F , p27 suppresses BIR / RMD and SSA / RMD . U2OS ( EGFP - SSA - 3427 - 3 ) cells carrying the Tet - on - cyclin E allele were infected with and without p27 lentiviral viruses . RMD was determined before and after induction of cyclin E overexpression by DOX ( 0 . 5 µg / mL ) added 24 hours before transfection with the indicated sgRNAs / Cas9 . Cyclin E expression levels and cell cycle profiles are shown in Appendix Fig S6 .

O O B-GENEPROD O O O O O O O O O B-CELL_LINE O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD O O O O O O B-GENEPROD B-ORGANISM O O O O O O O O O O B-GENEPROD I-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O

F, p27 suppresses BIR/RMD and SSA/RMD. U2OS (EGFP-SSA-3427-3) cells carrying the Tet-on-cyclin E allele were infected with and without p27 lentiviral viruses. RMD was determined before and after induction of cyclin E overexpression by DOX (0.5 µg/mL) added 24 hours before transfection with the indicated sgRNAs/Cas9. Cyclin E expression levels and cell cycle profiles are shown in Appendix Fig S6.


B , Impact of the 5 ' DSB / repeat distance on RMD frequency when the 3 ' DSB / repeat distance is fixed . RMD assays were performed using mES cells carrying the RMD - GFP reporter , and RMD frequency was determined by the number of GFP + cells normalized to transfection efficiency . The distance between the sgRNA targeting sites and the repeats is indicated .

O O O O O O O B-SUBCELLULAR O O O O O B-EXP_ASSAY O O O O B-SUBCELLULAR O O O O O O O O O O O B-CELL_TYPE O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O

B, Impact of the 5' DSB/repeat distance on RMD frequency when the 3' DSB/repeat distance is fixed. RMD assays were performed using mES cells carrying the RMD-GFP reporter, and RMD frequency was determined by the number of GFP+ cells normalized to transfection efficiency. The distance between the sgRNA targeting sites and the repeats is indicated.


C , Long - range RMD is dependent on POLD3 when one DSB is close to a repeat . mES cells carrying the RMD - GFP reporter were transfected with nontargeting siRNA ( Ctrl ) or a pool of POLD3 siRNAs ( siPOLD3 ) 24 hours before for the first time , and with transfection of sgRNAs / Cas9 the second time . The graphs show the frequency of GFP + cells normalized to the transfection efficiency . The distance between the sgRNA targeting sites and the repeats is indicated .

O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O B-CELL_TYPE O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O

C, Long-range RMD is dependent on POLD3 when one DSB is close to a repeat. mES cells carrying the RMD-GFP reporter were transfected with nontargeting siRNA (Ctrl) or a pool of POLD3 siRNAs (siPOLD3) 24 hours before for the first time, and with transfection of sgRNAs/Cas9 the second time. The graphs show the frequency of GFP+ cells normalized to the transfection efficiency. The distance between the sgRNA targeting sites and the repeats is indicated.


D and E , Impact of the 3 ' DSB / repeat distance on RMD frequency when the 5 ' DSB / repeat distance is fixed . RMD assays were performed using mES cells carrying the RMD - GFP reporter , and RMD frequency was determined by the number of GFP + cells normalized to transfection efficiency . The distance between the sgRNA targeting sites and the repeats is indicated .

O O O O O O O O O B-SUBCELLULAR O O O O O B-EXP_ASSAY O O O O B-SUBCELLULAR O O O O O O O O O O O B-CELL_TYPE O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O

D and E, Impact of the 3' DSB/repeat distance on RMD frequency when the 5' DSB/repeat distance is fixed. RMD assays were performed using mES cells carrying the RMD-GFP reporter, and RMD frequency was determined by the number of GFP+ cells normalized to transfection efficiency. The distance between the sgRNA targeting sites and the repeats is indicated.


F , RMD frequency of two far - apart repeats that are a short distance from the DSB . RMD was determined in mES cells carrying the RMD - GFP reporter using the indicated sgRNAs / Cas9 , with the frequency of GFP + cells normalized to transfection efficiency . The distance between the sgRNA targeting sites and the repeats is indicated .

O O O B-EXP_ASSAY O O O O O O O O O O O O O B-SUBCELLULAR O O O O O B-CELL_TYPE O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O

F, RMD frequency of two far-apart repeats that are a short distance from the DSB. RMD was determined in mES cells carrying the RMD-GFP reporter using the indicated sgRNAs/Cas9, with the frequency of GFP+ cells normalized to transfection efficiency. The distance between the sgRNA targeting sites and the repeats is indicated.


G , The roles of POLD3 , RAD51 and RAD52 in RMD of two far - apart repeats that are a short distance from the DSB . RMD was determined in mES cells carrying the RMD - GFP reporter using the indicated sgRNAs / Cas9 after POLD3 ( left ) or RAD51 ( middle ) was depleted by siRNA or RAD52 was knocked out ( right ) . The distance between the sgRNA targeting sites and the repeats is indicated .

O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O B-CELL_TYPE O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O

G, The roles of POLD3, RAD51 and RAD52 in RMD of two far-apart repeats that are a short distance from the DSB. RMD was determined in mES cells carrying the RMD-GFP reporter using the indicated sgRNAs/Cas9 after POLD3 (left) or RAD51 (middle) was depleted by siRNA or RAD52 was knocked out (right). The distance between the sgRNA targeting sites and the repeats is indicated.


SSA / RMD and BIR / SSA are both dependent on ATM . SSA / RMD and BIR / SSA frequencies in U2OS ( EGFP - SSA - 3427 - 17 ) cells were determined after the addition of ATM inhibitor ( 20mM ku55933 ) or DMSO to the cell culture medium 24 hours before transfection of the indicated sgRNA /

O O O O O O O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O B-CELL_LINE O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O

SSA/RMD and BIR/SSA are both dependent on ATM. SSA/RMD and BIR/SSA frequencies in U2OS (EGFP-SSA-3427-17) cells were determined after the addition of ATM inhibitor (20mM ku55933) or DMSO to the cell culture medium 24 hours before transfection of the indicated sgRNA/Cas9


BIR / SSA are both dependent on ATM . BIR / RMD in mES cells carrying the RMD - GFP reporter were determined after the addition of ATM inhibitor ( 20mM ku55933 ) or DMSO to the cell culture medium 24 hours before transfection of the indicated sgRNA / Cas9 or in ATM KO mES cells carrying the RMD - GFP reporter ( right ) .

O O O O O O O B-GENEPROD O O O O O B-CELL_TYPE O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-CELL_TYPE O O O O O B-GENEPROD O O O O O

BIR/SSA are both dependent on ATM. BIR/RMD in mES cells carrying the RMD-GFP reporter were determined after the addition of ATM inhibitor (20mM ku55933) or DMSO to the cell culture medium 24 hours before transfection of the indicated sgRNA/Cas9 or in ATM KO mES cells carrying the RMD-GFP reporter (right).


Dependence of RMD on H2AX . SSA / RMD and BIR / RMD frequencies in U2OS ( EGFP - SSA - 3427 - 17 ) cells were determined using the indicated sgRNA / Cas9 after depletion of H2AX by lentivirus expressed shRNA Depletion of H2AX by shRNA in was determined by

O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O B-CELL_LINE O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-ORGANISM O O O O B-GENEPROD O O O O O O

Dependence of RMD on H2AX. SSA/RMD and BIR/RMD frequencies in U2OS (EGFP-SSA-3427-17) cells were determined using the indicated sgRNA/Cas9 after depletion of H2AX by lentivirus expressed shRNA Depletion of H2AX by shRNA in was determined by qPCR


D , Dependence of RMD on H2AX . BIR / RMD in mES cells carrying the RMD - GFP reporter , or by transfection with H2AX siRNA or in RAD52 KO

O O O O O O B-GENEPROD O O O O O B-CELL_TYPE O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O

D, Dependence of RMD on H2AX. BIR/RMD in mES cells carrying the RMD-GFP reporter , or by transfection with H2AX siRNA or in RAD52 KO cells


A , Frequency of long - range RMD is increased by an additional DSB downstream of the 3 ' repeat . Illustrations show the GFP - RMD reporter , with sgRNAs / Cas9 cleavage sites indicated as red arrows ( left ) . The frequency of BIR / SSA in mES cells carrying the RMD - GFP reporter was determined after cleavage of the reporter by the indicated sgRNAs / Cas9 ( right ) .

O O B-EXP_ASSAY O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O

A, Frequency of long-range RMD is increased by an additional DSB downstream of the 3' repeat. Illustrations show the GFP-RMD reporter, with sgRNAs/Cas9 cleavage sites indicated as red arrows (left). The frequency of BIR/SSA in mES cells carrying the RMD-GFP reporter was determined after cleavage of the reporter by the indicated sgRNAs/Cas9 (right).


B and C , The effects of RAD52 , POLD3 , ATM and H2AX inactivation on BIR / RMD with cleavage at L17 / D10k . The frequency of BIR / RMD in mES cells carrying the RMD - GFP reporter was determined using the sgRNAs / Cas9 at L17 / D10k in RAD52 KO cells or after POLD3 depletion by siRNA ( B ) , or after treatment with ATM inhibitor ( ku55933 , 15mM ) or H2AX depletion by siRNA ( C ) .

O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-CELL_TYPE O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O

B and C, The effects of RAD52, POLD3, ATM and H2AX inactivation on BIR/RMD with cleavage at L17/D10k. The frequency of BIR/RMD in mES cells carrying the RMD-GFP reporter was determined using the sgRNAs/Cas9 at L17/D10k in RAD52 KO cells or after POLD3 depletion by siRNA (B), or after treatment with ATM inhibitor (ku55933, 15mM) or H2AX depletion by siRNA (C).


A , Long - range BIR / RMD with identical repeats or divergent repeats . The frequency of BIR / RMD in mES cells carrying the RMD - GFP reporter with identical repeats ( 0 % ) or repeats with 1 % and 3 % divergence was determined after cleavage at L17 / R3 . 3k or L17 / R28 . 4k .

O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-CELL_TYPE O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, Long-range BIR/RMD with identical repeats or divergent repeats. The frequency of BIR/RMD in mES cells carrying the RMD-GFP reporter with identical repeats (0%) or repeats with 1% and 3% divergence was determined after cleavage at L17/R3.3k or L17/R28.4k.


