C - F . WT ( C ) , FIP200 knockout ( KO ) ( D ) , ULK1 / 2 knockout ( E ) MEF or ATG13 knockout 4T1 ( F ) were subjected to starvation conditions before immunoprecipitation against control Immunoglobulin G ( IgG ) or anti - TRIM27 before blotting against indicated endogenous forms of TRIM27 , FIP200 , ULK1 , ATG13 , USP7 . Data information : experiments were performed at least three times independently with similar results .

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C-F. WT (C), FIP200 knockout (KO) (D), ULK1/2 knockout (E) MEF cells or ATG13 knockout 4T1 cells (F) were subjected to starvation conditions before immunoprecipitation against control Immunoglobulin G (IgG) or anti-TRIM27 before blotting against the indicated endogenous forms of TRIM27, FIP200, ULK1, ATG13, and USP7. Data information: the experiments were performed at least three times independently with similar results.


G . Co - immunoprecipitation assays in HEK293T after co - transfection with Flag - TRIM27 either full ULK ( HA - ULK1 1 - 1051 ) or indicated various truncated forms of ULK1 designated HA - ULK1 1 - 828 , HA - ULK1 1 - 600 , HA - ULK1 1 - 500 ; AA , . Data information : , experiments were performed at least three times independently with similar results .

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G. Co-immunoprecipitation assays in HEK293T cells after co-transfection with Flag-TRIM27 and either full length ULK (HA-ULK1 1-1051) or the indicated various truncated forms of ULK1 designated HA-ULK1 1-828, HA-ULK1 1-600, and HA-ULK1 1-500; AA, amino acids. Data information: , the experiments were performed at least three times independently with similar results.


H . Co - immunoprecipitation assays in HeLa stably expressing HA - ULK1 immunoprecipitation against IgG or HA followed by Western blotting as indicated . Data information : experiments were performed at least three times independently with similar results .

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H. Co-immunoprecipitation assays in HeLa cells stably expressing HA-ULK1 and immunoprecipitation against IgG or HA followed by Western blotting as indicated. Data information: the experiments were performed at least three times independently with similar results.


HeLa stably expressing Flag - TRIM27 were starved as indicated or starved for 2h before replenishing with complete medium . Anti - Flag immunoprecipitates ( top panel ) input samples ( bottom panel ) were then subjected to blotting against Flag ULK1 . Data information : experiments were performed at least three times independently with similar results .

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HeLa cells stably expressing Flag-TRIM27 were starved as indicated or starved for 2h before replenishing with complete medium. Anti-Flag immunoprecipitates (top panel) and input samples (bottom panel) were then subjected to blotting against Flag and ULK1. Data information: the experiments were performed at least three times independently with similar results.


Purified recombinant GST or GST - TRIM27 was incubated with recombinant HA - ULK1 bound ULK1 analyzed by Western blotting . Data information : experiments were performed at least three times independently with similar results .

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Purified recombinant GST or GST-TRIM27 was incubated with recombinant HA-ULK1 and bound ULK1 analyzed by Western blotting. Data information: the experiments were performed at least three times independently with similar results.


Ubiquitination assays in HEK293T after co - transfection with HA - ULK1 His - Ub along with Flag - TRIM27 ( A ) Samples recovered with Ni - NTA ( top panel ) input samples ( bottom panel ) were then subjected to blotting against anti - HA , Flag or His as indicated . Data information : , experiments were performed at least three times independently with similar results .

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Ubiquitination assays in HEK293T cells after co-transfection with HA-ULK1 and His-Ub along with Flag-TRIM27 (A) Samples recovered with Ni-NTA (top panel) and input samples (bottom panel) were then subjected to blotting against anti-HA, Flag or His as indicated. Data information: , the experiments were performed at least three times independently with similar results.


Ubiquitination assays in HEK293T after knockdown of TRIM27 using independent shRNAs ( shTRIM27 - 1 - 2 ) ( B ) . Samples recovered with Ni - NTA ( top panel ) input samples ( bottom panel ) were then subjected to blotting against anti - HA or His as indicated . Data information : , experiments were performed at least three times independently with similar results .

