Figure 2MDM were transfected with control or pool of SAMHD1 ( KD ) siRNA and 3 days later treated with 5μM ETO and infected in the presence of ETO with VSV ‐ G pseudotyped HIV ‐ 1 GFP 18h later . Cells from a representative donor were used for immunoblotting . The percentage of infected cells was quantified by the automated cell ‐ imaging system Hermes WiScan and ImageJ 48h post - infection . ( n = 3 , mean ± s . e . m . ; * * P ‐ value ≤ 0 . 01 ; ( ns ) non ‐ significant , paired t ‐ test ) . MDM were treated with 5μM ETO for 18h and co ‐ infected in the presence of ETO with VSV ‐ G HIV ‐ 1 GFP and SIVmac virus ‐ like particles containing Vpx / Vpr ( SIV VLP ) . Cells from a representative donor were used for immunoblotting . The percentage of infected cells was quantified by the Hermes WiScan and ImageJ 48h post - infection . ( n = 3 , mean ± s . e . m . ; * * P ‐ value ≤ 0 . 01 ; ( ns ) non ‐ significant , paired t ‐ test ) . MDM were treated with 5μM ETO for 18h and co ‐ infected in the presence of ETO with VSV ‐ G HIV ‐ 1 GFP and SIVmac virus ‐ like particles containing Vpx / Vpr ( SIV VLP ) or deleted for Vpx ( SIV VLP del Vpx ) or deleted for Vpr ( SIV VLP del Vpr ) . Cells from a representative donor were used for immunoblotting . The percentage of infected cells was quantified by the automated cell ‐ imaging system Hermes WiScan and ImageJ 48h post - infection . ( n = 4 , mean ± s . e . m . ; * * P ‐ value ≤ 0 . 01 ; ( ns ) non ‐ significant , paired t ‐ test ) . D - F . MDM were treated with 5μM ETO for 18h and co ‐ infected in the presence of ETO with HIV - 1 BaL and SIVmac virus ‐ like particles containing Vpx / Vpr ( SIV VLP ) . DNA was isolated 18 h post ‐ infection for qPCR quantification of F . Late RT products ; G . 2LTR circles ; H integrated viral DNA . Late viral RT products . AZT : MDM treated with 20μM AZT , a reverse - transcriptase inhibitor , were used as control . ( n = 3 , mean ± s . e . m . ; ( ns ) non ‐ significant , paired t ‐ test ) . D - F . MDM were treated with 5μM ETO for 18h and co ‐ infected in the presence of ETO with HIV - 1 BaL and SIVmac virus ‐ like particles containing Vpx / Vpr ( SIV VLP ) . DNA was isolated 18 h post ‐ infection for qPCR quantification of F . Late RT products ; G . 2LTR circles ; H integrated viral DNA . 2LTR circles . ( n = 3 , mean ± s . e . m . ; ( ns ) non ‐ significant ; * P ‐ value ≤ 0 . 05 , paired t ‐ test ) . D - F . MDM were treated with 5μM ETO for 18h and co ‐ infected in the presence of ETO with HIV - 1 BaL and SIVmac virus ‐ like particles containing Vpx / Vpr ( SIV VLP ) . DNA was isolated 18 h post ‐ infection for qPCR quantification of F . Late RT products ; G . 2LTR circles ; H integrated viral DNA . Integrated copies of viral DNA , Alu - Gag qPCR . ( n = 3 , mean ± s . e . m . ; ( ns ) non ‐ significant ; * * P ‐ value ≤ 0 . 01 , paired t ‐ test ) . The percentage of infected cells was quantified by Hermes WiScan and ImageJ 48h post - infection . ( n = 3 , mean ± s . e . m . ; ( ns ) non ‐ significant ; * * P ‐ value ≤ 0 . 01 , paired t ‐ test ) .

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

Figure 2MDM were transfected with control or pool of SAMHD1 (KD) siRNA and 3 days later treated with 5μM ETO and infected in the presence of ETO with VSV‐G pseudotyped HIV‐1 GFP 18h later. Cells from a representative donor were used for immunoblotting. The percentage of infected cells was quantified by the automated cell‐imaging system Hermes WiScan and ImageJ 48h post-infection. (n = 3, mean ± s.e.m.; **P‐value ≤ 0.01; (ns) non‐significant, paired t‐test).MDM were treated with 5μM ETO for 18h and co‐infected in the presence of ETO with VSV‐G HIV‐1 GFP and SIVmac virus‐like particles containing Vpx/Vpr (SIV VLP). Cells from a representative donor were used for immunoblotting. The percentage of infected cells was quantified by the Hermes WiScan and ImageJ 48h post-infection. (n = 3, mean ± s.e.m.; **P‐value ≤ 0.01; (ns) non‐significant, paired t‐test).MDM were treated with 5μM ETO for 18h and co‐infected in the presence of ETO with VSV‐G HIV‐1 GFP and SIVmac virus‐like particles containing Vpx/Vpr (SIV VLP) or deleted for Vpx (SIV VLP del Vpx) or deleted for Vpr (SIV VLP del Vpr). Cells from a representative donor were used for immunoblotting. The percentage of infected cells was quantified by the automated cell‐imaging system Hermes WiScan and ImageJ 48h post-infection. (n = 4, mean ± s.e.m.; **P‐value ≤ 0.01; (ns) non‐significant, paired t‐test).D-F. MDM were treated with 5μM ETO for 18h and co‐infected in the presence of ETO with HIV-1 BaL and SIVmac virus‐like particles containing Vpx/Vpr (SIV VLP). DNA was isolated 18 h post‐infection for qPCR quantification of F. Late RT products; G. 2LTR circles; H integrated viral DNA.Late viral RT products. AZT: MDM treated with 20μM AZT, a reverse-transcriptase inhibitor, were used as control. (n = 3, mean ± s.e.m.; (ns) non‐significant, paired t‐test).D-F. MDM were treated with 5μM ETO for 18h and co‐infected in the presence of ETO with HIV-1 BaL and SIVmac virus‐like particles containing Vpx/Vpr (SIV VLP). DNA was isolated 18 h post‐infection for qPCR quantification of F. Late RT products; G. 2LTR circles; H integrated viral DNA.2LTR circles. (n = 3, mean ± s.e.m.; (ns) non‐significant; *P‐value ≤ 0.05, paired t‐test).D-F. MDM were treated with 5μM ETO for 18h and co‐infected in the presence of ETO with HIV-1 BaL and SIVmac virus‐like particles containing Vpx/Vpr (SIV VLP). DNA was isolated 18 h post‐infection for qPCR quantification of F. Late RT products; G. 2LTR circles; H integrated viral DNA.Integrated copies of viral DNA, Alu-Gag qPCR. (n = 3, mean ± s.e.m.; (ns) non‐significant; **P‐value ≤ 0.01, paired t‐test).The percentage of infected cells was quantified by Hermes WiScan and ImageJ 48h post-infection. (n = 3, mean ± s.e.m.; (ns) non‐significant; **P‐value ≤ 0.01, paired t‐test).


Figure 3A - B . MDM were treated with 5μM ETO for 18h and co ‐ infected in the presence of ETO with VSV ‐ G HIV ‐ 1 GFP and SIVmac virus ‐ like particles containing Vpx wild type ( WT ) / Vpr or Vpx Q76A mutant / Vpr ( SIV VLP Q76A ) . ( n = 3 , mean ± s . e . m . ; ( ns ) non ‐ significant ; * P ‐ value ≤ 0 . 05 ; * * P ‐ value ≤ 0 . 01 , paired t ‐ test ) . MDM were differentiated and cultured in Human serum instead of FCS . A - B . MDM were treated with 5μM ETO for 18h and co ‐ infected in the presence of ETO with VSV ‐ G HIV ‐ 1 GFP and SIVmac virus ‐ like particles containing Vpx wild type ( WT ) / Vpr or Vpx Q76A mutant / Vpr ( SIV VLP Q76A ) . ( n = 3 , mean ± s . e . m . ; ( ns ) non ‐ significant ; * P ‐ value ≤ 0 . 05 ; * * P ‐ value ≤ 0 . 01 , paired t ‐ test ) . MDM were differentiated and cultured in FCS . A standard culture condition used in all experiments . See Materials and Methods . C - F . MDM we treated with 5μM ETO for 18h and co ‐ infected in the presence of ETO with HIV - 1 BaL and SIV VLP Q76A . DNA was isolated 18 h post ‐ infection for qPCR quantification of D . Late RT products ; E . 2LTR circles ; F . integrated viral DNA . The percentage of infected cells was quantified by the automated cell ‐ imaging system Hermes WiScan and ImageJ 48h post - infection . Cells from a representative donor were used for immunoblotting . ( n = 3 , mean ± s . e . m . ; * P ‐ value ≤ 0 . 05 ; * * P ‐ value ≤ 0 . 01 , paired t ‐ test ) . C - F . MDM we treated with 5μM ETO for 18h and co ‐ infected in the presence of ETO with HIV - 1 BaL and SIV VLP Q76A . DNA was isolated 18 h post ‐ infection for qPCR quantification of D . Late RT products ; E . 2LTR circles ; F . integrated viral DNA . Late viral RT products . AZT : MDM treated with 20μM AZT , a reverse - transcriptase inhibitor , were used as control . ( n = 3 , mean ± s . e . m . ; ( ns ) non ‐ significant ; * P ‐ value ≤ 0 . 05 , paired t ‐ test ) . C - F . MDM we treated with 5μM ETO for 18h and co ‐ infected in the presence of ETO with HIV - 1 BaL and SIV VLP Q76A . DNA was isolated 18 h post ‐ infection for qPCR quantification of D . Late RT products ; E . 2LTR circles ; F . integrated viral DNA . 2LTR circles . ( n = 3 , mean ± s . e . m . ; ( ns ) non ‐ significant ; * P ‐ value ≤ 0 . 05 , paired t ‐ test ) . C - F . MDM we treated with 5μM ETO for 18h and co ‐ infected in the presence of ETO with HIV - 1 BaL and SIV VLP Q76A . DNA was isolated 18 h post ‐ infection for qPCR quantification of D . Late RT products ; E . 2LTR circles ; F . integrated viral DNA . Integrated copies of viral DNA , Alu - Gag qPCR . ( n = 3 , mean ± s . e . m . ; * P ‐ value ≤ 0 . 05 ; * * P ‐ value ≤ 0 . 01 , paired t ‐ test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-B. MDM were treated with 5μM ETO for 18h and co‐infected in the presence of ETO with VSV‐G HIV‐1 GFP and SIVmac virus‐like particles containing Vpx wild type (WT)/Vpr or Vpx Q76A mutant/Vpr (SIV VLP Q76A). (n = 3, mean ± s.e.m.; (ns) non‐significant; *P‐value ≤ 0.05; **P‐value ≤ 0.01, paired t‐test).MDM were differentiated and cultured in Human serum instead of FCS.A-B. MDM were treated with 5μM ETO for 18h and co‐infected in the presence of ETO with VSV‐G HIV‐1 GFP and SIVmac virus‐like particles containing Vpx wild type (WT)/Vpr or Vpx Q76A mutant/Vpr (SIV VLP Q76A). (n = 3, mean ± s.e.m.; (ns) non‐significant; *P‐value ≤ 0.05; **P‐value ≤ 0.01, paired t‐test).MDM were differentiated and cultured in FCS. A standard culture condition used in all experiments. See Materials and Methods. C-F. MDM we treated with 5μM ETO for 18h and co‐infected in the presence of ETO with HIV-1 BaL and SIV VLP Q76A. DNA was isolated 18 h post‐infection for qPCR quantification of D. Late RT products; E. 2LTR circles; F. integrated viral DNA.  The percentage of infected cells was quantified by the automated cell‐imaging system Hermes WiScan and ImageJ 48h post-infection. Cells from a representative donor were used for immunoblotting. (n = 3, mean ± s.e.m.; *P‐value ≤ 0.05; **P‐value ≤ 0.01, paired t‐test).  C-F. MDM we treated with 5μM ETO for 18h and co‐infected in the presence of ETO with HIV-1 BaL and SIV VLP Q76A. DNA was isolated 18 h post‐infection for qPCR quantification of D. Late RT products; E. 2LTR circles; F. integrated viral DNA.Late viral RT products. AZT: MDM treated with 20μM AZT, a reverse-transcriptase inhibitor, were used as control. (n = 3, mean ± s.e.m.; (ns) non‐significant; *P‐value ≤ 0.05, paired t‐test).C-F. MDM we treated with 5μM ETO for 18h and co‐infected in the presence of ETO with HIV-1 BaL and SIV VLP Q76A. DNA was isolated 18 h post‐infection for qPCR quantification of D. Late RT products; E. 2LTR circles; F. integrated viral DNA.2LTR circles. (n = 3, mean ± s.e.m.; (ns) non‐significant; *P‐value ≤ 0.05, paired t‐test).C-F. MDM we treated with 5μM ETO for 18h and co‐infected in the presence of ETO with HIV-1 BaL and SIV VLP Q76A. DNA was isolated 18 h post‐infection for qPCR quantification of D. Late RT products; E. 2LTR circles; F. integrated viral DNA.Integrated copies of viral DNA, Alu-Gag qPCR. (n = 3, mean ± s.e.m.; *P‐value ≤ 0.05; **P‐value ≤ 0.01, paired t‐test).