B and C , The effect of MSH2 on BIR / RMD when repeat sequences are divergent . The frequency of BIR / RMD was determined after cleavage at L17 / R3 . 3k or L17 / R28 . 4k in mES cells carrying the RMD - GFP reporter with 1 % and 3 % divergent repeats , and with and without MSH2 KO ( B ) or with MSH2 KO and POLD3 depletion by siRNAs ( C ) . The MSH2 KO was validated by Western blot , using KU70 as the loading control as shown in ( B ) . Data information : Error bars represent standard deviation ( SD ) of at least three independent experiments . * P < 0 . 05 , * * P < 0 . 01 , two - tailed non - paired t - test .

O O O O O O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and C, The effect of MSH2 on BIR/RMD when repeat sequences are divergent. The frequency of BIR/RMD was determined after cleavage at L17/R3.3k or L17/R28.4k in mES cells carrying the RMD-GFP reporter with 1% and 3% divergent repeats, and with and without MSH2 KO (B) or with MSH2 KO and POLD3 depletion by siRNAs (C). The MSH2 KO was validated by Western blot, using KU70 as the loading control as shown in (B). Data information: Error bars represent standard deviation (SD) of at least three independent experiments. *P < 0.05, **P < 0.01, two-tailed non-paired t-test.


( A ) Kaplan Meier plot for survival in NeuT ; Apln + / + ( n = 11 ) and NeuT ; Apln - / - ( n = 10 ) mice with mammary cancer after tumor onset . * P = 0 . 0185 ; Log rank test .

O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-ORGANISM O B-DISEASE I-DISEASE O O O O O O O O O O O O O O O

(A) Kaplan Meier plot for survival in NeuT;Apln+/+ (n=11) and NeuT;Apln-/- (n=10) mice with mammary cancer after tumor onset. *P=0.0185; Log rank test.


( B ) Tumor volumes , followed over time , of control mammary tumor E0771 cells ( shRenilla ) and Apelin - depleted ( shApln ) E0771 cells orthotopically - injected into both syngeneic C57BL / 6J Apln + / + and Apln - / - mice ( 5x105 cells / mouse ) , respectively . Tumor volumes were determined using calipers and are shown as mean tumor volumes ± S . E . M . Data shown is pooled from 2 independent experiments . Apln + / + ; shRenilla ( n = 18 ) , Apln + / + ; shApln ( n = 17 ) , Apln - / - ; shRenilla ( n = 14 ) , Apln - / - ; shApln ( n = 15 ) ; * * P < 0 . 01 , * * * P < 0 . 001 ; two - way ANOVA .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-DISEASE I-DISEASE B-CELL_LINE O O O O O B-GENEPROD O O O O O B-CELL_LINE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM O O O O B-ORGANISM O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O 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) Tumor volumes, followed over time, of control mammary tumor E0771 cells (shRenilla) and Apelin-depleted (shApln) E0771 cells orthotopically-injected into both syngeneic C57BL/6J Apln+/+ and Apln-/- mice (5x105 cells/mouse), respectively. Tumor volumes were determined using calipers and are shown as mean tumor volumes ± S.E.M. Data shown is pooled from 2 independent experiments. Apln+/+;shRenilla (n=18), Apln+/+;shApln (n=17), Apln-/-;shRenilla (n=14), Apln-/-;shApln (n=15); ** P<0.01, *** P<0.001; two-way ANOVA.


( C ) Mean percentages ( ± S . E . M . ) of CD31 + area in E0771 shRenilla ( n = 3 ) or shApln ( n = 3 ) mammary tumors , assessed on day 23 post - orthotopic injection into C57BL / 6J Apln + / + or Apln - / - mice , respectively . * * P < 0 . 01 ; t test . ( D ) Mean percentages ( ± S . E . M . ) of extravasated Dextran in E0771 shRenilla ( n = 9 ) or shApln ( n = 12 ) mammary tumors , assessed on day 19 post - orthotopic injection into C57BL / 6J Apln + / + or Apln - / - mice , respectively . * * P < 0 . 01 ; t test . Right panel shows representative immunofluorescence of Dextran ( red ) , CD31 + vessels ( green ) , and DAPI ( blue ) . The white arrows indicate regions of Dextran extravasation . Scale bars = 100

O O O O O O O O O O O O O O O B-GENEPROD O O O B-CELL_LINE O O O O O O O O O O O O O B-DISEASE I-DISEASE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM O O O O O O 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-SMALL_MOLECULE O B-CELL_LINE O O O O O O O O O O O O O B-DISEASE I-DISEASE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O B-GENEPROD O B-TISSUE O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O

(C) Mean percentages (± S.E.M.) of CD31+ area in E0771 shRenilla (n=3) or shApln (n=3) mammary tumors, assessed on day 23 post-orthotopic injection into C57BL/6J Apln+/+ or Apln-/- mice, respectively. **P<0.01; t test. (D) Mean percentages (± S.E.M.) of extravasated Dextran in E0771 shRenilla (n=9) or shApln (n=12) mammary tumors, assessed on day 19 post-orthotopic injection into C57BL/6J Apln+/+ or Apln-/- mice, respectively. **P<0.01; t test. Right panel shows representative immunofluorescence of Dextran (red), CD31+ vessels (green), and DAPI (blue). The white arrows indicate regions of Dextran extravasation. Scale bars = 100 μm


( E ) Mean counts ( ± S . E . M . ) of pimonidazole positive foci , assessed on day 26 post - orthotopic injection of E0771 shRenilla ( n = 6 ) or shApln ( n = 4 ) into C57BL / 6J wild - type mice ( 5x105 cells / mouse ) . * P < 0 . 05 ; t test . Right panels show representative immunohistochemical pimonidazole staining at two different magnifications ; Scale bars = 200 μm ( upper panels ) and 50 μm ( lower panels ) .

O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O B-ORGANISM O O O O O O O O O O O O O O O O B-EXP_ASSAY B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O

(E) Mean counts (± S.E.M.) of pimonidazole positive foci, assessed on day 26 post-orthotopic injection of E0771 shRenilla (n=6) or shApln (n=4) into C57BL/6J wild-type mice (5x105 cells/mouse). *P<0.05; t test. Right panels show representative immunohistochemical pimonidazole staining at two different magnifications; Scale bars = 200 μm (upper panels) and 50 μm (lower panels).


( F ) Mean percentage ( ± S . E . M . ) of tumor - infiltrating immune cells normalized to CD45 + . E0771 shRenilla ( n = 8 ) or shApln ( n = 6 ) were orthotopically injected into C57BL / 6J Apln + / + or Apln - / - mice ( 5x105 cells / mouse ) , respectively and tumors were harvested day 25 post - injection . * P < 0 . 05 , * * P < 0 . 01 ; t test . All immune cell populations were gated for viable CD45 + cells and then further defined as : CD8 T cells ( Thy1 . 2 + , CD8 + ) , CD4 T cells , ( Thy1 . 2 + , CD4 + ) , Inflammatory Monocytes ( Ly6C + , Cd11b + , Ly6G - ) , PMN - MDSCs ( Ly6G + , Cd11b + ) , natural killer cells ( Thy1 . 2 - , Ly6G - , NK1 . 1 + ) , natural killer T cells ( Thy1 . 2 + , CD4 - , CD8 - , NK1 . 1 + ) and peripheral dendritic cells ( Ly6G - , Ly6C + , Cd11b - , PDAC1 + , B220 + ) .

O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-CELL_LINE O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM O O O O B-ORGANISM O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O

(F) Mean percentage (± S.E.M.) of tumor-infiltrating immune cells normalized to CD45+. E0771 shRenilla (n=8) or shApln (n=6) were orthotopically injected into C57BL/6J Apln+/+ or Apln-/- mice (5x105 cells/mouse), respectively and tumors were harvested day 25 post-injection. *P<0.05, **P<0.01; t test. All immune cell populations were gated for viable CD45+ cells and then further defined as: CD8 T cells (Thy1.2+, CD8+), CD4 T cells, (Thy1.2+, CD4+), Inflammatory Monocytes (Ly6C+, Cd11b+, Ly6G-), PMN-MDSCs (Ly6G+, Cd11b+), natural killer cells (Thy1.2-, Ly6G-, NK1.1+), natural killer T cells (Thy1.2+, CD4-, CD8-, NK1.1+) and peripheral dendritic cells (Ly6G- ,Ly6C+, Cd11b- , PDAC1+, B220+).


( A ) Left - Heatmap of RNA - Seq transcriptome analysis of CD31 + / CD105 + endothelial cells sorted from tumors established by E0771 shRenilla ( n = 6 ) or shApln ( n = 3 ) cells orthotopically injected into C57BL / 6J Apln + / + or Apln - / - mice , respectively . Tumors were harvested day 25 post - injection and genes displayed are significantly deregulated at the adjusted p - value cut - off of 0 . 05 . Right - Ingenuity Pathway Analysis for biological processes predicted to be decreased downstream of the differentially expressed genes .

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O B-CELL_LINE O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Left - Heatmap of RNA-Seq transcriptome analysis of CD31+/CD105+ endothelial cells sorted from tumors established by E0771 shRenilla (n=6) or shApln (n=3) cells orthotopically injected into C57BL/6J Apln+/+ or Apln-/- mice, respectively. Tumors were harvested day 25 post-injection and genes displayed are significantly deregulated at the adjusted p-value cut-off of 0.05. Right - Ingenuity Pathway Analysis for biological processes predicted to be decreased downstream of the differentially expressed genes.


