorylated at S897 or Y588) in OVCAR3, OVCAR4 and OVCAR8 after treatment with 0-20 μM cisplatin for 72 h was assessed by immunoblotting (A) and quantified for pS897/pY588 ratio (B). N = 4.EphA2 (total and phosphorylated) in corresponding untreated cells.Cytotoxicity assay results after cell treatment with 0-20 μM cisplatin for 72 h. N = 6.Quantitative assessment of EphA2 (total and phosphorylated) and pS897/pY588 ratio in early passage patient-derived HGSC cultures treated with 0-20 μM cisplatin for 72 h (see immunoblots in Appendix Fig S2A). N = 6 patients, pooled.Corresponding EphA2 pS897/pY588 ratios for individual patient cells.EphA2 (total and phosphorylated) in OVCAR4 and OVCAR8 after treatment with 0-80 μM carboplatin for 72 h (G) along with pS897/pY588 quantification (H). N = 3.


Figure 3Bioluminescence images visualize mock and carboplatin treated OVCAR4 xenograft tumors ( day 53 ; after 13 d i . p . treatment ) . Color scale unit : p / sec / cm2 / sr . Charts illustrate the tumor growth ( B ) and ascites volumes at the end of the follow - up ( C ) . Carboplatin treatment started on day 35 . Bioluminescent unit : p / sec / cm2 / sr . N = 5 mice / group . Representative micrographs of hematoxylin - eosin staining in the xenografts tumors . Scale bars : 100 μmEphA2 - pS897 immunofluorescence in the xenografts tumors . Scale bars : 10 μmQuantitative assessment of tumor EphA2 ( F ) and EphA2 - pS897 ( G ) . N = 5 mice / group . Confocal micrographs of tumor EphA2 - pS897 ( red ) and cleaved caspase - 3 ( H ; clCasp3 , green ) and corresponding clCasp3 quantification ( I ) . Scale bars : 20 μm . N = 5 mice / group .

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Figure 3Bioluminescence images visualize mock and carboplatin treated OVCAR4 xenograft tumors (day 53; after 13 d i.p. treatment). Color scale unit: p/sec/cm2/sr. Charts illustrate the tumor growth (B) and ascites volumes at the end of the follow-up (C). Carboplatin treatment started on day 35. Bioluminescent unit: p/sec/cm2/sr. N = 5 mice/group.Representative micrographs of hematoxylin-eosin staining in the xenografts tumors. Scale bars: 100 μmEphA2-pS897 immunofluorescence in the xenografts tumors. Scale bars: 10 μmQuantitative assessment of tumor EphA2 (F) and EphA2-pS897 (G). N = 5 mice/group.Confocal micrographs of tumor EphA2-pS897 (red) and cleaved caspase-3 (H; clCasp3, green) and corresponding clCasp3 quantification (I). Scale bars: 20 μm. N = 5 mice/group.


Figure 4Immunoblot images show EphA2 ( A ; total , pS897 and pY588 ) and corresponding pS897 / pY588 ratios ( B ) of TYK - nu and TYK - nu . R after treatment with 0 - 10 μM cisplatin for 72 h . N = 5 . EphA2 ( total and phosphorylated ) in the cells treated with 0 - 80 μM carboplatin for 72 h . EphA2 ( total and phosphorylated ) in the cellsEphA2 ( total and pS897 ) in TYK - nu after treatment with inhibitors against MEK ( UO126 , 10 μM ) , PKA ( H89 . 2HCl , 10 μM ) , PI3K ( LY294002 , 10 μM ) or its alpha subunit ( BYL - 719 , 5 μM ) and RSK ( BI - D1870 , 10 μM ) in combination with 0 - 10 μM cisplatin for 72 h ( E ) . Corresponding EphA2 - pS897 quantification for BI - D1870 treatment ( F ) N = 5 . EphA2 ( total and pS897 ) in TYK - nu . R treated with 0 - 20 μM cisplatin without or with 10 μM BI - D1870 for 72 h . EphA2 , RSK and ERK1 / 2 ( total and phosphorylated ) in TYK - nu ( H ) and OVCAR4 ( I ) treated with 0 - 30 μM carboplatin without or with 50 μM LJH685 for 72 h .