Figure 4MDM were treated with 5μM ETO for 18h , 3μg / ml cGAMP and 10ng / ml IFNβfor 18h . RNA was isolated and qPCR performed for selected genes using TaqMan assays . Expression levels of target genes were normalised to GAPDH . ( n = 3 , mean ± s . e . m . ; * P ‐ value ≤ 0 . 05 ; ( ns ) non ‐ significant , paired t ‐ test ) . MDM were treated with 5μM ETO or cGAMP for 18h or 100ng / ml LPS for 2h . Cells were stained and analysed for IRF3 translocation into nucleus . Scale bars : 10μm . Quantification of nuclei positive for IRF3 staining . ( n = 3 , mean ± s . e . m . ) .

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Figure 4MDM were treated with 5μM ETO for 18h, 3μg/ml cGAMP and 10ng/ml IFNβfor 18h. RNA was isolated and qPCR performed for selected genes using TaqMan assays. Expression levels of target genes were normalised to GAPDH. (n = 3, mean ± s.e.m.; *P‐value ≤ 0.05; (ns) non‐significant, paired t‐test).MDM were treated with 5μM ETO or cGAMP for 18h or 100ng/ml LPS for 2h. Cells were stained and analysed for IRF3 translocation into nucleus. Scale bars: 10μm.Quantification of nuclei positive for IRF3 staining. (n = 3, mean ± s.e.m.).


Figure 5MDM were treated with increasing concentrations of ETO and CTH . Cells were stained for MCM2 expression , acquired and analysed using the automated cell ‐ imaging system Hermes WiScan and ImageJ . On average 104 cells were acquired . ( n = 3 , mean ± s . e . m . ) . MDM were treated with 5μM ETO , lysed and immunoblotting performed to detect cell cycle / cell cycle arrest and DNA damage associated proteins . MDM were treated with 5μM ETO or 0 . 01μM CTH for 18h , lysed and immunoblotting performed to detect cell cycle / cell cycle arrest and DNA damage associated proteins .

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Figure 5MDM were treated with increasing concentrations of ETO and CTH. Cells were stained for MCM2 expression, acquired and analysed using the automated cell‐imaging system Hermes WiScan and ImageJ. On average 104 cells were acquired. (n = 3, mean ± s.e.m.).MDM were treated with 5μM ETO, lysed and immunoblotting performed to detect cell cycle/cell cycle arrest and DNA damage associated proteins.MDM were treated with 5μM ETO or 0.01μM CTH for 18h, lysed and immunoblotting performed to detect cell cycle/cell cycle arrest and DNA damage associated proteins.


Figure 1C Relative mRNA expression of Zeb1 , Acp5 , and Ctsk during the differentiation from BMDMs to osteoclasts in culture ( n = 3 ) . D Western blot of Zeb1 , c - Src , and Ctsk expression in BMDMs and mature osteoclasts ( n = 3 ) . F Zeb1 ( red ) and TRAP ( green ) immunofluorescence of a femur section including the growth plate ( GP ) and metaphysis from a wild - type mouse . The multinucleated cells associated with the surface of the bone marrow cavity ( BM cavity ) that are shown in the red box are further enlarged in the panels to the right . Scale bar , 10 μm .

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Figure 1C Relative mRNA expression of Zeb1, Acp5, and Ctsk during the differentiation from BMDMs to osteoclasts in culture (n = 3).D Western blot of Zeb1, c-Src, and Ctsk expression in BMDMs and mature osteoclasts (n = 3).F Zeb1 (red) and TRAP (green) immunofluorescence of a femur section including the growth plate (GP) and metaphysis from a wild-type mouse. The multinucleated cells associated with the surface of the bone marrow cavity (BM cavity) that are shown in the red box are further enlarged in the panels to the right. Scale bar, 10 μm.


Figure 2A Representative nanoCT of sagittal sections of femurs with 3D reconstruction of the distal femur trabeculae of 3 - month - old wild - type and Zeb1ΔM / ΔM male mice are shown . The genotype of Zeb1 + / + , Zeb1f / + , Zebf / f mice is defined as " wild - type " control . Scale bar , 500 μm . B Quantification of BV / TV , Tb . Th , Tb . N , and Tb . Sp as determined by nanoCT in 3 - month - old wild - type and Zeb1ΔM / ΔM male mice ( n = 8 ) . CC H & E and TRAP staining of the distal femurs of 3 - month - old male wild - type and Zeb1ΔM / ΔM mice . Scale bar , 200 μm . D Quantification of osteoclast number per bone surface ( N . Oc / BS ) and eroded surface per bone surface ( ES / BS ) , as well as serum CTX - I and OCN levels in 3 - month - old wild - type and Zeb1ΔM / ΔM male mice are shown ( n = 6 ) . EE Golden ' s trichrome staining illuminating howship ' s lacunae ( arrow ) of the distal femurs of 3 - month - old male wild - type and Zeb1ΔM / ΔM mice were performed along with double - calcein bone labeling and confocal imaging to assess bone formation . Scale bar , 10 μm . F Quantification of MAR and BFR as assessed in 3 - month - old male wild - type and Zeb1ΔM / ΔM mice ( n = 6 ) .

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Figure 2A Representative nanoCT of sagittal sections of femurs with 3D reconstruction of the distal femur trabeculae of 3-month-old wild-type and Zeb1ΔM/ΔM male mice are shown. The genotype of Zeb1+/+, Zeb1f/+, Zebf/f mice is defined as "wild-type" control. Scale bar, 500 μm. B Quantification of BV/TV, Tb.Th, Tb.N, and Tb.Sp as determined by nanoCT in 3-month-old wild-type and Zeb1ΔM/ΔM male mice (n = 8). CC H&E and TRAP staining of the distal femurs of 3-month-old male wild-type and Zeb1ΔM/ΔM mice. Scale bar, 200 μm. D Quantification of osteoclast number per bone surface (N.Oc/BS) and eroded surface per bone surface (ES/BS), as well as serum CTX-I and OCN levels in 3-month-old wild-type and Zeb1ΔM/ΔM male mice are shown (n = 6). EE Golden's trichrome staining illuminating howship's lacunae (arrow) of the distal femurs of 3-month-old male wild-type and Zeb1ΔM/ΔM mice were performed along with double-calcein bone labeling and confocal imaging to assess bone formation. Scale bar, 10 μm. F Quantification of MAR and BFR as assessed in 3-month-old male wild-type and Zeb1ΔM/ΔM mice (n = 6). D


Figure 3B Zeb1 , NFATc1 , c - Fos , c - Src , β3 integrin , Mmp9 , Mmp14 , and Ctsk expression as assessed by Western blot in wild - type and Zeb1ΔM / ΔM BMDMs during osteoclast differentiation ( n = 3 ) . G Supernatant CTX - I determined by ELISA ( n = 6 ) . H , I After a 6 - day culture atop bone slice , phalloidin staining ( red ) was performed in wild - type versus Zeb1ΔM / ΔM osteoclasts ( I ) , and actin ring area per cell quantified ( H ) . Scale bar , 20 μm .