( B ) Quantification of vessel sprouts ( mean values ± S . E . M . ) upon VEGF treatment ( 30ng / mL ) of embryoid bodies ( EB ) derived from murine ES cells ( mESCs ) with sense integrations in the Apelin gene ( Apln STOP ) of the splice acceptor described in Appendix Figure S1B or Cre - reverted antisense ( Apln GO ) sister cells . Apln GO ( Day 2 n = 6 ; Day 4 n = 40 , Day 6 n = 54 , Day 8 n = 32 , Day 10 n = 35 , Day 11 n = 31 ) , Apln STOP ( Day 2 n = 4 ; Day 4 n = 15 , Day 6 n = 31 , Day 8 n = 28 , Day 10 n = 32 , Day 11 n = 37 ) ; * * P < 0 . 01 , * * * P < 0 . 001 , two - way ANOVA . Representative brightfield images and automated analysis of vessel sprouts by Definiens software are shown in the bottom panels . Scale bars = 200

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O O O O O O O B-TISSUE I-TISSUE O O O O O B-ORGANISM B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O

(B) Quantification of vessel sprouts (mean values ± S.E.M.) upon VEGF treatment (30ng/mL) of embryoid bodies (EB) derived from murine ES cells (mESCs) with sense integrations in the Apelin gene (Apln STOP) of the splice acceptor described in Appendix Figure S1B or Cre-reverted antisense (Apln GO) sister cells. Apln GO (Day 2 n=6; Day 4 n=40, Day 6 n=54, Day 8 n= 32, Day 10 n=35, Day 11 n=31), Apln STOP (Day 2 n=4; Day 4 n=15, Day 6 n=31, Day 8 n= 28, Day 10 n=32, Day 11 n=37); ** P<0.01, *** P<0.001, two-way ANOVA. Representative brightfield images and automated analysis of vessel sprouts by Definiens software are shown in the bottom panels. Scale bars = 200 μm


( C ) Differentially expressed genes using RNAseq transcriptome analysis of CD31 + endothelial cells ( ECs ) sorted from sprouting EBs from Apelin STOP cells stimulated with VEGF ( 30ng / mL ) and DMSO ( - Apln ) and repaired Apelin GO sister cells stimulated with VEGF and Apelin ( 1000nM ) ( + Apln ) . VEGF target genes and angiogenesis - related genes , predicted by Ingenuity Pathway Analysis ( IPA ) software , are indicated by bars on the upper axis of the heatmap . GO terms were analyzed by DAVID on - line software .

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O B-TISSUE O B-GENEPROD O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Differentially expressed genes using RNAseq transcriptome analysis of CD31+ endothelial cells (ECs) sorted from sprouting EBs from Apelin STOP cells stimulated with VEGF (30ng/mL) and DMSO (-Apln) and repaired Apelin GO sister cells stimulated with VEGF and Apelin (1000nM) (+Apln). VEGF target genes and angiogenesis-related genes, predicted by Ingenuity Pathway Analysis (IPA) software, are indicated by bars on the upper axis of the heatmap. GO terms were analyzed by DAVID on-line software.


( A ) Experimental set up and ( right ) Kaplan Meier survival plot of NeuT ; Apln + / + and NeuT ; Apln - / - mice with mammary cancer , left untreated ( control ) or treated with the indicate dose of the broad VEGFR blocker sunitinib . NeuT ; Apln + / + Control ( n = 8 ) , NeuT ; Apln - / - Control ( n = 11 ) , NeuT ; Apln + / + Sunitinib ( n = 11 ) , NeuT ; Apln - / - Sunitinib ( n = 12 ) ; * P < 0 . 05 ; Log rank test . Mice were sacrificed when the tumor size reached 1cm3 , following ethical guidelines . The dotted line indicates 50 % of survival .

O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O B-DISEASE I-DISEASE O O O O O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O

(A) Experimental set up and (right) Kaplan Meier survival plot of NeuT;Apln+/+ and NeuT;Apln-/- mice with mammary cancer, left untreated (control) or treated with the indicate dose of the broad VEGFR blocker sunitinib. NeuT;Apln+/+ Control (n=8), NeuT;Apln-/- Control (n=11), NeuT;Apln+/+ Sunitinib (n=11), NeuT;Apln-/- Sunitinib (n=12); *P<0.05; Log rank test. Mice were sacrificed when the tumor size reached 1cm3, following ethical guidelines. The dotted line indicates 50% of survival.


( B ) Percentages ( mean ± S . E . M . ) of tumor burden in mammary glands of untreated ( control ) and sunitinib - treated NeuT ; Apln + / + and NeuT ; Apln - / - mice , assessed 4 weeks after tumor onset . NeuT ; Apln + / + Control ( n = 6 ) , NeuT ; Apln - / - Control ( n = 8 ) , NeuT ; Apln + / + Sunitinib ( n = 5 ) , NeuT ; Apln - / - Sunitinib ( n = 5 ) ; * P < 0 . 05 ; * * * P < 0 . 001 ; one - way ANOVA . Representative H & E images are shown for each genotype in the right panels . Scale bars = 1000 μm ( large panels ) and 50 μm ( insets ) .

O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE I-TISSUE O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O 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) Percentages (mean ± S.E.M.) of tumor burden in mammary glands of untreated (control) and sunitinib-treated NeuT;Apln+/+ and NeuT;Apln-/- mice, assessed 4 weeks after tumor onset. NeuT;Apln+/+ Control (n=6), NeuT;Apln-/- Control (n=8), NeuT;Apln+/+ Sunitinib (n=5), NeuT;Apln-/- Sunitinib (n=5); *P<0.05; ***P<0.001; one-way ANOVA. Representative H&E images are shown for each genotype in the right panels. Scale bars = 1000 μm (large panels) and 50 μm (insets).


( C ) Tumor volumes of untreated ( control ) and sunitinib - treated NeuT ; Apln + / + and NeuT ; Apln - / - mammary tumors , size - matched at 20 - 70mm3 and followed over time by MRI analysis ; NeuT ; Apln + / + Control ( n = 7 ) , NeuT ; Apln - / - Control ( n = 5 ) , NeuT ; Apln + / + Sunitinib ( n = 5 ) , NeuT ; Apln - / - Sunitinib ( n = 6 ) mice per group ; Lines represent nonlinear fit of tumor growth . Box and arrow indicate the time point used for analysis in panel D . ( D ) Mitotic counts ( mean ± S . E . M . ) and representative H & E images of mammary tumors in untreated ( control ) and sunitinib - treated NeuT ; Apln + / + and NeuT ; Apln - / - mice , assessed 6 weeks after tumor onset . ; NeuT ; Apln + / + Control ( n = 7 ) , NeuT ; Apln - / - Control ( n = 6 ) , NeuT ; Apln + / + Sunitinib ( n = 9 ) , NeuT ; Apln - / - Sunitinib ( n = 4 ) tumors per group ; * P < 0 . 05 ; one - way ANOVA to sunitinib - treated NeuT ; Apln - / - . White arrows indicate mitotic figures . Scale bars = 50

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-DISEASE I-DISEASE O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O B-ORGANISM O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-DISEASE I-DISEASE O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O

(C) Tumor volumes of untreated (control) and sunitinib-treated NeuT;Apln+/+ and NeuT;Apln-/- mammary tumors, size-matched at 20-70mm3 and followed over time by MRI analysis; NeuT;Apln+/+ Control (n=7), NeuT;Apln-/- Control (n=5), NeuT;Apln+/+ Sunitinib (n=5), NeuT;Apln-/- Sunitinib (n=6) mice per group; Lines represent nonlinear fit of tumor growth. Box and arrow indicate the time point used for analysis in panel D. (D) Mitotic counts (mean ± S.E.M.) and representative H&E images of mammary tumors in untreated (control) and sunitinib-treated NeuT;Apln+/+ and NeuT;Apln-/- mice, assessed 6 weeks after tumor onset.; NeuT;Apln+/+ Control (n=7), NeuT;Apln-/- Control (n=6), NeuT;Apln+/+ Sunitinib (n=9), NeuT;Apln-/- Sunitinib (n=4) tumors per group; *P<0.05; one-way ANOVA to sunitinib-treated NeuT;Apln-/-. White arrows indicate mitotic figures. Scale bars = 50 μm


( E ) Tumor volumes , followed over the indicated time , of orthotopically injected E0771 shRenilla cells left untreated ( control ) or treated three times a week from day 8 after tumor injection with an Apelin antagonist alone ( MM54 , 0 . 4μg / g ) , sunitinib alone ( 60mg / kg ) or a combination of both . Tumor volumes were measured using calipers and are shown as mean tumor volumes ± S . E . M . E0771 shRenilla Control ( n = 6 ) , MM54 ( n = 5 ) , Sunitinib ( n = 5 ) , Sunitinib + MM54 ( n = 5 ) ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , two - way ANOVA .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-CELL_LINE O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Tumor volumes, followed over the indicated time, of orthotopically injected E0771 shRenilla cells left untreated (control) or treated three times a week from day 8 after tumor injection with an Apelin antagonist alone (MM54, 0.4μg/g), sunitinib alone (60mg/kg) or a combination of both. Tumor volumes were measured using calipers and are shown as mean tumor volumes ± S.E.M. E0771 shRenilla Control (n=6), MM54 (n=5), Sunitinib (n=5), Sunitinib+MM54 (n=5); *P<0.05, **P<0.01, ***P<0.001, two-way ANOVA.


( B ) Representative MRI images ; Scale bar = 5mm and ( C ) quantification ( mean ± S . E . M . ) of relative tumor blood volume ( rTBV ) over time after NeuT + mammary tumors were size - matched at 20 - 70mm3 ( 0 weeks ) . Treatments and genotypes are indicated . NeuT ; Apln + / + Control ( n = 4 ) , NeuT ; Apln - / - Control ( n = 4 ) , NeuT ; Apln + / + Sunitinib ( n = 5 ) , NeuT ; Apln - / - Sunitinib ( n = 5 ) ; * * P < 0 . 01 , and * * * P < 0 . 001 compared to untreated NeuT ; Apln + / + mice and # P < 0 . 05 compared to untreated control NeuT ; Apln - / - mice ; two - way ANOVA . Of note , in NeuT ; Apln + / + mice , only 2 tumors could be analyzed after 6 weeks as some mice had to be sacrificed due to the large tumor sizes following ethical guidelines . Thus , we did not perform any statistical analysis on the 6 weeks timepoints .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O B-DISEASE I-DISEASE O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O 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-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O B-ORGANISM O O O O O 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) Representative MRI images; Scale bar = 5mm and (C) quantification (mean ± S.E.M.) of relative tumor blood volume (rTBV) over time after NeuT+ mammary tumors were size-matched at 20-70mm3 (0 weeks). Treatments and genotypes are indicated. NeuT;Apln+/+ Control (n=4), NeuT;Apln-/- Control (n=4), NeuT;Apln+/+ Sunitinib (n=5), NeuT;Apln-/- Sunitinib (n=5); **P<0.01, and ***P<0.001 compared to untreated NeuT;Apln+/+ mice and #P<0.05 compared to untreated control NeuT;Apln-/- mice; two-way ANOVA. Of note, in NeuT;Apln+/+ mice, only 2 tumors could be analyzed after 6 weeks as some mice had to be sacrificed due to the large tumor sizes following ethical guidelines. Thus, we did not perform any statistical analysis on the 6 weeks timepoints.