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Figure 4Immunoblot images show EphA2 (A; total, pS897 and pY588) and corresponding pS897/pY588 ratios (B) of TYK-nu and TYK-nu.R after treatment with 0-10 μM cisplatin for 72 h . N = 5.EphA2 (total and phosphorylated) in the cells treated with 0-80 μM carboplatin for 72 h.EphA2 (total and phosphorylated) in the cellsEphA2 (total and pS897) in TYK-nu after treatment with inhibitors against MEK (UO126, 10 μM), PKA (H89.2HCl, 10 μM), PI3K (LY294002, 10 μM) or its alpha subunit (BYL-719, 5 μM) and RSK (BI-D1870, 10 μM) in combination with 0-10 μM cisplatin for 72 h (E). Corresponding EphA2-pS897 quantification for BI-D1870 treatment (F) N = 5.EphA2 (total and pS897) in TYK-nu.R treated with 0-20 μM cisplatin without or with 10 μM BI-D1870 for 72 h.EphA2, RSK and ERK1/2 (total and phosphorylated) in TYK-nu (H) and OVCAR4 (I) treated with 0-30 μM carboplatin without or with 50 μM LJH685 for 72 h.


Figure 5Charts show OC cell viability after 72 h treatment with 0 - 20 μM cisplatin and 10 μM BI - D1870Charts show OC cell viability after 72 h treatment with 0 - 20 μM cisplatin and 50 μM LJH685RSK and EphA2 ( total and phosphorylated ) in OC cells treated with 10 µM cisplatin and 25 µM LJH685 as indicated for 72 h . Charts illustrate the corresponding quantified inhibition of EphA2 - pS897 and increase of EphA2 - pY588 shown in ( C ) .

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Figure 5Charts show OC cell viability after 72 h treatment with 0-20 μM cisplatin and 10 μM BI-D1870Charts show OC cell viability after 72 h treatment with 0-20 μM cisplatin and 50 μM LJH685RSK and EphA2 (total and phosphorylated) in OC cells treated with 10 µM cisplatin and 25 µM LJH685 as indicated for 72 h.Charts illustrate the corresponding quantified inhibition of EphA2-pS897 and increase of EphA2-pY588 shown in (C).


Figure 6Immunoblot images ( A ) and quantification ( B ) show GPRC5A 46 / 41 kDa ratios in OC cells treated with 0 - 10 µM cisplatin and with 25 µM LJH685 alone or as a combination for 72 h . The ratio in mock cells was set to 1 . N = 4 . Confocal micrographs of EphA2 ( red ) and GPRC5A ( green ) in TYK - nu and TYK - nu . R treated with 5 µM cisplatin and 10 μM LJH685 as indicated for 72 h . Arrows point co - localization of the receptors . Scale bars : 20 μm . EphA2 ( total and phosphorylated ) , RSK1 / 2 and GPRC5A in TYK - nu were assessed by immunoblotting after RSK1 / 2 knockdown for 2 d and following treatment with 0 - 5 μM cisplatin for 72 h . Asterisks indicate unspecific bands . EphA2 ( total and phosphorylated ) , RSK1 / 2 and GPRC5A and these proteins coupled with PARP and PCNA in TYK - nu . R were assessed by immunoblotting after RSK1 / 2 knockdown for 2 d and following treatment with 0 - 5 μM cisplatin for 72 h . Asterisks indicate unspecific bands . Chart illustrates the viability of the RSK1 / 2 depleted TYK - nu . R after cisplatin treatment . N = 3 .