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Figure 3B Zeb1, NFATc1, c-Fos, c-Src, β3 integrin, Mmp9, Mmp14, and Ctsk expression as assessed by Western blot in wild-type and Zeb1ΔM/ΔM BMDMs during osteoclast differentiation (n = 3).G Supernatant CTX-I determined by ELISA (n = 6).H, I After a 6-day culture atop bone slice, phalloidin staining (red) was performed in wild-type versus Zeb1ΔM/ΔM osteoclasts (I), and actin ring area per cell quantified (H). Scale bar, 20 μm.


Figure 4E Relative mRNA expression of Ckmt1 in BMDMs and osteoclasts generated from wild - type or Zeb1ΔM / ΔM mice . F MtCK1 , Ckb , VDAC , and Tomm20 expression as assessed by Western blot in BMDMs and osteoclasts generated from wild - type or Zeb1ΔM / ΔM mice ( n = 3 ) . H MtCK1 ( green ) immunofluorescence of wild - type and Zeb1ΔM / ΔM osteoclasts . Scale bar , 20

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Figure 4E Relative mRNA expression of Ckmt1 in BMDMs and osteoclasts generated from wild-type or Zeb1ΔM/ΔM mice.F MtCK1, Ckb, VDAC, and Tomm20 expression as assessed by Western blot in BMDMs and osteoclasts generated from wild-type or Zeb1ΔM/ΔM mice (n = 3).H MtCK1 (green) immunofluorescence of wild-type and Zeb1ΔM/ΔM osteoclasts. Scale bar, 20 μ


Figure 5A Phosphocreatine ( PCr ) and creatine ( Cr ) level , as well as PCr / Cr ratio of wild - type or Zeb1ΔM / ΔM osteoclasts cultured atop plastic substrata as determined by LC / MS analysis ( n = 3 ) . B , C Oxygen consumption rate ( OCR ) profile plot ( B ) and mitochondrial function parameters ( C ) analyzed by XF Cell Mito Stress Assay in osteoclasts from wild - type or Zeb1ΔM / ΔM mice after sequential treatment of compounds modulating mitochondrial function ( n = 4 ) .

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Figure 5A Phosphocreatine (PCr) and creatine (Cr) level, as well as PCr/Cr ratio of wild-type or Zeb1ΔM/ΔM osteoclasts cultured atop plastic substrata as determined by LC/MS analysis (n = 3).B, C Oxygen consumption rate (OCR) profile plot (B) and mitochondrial function parameters (C) analyzed by XF Cell Mito Stress Assay in osteoclasts from wild-type or Zeb1ΔM/ΔM mice after sequential treatment of compounds modulating mitochondrial function (n = 4).


Figure 6C Wild - type BMDMs were transduced with either a mock vector , wild - type human MtCK1 , or a catalytically - inactive MtCK1C316G mutant expression vector , differentiated into osteoclasts , and cell lysates collected for MtCK1 , Ckb , Tomm20 , VDAC , Zeb1 , c - Src , and Ctsk immunoblotting ( n = 3 ) . E PCr / Cr ratio of mock vector - , MtCK1 - , or MtCK1C316G - transduced wild - type osteoclasts cultured on plastic substrata as determined by LC / MS analysis ( n = 3 ) . OCR profile plot ( H ) analyzed by XF Cell Mito Stress Assay in mock vector - , MtCK1 - , or MtCK1C316G - transduced wild - type osteoclasts after sequential treatment of compounds modulating mitochondrial function ( n = 4 ) . J , K Mock vector - , MtCK1 - , or MtCK1C316G - transduced wild - type pre - osteoclasts were cultured atop bone slices for 3 days , stained with phalloidin ( red ) . Osteoclasts were removed and resorption pits visualized by WGA - DAB staining ( J ) . The actin ring area per cell and resorption pit area were quantified ( K ; n = 6 ) . Scale bar , upper 20 μm , lower 100 μm .

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 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 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

Figure 6C Wild-type BMDMs were transduced with either a mock vector, wild-type human MtCK1, or a catalytically-inactive MtCK1C316G mutant expression vector, differentiated into osteoclasts, and cell lysates collected for MtCK1, Ckb, Tomm20, VDAC, Zeb1, c-Src, and Ctsk immunoblotting (n = 3).E PCr/Cr ratio of mock vector-, MtCK1-, or MtCK1C316G-transduced wild-type osteoclasts cultured on plastic substrata as determined by LC/MS analysis (n = 3).OCR profile plot (H) analyzed by XF Cell Mito Stress Assay in mock vector-, MtCK1-, or MtCK1C316G-transduced wild-type osteoclasts after sequential treatment of compounds modulating mitochondrial function (n = 4).J, K Mock vector-, MtCK1-, or MtCK1C316G-transduced wild-type pre-osteoclasts were cultured atop bone slices for 3 days, stained with phalloidin (red). Osteoclasts were removed and resorption pits visualized by WGA-DAB staining (J). The actin ring area per cell and resorption pit area were quantified (K; n = 6). Scale bar, upper 20 μm, lower 100 μm.


Figure 7A , B Human monocyte - derived macrophages ( hMDMs ) were transfected with either shCTRL or siRNA targeting ZEB1 , differentiated into osteoclasts , and relative mRNA expression of CKMT1 ( A ) and mitochondrial creatine kinase activity determined ( B ; n = 3 ) . F Mitochondrial function parameters analyzed by XF Cell Mito Stress Assay in non - targeting siCTRL - or siZEB1 - transfected osteoclasts after sequential treatment of compounds modulating mitochondrial function ( n = 4 ) . G , H siCTRL - or siZEB1 - transfected human osteoclasts were cultured atop bone slices for 6 days and stained with phalloidin ( red ) . Osteoclasts were removed and resorption pits visualized by WGA - DAB staining ( G ) . Scale bar , upper 20 μm , lower 100 μm . The actin ring area per cell , resorption pit area , and the number of TRAP + MNCs were quantified ( H ; n = 6 ) . I - L Calvaria isolated from wild - type and Zeb1ΔM / ΔM mice were cultured in the presence or absence of 200 μM cyclocreatine , and stained for phalloidin ( red ) and Vpp3 ( green ) ( I ) . Scale bar , 10 μm . The TRAP activity of whole cell lysates , actin ring area per cell , and supernatant CTX - I were quantified ( J - L ; n = 6 ) .

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Figure 7A, B Human monocyte-derived macrophages (hMDMs) were transfected with either shCTRL or siRNA targeting ZEB1, differentiated into osteoclasts, and relative mRNA expression of CKMT1 (A) and mitochondrial creatine kinase activity determined (B; n = 3).F Mitochondrial function parameters analyzed by XF Cell Mito Stress Assay in non-targeting siCTRL- or siZEB1-transfected osteoclasts after sequential treatment of compounds modulating mitochondrial function (n = 4).G, H siCTRL- or siZEB1-transfected human osteoclasts were cultured atop bone slices for 6 days and stained with phalloidin (red). Osteoclasts were removed and resorption pits visualized by WGA-DAB staining (G). Scale bar, upper 20 μm, lower 100 μm. The actin ring area per cell, resorption pit area, and the number of TRAP+ MNCs were quantified (H; n = 6).I-L Calvaria isolated from wild-type and Zeb1ΔM/ΔM mice were cultured in the presence or absence of 200 μM cyclocreatine, and stained for phalloidin (red) and Vpp3 (green) (I). Scale bar, 10 μm. The TRAP activity of whole cell lysates, actin ring area per cell, and supernatant CTX-I were quantified (J-L; n = 6).


Figure 1A panel of 42 breast cancer cell lines was challenged with increasing concentrations of ATRA ( 11 nM - 10 μM ) for 3 , 6 , and 9 days , and cell growth was determined . The graphs show the growth - inhibitory effect exerted by the indicated concentrations of ATRA in SKBR3 , HCC - 1954 , and MDA - MB436 cells which are representative of lines characterized by a high , intermediate and low ATRA score , respectively . Each point is the mean ± SD of three replicate cultures . * * Significantly lower than the corresponding vehicle - treated group ( P - value < 0 . 01 , Student ' s t - test ) . Cell lines are ranked in ascending order according to the ATRA score . The plot distinguishes four separate groups of cell lines ( D - A ) with increasing ATRA scores , as indicated by the colored scale on the right . The cell lines are also grouped in tertiles , T1 - T3 , according to an ascending ATRA score . Each calculated value is representative of at least two independent experiments .

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Figure 1A panel of 42 breast cancer cell lines was challenged with increasing concentrations of ATRA (11 nM-10 μM) for 3, 6, and 9 days, and cell growth was determined.The graphs show the growth-inhibitory effect exerted by the indicated concentrations of ATRA in SKBR3, HCC-1954, and MDA-MB436 cells which are representative of lines characterized by a high, intermediate and low ATRA score, respectively. Each point is the mean ± SD of three replicate cultures. **Significantly lower than the corresponding vehicle-treated group (P-value < 0.01, Student's t-test).Cell lines are ranked in ascending order according to the ATRA score. The plot distinguishes four separate groups of cell lines (D-A) with increasing ATRA scores, as indicated by the colored scale on the right. The cell lines are also grouped in tertiles, T1-T3, according to an ascending ATRA score. Each calculated value is representative of at least two independent experiments.


Figure 2SCID mice were xenografted subcutaneously with 10 × 106HCC - 1599 cells on both sides . One week after transplantation 10 animals / experimental group were treated intraperitoneally with vehicle ( DMSO ) or two doses of ATRA ( 7 . 5 and 15 . 0 mg / kg ) once / day , 5 days a week for a total of 24 days . At the end of this period , treatment was discontinued until sacrifice . The size of the tumors was determined with a caliper and the volume plotted . Each point is the mean ± SE of 20 tumors . * Significantly lower than the corresponding vehicle - treated group ( P - value < 0 . 05 , Student ' s t - test ) . * * Significantly lower than the corresponding vehicle - treated group ( P - value < 0 . 01 , Student ' s t - test ) . Magnetic resonance imaging ( MRI ) analysis was performed on five animals / experimental group on day 24 . The picture shows representative 2D images of tumor sections from one animal treated with vehicle and one animal treated with 15 . 0 mg / kg ATRA . The bar graph shows the volume of the tumors calculated after 3D reconstruction of the MRI images . Each point is the mean ± SE of five tumors . The P - value of the comparisons of ATRA versus vehicle is shown .