( D ) Analysis ( mean values ± S . E . M . ) of CD31 + area ( x104 μm2 ) / field , number of dilated tumor vessels and percentage of alphaSMA + area per CD31 + blood vessels in mammary tumors of untreated ( control ) and sunitinib - treated NeuT ; Apln + / + and NeuT ; Apln - / - mice , assessed 6 weeks after mammary tumors were size - matched . NeuT ; Apln + / + Control ( n = 4 ) , NeuT ; Apln - / - Control ( n = 4 ) , NeuT ; Apln + / + Sunitinib ( n = 5 ) , NeuT ; Apln - / - Sunitinib ( n = 4 ) ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; one - way ANOVA and Kruskal - Wallis test . ( E ) Representative immunofluorescence and immunohistochemistry images from Fig . 5D quantification . Dilated blood vessels are marked by a red asterisk . DAPI ( blue ) is shown as a counterstain to visualize nuclei . Scale bars = 100 μm ( upper panels ) , 50 μm ( middel panels ) and 20 μm ( lower panels ) .

O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-TISSUE I-TISSUE I-TISSUE O O O B-GENEPROD O O O B-GENEPROD O B-TISSUE I-TISSUE O B-DISEASE I-DISEASE O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O O O B-DISEASE I-DISEASE O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O B-EXP_ASSAY O O O O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Analysis (mean values ± S.E.M.) of CD31+ area (x104 μm2)/field, number of dilated tumor vessels and percentage of alphaSMA+ area per CD31+ blood vessels in mammary tumors of untreated (control) and sunitinib-treated NeuT;Apln+/+ and NeuT;Apln-/- mice, assessed 6 weeks after mammary tumors were size-matched. NeuT;Apln+/+ Control (n=4), NeuT;Apln-/- Control (n=4), NeuT;Apln+/+ Sunitinib (n=5), NeuT;Apln-/- Sunitinib (n=4); *P<0.05; **P<0.01; ***P<0.001; one-way ANOVA and Kruskal-Wallis test. (E) Representative immunofluorescence and immunohistochemistry images from Fig. 5D quantification. Dilated blood vessels are marked by a red asterisk. DAPI (blue) is shown as a counterstain to visualize nuclei. Scale bars = 100 μm (upper panels), 50 μm (middel panels) and 20 μm (lower panels).


( A ) Percentages of CA9 + cells adjacent to CD31 + tumor blood vessels in untreated ( control ) and sunitinib - treated NeuT ; Apln + / + and NeuT ; Apln - / - mice , 6 weeks after mammary tumors were size - matched . NeuT ; Apln + / + Control ( n = 4 ) , NeuT ; Apln - / - Control ( n = 4 ) , NeuT ; Apln + / + Sunitinib ( n = 4 ) , NeuT ; Apln - / - Sunitinib ( n = 4 ) ; 100 - 200 peri - vascular intra - tumoral regions per group were counted . * * * P < 0 . 001 ; one - way ANOVA . Right panels show representative immunofluorescent images . Areas limited by dotted white lines indicate CA9 + areas . Scale bars = 50 μm .

O O O O O B-GENEPROD O O O O B-GENEPROD O B-TISSUE I-TISSUE I-TISSUE O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O O B-DISEASE I-DISEASE O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O O O O O O

(A) Percentages of CA9+ cells adjacent to CD31+ tumor blood vessels in untreated (control) and sunitinib-treated NeuT;Apln+/+ and NeuT;Apln-/- mice, 6 weeks after mammary tumors were size-matched. NeuT;Apln+/+ Control (n=4), NeuT;Apln-/- Control (n=4), NeuT;Apln+/+ Sunitinib (n=4), NeuT;Apln-/- Sunitinib (n=4); 100-200 peri-vascular intra-tumoral regions per group were counted. ***P<0.001; one-way ANOVA. Right panels show representative immunofluorescent images. Areas limited by dotted white lines indicate CA9+ areas. Scale bars = 50 μm.


( B ) Representative MRI images ; Scale bar = 5mm and ( C ) quantification ( mean ± S . E . M . ) of vessel permeability ( K2 ) in NeuT + mammary tumors followed over time . Treatments and genotypes are indicated . NeuT ; Apln + / + Control ( n = 4 ) , NeuT ; Apln - / - Control ( n = 5 ) , NeuT ; Apln + / + Sunitinib ( n = 4 ) , NeuT ; Apln - / - Sunitinib ( n = 5 ) ; * * * P < 0 . 001 , compared to untreated control NeuT ; Apln + / + mice and § P < 0 . 05 compared to sunitinib - treated NeuT ; Apln + / + mice ; two - way ANOVA . Of note , in NeuT ; Apln + / + mice , only 2 tumors could be analysed after 6 weeks as some mice had to be sacrificed due to the large tumor sizes following ethical guidelines . Thus we did not perform any statistical analysis on the 6 weeks timepoints .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-TISSUE B-EXP_ASSAY O O O O B-GENEPROD O B-DISEASE I-DISEASE O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O B-ORGANISM O O O O 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) Representative MRI images; Scale bar = 5mm and (C) quantification (mean ± S.E.M.) of vessel permeability (K2) in NeuT+ mammary tumors followed over time. Treatments and genotypes are indicated. NeuT;Apln+/+ Control (n=4), NeuT;Apln-/- Control (n=5), NeuT;Apln+/+ Sunitinib (n=4), NeuT;Apln-/- Sunitinib (n=5); ***P<0.001, compared to untreated control NeuT;Apln+/+ mice and §P<0.05 compared to sunitinib-treated NeuT;Apln+/+ mice; two-way ANOVA. Of note, in NeuT;Apln+/+ mice, only 2 tumors could be analysed after 6 weeks as some mice had to be sacrificed due to the large tumor sizes following ethical guidelines. Thus we did not perform any statistical analysis on the 6 weeks timepoints.


( A ) Number of metastatic lung foci in untreated ( control ) and sunitinib - treated ( 60mg / kg , three times a week from tumor initiation ) NeuT ; Apln + / + or NeuT ; Apln - / - mice , assessed 6 weeks after mammary tumors were size - matched . Data of individual lung sections and means ( black bars ) are shown . Right panels show representative H & E images , where black arrows and insets indicate metastatic foci . Scale bars = 1000 μm ( large panels ) and 50 μm ( insets ) . * P < 0 . 05 , * * P < 0 . 01 ; * * * P < 0 . 001 ; Kruskal - Wallis test ; n = 3 mice per cohort and three sections per lung were analysed .

O O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O O O B-DISEASE I-DISEASE O O O O O O O O B-TISSUE O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O 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-ORGANISM O O O O O O B-TISSUE O O O

(A) Number of metastatic lung foci in untreated (control) and sunitinib-treated (60mg/kg, three times a week from tumor initiation) NeuT;Apln+/+ or NeuT;Apln-/- mice, assessed 6 weeks after mammary tumors were size-matched. Data of individual lung sections and means (black bars) are shown. Right panels show representative H&E images, where black arrows and insets indicate metastatic foci. Scale bars = 1000 μm (large panels) and 50 μm (insets). *P<0.05, **P<0.01; ***P<0.001; Kruskal-Wallis test; n=3 mice per cohort and three sections per lung were analysed.


( B ) Kaplan Meier survival plot for distant metastasis free survival from the KM - plotter database ( Data Ref : Győrffy et al , 2013 ) for high and low Apelin ( APLN ) - expressing groups in human breast cancer . Patients were split by the median . Affymetrix Apelin ID 222856 _ at .

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(B) Kaplan Meier survival plot for distant metastasis free survival from the KM-plotter database (Data Ref: Győrffy et al, 2013) for high and low Apelin (APLN)-expressing groups in human breast cancer. Patients were split by the median. Affymetrix Apelin ID 222856_at.


( C ) Kaplan - Meier plot for progression - free survival stratifying RCC patients with high and low APELIN serum levels 3 - 5 months after the start date of sunitinib treatment . * P = 0 . 0367 ; Log rank test .

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(C) Kaplan-Meier plot for progression-free survival stratifying RCC patients with high and low APELIN serum levels 3-5 months after the start date of sunitinib treatment. *P=0.0367; Log rank test.


( D ) Kaplan Meier plots for progression - free survival in RCC patients stratified into groups of high or low levels of APELIN and VEGF . Cut - off levels were set by the median . Serum was analysed 3 - 5 months after the start date of sunitinib treatment . * P < 0 . 05 , * * P < 0 . 01 ; Log rank test .

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(D) Kaplan Meier plots for progression-free survival in RCC patients stratified into groups of high or low levels of APELIN and VEGF. Cut-off levels were set by the median. Serum was analysed 3-5 months after the start date of sunitinib treatment. *P<0.05, **P<0.01; Log rank test.


( A ) Localization of endogenous SHP - 1 in hematopoietic and non - hematopoietic cells was determined by confocal imaging after staining with SHP - 1 antibody . DAPI was used for nuclear staining , Scale bar : 5µm . Fluorescence intensities along the line drawn in a representative cell were obtained using Zen software and the co - localization plots were made using GraphPad ( Red : SHP - 1 , Blue : DAPI ) .

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(A) Localization of endogenous SHP-1 in hematopoietic and non-hematopoietic cells was determined by confocal imaging after staining with SHP-1 antibody. DAPI was used for nuclear staining, Scale bar:5µm. Fluorescence intensities along the line drawn in a representative cell were obtained using Zen software and the co-localization plots were made using GraphPad (Red: SHP-1, Blue: DAPI).


( C ) SFB tagged PAF1 , SFB - LEO1 , SFB - CTR9 , SFB - CDC73 , SFB - RTF1 , SFB - WDR61 along with Myc SHP - 1 or Myc PTPN7 were transfected in 293T cells and interaction of SHP - 1 with PAF complex components was detected by immunoblotting with Myc antibody after S protein beads pulldown in presence of TurboNuclease .