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Figure 6Immunoblot images (A) and quantification (B) show GPRC5A 46/41 kDa ratios in OC cells treated with 0-10 µM cisplatin and with 25 µM LJH685 alone or as a combination for 72 h. The ratio in mock cells was set to 1. N = 4.Confocal micrographs of EphA2 (red) and GPRC5A (green) in TYK-nu and TYK-nu.R treated with 5 µM cisplatin and 10 μM LJH685 as indicated for 72 h. Arrows point co-localization of the receptors. Scale bars: 20 μm.EphA2 (total and phosphorylated), RSK1/2 and GPRC5A in TYK-nu were assessed by immunoblotting after RSK1/2 knockdown for 2 d and following treatment with 0-5 μM cisplatin for 72 h. Asterisks indicate unspecific bands.EphA2 (total and phosphorylated), RSK1/2 and GPRC5A and these proteins coupled with PARP and PCNA in TYK-nu.R were assessed by immunoblotting after RSK1/2 knockdown for 2 d and following treatment with 0-5 μM cisplatin for 72 h. Asterisks indicate unspecific bands.Chart illustrates the viability of the RSK1/2 depleted TYK-nu.R after cisplatin treatment. N = 3.


Figure 7Confocal micrographs show cleaved caspase - 3 ( clCasp3 , green ) , mRFP ( orange ) and phalloidin ( F - actin , red ; only shown for CAF ) in 3D OVCAR8 - RFP and OCKI _ p22 CAF mono - and co - cultures treated without or with 20 µM cisplatin or 25 µM LJH685 alone or in combination for 20 h . Scale bars : 100 μm . Quantitative assessment of clCasp3 + cells in the 3D cultures . N = 4 . Confocal micrographs show cytokeratin 7 ( CK7 , green ) and phalloidin ( F - actin , red ) in 3D patient OCKI _ p13 mono - and co - culture with OCKI _ p22 CAF treated without or with 20 µM cisplatin or 25 µM LJH685 alone or in combination for 20 h . Scale bars : 50 μm . Quantitative assessment of clCasp3 + OCKI _ p13 cells in 3D co - cultures of patient - derived cells . N = 4 . Immunohistochemistry images and quantifications of TUNEL in OVCAR4 xenografts in mock ( N = 4 ) , carboplatin ( N = 4 ) and carboplatin + BI - D1870 ( N = 5 ) treated mice . Scale bars : 100 μm . Immunohistochemistry images and quantifications of Ki67 in OVCAR4 xenografts in mock ( N = 4 ) , carboplatin ( N = 4 ) and carboplatin + BI - D1870 ( N = 5 ) treated mice . Scale bars : 100 μm .

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Figure 7Confocal micrographs show cleaved caspase-3 (clCasp3, green), mRFP (orange) and phalloidin (F-actin, red; only shown for CAF) in 3D OVCAR8-RFP and OCKI_p22 CAF mono- and co-cultures treated without or with 20 µM cisplatin or 25 µM LJH685 alone or in combination for 20 h. Scale bars: 100 μm.Quantitative assessment of clCasp3+ cells in the 3D cultures. N = 4.Confocal micrographs show cytokeratin 7 (CK7, green) and phalloidin (F-actin, red) in 3D patient OCKI_p13 mono- and co-culture with OCKI_p22 CAF treated without or with 20 µM cisplatin or 25 µM LJH685 alone or in combination for 20 h. Scale bars: 50 μm. Quantitative assessment of clCasp3+ OCKI_p13 cells in 3D co-cultures of patient-derived cells. N = 4.Immunohistochemistry images and quantifications of TUNEL in OVCAR4 xenografts in mock (N = 4), carboplatin (N = 4) and carboplatin+BI-D1870 (N = 5) treated mice. Scale bars: 100 μm.Immunohistochemistry images and quantifications of Ki67 in OVCAR4 xenografts in mock (N = 4), carboplatin (N = 4) and carboplatin+BI-D1870 (N = 5) treated mice. Scale bars: 100 μm.