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Figure 2SCID mice were xenografted subcutaneously with 10 × 106HCC-1599 cells on both sides. One week after transplantation 10 animals/experimental group were treated intraperitoneally with vehicle (DMSO) or two doses of ATRA (7.5 and 15.0 mg/kg) once/day, 5 days a week for a total of 24 days. At the end of this period, treatment was discontinued until sacrifice.The size of the tumors was determined with a caliper and the volume plotted. Each point is the mean ± SE of 20 tumors. *Significantly lower than the corresponding vehicle-treated group (P-value < 0.05, Student's t-test). **Significantly lower than the corresponding vehicle-treated group (P-value < 0.01, Student's t-test).Magnetic resonance imaging (MRI) analysis was performed on five animals/experimental group on day 24. The picture shows representative 2D images of tumor sections from one animal treated with vehicle and one animal treated with 15.0 mg/kg ATRA. The bar graph shows the volume of the tumors calculated after 3D reconstruction of the MRI images. Each point is the mean ± SE of five tumors. The P-value of the comparisons of ATRA versus vehicle is shown.


Figure 3ATRA - dependent anti - tumor activity in short - term cultures of primary breast tumorsTissue slices deriving from surgical specimens were challenged with vehicle ( DMSO ) or ATRA ( 0 . 1 μM ) for 48 h . The panel illustrates examples of the immuno - histochemical data obtained in four representative cases : ( i ) Luminal - A ( Lum A ) ; ( ii ) Luminal - B ( Lum B ) ; ( iii ) Her2 + with RARA coamplification ( Her2 + / RARA + ) and ( iv ) triple negative ( TN ) . The percentage of Ki67 - positive tumor cells in the 45 samples considered are illustrated by the bar graphs . Each value represents the mean ± SE of at least five separate fields for each experimental sample . * Significantly lower than the corresponding vehicle - treated control ( P - value < 0 . 05 , Student ' s t - test ) . * * Significantly lower than the corresponding vehicle - treated control ( P - value < 0 . 01 , Student ' s t - test ) .

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Figure 3ATRA-dependent anti-tumor activity in short-term cultures of primary breast tumorsTissue slices deriving from surgical specimens were challenged with vehicle (DMSO) or ATRA (0.1 μM) for 48 h.The panel illustrates examples of the immuno-histochemical data obtained in four representative cases: (i) Luminal-A (Lum A); (ii) Luminal-B (Lum B); (iii) Her2+ with RARA coamplification (Her2+/RARA+) and (iv) triple negative (TN).The percentage of Ki67-positive tumor cells in the 45 samples considered are illustrated by the bar graphs. Each value represents the mean ± SE of at least five separate fields for each experimental sample. *Significantly lower than the corresponding vehicle-treated control (P-value < 0.05, Student's t-test). **Significantly lower than the corresponding vehicle-treated control (P-value < 0.01, Student's t-test).


Figure 4Associations between components of the retinoid signaling pathway and the phenotype in breast cancer cell linesThe figure illustrates the associations between the indicated retinoid receptors / binding proteins and the Luminal versus Basal phenotype ( left panels ) , ER positivity versus ER negativity ( middle panels ) as well as HER2 positivity versus HER2 negativity ( right panels ) . The gene - expression microarray and RNA - seq data refer to 42 and 40 breast cancer cell lines , respectively . The P - values of the indicated comparisons after Student ' s t - test are shown in red . L ( red ) = Luminal cell lines ; B ( blue ) = Basal cell lines ; ER + ( red ) = ER - positive cell lines ; ER − ( red ) = ER - negative cell lines ; ER − ( blue ) = ER - negative Luminal cell lines ; H + ( red ) = HER2 - positive cell lines ; H − ( red ) = HER2 - negative cell lines ; H + ( blue ) = HER2 - positive Luminal cell lines ; H − ( blue ) = HER2 - negative Luminal cell lines . fpkm = fragments per kilobase of exon per million fragments mapped .

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Figure 4Associations between components of the retinoid signaling pathway and the phenotype in breast cancer cell linesThe figure illustrates the associations between the indicated retinoid receptors/binding proteins and the Luminal versus Basal phenotype (left panels), ER positivity versus ER negativity (middle panels) as well as HER2 positivity versus HER2 negativity (right panels). The gene-expression microarray and RNA-seq data refer to 42 and 40 breast cancer cell lines, respectively. The P-values of the indicated comparisons after Student's t-test are shown in red. L (red) = Luminal cell lines; B (blue) = Basal cell lines; ER+ (red) = ER-positive cell lines; ER− (red) = ER-negative cell lines; ER− (blue) = ER-negative Luminal cell lines; H+ (red) = HER2-positive cell lines; H− (red) = HER2-negative cell lines; H+ (blue) = HER2-positive Luminal cell lines; H− (blue) = HER2-negative Luminal cell lines. fpkm = fragments per kilobase of exon per million fragments mapped.


Figure 5Associations between components of the retinoid pathway and breast cancer phenotypeAssociations between the expression of the indicated members of the retinoid pathway in the TCGA gene - expression database are shown . Mammary tumors are classified into Luminal - A , Luminal - B , HER2 - like , Normal - like , and Basal according to the PAM50 fingerprint . The average expression levels and the corresponding SD values of the indicated members of the retinoid pathway are shown by the upper box plots . For each member of the retinoid pathway , significant differences between the indicated groups of tumors are shown in the lower table . Significant P - values for the indicated comparisons ( Student ' s t - test ) are shown in red .

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Figure 5Associations between components of the retinoid pathway and breast cancer phenotypeAssociations between the expression of the indicated members of the retinoid pathway in the TCGA gene-expression database are shown. Mammary tumors are classified into Luminal-A, Luminal-B, HER2-like, Normal-like, and Basal according to the PAM50 fingerprint. The average expression levels and the corresponding SD values of the indicated members of the retinoid pathway are shown by the upper box plots. For each member of the retinoid pathway, significant differences between the indicated groups of tumors are shown in the lower table. Significant P-values for the indicated comparisons (Student's t-test) are shown in red.


Figure 6The gene - expression microarray and RNA - seq data associated with 40 of the breast cancer cell lines were used for the analyses . The panels illustrate the associations of RARα and PPARβ / δ with ATRA sensitivity . The left panels show the basal average levels of the indicated transcript in the cell lines belonging to the T1 and T3 groups ( 13 cell lines in each of the T1 and T3 groups ) defined by ascending ATRA scores . The intermediate and right panels indicate the same results after stratification for the Luminal ( microarray and RNA - seq data = 7 cell lines in each of the T1 and T3 groups ) and the Basal ( microarray and RNA - seq data = 7 cell lines in each of the T1 and T3 groups ) phenotype , respectively . fpkm = fragments per kilobase of exon per million fragments mapped . The basal expression levels of the indicated RAR - isoform variants were determined with the use of specific Taqman assays . The results are associated with ATRA sensitivity before ( TOTAL ) and after stratification of the cell lines for the Luminal and Basal phenotype as in ( A ) . Data information : Significant P - values ( Student ' s t - test ) are indicated in red .

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Figure 6The gene-expression microarray and RNA-seq data associated with 40 of the breast cancer cell lines were used for the analyses. The panels illustrate the associations of RARα and PPARβ/δ with ATRA sensitivity. The left panels show the basal average levels of the indicated transcript in the cell lines belonging to the T1 and T3 groups (13 cell lines in each of the T1 and T3 groups) defined by ascending ATRA scores. The intermediate and right panels indicate the same results after stratification for the Luminal (microarray and RNA-seq data = 7 cell lines in each of the T1 and T3 groups) and the Basal (microarray and RNA-seq data = 7 cell lines in each of the T1 and T3 groups) phenotype, respectively. fpkm = fragments per kilobase of exon per million fragments mapped.The basal expression levels of the indicated RAR-isoform variants were determined with the use of specific Taqman assays. The results are associated with ATRA sensitivity before (TOTAL) and after stratification of the cell lines for the Luminal and Basal phenotype as in (A).Data information: Significant P-values (Student's t-test) are indicated in red.


Figure 7Basal levels of RARα , RARβ , and RARγ mRNA splicing variants in mammary tumorsTotal RNA was extracted from the tissue slices deriving from the surgical specimens of breast cancer patients used in Fig3 before any treatment with DMSO or ATRA . RNA was subjected to RT - PCR analysis to determine the basal expression of the indicated RAR splicing variants . Each value represents the mean ± SD of two replicate measurements . The table shows the statistical significance of the indicated comparisons . * Significantly different ( P - value < 0 . 05 , Student ' s t - test ) . * * Significantly different ( P - value < 0 . 01 , Student ' s t - test ) . The plots illustrate the average expression levels of the indicated mRNAs ( mean ± SD of two replicates ) in tumor samples classified as ATRA - sensitive ( Sens ) and ATRA - resistant ( Res ) according to the response of Ki67 . * Significantly different ( P - value < 0 . 05 , Student ' s t - test ) .

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Figure 7Basal levels of RARα, RARβ, and RARγ mRNA splicing variants in mammary tumorsTotal RNA was extracted from the tissue slices deriving from the surgical specimens of breast cancer patients used in Fig3 before any treatment with DMSO or ATRA. RNA was subjected to RT-PCR analysis to determine the basal expression of the indicated RAR splicing variants.Each value represents the mean ± SD of two replicate measurements. The table shows the statistical significance of the indicated comparisons. *Significantly different (P-value < 0.05, Student's t-test). **Significantly different (P-value < 0.01, Student's t-test).The plots illustrate the average expression levels of the indicated mRNAs (mean ± SD of two replicates) in tumor samples classified as ATRA-sensitive (Sens) and ATRA-resistant (Res) according to the response of Ki67. *Significantly different (P-value < 0.05, Student's t-test).