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(C) SFB tagged PAF1, SFB-LEO1, SFB-CTR9, SFB-CDC73, SFB-RTF1, SFB-WDR61 along with Myc SHP-1 or Myc PTPN7 were transfected in 293T cells and interaction of SHP-1 with PAF complex components was detected by immunoblotting with Myc antibody after S protein beads pulldown in presence of TurboNuclease.


( D ) SFB empty vector , SFB SHP - 1 or SFB PTPN7 was co - transfected with Myc tagged H2B in 293T cells . The interaction of H2B with SHP - 1 was detected through immunoblotting using Myc antibody after pull down with streptavidin sepharose beads .

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(D) SFB empty vector, SFB SHP-1 or SFB PTPN7 was co-transfected with Myc tagged H2B in 293T cells. The interaction of H2B with SHP-1 was detected through immunoblotting using Myc antibody after pull down with streptavidin sepharose beads.


( E ) Endogenous association of SHP - 1 with Paf1 complex subunits ( CDC73 , RTF1 , WDR61 , LEO1 , Paf1 , CTR9 ) was detected by immunoblotting after performing immunoprecipitation with either IgG or SHP - 1 antibody .

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(E) Endogenous association of SHP-1 with Paf1 complex subunits (CDC73, RTF1, WDR61, LEO1, Paf1, CTR9) was detected by immunoblotting after performing immunoprecipitation with either IgG or SHP-1 antibody.


( F ) Immunoprecipitation ( IP ) with control IgG or anti - SHP - 1 antibody was performed with extracts prepared from MCF7 cells and endogenous association of H2B with SHP - 1 was detected by immunoblotting .

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(F) Immunoprecipitation (IP) with control IgG or anti-SHP-1 antibody was performed with extracts prepared from MCF7 cells and endogenous association of H2B with SHP-1 was detected by immunoblotting.


( G ) Glutathione Sepharose beads immobilized with bacterially expressed recombinant GST or GST - H2B proteins were incubated with bacterially purified recombinant MBP - SHP - 1 . The association of SHP - 1 with H2B was detected by immunoblotting with MBP antibody . Expression of GST , GST - H2B and MBP SHP - 1 was shown by Coomassie staining .

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(G) Glutathione Sepharose beads immobilized with bacterially expressed recombinant GST or GST-H2B proteins were incubated with bacterially purified recombinant MBP-SHP-1. The association of SHP-1 with H2B was detected by immunoblotting with MBP antibody. Expression of GST, GST-H2B and MBP SHP-1 was shown by Coomassie staining.


( A ) Nuclear extracts of WT and SHP - 1 knockout cells ( derived from two independent guide RNAs ) were made and the levels of H2BUb were detected .

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(A) Nuclear extracts of WT and SHP-1 knockout cells (derived from two independent guide RNAs) were made and the levels of H2BUb were detected.


( B ) Wild type ( WT ) 293T cells along with SHP - 1 KO cells transfected with either Myc vector , Myc SHP - 1 ∆ SH2 or Myc SHP - 1 NLS mutant were lysed . H2BUb levels were determined using specific antibody .

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(B) Wild type (WT) 293T cells along with SHP-1 KO cells transfected with either Myc vector, Myc SHP-1 ∆SH2 or Myc SHP-1 NLS mutant were lysed. H2BUb levels were determined using specific antibody.


( C ) WT and SHP - 1 KO 293T cells transfected with Myc vector , Myc SHP - 1 or Myc SHP - 1 C / S ( C453S ) mutant were lysed . H2BUb levels were determined using specific antibody .

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(C) WT and SHP-1 KO 293T cells transfected with Myc vector, Myc SHP-1 or Myc SHP-1 C/S (C453S) mutant were lysed. H2BUb levels were determined using specific antibody.


( D ) H2B WT along with the indicated Y / F and Y / D mutants were transfected into 293T cells . Histone extract was prepared from cells transfected with each construct and subjected to western blotting with anti - H2B and anti - H2BUb antibodies .

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(D) H2B WT along with the indicated Y/F and Y/D mutants were transfected into 293T cells. Histone extract was prepared from cells transfected with each construct and subjected to western blotting with anti-H2B and anti-H2BUb antibodies.


( E ) In vitro ubiquitination assay was performed using bacterially purified GST , GST tagged H2B and its mutants as substrates . Recombinant E1 , E2 ( UBE2A ) and ubiquitin were included in each reaction . Ubiquitination was detected by H2BUb antibody .

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(E) In vitro ubiquitination assay was performed using bacterially purified GST, GST tagged H2B and its mutants as substrates. Recombinant E1, E2(UBE2A) and ubiquitin were included in each reaction. Ubiquitination was detected by H2BUb antibody.


( F ) In vitro ubiquitination was performed using biotin tagged non - phospho and phospho ( pY121 ) peptide of H2B as substrates in the presence of E1 and E2 . H2BUb was detected by immunoblotting with specific antibody .

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(F) In vitro ubiquitination was performed using biotin tagged non-phospho and phospho (pY121) peptide of H2B as substrates in the presence of E1 and E2. H2BUb was detected by immunoblotting with specific antibody.


( A ) In vitro phosphorylated GST H2B by Syk kinase was incubated with bacterially purified recombinant wild type SHP - 1 or elution buffer and the amount of released phosphate was assayed colorimetrically using the malachite green reagent ( A620 nm ) . Data represent mean absorbance from four independent experiments . Error bar indicates SD ; * p < 0 . 05 , by Student ' s t test .

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(A) In vitro phosphorylated GST H2B by Syk kinase was incubated with bacterially purified recombinant wild type SHP-1 or elution buffer and the amount of released phosphate was assayed colorimetrically using the malachite green reagent (A620 nm). Data represent mean absorbance from four independent experiments. Error bar indicates SD; *p<0.05, by Student's t test.


( B ) Bacterially purified GST H2B was subjected to in vitro kinase assay using HEK293T cell lysate , and then phosphorylated proteins were incubated with either bacterially purified MBP tagged constitutively active ( SHP - 1 ΔSH2 ) , or catalytically inactive SHP - 1 C / S mutant . The phosphorylation status of recombinant H2B was detected by immunoblotting with phospho - tyrosine antibody .

O O O O O B-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O

(B) Bacterially purified GST H2B was subjected to in vitro kinase assay using HEK293T cell lysate, and then phosphorylated proteins were incubated with either bacterially purified MBP tagged constitutively active (SHP-1 ΔSH2), or catalytically inactive SHP-1 C/S mutant. The phosphorylation status of recombinant H2B was detected by immunoblotting with phospho-tyrosine antibody.


( C ) Phospho ( pH2B ) Y121 and non - phospho peptide of H2B was incubated with constitutively active ( SHP - 1 ΔSH2 ) or catalytically inactive mutant ( C / S ) of SHP - 1 and the released phosphate was assayed colorimetrically using the malachite green reagent ( A620nm ) . n = 3 independent experiments , Error bars indicate SD ; * * * p < 0 . 001 ( one - way ANOVA , Tukey ' s multiple comparison test ) .

O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE O O B-EXP_ASSAY O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Phospho (pH2B) Y121 and non-phospho peptide of H2B was incubated with constitutively active (SHP-1 ΔSH2) or catalytically inactive mutant (C/S) of SHP-1 and the released phosphate was assayed colorimetrically using the malachite green reagent (A620nm). n=3 independent experiments, Error bars indicate SD; ***p<0.001 (one-way ANOVA, Tukey's multiple comparison test).


( D ) 293T cells were transfected with vector , Myc tagged SHP - 1 WT or C / S mutant . The phosphorylation status of endogenous H2B was detected by immunoblotting with pY121 - H2B antibody .

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(D) 293T cells were transfected with vector, Myc tagged SHP-1 WT or C/S mutant. The phosphorylation status of endogenous H2B was detected by immunoblotting with pY121-H2B antibody.


( E ) Nuclear extracts of 293T WT and SHP - 1 KO cells were made and the levels of H2B phosphorylation were detected using pY121 - H2B antibody .

O O O B-SUBCELLULAR O O B-CELL_LINE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O B-GENEPROD O O

(E) Nuclear extracts of 293T WT and SHP-1 KO cells were made and the levels of H2B phosphorylation were detected using pY121-H2B antibody.


( F ) Wild type ( WT ) along with SHP - 1 KO 293T cells transfected with either Myc vector , Myc SHP - 1 or Myc SHP - 1 C / S mutant were lysed . Levels of pY121 - H2B and H2BUb were determined using specific antibodies .

O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O

(F) Wild type (WT) along with SHP-1 KO 293T cells transfected with either Myc vector, Myc SHP-1 or Myc SHP-1 C/S mutant were lysed. Levels of pY121-H2B and H2BUb were determined using specific antibodies.


( G ) 293T Cells were treated with SHP - 1 inhibitor TPI ( 50nm ) for 1hr and levels of pY121 - H2B was determined using pY121H2B antibody .

O O O B-CELL_LINE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O O O O

(G) 293T Cells were treated with SHP-1 inhibitor TPI (50nm) for 1hr and levels of pY121-H2B was determined using pY121H2B antibody.


MCF7 cells were transduced with ( H ) CTR9 shRNA along with control shRNA . Post 72 h , cells were collected and lysed to isolate soluble and nuclear fractions . Levels of phosphorylated and ubiquitinated H2B were detected by immunoblotting with indicated specific antibodies .

B-CELL_LINE O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O B-EXP_ASSAY O B-EXP_ASSAY B-GENEPROD O O O B-EXP_ASSAY O O O O O

MCF7 cells were transduced with (H) CTR9 shRNA along with control shRNA. Post 72 h, cells were collected and lysed to isolate soluble and nuclear fractions. Levels of phosphorylated and ubiquitinated H2B were detected by immunoblotting with indicated specific antibodies.


MCF7 cells were transduced with ( I ) Paf1 shRNA , along with control shRNA . Post 72 h , cells were collected and lysed to isolate soluble and nuclear fractions . Levels of phosphorylated and ubiquitinated H2B were detected by immunoblotting with indicated specific antibodies .

B-CELL_LINE O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O B-EXP_ASSAY O B-EXP_ASSAY B-GENEPROD O O O B-EXP_ASSAY O O O O O

MCF7 cells were transduced with (I) Paf1 shRNA, along with control shRNA. Post 72 h, cells were collected and lysed to isolate soluble and nuclear fractions. Levels of phosphorylated and ubiquitinated H2B were detected by immunoblotting with indicated specific antibodies.