Figure 8Representative confocal micrographs show EphA2 ( red ) , RSK ( green : top ) and GPRC5A ( green : bottom ) in frozen sections of HGSC patient tumors . S indicates the stroma . Scale bars : 50 μm . Indicated proteins and EphA2 phosphorylation were assessed by immunoblotting in patient - derived HGSC cells and CAFs . GPRC5A in earlier ( top ) and later passage ( bottom ) HGSC cells . See normalized GPRC5A relative to OCKI _ p01 below the immunoblot images . Charts illustrate EphA2 - pS897 inhibition ( circles ) and EphA2 - pY588 increase ( squares ) by 10 µM BI - D1870 alone ( unbroken line ) or in combination with 0 - 10 µM cisplatin ( dotted line ) in the GPRC5Ahigh HGSC cells . See Appendix S7B for immunoblots . Cell viability of the GPRC5Ahigh OCKI _ p02 and OCKI _ p06 after treatment with a combination of 0 - 20 µM cisplatin and 10 µM BI - D1870 for 72 h . N = 3 . Cell viability upon 72 h combinatorial treatment with 0 - 20 µM cisplatin and 25 µM LJH685 in patient - derived CAFsCell viability upon 72 h combinatorial treatment with 0 - 20 µM cisplatin and 10 µM BI - D1870 in mesothelial cells ( G ; N = 3 ) . Chart illustrates the changes of EphA2 - pS897 and EphA2 - pY588 by BI - D1870 alone and with cisplatin in mesothelial cells . Upon combination with 10 µM cisplatin for 72 h , 10 µM BI - D1870 decreased EphA2 - pS897 and EphA2 - pY588 . See Appendix Fig S7J for immunoblot . Confocal micrographs of EphA2 ( red ) and GPRC5A ( green ) in primary OCKI _ p06 patient cells cultured in 3D collagen for 4 d before 72 h treatment with or without 10 µM cisplatin . Scale bars : 20 μm .

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Figure 8Representative confocal micrographs show EphA2 (red), RSK (green: top) and GPRC5A (green: bottom) in frozen sections of HGSC patient tumors. S indicates the stroma. Scale bars: 50 μm.Indicated proteins and EphA2 phosphorylation were assessed by immunoblotting in patient-derived HGSC cells and CAFs.GPRC5A in earlier (top) and later passage (bottom) HGSC cells. See normalized GPRC5A relative to OCKI_p01 below the immunoblot images.Charts illustrate EphA2-pS897 inhibition (circles) and EphA2-pY588 increase (squares) by 10 µM BI-D1870 alone (unbroken line) or in combination with 0-10 µM cisplatin (dotted line) in the GPRC5Ahigh HGSC cells. See Appendix S7B for immunoblots.Cell viability of the GPRC5Ahigh OCKI_p02 and OCKI_p06 after treatment with a combination of 0-20 µM cisplatin and 10 µM BI-D1870 for 72 h. N = 3.Cell viability upon 72 h combinatorial treatment with 0-20 µM cisplatin and 25 µM LJH685 in patient-derived CAFsCell viability upon 72 h combinatorial treatment with 0-20 µM cisplatin and 10 µM BI-D1870 in mesothelial cells (G; N = 3).Chart illustrates the changes of EphA2-pS897 and EphA2-pY588 by BI-D1870 alone and with cisplatin in mesothelial cells. Upon combination with 10 µM cisplatin for 72 h, 10 µM BI-D1870 decreased EphA2-pS897 and EphA2-pY588. See Appendix Fig S7J for immunoblot.Confocal micrographs of EphA2 (red) and GPRC5A (green) in primary OCKI_p06 patient cells cultured in 3D collagen for 4 d before 72 h treatment with or without 10 µM cisplatin. Scale bars: 20 μm.