Figure 8RARα protein and ATRA sensitivity in breast cancer cell linesTotal proteins were extracted from logarithmically growing cell lines and subjected to Western blot analysis . The panel illustrates representative Western blots for a number of cell lines . The blots were sequentially developed with RARα - specific and control tubulin antibodies . The positions of the RARα and tubulin bands ( right ) along with the position of a relevant molecular weight marker ( left ) are indicated . To normalize the Western blot signals in different gels , the same preparation of RARα - transfected COS - 7cell extracts ( COS - 7 ) and MCF7 extracts was loaded in each gel . The blots are representative of at least two independent experiments providing similar results . The quantitative results obtained after densitometric analysis of the RARα bands are plotted against the ATRA scores . Cell lines are grouped according to the ATRA score ( A - D groups and T1 - T3 tertiles ) . Left : The upper graph indicates the averages expression levels of the RARα protein in T1 - T3 cell lines . The middle and lower graphs indicate the average expression levels after stratification of the cell lines for the Luminal ( six cell lines in each of the T1 and T3 groups ) and the Basal ( six cell lines in each of the T1 and T3 groups ) phenotype , respectively . Right : The levels of the RARα protein in Basal and Luminal cell lines ( upper graph ) , in ER + , ER − , and Luminal ER − cell lines ( middle graph ) and the indicated classes of HER2 + and HER2 − cell lines ( lower graph ) are shown . * Significantly different ( P - value < 0 . 05 , Student ' s t - test ) . * * Significantly different ( P - value < 0 . 01 , Student ' s t - test ) . The plots show the correlation curves between the levels of the indicated RARα - variant transcripts and the RARα protein .

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Figure 8RARα protein and ATRA sensitivity in breast cancer cell linesTotal proteins were extracted from logarithmically growing cell lines and subjected to Western blot analysis.The panel illustrates representative Western blots for a number of cell lines. The blots were sequentially developed with RARα-specific and control tubulin antibodies. The positions of the RARα and tubulin bands (right) along with the position of a relevant molecular weight marker (left) are indicated. To normalize the Western blot signals in different gels, the same preparation of RARα-transfected COS-7cell extracts (COS-7) and MCF7 extracts was loaded in each gel. The blots are representative of at least two independent experiments providing similar results.The quantitative results obtained after densitometric analysis of the RARα bands are plotted against the ATRA scores. Cell lines are grouped according to the ATRA score (A-D groups and T1-T3 tertiles).Left: The upper graph indicates the averages expression levels of the RARα protein in T1-T3 cell lines. The middle and lower graphs indicate the average expression levels after stratification of the cell lines for the Luminal (six cell lines in each of the T1 and T3 groups) and the Basal (six cell lines in each of the T1 and T3 groups) phenotype, respectively. Right: The levels of the RARα protein in Basal and Luminal cell lines (upper graph), in ER+, ER−, and Luminal ER−cell lines (middle graph) and the indicated classes of HER2+ and HER2−cell lines (lower graph) are shown. *Significantly different (P-value < 0.05, Student's t-test). **Significantly different (P-value < 0.01, Student's t-test).The plots show the correlation curves between the levels of the indicated RARα-variant transcripts and the RARα protein.


Figure 9Effects of RAR agonists on the growth of Luminal and Basal breast cancer cell linesThe indicated Luminal and Basal cell lines were challenged with increasing concentrations of ATRA , the RARα agonist , AM580 , the RARβ agonist , UVI2003 , and the RARγ agonist , BMS961 , for 6 days . The complement of RAR - variant transcripts expressed in each cell line is shown in the left bar graphs ( mean ± SD of two replicate measurements ) . The growth curves ( sulforhodamine assay ) of the cell lines are illustrated by the right linear plots . The results are expressed in % values relative to the corresponding control dishes treated with vehicle alone ( right graphs ) . Each result is the mean ± SD of five replicate wells . ATRA sc = ATRA score .

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

Figure 9Effects of RAR agonists on the growth of Luminal and Basal breast cancer cell linesThe indicated Luminal and Basal cell lines were challenged with increasing concentrations of ATRA, the RARα agonist, AM580, the RARβ agonist, UVI2003, and the RARγ agonist, BMS961, for 6 days. The complement of RAR-variant transcripts expressed in each cell line is shown in the left bar graphs (mean ± SD of two replicate measurements). The growth curves (sulforhodamine assay) of the cell lines are illustrated by the right linear plots. The results are expressed in % values relative to the corresponding control dishes treated with vehicle alone (right graphs). Each result is the mean ± SD of five replicate wells. ATRA sc = ATRA score.


Figure 10A RARα1 / 3 plasmid construct and the corresponding control void vector were stably transfected into ATRA - resistantMDA - MB453 cells . Two cell clones over - expressing RARα ( RARA - C5 andRARA - C7 ) and two appropriate control clones ( Vect - C1 andVect - C2 ) were isolated . A RARα3 shRNA plasmid construct and the corresponding void vector were stably transfected into ATRA - sensitiveSKBR3 cells . After selection , two cell clones silenced for RARα3 ( RARA - sh18 andRARA - sh19 ) and two appropriate control clones ( Vect - C6 andVect - C8 ) were isolated . A , D The indicated clones and the parental cell line ( WT ) were transiently transfected with the RARE - DR5 - Lucretinoid reporter construct and the level of luciferase activity was measured 24 h after treatment with vehicle ( DMSO ) and ATRA ( 100 nM ) , as illustrated in the upper bar graph . Each value is the mean ± SD of three replicate cultures . The levels of the RARα protein measured in the indicated clones by Western blotanalysis is shown under the bar graph . To demonstrate that similar levels of total proteins were loaded in each lane , the β - actin band signal obtained after re - blotting of the gel is shown . FI = Fluorescence intensity . B , E The panels illustrate the growth curves of the indicated MDA - MB453 and SKBR3 clones and the SKBR3 parental cell lines ( WT ) measured with the sulforhodamine assay . C The bar graphs illustrate the effect of increasing concentrations of ATRA on the growth of the indicated MDA - MB453 clones and the parental cell line . The cell lines were challenged with vehicle ( DMSO ) or ATRA for 3 , 6 , and 9 days prior to the sulforhodamine assay . OD = optical density at 540 nm . Each value is the mean ± SD of five replicate culture wells . A , D The indicated clones and the parental cell line ( WT ) were transiently transfected with the RARE - DR5 - Lucretinoid reporter construct and the level of luciferase activity was measured 24 h after treatment with vehicle ( DMSO ) and ATRA ( 100 nM ) , as illustrated in the upper bar graph . Each value is the mean ± SD of three replicate cultures . The levels of the RARαprotein measured in the indicated clones by Western blot analysis is shown under the bar graph . To demonstrate that similar levels of total proteins were loaded in each lane , the β - actin band signal obtained after re - blotting of the gel is shown . FI = Fluorescence intensity . B , E The panels illustrate the growth curves of the indicated MDA - MB453 and SKBR3 clones and the SKBR3 parental cell lines ( WT ) measured with the sulforhodamine assay . C The bar graphs illustrate the effect of increasing concentrations of ATRA on the growth of the indicated MDA - MB453 clones and the parental cell line . The cell lines were challenged with vehicle ( DMSO ) or ATRA for 3 , 6 , and 9 days prior to the sulforhodamine assay . OD = optical density at 540 nm . Each value is the mean ± SD of five replicate culture wells . A , D The indicated clones and the parental cell line ( WT ) were transiently transfected with the RARE - DR5 - Lucretinoid reporter construct and the level of luciferase activity was measured 24 h after treatment with vehicle ( DMSO ) and ATRA ( 100 nM ) , as illustrated in the upper bar graph . Each value is the mean ± SD of three replicate cultures . The levels of the RARαprotein measured in the indicated clones by Western blotanalysis is shown under the bar graph . To demonstrate that similar levels of total proteins were loaded in each lane , the β - actin band signal obtained after re - blotting of the gel is shown . FI = Fluorescence intensity . B , E The panels illustrate the growth curves of the indicated MDA - MB453 and SKBR3 clones and the SKBR3 parental cell lines ( WT ) measured with the sulforhodamine assay . C The bar graphs illustrate the effect of increasing concentrations of ATRA on the growth of the indicated MDA - MB453 clones and the parental cell line . The cell lines were challenged with vehicle ( DMSO ) or ATRA for 3 , 6 , and 9 days prior to the sulforhodamine assay . OD = optical density at 540 nm . Each value is the mean ± SD of five replicate culture wells . F The graphs illustrate the effect of increasing concentrations of ATRA on the growth of the indicated SKBR3 clones and the parental cell line . The cell lines were challenged with vehicle ( DMSO ) or ATRA for 3 and 6 days prior to the sulforhodamine assay . Each value is the mean ± SD of five replicate culture wells . G The panel illustrates the effects exerted by ATRA ( 0 . 1 μM ) on random cell motility of the indicated MDA - MB453 and SKBR3 clones . The results are representative of two independent experiments . Each value is the mean ± SE of the motility of at least 60 cells .