MCF7 cells were transduced with ( J ) RTF1 shRNA along with control shRNA . Post 72 h , cells were collected and lysed to isolate soluble and nuclear fractions . Levels of phosphorylated and ubiquitinated H2B were detected by immunoblotting with indicated specific antibodies .

MCF7 cells were transduced with (J) RTF1 shRNA along with control shRNA. Post 72 h, cells were collected and lysed to isolate soluble and nuclear fractions. Levels of phosphorylated and ubiquitinated H2B were detected by immunoblotting with indicated specific antibodies.


( A ) Cell lysates derived from 293T cells transfected with empty vector , SFB - tagged H2B , H2B Y121F or H2B Y121D constructs were pulled down with S - protein agarose beads . RNF20 interaction with H2B was detected by immunoblotting with specific antibody .

O O O O O O O B-CELL_LINE O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD B-SMALL_MOLECULE O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O O O B-EXP_ASSAY O O O O

(A) Cell lysates derived from 293T cells transfected with empty vector, SFB-tagged H2B, H2B Y121F or H2B Y121D constructs were pulled down with S-protein agarose beads. RNF20 interaction with H2B was detected by immunoblotting with specific antibody.


( B ) HEK293T cells were co - transfected with the SFB - tagged H2B , H2B Y121F or H2B Y121D constructs along with Myc - tagged UBE2A . Cell lysates were pulled down with S - protein beads , and interaction was detected by immunoblotting with Myc antibody .

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(B) HEK293T cells were co-transfected with the SFB-tagged H2B, H2B Y121F or H2B Y121D constructs along with Myc-tagged UBE2A. Cell lysates were pulled down with S-protein beads, and interaction was detected by immunoblotting with Myc antibody.


( C ) Glutathione Sepharose beads bound with bacterially expressed recombinant GST - H2B , GST H2B Y121F and GST - H2B Y121D proteins were incubated with bacterially purified recombinant His UBE2A , and interaction of UBE2A with H2B was detected by immunoblotting with His antibody . Recombinant protein expression and pulldown was shown by Coomassie staining .

O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O

(C) Glutathione Sepharose beads bound with bacterially expressed recombinant GST-H2B, GST H2B Y121F and GST-H2B Y121D proteins were incubated with bacterially purified recombinant His UBE2A, and interaction of UBE2A with H2B was detected by immunoblotting with His antibody. Recombinant protein expression and pulldown was shown by Coomassie staining.


( D ) Bacterially purified GST , GST H2B and GST H2B - p ( phosphorylated using Syk ) was incubated with recombinant His UBE2A , and interaction of UBE2A with H2B was detected by immunoblotting with His antibody . Recombinant protein expression and pulldown was shown by Coomassie staining .

O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O

(D) Bacterially purified GST, GST H2B and GST H2B-p (phosphorylated using Syk) was incubated with recombinant His UBE2A, and interaction of UBE2A with H2B was detected by immunoblotting with His antibody. Recombinant protein expression and pulldown was shown by Coomassie staining.


( E ) WT 293T cells along with SHP - 1 KO were co - transfected with SFB vector or SFB H2B along with Myc UbE2A . Cell lysates were pulldown using S - protein beads , and interaction of UBE2A with H2B was determined by Western blotting with the indicated antibodies .

O O O O B-CELL_LINE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O

(E) WT 293T cells along with SHP-1 KO were co-transfected with SFB vector or SFB H2B along with Myc UbE2A. Cell lysates were pulldown using S-protein beads, and interaction of UBE2A with H2B was determined by Western blotting with the indicated antibodies.


( F ) HEK293T cells were transfected with SFB vector or SFB H2B along with Myc UBE2A . Cells were treated with SHP - 1 inhibitor TPI ( 50nm ) for 1hr after 24hrs of transfection . Cells lysates were subjected to pull - down with S - protein beads , and western blotting was performed using specific antibodies .

O O O B-CELL_LINE O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O

(F) HEK293T cells were transfected with SFB vector or SFB H2B along with Myc UBE2A.Cells were treated with SHP-1 inhibitor TPI (50nm) for 1hr after 24hrs of transfection. Cells lysates were subjected to pull-down with S-protein beads, and western blotting was performed using specific antibodies.


( A ) Representative super - resolution images of MCF7 cells immunostained with SHP - 1 in combination with CDK9 , total RNA Polymerse II , RNA polymerase II Ser2P , and RNA Polymerase II Ser5P . Scale bar : 5µm . Fluorescence intensities along the line drawn in a representative section of cell were obtained using Zen software and the co - localization plots were made using GraphPad .

O O O O O O O O O B-CELL_LINE O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O

(A) Representative super-resolution images of MCF7 cells immunostained with SHP-1 in combination with CDK9, total RNA Polymerse II, RNA polymerase II Ser2P, and RNA Polymerase II Ser5P. Scale bar: 5µm. Fluorescence intensities along the line drawn in a representative section of cell were obtained using Zen software and the co-localization plots were made using GraphPad.


( B ) Co - localization of SHP - 1 with CDK9 , total RNA Polymerase II , RNA Polymerase II S2P , and RNA Polymerase II S5P was measured by Pearson correlation coefficient using ImageJ . Values presented as the mean ± SD , n = 10 ( from three independent experiments ) . * * * p value < 0 . 001 . ( One - way ANOVA ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O 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) Co-localization of SHP-1 with CDK9, total RNA Polymerase II, RNA Polymerase II S2P, and RNA Polymerase II S5P was measured by Pearson correlation coefficient using ImageJ. Values presented as the mean ± SD, n=10 (from three independent experiments). ***p value < 0.001. (One-way ANOVA).


( C ) Endogenous interaction of SHP - 1 with RNA Polymerase II , RNA polymerase II Ser2P , and RNA Polymerase II Ser5P and CDK9 was detected by immunoblotting after performing immunoprecipitation with either IgG or SHP - 1 antibody using TurboNuclease treated cell lysate .

O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-GENEPROD O O O B-EXP_ASSAY O O B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O

(C) Endogenous interaction of SHP-1 with RNA Polymerase II, RNA polymerase II Ser2P, and RNA Polymerase II Ser5P and CDK9 was detected by immunoblotting after performing immunoprecipitation with either IgG or SHP-1 antibody using TurboNuclease treated cell lysate.


( D ) Nuclear extracts of MCF - 7 WT and SHP - 1 knockout cells were made and the levels of RNA Polymerase II , RNA Polymerase II Ser2P and RNA Polymerase II Ser5P were detected by immunoblotting using specific antibodies .

O O O B-SUBCELLULAR O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O B-EXP_ASSAY O O O O

(D) Nuclear extracts of MCF-7 WT and SHP-1 knockout cells were made and the levels of RNA Polymerase II, RNA Polymerase II Ser2P and RNA Polymerase II Ser5P were detected by immunoblotting using specific antibodies.


( E ) Nuclear extracts of cells treated with either DMSO or SHP - 1 inhibitor TPI ( 50nm ) were used to detect total RNA Polymerase II and its modifications by immunoblotting using specific antibodies .

O O O B-SUBCELLULAR O O O O O O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O B-EXP_ASSAY O O O O

(E) Nuclear extracts of cells treated with either DMSO or SHP-1 inhibitor TPI (50nm) were used to detect total RNA Polymerase II and its modifications by immunoblotting using specific antibodies.


( F ) Nuclear extracts of MCF - 7 WT and SHP - 1 knockout cells transfected with Myc vector , H2B Y121F or H2B Y121D constructs were made and the levels of RNA Polymerase II , RNA Polymerase II Ser2P , RNA Polymerase II Ser5P and Lamin B1 were detected by immunoblotting using specific antibodies .

O O O B-SUBCELLULAR O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O O O

(F) Nuclear extracts of MCF-7 WT and SHP-1 knockout cells transfected with Myc vector, H2B Y121F or H2B Y121D constructs were made and the levels of RNA Polymerase II, RNA Polymerase II Ser2P, RNA Polymerase II Ser5P and Lamin B1 were detected by immunoblotting using specific antibodies.


( G ) Immunofluorescence of WT or SHP - 1 knockout MCF7 cells labelled with the 5 - Ethynyl Uridine ( EU ) for 60 minutes to capture nascent RNA synthesis . Scale bars 10μm . ( H ) The EU incorporation in MCF - 7 WT and SHP - 1 KO cells was quantified in three independent experiments . Values presented as the mean ± SD , * * * indicates p < 0 . 001 ( One - way ANOVA ) .

O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Immunofluorescence of WT or SHP-1 knockout MCF7 cells labelled with the 5-Ethynyl Uridine (EU) for 60 minutes to capture nascent RNA synthesis. Scale bars 10μm. (H) The EU incorporation in MCF-7 WT and SHP-1 KO cells was quantified in three independent experiments. Values presented as the mean ± SD, ***indicates p<0.001 (One-way ANOVA).


( I ) Representative images of immunofluorescence of DMSO or TPI treated ( 50nm ) MCF7 cells labelled with EU for 60 minutes was shown . Scale bar : 40µm . ( J ) The EU incorporation in MCF7 cells treated with DMSO or TPI was quantified in three independent experiments . Values presented as the mean ± SD , * * * indicates p < 0 . 001 ( One - way ANOVA ) .

O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-CELL_LINE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O B-CELL_LINE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(I) Representative images of immunofluorescence of DMSO or TPI treated (50nm) MCF7 cells labelled with EU for 60 minutes was shown. Scale bar: 40µm. (J) The EU incorporation in MCF7 cells treated with DMSO or TPI was quantified in three independent experiments. Values presented as the mean ± SD, ***indicates p<0.001 (One-way ANOVA).


( C , D ) MCF - 7 WT cells along with SHP - 1 KO cells transfected with either vector , H2B Y121F or H2B Y121D mutant were used for ChIP q - PCR analysis of the H2BUb at indicated genes . The data shown is derived from two independent experiments . Error bars indicate the mean ± SD .

O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

(C, D) MCF-7 WT cells along with SHP-1 KO cells transfected with either vector, H2B Y121F or H2B Y121D mutant were used for ChIP q-PCR analysis of the H2BUb at indicated genes. The data shown is derived from two independent experiments. Error bars indicate the mean ± SD.