Figure 9Representative immunostainings for GPRC5A scoring in HGSC primary and metastatic tumors . For maximum intensity scores an optical 4 - point scale ( 0 : negative , 1 : mild , 2 : moderate , 3 : high ) was used . For survival analyses a 2 - point scale ( low : scores 0 - 2 , high : score 3 ) was used . Scale bars : 200 μm . Kaplan Meier survival curves illustrate the overall ( OS ) and progression - free ( PFS ) survival of patients with high or low GPRC5A . In primary tumors , mean OS for GPRC5Ahigh was 39 months vs 56 months for GPRC5Alow ( C ) . In patients with metastatic tumors , mean OS for GPRC5Ahigh was 37 months vs 60 months for GPRC5Alow . Mean PFS in metastatic tumors was 20 months for GPRC5Ahigh vs 35 months for GPRC5Alow ( D ) . No significant difference in mean PFS in primary tumor . Kaplan Meier survival estimates for the PFS of patients with high or low ( top 25 % vs bottom 25 % ) GPRC5A ( F ) , EPHA2 ( G ) , GPRC5A + EPHA2 ( H ) and GPRC5A + EPHA2 + RSK1 ( RPS6KA1 ) + RSK2 ( RPS6KA3 ) combined ( I ) mRNA expression . Mean PFS for GPRC5A + EPHA2high was 15 months vs 26 months for GPRC5A + EPHA2low ( H ) . Mean PFS for GPRC5A + EPHA2 + RPS6KA1 / 3 high was 14 months vs 31 months for GPRC5A + EPHA2 + RPS6KA1 / 3low ( I ) .

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Figure 9Representative immunostainings for GPRC5A scoring in HGSC primary and metastatic tumors. For maximum intensity scores an optical 4-point scale (0: negative, 1: mild, 2: moderate, 3: high) was used. For survival analyses a 2-point scale (low: scores 0-2, high: score 3) was used. Scale bars: 200 μm.Kaplan Meier survival curves illustrate the overall (OS) and progression-free (PFS) survival of patients with high or low GPRC5A. In primary tumors, mean OS for GPRC5Ahigh was 39 months vs 56 months for GPRC5Alow (C). In patients with metastatic tumors, mean OS for GPRC5Ahigh was 37 months vs 60 months for GPRC5Alow. Mean PFS in metastatic tumors was 20 months for GPRC5Ahigh vs 35 months for GPRC5Alow (D). No significant difference in mean PFS in primary tumor.Kaplan Meier survival estimates for the PFS of patients with high or low (top 25% vs bottom 25%) GPRC5A (F), EPHA2 (G), GPRC5A+EPHA2 (H) and GPRC5A+EPHA2+RSK1(RPS6KA1)+RSK2(RPS6KA3) combined (I) mRNA expression. Mean PFS for GPRC5A+EPHA2high was 15 months vs 26 months for GPRC5A+EPHA2low (H). Mean PFS for GPRC5A+EPHA2+RPS6KA1/3 high was 14 months vs 31 months for GPRC5A+EPHA2+RPS6KA1/3low (I).


Figure 1D H & E ( hematoxylin - eosin ) staining ( left ) and oil red O staining ( right ) of livers . Scale bar , 50μm . E Serum cholesterol concentrations of each group . Liver cholesterol concentrations ( F ) of each group were examined . H and I Cholesterol levels in p53 + / + and p53 - / - HepG2 cells ( H ) or HCT116 cells ( I ) were examined . Protein expression was shown by western blotting ( bottom panel ) .

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Figure 1D H&E (hematoxylin-eosin) staining (left) and oil red O staining (right) of livers. Scale bar, 50μm.E Serum cholesterol concentrations of each group. Liver cholesterol concentrations (F) of each group were examined.H and I Cholesterol levels in p53+/+ and p53-/- HepG2 cells (H) or HCT116 cells (I) were examined. Protein expression was shown by western blotting (bottom panel).


Figure 2C mRNA and protein levels of SQLE in different tissues of p53 + / + or p53 - / - mice were determined by qRT - PCR and western blot respectively . F and G p53 + / + and p53 - / - HepG2 cells were cultured for 48 hours in medium containing fetal bovine serum ( Serum ) or lipoprotein - depleted FBS ( LPDS ) . Protein expression was shown by western blotting ( F ) . mRNA levels of SQLE were examined by qRT - PCR ( G ) . P : premature SREBP2 ; M : mature SREBP2 . Protein expression and mRNA levels of p53 + / + and p53 - / - HepG2 cells treated with control siRNA or three separated sets of SREBP2 siRNAs for 72 hours as indicated .