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

Figure 10A RARα1/3 plasmid construct and the corresponding control void vector were stably transfected into ATRA-resistantMDA-MB453 cells. Two cell clones over-expressing RARα (RARA-C5 andRARA-C7) and two appropriate control clones (Vect-C1 andVect-C2) were isolated. A RARα3 shRNA plasmid construct and the corresponding void vector were stably transfected into ATRA-sensitiveSKBR3 cells. After selection, two cell clones silenced for RARα3 (RARA-sh18 andRARA-sh19) and two appropriate control clones (Vect-C6 andVect-C8) were isolated.A, D The indicated clones and the parental cell line (WT) were transiently transfected with the RARE-DR5-Lucretinoid reporter construct and the level of luciferase activity was measured 24 h after treatment with vehicle (DMSO) and ATRA (100 nM), as illustrated in the upper bar graph. Each value is the mean ± SD of three replicate cultures. The levels of the RARα protein measured in the indicated clones by Western blotanalysis is shown under the bar graph. To demonstrate that similar levels of total proteins were loaded in each lane, the β-actin band signal obtained after re-blotting of the gel is shown. FI = Fluorescence intensity.B, E The panels illustrate the growth curves of the indicated MDA-MB453 and SKBR3 clones and the SKBR3 parental cell lines (WT) measured with the sulforhodamine assay.C The bar graphs illustrate the effect of increasing concentrations of ATRA on the growth of the indicated MDA-MB453 clones and the parental cell line. The cell lines were challenged with vehicle (DMSO) or ATRA for 3, 6, and 9 days prior to the sulforhodamine assay. OD = optical density at 540 nm. Each value is the mean ± SD of five replicate culture wells.A, D The indicated clones and the parental cell line (WT) were transiently transfected with the RARE-DR5-Lucretinoid reporter construct and the level of luciferase activity was measured 24 h after treatment with vehicle (DMSO) and ATRA (100 nM), as illustrated in the upper bar graph. Each value is the mean ± SD of three replicate cultures. The levels of the RARαprotein measured in the indicated clones by Western blot analysis is shown under the bar graph. To demonstrate that similar levels of total proteins were loaded in each lane, the β-actin band signal obtained after re-blotting of the gel is shown. FI = Fluorescence intensity.B, E The panels illustrate the growth curves of the indicated MDA-MB453 and SKBR3 clones and the SKBR3 parental cell lines (WT) measured with the sulforhodamine assay.C The bar graphs illustrate the effect of increasing concentrations of ATRA on the growth of the indicated MDA-MB453 clones and the parental cell line. The cell lines were challenged with vehicle (DMSO) or ATRA for 3, 6, and 9 days prior to the sulforhodamine assay. OD = optical density at 540 nm. Each value is the mean ± SD of five replicate culture wells.A, D The indicated clones and the parental cell line (WT) were transiently transfected with the RARE-DR5-Lucretinoid reporter construct and the level of luciferase activity was measured 24 h after treatment with vehicle (DMSO) and ATRA (100 nM), as illustrated in the upper bar graph. Each value is the mean ± SD of three replicate cultures. The levels of the RARαprotein measured in the indicated clones by Western blotanalysis is shown under the bar graph. To demonstrate that similar levels of total proteins were loaded in each lane, the β-actin band signal obtained after re-blotting of the gel is shown. FI = Fluorescence intensity.B, E The panels illustrate the growth curves of the indicated MDA-MB453 and SKBR3 clones and the SKBR3 parental cell lines (WT) measured with the sulforhodamine assay.C The bar graphs illustrate the effect of increasing concentrations of ATRA on the growth of the indicated MDA-MB453 clones and the parental cell line. The cell lines were challenged with vehicle (DMSO) or ATRA for 3, 6, and 9 days prior to the sulforhodamine assay. OD = optical density at 540 nm. Each value is the mean ± SD of five replicate culture wells.F The graphs illustrate the effect of increasing concentrations of ATRA on the growth of the indicated SKBR3 clones and the parental cell line. The cell lines were challenged with vehicle (DMSO) or ATRA for 3 and 6 days prior to the sulforhodamine assay. Each value is the mean ± SD of five replicate culture wells.G The panel illustrates the effects exerted by ATRA (0.1 μM) on random cell motility of the indicated MDA-MB453 and SKBR3 clones. The results are representative of two independent experiments. Each value is the mean ± SE of the motility of at least 60 cells.


Figure 1B , C GOT2 can be acetylated . Flag ‐ tagged GOT2 was ectopically expressed in HEK293T cells treated with NAM ( 5 mM ) and / or TSA ( 0 . 5 mM ) for the indicated time period . Acetylation levels of Flag ‐ bead ‐ purified GOT2 were determined by Western blot analysis using a pan ‐ anti ‐ acetyllysine antibody ( α ‐ Ac ) . IB and IP denote immunoblotting and immunoprecipitation , respectively . Relative GOT2 acetylation ratios were calculated after normalizing against Flag . D GOT2 acetylation enhances the interaction between ectopically expressed GOT2 and MDH2 . Flag ‐ GOT2 and Myc ‐ MDH2 were co ‐ overexpressed in HEK293T cells treated without or with NAM ( 5 mM ) for the indicated time period . The acetylation level of Flag ‐ bead ‐ purified GOT2 and the protein association between ectopic proteins of GOT2 and MDH2 were determined by Western blot analysis . E Acetylation enhances the interaction between ectopically expressed GOT2 and endogenous MDH2 . Flag ‐ GOT2 was co ‐ overexpressed in HEK293T cells treated without or with NAM ( 5 mM ) for the indicated time period . The acetylation level of Flag ‐ GOT2 and its association with endogenous MDH2 were determined by Western blot analysis . F Acetylation enhances the interaction between ectopically expressed MDH2 and endogenous GOT2 . Flag ‐ MDH2 was co ‐ overexpressed in HEK293T cells treated without or with NAM ( 5 mM ) for the indicated time period . The protein association between Flag ‐ MDH2 and endogenous GOT2 was determined by Western blot analysis . H Mapping the major lysine residue ( s ) of acetylation in GOT2 whose acetylation can affect protein interaction between GOT2 and MDH2 . Putative acetylated residues were divided into six groups according to their position in the structure of GOT2 . Each group of putative acetylated lysine ( K ) sites was mutated to arginine ( R ) , and the deacetylated mimic K ‐ to ‐ R mutants were examined for their protein association with ectopically expressed Myc ‐ MDH2 by Western blot analysis . I GOT2 coupled with MDH2 shows a high level of K159 acetylation . Flag ‐ GOT2 was overexpressed in HEK293T cells without or with co ‐ overexpression of Myc ‐ MDH2 . These transfected cells were treated with NAM ( 5 mM ) for 5 h . Double immunoprecipitation was performed to obtain the Flag ‐ GOT2 from the Myc ‐ MDH2 immunoprecipitates ( Ppt , supposed to be GOT2 bound with MDH2 ) . The remaining supernatants ( Sup , supposed to be GOT2 not bound with MDH2 ) were also harvested and precipitated by Flag antibody to test the K159 acetylation level of Flag ‐ GOT2 by Western blot analysis . J The 3KQ / R mutations do not change GOT2 enzyme activity . Wild ‐ type and 3K mutant GOT2 proteins were overexpressed and purified from E . coli , and the enzyme activity of GOT2 was determined as described in " " . Shown are average values with standard deviation ( SD ) of triplicated experiments . n . s . = not significant for the indicated comparison .

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 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 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 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 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

Figure 1B, C GOT2 can be acetylated. Flag‐tagged GOT2 was ectopically expressed in HEK293T cells treated with NAM (5 mM) and/or TSA (0.5 mM) for the indicated time period. Acetylation levels of Flag‐bead‐purified GOT2 were determined by Western blot analysis using a pan‐anti‐acetyllysine antibody (α‐Ac). IB and IP denote immunoblotting and immunoprecipitation, respectively. Relative GOT2 acetylation ratios were calculated after normalizing against Flag.D GOT2 acetylation enhances the interaction between ectopically expressed GOT2 and MDH2. Flag‐GOT2 and Myc‐MDH2 were co‐overexpressed in HEK293T cells treated without or with NAM (5 mM) for the indicated time period. The acetylation level of Flag‐bead‐purified GOT2 and the protein association between ectopic proteins of GOT2 and MDH2 were determined by Western blot analysis.E Acetylation enhances the interaction between ectopically expressed GOT2 and endogenous MDH2. Flag‐GOT2 was co‐overexpressed in HEK293T cells treated without or with NAM (5 mM) for the indicated time period. The acetylation level of Flag‐GOT2 and its association with endogenous MDH2 were determined by Western blot analysis.F Acetylation enhances the interaction between ectopically expressed MDH2 and endogenous GOT2. Flag‐MDH2 was co‐overexpressed in HEK293T cells treated without or with NAM (5 mM) for the indicated time period. The protein association between Flag‐MDH2 and endogenous GOT2 was determined by Western blot analysis.H Mapping the major lysine residue(s) of acetylation in GOT2 whose acetylation can affect protein interaction between GOT2 and MDH2. Putative acetylated residues were divided into six groups according to their position in the structure of GOT2. Each group of putative acetylated lysine (K) sites was mutated to arginine (R), and the deacetylated mimic K‐to‐R mutants were examined for their protein association with ectopically expressed Myc‐MDH2 by Western blot analysis.I GOT2 coupled with MDH2 shows a high level of K159 acetylation. Flag‐GOT2 was overexpressed in HEK293T cells without or with co‐overexpression of Myc‐MDH2. These transfected cells were treated with NAM (5 mM) for 5 h. Double immunoprecipitation was performed to obtain the Flag‐GOT2 from the Myc‐MDH2 immunoprecipitates (Ppt, supposed to be GOT2 bound with MDH2). The remaining supernatants (Sup, supposed to be GOT2 not bound with MDH2) were also harvested and precipitated by Flag antibody to test the K159 acetylation level of Flag‐GOT2 by Western blot analysis.J The 3KQ/R mutations do not change GOT2 enzyme activity. Wild‐type and 3K mutant GOT2 proteins were overexpressed and purified from E. coli, and the enzyme activity of GOT2 was determined as described in "". Shown are average values with standard deviation (SD) of triplicated experiments. n.s. = not significant for the indicated comparison.


Figure 2A , B Glucose and glutamine increase GOT2 K159 acetylation and GOT2 - MDH2 association in HEK293T cells . Flag ‐ GOT2 and Myc ‐ MDH2 were overexpressed in cells treated with increased concentrations of glucose ( A ) or glutamine ( B ) for 6 h . GOT2 proteins were purified by Flag beads , and the K159 acetylation level of GOT2 and its protein association with Myc ‐ MDH2 were determined by Western blot analysis . C , D Glucose and glutamine increase GOT2 K159 acetylation in Panc ‐ 1 cells . Flag ‐ GOT2 was overexpressed in cells treated with different concentrations of glucose ( C ) and glutamine ( D ) for 4 h . GOT2proteins were purified by Flag beads , and the K159 acetylation level of GOT2 was determined by Western blot analysis . Relative GOT2 K159 acetylation levels were normalized against Flagprotein levels . E , F Quantification of the percentage of K159 ‐ acetylated endogenous GOT2 in Panc ‐ 1 cells . Recombinant fully K159 ‐ acetylated GOT2 was loaded onto the same gel , together with endogenous GOT2 from Panc ‐ 1 cells treated without or with glucose ( 12 mM ) ( E ) or glutamine ( 2 mM ) ( F ) for 4 h . GOT2 protein and K159 acetylation were detected by Western blot . The percentages of K159 acetylation in GOT2 were calculated after normalizing against GOT2 protein levels . G , H 3K mutant GOT2 displays negligible response in changing protein association with MDH2 after glucose or glutamine treatment . Panc ‐ 1 cells with GOT2 knockdown and re ‐ expression of wild ‐ type or 3K mutant GOT2 were treated with glucose ( G ) or glutamine ( H ) at the indicated concentrations for 4 h . The protein association between Flag ‐ tagged wild ‐ type or 3K mutant GOT2 and endogenous MDH2 was determined by Western blot analysis .