( A ) Lysates of WT and SHP - 1 knockout MCF7 cells were made and the levels of LC3 - I and II were detected by immunoblotting . ( B ) Levels of LC3 II were normalized against total LC3 ( LC3 I + LC3 II ) . The data shown is derived from three independent experiments . Error bars indicate the mean ± SD . * p value < 0 . 05 , Student ' s t - test .

O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Lysates of WT and SHP-1 knockout MCF7 cells were made and the levels of LC3-I and II were detected by immunoblotting. (B) Levels of LC3 II were normalized against total LC3 (LC3 I+LC3 II). The data shown is derived from three independent experiments. Error bars indicate the mean ± SD. *p value < 0.05, Student's t-test.


( C ) Representative super - resolution images of WT and SHP - 1 KO MCF7 cells immunostained with LC3 antibody , ( Scale bar : 5µm ) . ( D ) Quantification of LC3 puncta accumulation per cell done by using ImageJ was shown . Bars represents mean ± SD . ( N = 60 ) * * * p value < 0 . 001 , Student ' s t - test .

O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Representative super- resolution images of WT and SHP-1 KO MCF7 cells immunostained with LC3 antibody, (Scale bar: 5µm). (D) Quantification of LC3 puncta accumulation per cell done by using ImageJ was shown. Bars represents mean ± SD.(N=60) ***p value < 0.001, Student's t-test.


( E ) WT along with SHP - 1 KO MCF7 cells transfected with either Myc vector , Myc SHP - 1 or Myc SHP - 1 NLS mutant were lysed . Levels of LC3 and actin were determined using specific antibodies .

O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O

(E) WT along with SHP-1 KO MCF7 cells transfected with either Myc vector, Myc SHP-1 or Myc SHP-1 NLS mutant were lysed. Levels of LC3 and actin were determined using specific antibodies.


( F ) WT and SHP - 1 knockout MCF7 cells transfected with the Myc vector or Myc - tagged H2B , H2B Y121F or H2B Y121D constructs were immunostained with endogenous p62 and Myc antibody . Image acquisition was done using LSM 700 , ( Scale bar : 20µm ) . ( G ) Accumulation of p62 was quantified by counting p62 positive puncta per cell . ( N = 25 ) ; values presented as the mean ± SD . * * * p value < 0 . 001 ( One - way ANOVA ) .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) WT and SHP-1 knockout MCF7 cells transfected with the Myc vector or Myc-tagged H2B, H2B Y121F or H2B Y121D constructs were immunostained with endogenous p62 and Myc antibody. Image acquisition was done using LSM 700, (Scale bar: 20µm). (G) Accumulation of p62 was quantified by counting p62 positive puncta per cell. (N= 25); values presented as the mean ± SD. ***p value < 0.001 (One-way ANOVA).


( H ) Representative super - resolution images of LC3 and LAMP2 immunostaining in WT and SHP - 1 KO MCF7 cells . ( Scale bar : 20 µm for merge & 5 µm for Zoom ) . ( I ) LC3 puncta co - localizing with LAMP2 per cell were quantified using ImageJ . ( N = 45 ) ; values presented as the mean ± SD . * * * p value < 0 . 001 , Student ' s t - test .

O O O O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) Representative super-resolution images of LC3 and LAMP2 immunostaining in WT and SHP-1 KO MCF7 cells. (Scale bar: 20 µm for merge & 5 µm for Zoom). (I) LC3 puncta co-localizing with LAMP2 per cell were quantified using ImageJ. (N=45); values presented as the mean ± SD. ***p value < 0.001, Student's t-test.


B Chromatin accessibility as measured by the number of all ( TSS and distal ) ATAC - Seq peaks ( read count ≥ 10 ) detected in DN2 ( n = 5 ) , DN3 ( n = 5 ) , ISP ( n = 5 ) , DPCD3 - ( n = 6 ) , DPCD3 + ( n = 6 ) , SPCD4 + ( n = 5 ) , SPCD8 + ( n = 5 ) populations . Horizontal lines of the box plot indicate the median , lower and upper limits of each box correspond to the first and third quartiles ( the 25th and 75th percentiles ) and the lower and upper whiskers extend from min to max ; pval = 0 . 035 ( Kruskal - Wallis Test ) .

O B-SUBCELLULAR O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Chromatin accessibility as measured by the number of all (TSS and distal) ATAC-Seq peaks (read count ≥10) detected in DN2 (n=5), DN3 (n=5), ISP (n=5), DPCD3- (n=6), DPCD3+ (n=6), SPCD4+ (n=5), SPCD8+ (n=5) populations. Horizontal lines of the box plot indicate the median, lower and upper limits of each box correspond to the first and third quartiles (the 25th and 75th percentiles) and the lower and upper whiskers extend from min to max; pval=0.035 (Kruskal-Wallis Test).


C Fraction of OCRs having increasing , decreasing , steady and fluctuating accessibility pattern during T - cell development ( x - axis ) . Average number of normalized reads in OCRs ( y - axis ) .

O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O

C Fraction of OCRs having increasing, decreasing, steady and fluctuating accessibility pattern during T-cell development (x-axis). Average number of normalized reads in OCRs (y-axis).


A Unsupervised hierarchical clustering based on the variance stabilizing transformation ( VST ) normalized read counts ( each row is also normalized by the row mean ) of the 2 , 021 signature open chromatin regions of 7 stages

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O

A Unsupervised hierarchical clustering based on the variance stabilizing transformation (VST) normalized read counts (each row is also normalized by the row mean) of the 2,021 signature open chromatin regions of 7 stages


A Unsupervised hierarchical clustering based on the variance stabilizing transformation ( VST ) normalized read counts ( each row is also normalized by the row mean ) of the 2 , 021 signature open chromatin regions of 7

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O


B ATAC - Seq genome tracks showing three signature peaks located near the genes SAE1 , RAG1 , and CD8A . Framed regions indicate cell - type - specific signature ATAC - Seq peaks . Blue triangles indicate known promoter or enhancer regions , which are annotated in the GeneHancer ( GH ) Regulatory Elements database

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O

B ATAC-Seq genome tracks showing three signature peaks located near the genes SAE1, RAG1, and CD8A. Framed regions indicate cell-type-specific signature ATAC-Seq peaks. Blue triangles indicate known promoter or enhancer regions, which are annotated in the GeneHancer (GH) Regulatory Elements database


B ATAC - Seq genome tracks showing three signature peaks located near the genes SAE1 , RAG1 , and CD8A . Framed regions indicate cell - type - specific signature ATAC - Seq peaks . Blue triangles indicate known promoter or enhancer regions , which are annotated in the GeneHancer ( GH ) Regulatory Elements

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O


A The rationale of footprinting analysis as illustrated by actual ATAC - Seq peaks , HINT - ATAC footprint hits , and example TF motif instances near RAG1 gene in DN2 population of thymus donor 6 .

O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O B-TISSUE O O O

A The rationale of footprinting analysis as illustrated by actual ATAC-Seq peaks, HINT-ATAC footprint hits, and example TF motif instances near RAG1 gene in DN2 population of thymus donor 6.


B Heatmap of ln ( odds ratio ) of the 50 TFs showing the most differential abundance of binding motifs in the open chromatin regions . TFs with an SD of the motif counts < 10 are excluded .

O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O

B Heatmap of ln(odds ratio) of the 50 TFs showing the most differential abundance of binding motifs in the open chromatin regions. TFs with an SD of the motif counts <10 are excluded.


D Chromatin accessibility as assessed by the number of ATAC peaks identified in the sorted populations of normal thymic T - cell precursors and in T - ALLs , respectively ( n = 10 / 19 patients have two biological replicates , n = 9 / 19 patients have one replicate ) . Horizontal lines of the box plot indicate the median , lower and upper limits of each box correspond to the first and third quartiles ( the 25th and 75th percentiles ) and the lower and upper whiskers extend from min to max ; pval = 4 . 4e - 08 ( Kruskal - Wallis Test ) .

O B-SUBCELLULAR O O O O O O O B-EXP_ASSAY O O O O O O O O B-TISSUE B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O B-DISEASE I-DISEASE I-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Chromatin accessibility as assessed by the number of ATAC peaks identified in the sorted populations of normal thymic T-cell precursors and in T-ALLs, respectively (n=10/19 patients have two biological replicates, n=9/19 patients have one replicate). Horizontal lines of the box plot indicate the median, lower and upper limits of each box correspond to the first and third quartiles (the 25th and 75th percentiles) and the lower and upper whiskers extend from min to max; pval=4.4e-08 (Kruskal-Wallis Test).


E Heatmap representing the enrichment and depletion of binding motifs of top 50 TFs in OCRs of T - ALLs in comparison to healthy T - cell precursors . Patients on the x - axis ; same order as in panel ( C ) . Gray cells represent TFs having non - significant enrichment or depletion .

O O O O O O O O B-EXP_ASSAY O O O O O O O O B-DISEASE I-DISEASE I-DISEASE O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E Heatmap representing the enrichment and depletion of binding motifs of top 50 TFs in OCRs of T-ALLs in comparison to healthy T-cell precursors. Patients on the x-axis; same order as in panel (C). Gray cells represent TFs having non-significant enrichment or depletion.


C log2 fold changes of 292 genes which are differentially expressed ( x - axis , RNA - Seq : non - sorted bulk thymi vs . T - ALLs ) and differentially accessible ( y - axis , ATAC - Seq : sorted T - cell populations vs . T - ALLs ) in T - ALLs in comparison to healthy T - cell precursors . Dot size and color indicate the frequency ( number of patients having dysregulation of the gene ) . Genes with ≥ 5 / 19 frequency are shown on the plot . Protein coding genes with ≥ 12 / 19 frequency are labelled . For plotting the average LFC values of ATAC and RNA are used per gene per quartile . Q1 - 4 stands for the quadrant 1 - 4 .