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Figure 2C mRNA and protein levels of SQLE in different tissues of p53+/+ or p53-/- mice were determined by qRT-PCR and western blot respectively.F and G p53+/+ and p53-/- HepG2 cells were cultured for 48 hours in medium containing fetal bovine serum (Serum) or lipoprotein-depleted FBS (LPDS). Protein expression was shown by western blotting (F). mRNA levels of SQLE were examined by qRT-PCR (G). P: premature SREBP2; M: mature SREBP2.Protein expression and mRNA levels of p53+/+ and p53-/- HepG2 cells treated with control siRNA or three separated sets of SREBP2 siRNAs for 72 hours as indicated.


Figure 3F Luciferase reporter constructs containing RE1 , RE2 , RE1mut or RE2mut were transfected into p53 - / - HCT116 cells together with control ( PRK5 - flag vector ) or p53 expression vector ( PRK5 - flag p53 ) for 48 hours . Renilla vector pRL - CMV was used as a transfection internal control . Relative levels of luciferase are shown . Data represent three independent experiments . Protein expression is shown G Luciferase reporter constructs containing RE1 , RE2 , RE1mut or RE2mut were transfected into HEK293T cells together with control ( PRK5 - flag vector ) or p53 expression vector ( PRK5 - flag p53 ) for 48 hours . Renilla vector pRL - CMV was used as a transfection internal control . Relative levels of luciferase are shown . Protein expression is shown .

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Figure 3F Luciferase reporter constructs containing RE1, RE2, RE1mut or RE2mut were transfected into p53-/- HCT116 cells together with control (PRK5-flag vector) or p53 expression vector (PRK5-flag p53) for 48 hours. Renilla vector pRL-CMV was used as a transfection internal control. Relative levels of luciferase are shown. Data represent three independent experiments. Protein expression is shown G Luciferase reporter constructs containing RE1, RE2, RE1mut or RE2mut were transfected into HEK293T cells together with control (PRK5-flag vector) or p53 expression vector (PRK5-flag p53) for 48 hours. Renilla vector pRL-CMV was used as a transfection internal control. Relative levels of luciferase are shown. Protein expression is shown.


Figure 4A - C Squalene ( A ) , lanosterol ( B ) and cholesterol ( C ) abundance in p53 + / + and p53 - / - HepG2 cells transfected with control or SQLE siRNA for 48 hours were determined via ultra - high pressure liquid chromatography coupled to mass spectrometry ( UHPLC - MS ) . Protein levels were analyzed by western blotting with specific antibodies ( A bottom panel ) .

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Figure 4A-C Squalene (A), lanosterol (B) and cholesterol (C) abundance in p53+/+ and p53-/- HepG2 cells transfected with control or SQLE siRNA for 48 hours were determined via ultra-high pressure liquid chromatography coupled to mass spectrometry (UHPLC-MS). Protein levels were analyzed by western blotting with specific antibodies (A bottom panel).


Figure 5A Protein expression of SQLE in human liver tumors ( C ) and non - cancerous adjacent tissue ( N ) were analyzed by western blotting . B SQLE gene expression in HCC and adjacent normal tissue was analyzed in TCGA Liver Hepatocellular Carcinoma ( normal [ n = 50 ] versus tumors [ n = 371 ] ) . C SQLE gene expression in COAD and adjacent normal tissue was analyzed in TCGA Colon Cancer ( normal [ n = 41 ] versus tumors [ n = 286 ] ) . J - M p53 + / + HCT116 cells ( J and K ) and p53 + / + HepG2 cells ( L and M ) were transfected with SQLE siRNA or control siRNA in the presence or absence of exogenous SQLE cDNA for 48 hours . Cholesterol concentration ( upper panel ) and protein expression ( bottom panel ) were determined ( J and L ) . Cell proliferation is shown ( K and M ) .