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 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

Figure 2A, B Glucose and glutamine increase GOT2 K159 acetylation and GOT2-MDH2 association in HEK293T cells. Flag‐GOT2 and Myc‐MDH2 were overexpressed in cells treated with increased concentrations of glucose (A) or glutamine (B) for 6 h. GOT2 proteins were purified by Flag beads, and the K159 acetylation level of GOT2 and its protein association with Myc‐MDH2 were determined by Western blot analysis.C, D Glucose and glutamine increase GOT2 K159 acetylation in Panc‐1 cells. Flag‐GOT2 was overexpressed in cells treated with different concentrations of glucose (C) and glutamine (D) for 4 h. GOT2proteins were purified by Flag beads, and the K159 acetylation level of GOT2 was determined by Western blot analysis. Relative GOT2 K159 acetylation levels were normalized against Flagprotein levels.E, F Quantification of the percentage of K159‐acetylated endogenous GOT2 in Panc‐1 cells. Recombinant fully K159‐acetylated GOT2 was loaded onto the same gel, together with endogenous GOT2 from Panc‐1 cells treated without or with glucose (12 mM) (E) or glutamine (2 mM) (F) for 4 h. GOT2 protein and K159 acetylation were detected by Western blot. The percentages of K159 acetylation in GOT2 were calculated after normalizing against GOT2 protein levels.G, H 3K mutant GOT2 displays negligible response in changing protein association with MDH2 after glucose or glutamine treatment. Panc‐1 cells with GOT2 knockdown and re‐expression of wild‐type or 3K mutant GOT2 were treated with glucose (G) or glutamine (H) at the indicated concentrations for 4 h. The protein association between Flag‐tagged wild‐type or 3K mutant GOT2 and endogenous MDH2 was determined by Western blot analysis.


Figure 3A GOT2 interacts with SIRT3 , but not SIRT4 and SIRT5 . Flag ‐ tagged GOT2 was ectopically expressed in HEK293T cells together with the individual HA ‐ tagged SIRT as indicated . Proteins were purified by IP with Flag beads , following Western blot to detect SIRTs with an HA antibody . B Endogenous proteins of GOT2 and SIRT3 interact with each other . SIRT3 protein in HEK293T cells was purified by IP with an anti ‐ SIRT3 antibody , following Western blot to detect GOT2 with an anti ‐ GOT2 antibody . C GOT2 deacetylation is dependent on SIRT3 catalytic activity . Flag ‐ tagged GOT2 was ectopically expressed in HEK293T cells together with HA ‐ tagged wild ‐ type SIRT3 and a catalytically inactive mutant , SIRT3 H248Y . GOT2 proteins were purified by Flag beads , following Western blot to detect GOT2 K159 acetylation . D SIRT3 impairs the protein interaction between GOT2 and MDH2 . HEK293T cells were transfected with the plasmids as indicated . GOT2 proteins were purified by Flag beads , and their protein interaction with Myc ‐ MDH2 was determined by Western blot . E Knockdown of SIRT3 increases GOT2 K159 acetylation and enhances GOT2 - MDH2 association . HEK293T cells with or without transient SIRT3 knockdown were transfected with the plasmids as indicated . GOT2 proteins were purified by Flag beads , and the K159 acetylation level of GOT2 and its protein association with endogenous MDH2 were determined by Western blot analysis . F , G Knockdown of SIRT3 diminishes the effect of glucose or glutamine on changing GOT2 K159 acetylation . Flag ‐ tagged GOT2 was ectopically expressed in HEK293T cells without or with transient SIRT3 knockdown . These cells were treated with different concentrations of glucose ( F ) and glutamine ( G ) as indicated . GOT2 proteins were purified by Flag beads , and the K159 acetylation level of GOT2 was determined by Western blot analysis . H , I Up ‐ regulation of Sirt3 leads to decreased Got2 K159 acetylation in mouse tissues during fasting . C57BL / 6 mice ( male , 20 weeks ; n = 2 per group ) were fed a normal chow diet or fasted overnight . Upon sacrifice , mouse liver and white adipose tissues were harvested , and the levels of the indicated proteins were determined by Western blot analysis .

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Figure 3A GOT2 interacts with SIRT3, but not SIRT4 and SIRT5. Flag‐tagged GOT2 was ectopically expressed in HEK293T cells together with the individual HA‐tagged SIRT as indicated. Proteins were purified by IP with Flag beads, following Western blot to detect SIRTs with an HA antibody.B Endogenous proteins of GOT2 and SIRT3 interact with each other. SIRT3 protein in HEK293T cells was purified by IP with an anti‐SIRT3 antibody, following Western blot to detect GOT2 with an anti‐GOT2 antibody.C GOT2 deacetylation is dependent on SIRT3 catalytic activity. Flag‐tagged GOT2 was ectopically expressed in HEK293T cells together with HA‐tagged wild‐type SIRT3 and a catalytically inactive mutant, SIRT3 H248Y. GOT2 proteins were purified by Flag beads, following Western blot to detect GOT2 K159 acetylation.D SIRT3 impairs the protein interaction between GOT2 and MDH2. HEK293T cells were transfected with the plasmids as indicated. GOT2 proteins were purified by Flag beads, and their protein interaction with Myc‐MDH2 was determined by Western blot.E Knockdown of SIRT3 increases GOT2 K159 acetylation and enhances GOT2-MDH2 association. HEK293T cells with or without transient SIRT3 knockdown were transfected with the plasmids as indicated. GOT2 proteins were purified by Flag beads, and the K159 acetylation level of GOT2 and its protein association with endogenous MDH2 were determined by Western blot analysis.F, G Knockdown of SIRT3 diminishes the effect of glucose or glutamine on changing GOT2 K159 acetylation. Flag‐tagged GOT2 was ectopically expressed in HEK293T cells without or with transient SIRT3 knockdown. These cells were treated with different concentrations of glucose (F) and glutamine (G) as indicated. GOT2 proteins were purified by Flag beads, and the K159 acetylation level of GOT2 was determined by Western blot analysis.H, I Up‐regulation of Sirt3 leads to decreased Got2 K159 acetylation in mouse tissues during fasting. C57BL/6 mice (male, 20 weeks; n = 2 per group) were fed a normal chow diet or fasted overnight. Upon sacrifice, mouse liver and white adipose tissues were harvested, and the levels of the indicated proteins were determined by Western blot analysis.


Figure 4A The malate - aspartate shuttle , such as LDH , also plays a key role in regulating cytosolic NADH levels in Panc ‐ 1 cells . Cells were treated with AOA ( 1 . 6 mM , 20 min ) , oxamate ( 5 mM , 20 min ) , and EGCG ( 0 . 5 mM , 20 min ) . The cytosolic NADH level was measured as described in . B , C GOT2 3K acetylation promotes the net transfer of cytosolic NADH into mitochondria . In Panc ‐ 1 stable cells with GOT2 knockdown and re ‐ expressing the indicated proteins , the NADH level in the cytosol ( B ) and mitochondrion ( C ) was determined in cell extracts as described in . D GOT2 3K acetylation regulates mitochondrial NADH / NAD + redox in the cell . In Panc ‐ 1 stable cells with GOT2 knockdown and re ‐ expressing the indicated proteins , the ratio of NADH / NAD + in the mitochondria of cells was measured as described in . E GOT2 3K acetylation promotes ATP production . In Panc ‐ 1 stable cells with GOT2 knockdown and re ‐ expressing the indicated proteins , ATP production was determined in cell extracts as described in . F GOT2 3K acetylation promotes NADPH production . In Panc ‐ 1 stable cells with GOT2 knockdown and re ‐ expressing the indicated proteins , the ratio of NADPH / NADP + was determined in cell extracts as described in . G GOT2 3K acetylation promotes GSH production . In Panc ‐ 1 stable cells with GOT2 knockdown and re ‐ expressing the indicated proteins , the GSH / GSSG ratio was determined in cell extracts as described in . H GOT2 3K acetylation suppresses cellular ROS levels . In Panc ‐ 1 stable cells with GOT2 knockdown and re ‐ expressing the indicated proteins , ROS was determined as described in in cells under non ‐ stressed condition or exposed to menadione ( 50 μM for 30 min ) . I GOT2 3K acetylation protects cells from oxidative damage . Panc ‐ 1 stable cells with GOT2 knockdown and re ‐ expressing the indicated proteins were treated with increasing concentrations of menadione for 3 h as indicated , and cell viability was determined by counting the remaining adherent cells . J , K Panc ‐ 1 stable cells with GOT2 knockdown and re ‐ expressing the indicated proteins were seeded in a 6 ‐ well plate , and the cells were maintained under the condition of low glucose ( 0 . 5 mM , J ) or high glucose ( 12 mM , K ) . Culture medium was refreshed every day , and cell numbers were counted every 1 - 2 days over a period of 7 days . L , M Panc ‐ 1 stable cells in ( J , K ) were grown under low glucose ( 0 . 5 mM ) or high glucose ( 12 mM ) for 7 days . The level of histone H3 phosphorylation at Ser10 was determined by Western blot analysis ( L ) . The percentages of living cells and apoptotic cells were determined by flow cytometry as described in ( M ) .

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 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 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 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 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 O O O

Figure 4A The malate-aspartate shuttle, such as LDH, also plays a key role in regulating cytosolic NADH levels in Panc‐1 cells. Cells were treated with AOA (1.6 mM, 20 min), oxamate (5 mM, 20 min), and EGCG (0.5 mM, 20 min). The cytosolic NADH level was measured as described in .B, C GOT2 3K acetylation promotes the net transfer of cytosolic NADH into mitochondria. In Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins, the NADH level in the cytosol (B) and mitochondrion (C) was determined in cell extracts as described in .D GOT2 3K acetylation regulates mitochondrial NADH/NAD+ redox in the cell. In Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins, the ratio of NADH/NAD+ in the mitochondria of cells was measured as described in .E GOT2 3K acetylation promotes ATP production. In Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins, ATP production was determined in cell extracts as described in .F GOT2 3K acetylation promotes NADPH production. In Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins, the ratio of NADPH/NADP+ was determined in cell extracts as described in .G GOT2 3K acetylation promotes GSH production. In Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins, the GSH/GSSG ratio was determined in cell extracts as described in .H GOT2 3K acetylation suppresses cellular ROS levels. In Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins, ROS was determined as described in in cells under non‐stressed condition or exposed to menadione (50 μM for 30 min).I GOT2 3K acetylation protects cells from oxidative damage. Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins were treated with increasing concentrations of menadione for 3 h as indicated, and cell viability was determined by counting the remaining adherent cells.J, K Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins were seeded in a 6‐well plate, and the cells were maintained under the condition of low glucose (0.5 mM, J) or high glucose (12 mM, K). Culture medium was refreshed every day, and cell numbers were counted every 1-2 days over a period of 7 days.L, M Panc‐1 stable cells in (J, K) were grown under low glucose (0.5 mM) or high glucose (12 mM) for 7 days. The level of histone H3 phosphorylation at Ser10 was determined by Western blot analysis (L). The percentages of living cells and apoptotic cells were determined by flow cytometry as described in (M).