O O O O O O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-TISSUE O O B-DISEASE I-DISEASE I-DISEASE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O B-DISEASE I-DISEASE I-DISEASE O O B-DISEASE I-DISEASE I-DISEASE O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C log2 fold changes of 292 genes which are differentially expressed (x-axis, RNA-Seq: non-sorted bulk thymi vs. T-ALLs) and differentially accessible (y-axis, ATAC-Seq: sorted T-cell populations vs. T-ALLs) in T-ALLs in comparison to healthy T-cell precursors. Dot size and color indicate the frequency (number of patients having dysregulation of the gene). Genes with ≥5/19 frequency are shown on the plot. Protein coding genes with ≥12/19 frequency are labelled. For plotting the average LFC values of ATAC and RNA are used per gene per quartile. Q1 - 4 stands for the quadrant 1 - 4.


D Heatmap of VST normalized read counts ( each row is also normalized by the row mean ) of differentially expressed genes ( pval < 0 . 1 ; DESeq2 ) in three patients in comparison to remaining 16 . Orange triangles indicate three patients with no DAB1 but high SPI1 expression and motif counts .

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D Heatmap of VST normalized read counts (each row is also normalized by the row mean) of differentially expressed genes (pval<0.1; DESeq2) in three patients in comparison to remaining 16. Orange triangles indicate three patients with no DAB1 but high SPI1 expression and motif counts.


Levels of differential free amino acids in MERRF patients ' muscle ( B ) by LC - MS analysis , expressed relative to CTL . In panels the dashed line indicates the Mean CTL value set at 1 . In panels B data are presented as Mean ± SEM . Muscle : MERRF ( n = 10 ) , CTL ( n = 15 )

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Levels of differential free amino acids in MERRF patients' muscle (B) by LC-MS analysis, expressed relative to CTL. In panels the dashed line indicates the Mean CTL value set at 1. In panels B data are presented as Mean ± SEM. Muscle: MERRF (n=10), CTL (n=15)


( H ) Western blots of muscle lysates separated by denaturing SDS - PAGE and probed for OGDH , ALT2 , α - actin , and GOT2 ( top panel ) , ALT1 and GAPDH ( middle panel ) , GOT1 and GAPDH ( bottom panel ) , C ( CTL ) , M ( MERRF ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-DISEASE O O

(H) Western blots of muscle lysates separated by denaturing SDS-PAGE and probed for OGDH, ALT2, α-actin, and GOT2 (top panel), ALT1 and GAPDH (middle panel), GOT1 and GAPDH (bottom panel), C (CTL), M (MERRF).


( A - V ) Age - dependent muscle protein levels of ALT2 ( A ) , GOT2 ( C ) , OGDH ( E ) , GOT1 ( G ) , ALT1 ( I ) , AGC1 ( K ) , SUCLA2 ( M ) , BCAT2 ( O ) , BCKDH ( Q ) , PCCA ( S ) , and MUT ( U ) , estimated by band densitometry normalized by GAPDH , in COX10 KO expressed relative to same age CTL set at 1 . Representative western blots of muscle lysates from 170 - 200 days old mice separated by denaturing SDS - PAGE and probed for ALT2 ( B ) , GOT2 ( D ) , OGDH ( F ) , GOT1 ( H ) , ALT1 ( J ) , AGC1 ( L ) , SUCLA2 ( N ) , BCAT2 ( P ) , BCKDHB ( R ) , PCCA ( T ) , and MUT ( V ) and GAPDH ( B , D , F , H , J , L , N , P , R , T , V ) . : In panels data are presented as Mean ± SD . COX10 KO ( n = 4 ) , CTL ( n = 4 ) . Statistically significant differences between the two groups were estimated by unpaired two - tailed Student ' s test . * , p < 0 . 05 * * , p < 0 . 005 COX10 KO vs CTL .

O O O O O O O O B-TISSUE O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O O O O B-ORGANISM O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O

(A-V) Age-dependent muscle protein levels of ALT2 (A), GOT2 (C), OGDH (E), GOT1 (G), ALT1 (I), AGC1 (K), SUCLA2 (M), BCAT2 (O), BCKDH (Q), PCCA (S), and MUT (U), estimated by band densitometry normalized by GAPDH, in COX10 KO expressed relative to same age CTL set at 1. Representative western blots of muscle lysates from 170-200 days old mice separated by denaturing SDS-PAGE and probed for ALT2 (B), GOT2 (D), OGDH (F), GOT1 (H), ALT1 (J), AGC1 (L), SUCLA2 (N), BCAT2 (P), BCKDHB (R), PCCA (T), and MUT (V) and GAPDH (B, D, F, H, J, L, N, P, R, T, V). : In panels data are presented as Mean ± SD. COX10 KO (n=4), CTL (n=4). Statistically significant differences between the two groups were estimated by unpaired two-tailed Student's test. *, p<0.05 **, p<0.005 COX10 KO vs CTL.


( X ) Muscle fractional incorporation of M + 6 of glutamate , M + 5 of αKG , M + 4 of fumarate , M + 4 of malate , M + 3 of alanine and M + 3 of lactate , in 200 days COX10 KO and CTL mice IP injected with [ 13C5 , 15N ] - glutamate . In panels X data are presented as Mean ± SEM . In panel X , COX10 KO ( n = 4 ) , CTL ( n = 4 ) . Statistically significant differences between the two groups were estimated by unpaired two - tailed Student ' s test . * , p < 0 . 05 * * , p < 0 . 005 COX10 KO vs CTL .

O O O B-TISSUE B-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O B-ORGANISM O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O

(X) Muscle fractional incorporation of M+6 of glutamate, M+5 of αKG, M+4 of fumarate, M+4 of malate, M+3 of alanine and M+3 of lactate, in 200 days COX10 KO and CTL mice IP injected with [13C5, 15N]-glutamate. In panels X data are presented as Mean ± SEM. In panel X, COX10 KO (n=4), CTL (n=4). Statistically significant differences between the two groups were estimated by unpaired two-tailed Student's test. *, p<0.05 **, p<0.005 COX10 KO vs CTL.


( Z ) Muscle fractional incorporation of M + 6 of valine , M + 5 of α - KIV , M + 4 of β - ΗΙΒ , M + 3 of propionyl carnitine , in 200 days COX10 KO and CTL mice IP injected with [ 13C5 , 15N ] - valine . In panels Z , data are presented as Mean ± SEM . In panel Z , COX10 KO ( n = 3 ) , CTL ( n = 3 ) . Statistically significant differences between the two groups were estimated by unpaired two - tailed Student ' s test . * , p < 0 . 05 * * , p < 0 . 005 COX10 KO vs CTL .

O O O B-TISSUE B-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-GENEPROD O O O B-ORGANISM O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O

(Z) Muscle fractional incorporation of M+6 of valine, M+5 of α-KIV, M+4 of β-ΗΙΒ, M+3 of propionyl carnitine, in 200 days COX10 KO and CTL mice IP injected with [13C5, 15N]-valine. In panels Z, data are presented as Mean ± SEM. In panel Z, COX10 KO (n=3), CTL (n=3). Statistically significant differences between the two groups were estimated by unpaired two-tailed Student's test. *, p<0.05 **, p<0.005 COX10 KO vs CTL.


( B ) Age - dependent Ph - 4EBP1 / 4E - BP1 ratio in COX10 KO expressed relative to same age CTL set at 1 . ( C ) Representative western blot of muscle lysates from 200 days old mice separated by denaturing SDS - PAGE and probed for Ph - 4E - BP1 and for 4E - BP1 . In panels data are presented as Mean ± SD . COX10 KO ( n = 4 ) , CTL ( n = 4 ) . * , p < 0 . 05 COX10 KO vs . same age CTL .

O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O O B-ORGANISM O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O

(B) Age-dependent Ph-4EBP1/4E-BP1 ratio in COX10 KO expressed relative to same age CTL set at 1. (C) Representative western blot of muscle lysates from 200 days old mice separated by denaturing SDS-PAGE and probed for Ph-4E-BP1 and for 4E-BP1. In panels data are presented as Mean ± SD. COX10 KO (n=4), CTL (n=4). *, p<0.05 COX10 KO vs. same age CTL.


( D ) Age - dependent protein levels of Ph - 4E - BP1 in COX10 KO muscle estimated by band densitometry normalized by GAPDH expressed relative to 50 days COX10 KO set at 1 . ( E ) Western blots of muscle lysates from 50 , 100 , and 200 days old COX10 KO mice , separated by denaturing SDS - PAGE and probed for Ph - 4E - BP1 and GAPDH . In panel D , data are presented as Mean ± SD . # , p < 0 . 05 COX10 KO vs . COX10 KO at different ages . 50d ( n = 3 ) , 100d ( n = 3 ) , 200d ( n = 3 ) .

O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-TISSUE O O O B-EXP_ASSAY O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O O O O O O O B-GENEPROD O B-ORGANISM O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Age-dependent protein levels of Ph-4E-BP1 in COX10 KO muscle estimated by band densitometry normalized by GAPDH expressed relative to 50 days COX10 KO set at 1. (E) Western blots of muscle lysates from 50, 100, and 200 days old COX10 KO mice, separated by denaturing SDS-PAGE and probed for Ph-4E-BP1 and GAPDH. In panel D, data are presented as Mean ± SD. #, p<0.05 COX10 KO vs. COX10 KO at different ages. 50d (n=3), 100d (n=3), 200d (n=3).


Age - dependent protein levels of SESTRIN 2 ( F ) , REDD1 ( H ) , LC3II ( J ) estimated by band densitometry normalized by GAPDH , in COX10 KO expressed relative to same age CTL set at 1 . Representative western blot of muscle lysates from 100 and 170 - 200 days old mice separated by denaturing SDS - PAGE and probed for SESTRIN 2 ( G ) , REDD1 ( I ) , LC3II ( K ) In panels data are presented as Mean ± SD . COX10 KO ( n = 4 ) , CTL ( n = 4 ) . * , p < 0 . 05 COX10 KO vs . same age CTL .

O O O O O O B-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O O O O O O B-ORGANISM O O O O O O O O O B-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O

Age-dependent protein levels of SESTRIN 2 (F), REDD1 (H), LC3II (J) estimated by band densitometry normalized by GAPDH, in COX10 KO expressed relative to same age CTL set at 1. Representative western blot of muscle lysates from 100 and 170-200 days old mice separated by denaturing SDS-PAGE and probed for SESTRIN 2 (G), REDD1 (I), LC3II (K) In panels data are presented as Mean ± SD. COX10 KO (n=4), CTL (n=4). *, p<0.05 COX10 KO vs. same age CTL.