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Figure 5A Protein expression of SQLE in human liver tumors (C) and non-cancerous adjacent tissue (N) were analyzed by western blotting.B SQLE gene expression in HCC and adjacent normal tissue was analyzed in TCGA Liver Hepatocellular Carcinoma (normal [n=50] versus tumors [n=371]). C SQLE gene expression in COAD and adjacent normal tissue was analyzed in TCGA Colon Cancer (normal [n=41] versus tumors [n=286]). J - M p53+/+ HCT116 cells (J and K) and p53+/+ HepG2 cells (L and M) were transfected with SQLE siRNA or control siRNA in the presence or absence of exogenous SQLE cDNA for 48 hours. Cholesterol concentration (upper panel) and protein expression (bottom panel) were determined (J and L). Cell proliferation is shown (K and M).


Figure 6C Growth of p53 + / + and p53 - / - HepG2 cells transfected with and without exogenous SQLE ( HA - SQLE ) in LPDS medium . Protein expression is assayed after transfection 24 hours . G and H Cholesterol levels , protein expression ( G ) and cell growth ( H ) of SK - HEP - 1 cells stably expressing control shRNA or p53 shRNA in LPDS medium in presence or absence of 5μg cholesterol . L Percentage of SA - β - gal - positive cells of p53 + / + and p53 - / - HepG2 cells expressing SQLE siRNA or control siRNA ( upper panel ) . Protein expression ( middle panel ) and representative images ( lower panel ) are shown . Sacle bar , 100 μm .

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Figure 6C Growth of p53+/+ and p53-/- HepG2 cells transfected with and without exogenous SQLE (HA-SQLE) in LPDS medium. Protein expression is assayed after transfection 24 hours.G and H Cholesterol levels, protein expression (G) and cell growth (H) of SK-HEP-1 cells stably expressing control shRNA or p53 shRNA in LPDS medium in presence or absence of 5μg cholesterol.L Percentage of SA-β-gal-positive cells of p53+/+ and p53-/- HepG2 cells expressing SQLE siRNA or control siRNA (upper panel). Protein expression (middle panel) and representative images (lower panel) are shown . Sacle bar, 100 μm.


Figure 7p53 - / - C57BL / 6N male mice ( n = 7 ) were injected with a single dose of diethylnitrosamine ( DEN ) ( 25mg / kg ) at two weeks and then fed with HFD diet at 4 weeks . Oral administration of terbinafine were given at 13 weeks old as indicated . Shown are liver tumor incidence ( L ) and tumor numbers ( M ) . ( N ) H & E ( left ) , Ki - 67 ( middle ) and Oil Red O ( right ) staining of livers of mice . ( O ) Total liver cholesterol concentrations of mice were examined .

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Figure 7p53-/- C57BL/6N male mice (n=7) were injected with a single dose of diethylnitrosamine (DEN) (25mg/kg) at two weeks and then fed with HFD diet at 4 weeks. Oral administration of terbinafine were given at 13 weeks old as indicated. Shown are liver tumor incidence (L) and tumor numbers (M).(N) H&E (left), Ki-67 (middle) and Oil Red O (right) staining of livers of mice.(O) Total liver cholesterol concentrations of mice were examined.


Figure 8representative liver photos ( C , left ) , liver tumor numbers ( C , right ) , representative images ( D , left ) and quantification ( D , right ) of Ki - 67 staining are shown . Red arrows indicate the tumors in the liver ( C ) . E H & E ( left ) , Ki - 67 ( middle ) and Oil Red O ( right ) staining of livers of

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Figure 8representative liver photos (C, left), liver tumor numbers (C, right), representative images (D, left) and quantification (D, right) of Ki-67 staining are shown. Red arrows indicate the tumors in the liver (C).E H&E (left), Ki-67 (middle) and Oil Red O (right) staining of livers of mice