Figure 5A - C GOT2 3K acetylation promotes xenograft tumorgrowth . Panc ‐ 1 stable cells with GOT2 knockdown and re ‐ expressing the indicated proteins ( 2 × 106cells ) were injected subcutaneously into the flanks of nude mice . The tumor volume was carefully monitored over the indicated time period ( A ) . At 8 weeks after injection , tumors from six mice were extracted , photographed , and weighted ( B , C ) . D GOT2 3K acetylation promotes tumor cellproliferation . Tumor sections from xenografts were prepared for IHC staining , and the PCNA ‐ stained tumor sections were analyzed by quantifying the PCNA ‐ positive area . E Clinical cases with increased GOT2 K159 acetylation in humanpancreatictumor tissues . 10 pairs of humanpancreatictumor tissue ( shown as T ) and adjacent normal tissue ( shown as N ) were lysed . The acetylation level of GOT2 at K159 was compared against GOT2protein by Western blot . F Clinical cases with decreased SIRT3protein expression in humanpancreatictumor tissues . 10 pairs of humanpancreatictumor tissue ( shown as T ) and adjacent normal tissue ( shown as N ) were lysed . The SIRT3protein levels were compared against tubulin by Western blot . G , H Quantification of GOT2 K159 acetylation and SIRT3protein expression in clinical cases as described in ( E ) and ( F ) .

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 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

Figure 5A-C GOT2 3K acetylation promotes xenograft tumorgrowth. Panc‐1 stable cells with GOT2 knockdown and re‐expressing the indicated proteins (2 × 106cells) were injected subcutaneously into the flanks of nude mice. The tumor volume was carefully monitored over the indicated time period (A). At 8 weeks after injection, tumors from six mice were extracted, photographed, and weighted (B, C).D GOT2 3K acetylation promotes tumor cellproliferation. Tumor sections from xenografts were prepared for IHC staining, and the PCNA‐stained tumor sections were analyzed by quantifying the PCNA‐positive area.E Clinical cases with increased GOT2 K159 acetylation in humanpancreatictumor tissues. 10 pairs of humanpancreatictumor tissue (shown as T) and adjacent normal tissue (shown as N) were lysed. The acetylation level of GOT2 at K159 was compared against GOT2protein by Western blot.F Clinical cases with decreased SIRT3protein expression in humanpancreatictumor tissues. 10 pairs of humanpancreatictumor tissue (shown as T) and adjacent normal tissue (shown as N) were lysed. The SIRT3protein levels were compared against tubulin by Western blot.G, H Quantification of GOT2 K159 acetylation and SIRT3protein expression in clinical cases as described in (E) and (F).


Figure 1B . DIS3 expression in total cell lysates of control ( siCTRL ) and DIS3 U2OS silenced cells ( siDIS3 ) was analyzed by Western blot upon rescue with empty vector ( EV ) or DIS3 expressing vector ( DIS3 ) . Lamin B was used as the loading control . C . Representative images of comet assays performed under alkaline conditions in U2OS cells transfected with siCTRL or siDIS3 and then infected with a vector expressing DIS3 ( DIS3 ) or an empty vector ( EV ) . Quantitative analysis of comet tail lengths for each condition . Plots represent three biological replicates , with more than 150 cells for each point . The analysis is performed using Casplab software , error bars indicate s . e . m . ( n = 3 ) . * * * * p - value < 0 . 0001 . ns = not statistically significant , two - way ANOVA , scale bar is 200 µm . D . Immunostaining against RPA in U2OS cells transfected with siCTRL or with siDIS3 and 48 hours later infected with a vector expressing DIS3 ( DIS3 ) or an empty vector ( EV ) . The averages of foci number per cell were calculated from three independent experiments using Cell Profiler software . At least 150 cells were counted for each point , error bars indicate s . e . m . ( n = 3 ) . * p - value < 0 . 05 . ns = not statistically significant , two - way ANOVA , scale bar is 10 µm .

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Figure 1B. DIS3 expression in total cell lysates of control (siCTRL) and DIS3 U2OS silenced cells (siDIS3) was analyzed by Western blot upon rescue with empty vector (EV) or DIS3 expressing vector (DIS3). Lamin B was used as the loading control.C. Representative images of comet assays performed under alkaline conditions in U2OS cells transfected with siCTRL or siDIS3 and then infected with a vector expressing DIS3 (DIS3) or an empty vector (EV). Quantitative analysis of comet tail lengths for each condition. Plots represent three biological replicates, with more than 150 cells for each point. The analysis is performed using Casplab software, error bars indicate s.e.m. (n=3). **** p-value < 0.0001. ns= not statistically significant, two-way ANOVA, scale bar is 200 µm.D. Immunostaining against RPA in U2OS cells transfected with siCTRL or with siDIS3 and 48 hours later infected with a vector expressing DIS3 (DIS3) or an empty vector (EV). The averages of foci number per cell were calculated from three independent experiments using Cell Profiler software. At least 150 cells were counted for each point, error bars indicate s.e.m. (n=3). * p-value < 0.05. ns= not statistically significant, two-way ANOVA, scale bar is 10 µm.


Figure 2A . Immunostaining with S9 . 6 ( green ) and DAPI ( blue ) in U2OS cells , transfected for 48 hours with siCTRL or siDIS3 and then mock - treated ( UNTR ) or treated with RNase H or RNase III for 1 hour before fixation . Histograms show S9 . 6 signal intensity per cell , error bars indicate s . e . m . ( n = 3 , biological replicates ) . More than 150 cells were counted , and error bars indicate s . e . m . ( n = 3 biological replicates ) . ) . * * * * = p < 0 . 0001 , * * * = p < 0 . 001 , ns = not statistically significant , two - way ANOVA , scale bar is 10 µm . B . Immunostaining with J2 ( green ) and DAPI ( blue ) in U2OS cells , as in panel A ) . More than 150 cells were counted , and error bars indicate s . e . m . ( n = 3 biological replicates ) . For all conditions , differences between siCTRL and siDIS3 were not statistically significant , two - way ANOVA , scale bar is 10 µm . C . Dot blot analysis of DNA : RNA hybrids formation : serial dilutions ( 750 ng , 375 ng , 187 , 5 ng , 93 , 75 ng , 46 , 87 ng ) of genomic DNA extracted from WT or DIS3 silenced U2OS cells and pre - treated with RNase H , RNase III or mock treated , were arrayed on a membrane and probed using the S9 . 6 antibody . The histogram shows the S9 . 6 signal quantification in 3 biological replicates , error bars indicate s . e . m . , normalized on SYBR ® - Gold signal ( Adj . Vol . ) , using Image lab software . * * * = p < 0 . 001 , * * = p < 0 . 01 , ns = not statistically significant , two - way ANOVA . D . DRIP - qPCR analysis at four different R - loops sites , RPL13A , ACTB , ENSA , and TRIM33 in U2OS cells transduced with shScr or shDIS3 and treated or not with RNase H . Results are shown as mean + s . e . m of three independent experiments . * * * * = p < 0 . 0001 , * * * = p < 0 . 001 , * = p < 0 . 05 , two - way ANOVA . E . Representative pictures of comet assays performed under alkaline conditions in U2OS cells transfected for 48 hours with siCTRL or siDIS3 , untreated ( UNTR ) or treated with RNase H ( RNase H ) . Tail moments analysis was performed using Casplab software , error bars indicate s . e . m . Plots represent three biological replicates , with 150 cells for each cell line . * * * * = p < 0 . 0001 , ns = not statistically significant , two - way ANOVA , scale bar is 200 µm .

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 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 O O O O O 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 O O O O O

Figure 2A. Immunostaining with S9.6 (green) and DAPI (blue) in U2OS cells, transfected for 48 hours with siCTRL or siDIS3 and then mock-treated (UNTR) or treated with RNase H or RNase III for 1 hour before fixation. Histograms show S9.6 signal intensity per cell, error bars indicate s.e.m. (n=3, biological replicates). More than 150 cells were counted, and error bars indicate s.e.m. (n=3 biological replicates).). **** = p < 0.0001, *** = p < 0.001, ns = not statistically significant, two-way ANOVA, scale bar is 10 µm.B. Immunostaining with J2 (green) and DAPI (blue) in U2OS cells, as in panel A). More than 150 cells were counted, and error bars indicate s.e.m. (n=3 biological replicates). For all conditions, differences between siCTRL and siDIS3 were not statistically significant, two-way ANOVA, scale bar is 10 µm.C. Dot blot analysis of DNA:RNA hybrids formation: serial dilutions (750 ng, 375 ng, 187,5 ng, 93,75 ng, 46,87 ng) of genomic DNA extracted from WT or DIS3 silenced U2OS cells and pre-treated with RNase H, RNase III or mock treated, were arrayed on a membrane and probed using the S9.6 antibody. The histogram shows the S9.6 signal quantification in 3 biological replicates, error bars indicate s.e.m., normalized on SYBR®-Gold signal (Adj. Vol.), using Image lab software. *** = p < 0.001, ** = p < 0.01, ns= not statistically significant, two-way ANOVA.D. DRIP-qPCR analysis at four different R-loops sites, RPL13A, ACTB, ENSA, and TRIM33 in U2OS cells transduced with shScr or shDIS3 and treated or not with RNase H. Results are shown as mean + s.e.m of three independent experiments.**** = p < 0.0001, *** = p < 0.001, * = p < 0.05, two-way ANOVA.E. Representative pictures of comet assays performed under alkaline conditions in U2OS cells transfected for 48 hours with siCTRL or siDIS3, untreated (UNTR) or treated with RNase H (RNase H). Tail moments analysis was performed using Casplab software, error bars indicate s.e.m. Plots represent three biological replicates, with 150 cells for each cell line. **** = p < 0.0001, ns= not statistically significant, two-way ANOVA, scale bar is 200 µm.