Figure 1A . Serum levels of 15 - keto - dihydro - PGF2α in type 2 diabetic patients without diabetic retinopathy ( no DR ) , with non - proliferative diabetic retinopathy ( NPDR ) , and with proliferative diabetic retinopathy ( PDR ) ( n = 20 - 24 ) . Data information : n . s . stands for " not significant . " Data were analyzed by Kruskal - Wallis test with Dunn ' s multiple comparisons test ( A ) , Data are represented as mean ± SEM . C . Retinal PGF2α production in OIR mice during postnatal days 12 - 17 ( n = 4 ) . Data information : n . s . stands for " not significant . " Data were analyzed by , two - way ANOVA with Tukey ' s multiple comparisons test ( C , Data are represented as mean ± SEM . D . mRNA expression of PGF2α synthases in the retinas of OIR mice during postnatal days 12 - 17 ( n = E . mRNA expression of Ptgfr in the retinas of OIR mice during postnatal days 12 - 17 ( n = 6 - 8 ) . Data information : n . s . stands for " not significant . " Data were analyzed by , two - way ANOVA with Tukey ' s multiple comparisons test D , E , Data are represented as mean ± SEM . F . mRNA expression of Ptgfr in each retinal layer from normoxic and OIR mice on postnatal day 16 ( n = 6 ) . The left - hand image displays representative cross - sections from OIR and normoxic retinas , with isolectin B4 - stained vessels in green , DAPI - stained nuclei in blue , and LCM isolation locations circled by white dashed circles . INL , inner nuclear layer ; ONL , outer nuclear layer ; RGC , retinal ganglion cells . Data information : n . s . stands for " not significant . " Data were analyzed by two - way ANOVA with Tukey ' s multiple comparisons test F ) Scale bar : 20 μm . Data are represented as mean ± SEM . G . Ptgfr mRNA expression in retinal endothelial cells ( CD31 + CD45 - ) sorted by MACS from normoxic and OIR mice on postnatal day 16 ( n = 5 ) . Data information : n . s . stands for " not significant . " Data were analyzed Mann - Whitney test ( G ) . Scale bar : 20 μm . Data are represented as mean ± SEM .

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 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 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 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

Figure 1A. Serum levels of 15-keto-dihydro-PGF2α in type 2 diabetic patients without diabetic retinopathy (no DR), with non-proliferative diabetic retinopathy (NPDR), and with proliferative diabetic retinopathy (PDR) (n=20-24). Data information: n.s. stands for "not significant." Data were analyzed by Kruskal-Wallis test with Dunn's multiple comparisons test (A), Data are represented as mean ± SEM.C. Retinal PGF2α production in OIR mice during postnatal days 12-17 (n=4). Data information: n.s. stands for "not significant." Data were analyzed by , two-way ANOVA with Tukey's multiple comparisons test (C, Data are represented as mean ± SEM.D. mRNA expression of PGF2α synthases in the retinas of OIR mice during postnatal days 12-17 (n= E. mRNA expression of Ptgfr in the retinas of OIR mice during postnatal days 12-17 (n=6-8). Data information: n.s. stands for "not significant." Data were analyzed by , two-way ANOVA with Tukey's multiple comparisons test D, E, Data are represented as mean ± SEM.F. mRNA expression of Ptgfr in each retinal layer from normoxic and OIR mice on postnatal day 16 (n=6). The left-hand image displays representative cross-sections from OIR and normoxic retinas, with isolectin B4-stained vessels in green, DAPI-stained nuclei in blue, and LCM isolation locations circled by white dashed circles. INL, inner nuclear layer; ONL, outer nuclear layer; RGC, retinal ganglion cells. Data information: n.s. stands for "not significant." Data were analyzed by two-way ANOVA with Tukey's multiple comparisons test F) Scale bar: 20 μm. Data are represented as mean ± SEM.G. Ptgfr mRNA expression in retinal endothelial cells (CD31+CD45-) sorted by MACS from normoxic and OIR mice on postnatal day 16 (n=5). Data information: n.s. stands for "not significant." Data were analyzed Mann-Whitney test (G). Scale bar: 20 μm. Data are represented as mean ± SEM.