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Ubiquitination assays in HEK293T cells after knockdown of TRIM27 using independent shRNAs (shTRIM27-1 and -2) (B). Samples recovered with Ni-NTA (top panel) and input samples (bottom panel) were then subjected to blotting against anti-HA or His as indicated. Data information: , the experiments were performed at least three times independently with similar results.


C . In vitro ubiquitination assays performed with indicated combinations of recombinant GST , GST - TRIM27 , affinity - purified HA - ULK1 His - Ub in presence or absence of E1 , E2 , ATP . reactions were analyzed with anti - Ub , anti - HA , or anti - GST . Data information : experiments were performed at least three times independently with similar results .

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C. In vitro ubiquitination assays performed with the indicated combinations of recombinant GST, GST-TRIM27, affinity-purified HA-ULK1 and His-Ub in the presence or absence of E1, E2, and ATP. The reactions were analyzed with anti-Ub, anti-HA, or anti-GST. Data information: the experiments were performed at least three times independently with similar results.


Western blot of ULK1 in HeLa stably expressing control or TRIM27 shRNAs ( D ) Data information : , experiments were performed at least three times independently with similar results .

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Western blot analysis of ULK1 levels in HeLa cells stably expressing control or TRIM27 shRNAs (D) Data information: , the experiments were performed at least three times independently with similar results.


Western blot of HEK293T transfected with empty Flag vector ( - ) or Flag - TRIM27 after treatment with or without 10 μM MG132 for 4 hours ( E ) . Data information : experiments were performed at least three times independently with similar results .

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Western blot analysis of HEK293T cells transfected with empty Flag vector (-) or Flag-TRIM27 after treatment with or without 10 μM MG132 for 4 hours (E). Data information: the experiments were performed at least three times independently with similar results.


F . microscopy image of MEFs with stable expression of mCherry - ULK1 GFP - TRIM27 after treating with DMSO ( MOCK ) or 10 μM MG132 for 4 hours . Scale bar , 10 μm . Data information : experiments were performed at least three times independently with similar results .

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F. Fluorescence microscopy image of MEFs with stable expression of mCherry-ULK1 and GFP-TRIM27 after treating cells with DMSO (MOCK) or 10 μM MG132 for 4 hours. Scale bar, 10 μm. Data information: the experiments were performed at least three times independently with similar results.


HA - ULK1 in 50 μg / mL cycloheximide ( CHX ) - treated HEK293T with or without co - expression of Flag - TRIM27 ( G , H ) Western blots against HA - ULK1 actin control ( G , were subjected to densitometric quantitation ( H , Data information : experiments were performed at least three times independently with similar results . statistical data are presented as mean ± s . d . of three independent experiments . Statistical was performed using two - unpaired Student ' s t - ( H , * * P < 0 . 01 .

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HA-ULK1 levels in 50 μg/mL cycloheximide (CHX)-treated HEK293T cells with or without co-expression of Flag-TRIM27 (G, H) Western blots against HA-ULK1 and actin control (G, were subjected to densitometric quantitation (H, Data information: the experiments were performed at least three times independently with similar results. All the statistical data are presented as mean ± s.d. of three independent experiments. Statistical analysis was performed using two-tailed unpaired Student's t-tests (H , **P < 0.01.


endogenous ULK1 ( I , J ) in 50 μg / mL cycloheximide ( CHX ) - treated HEK293T or without TRIM27 RNAi ( I , J ) . actin control I ) were subjected to densitometric quantitation J ) . Data information : experiments were performed at least three times independently with similar results . statistical data are presented as mean ± s . d . of three independent experiments . Statistical was performed using two - unpaired Student ' s t - J ) , * * P < 0 . 01 .

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endogenous ULK1 levels (I, J) in 50 μg/mL cycloheximide (CHX)-treated HEK293T cells or without TRIM27 RNAi (I, J). and actin control I) were subjected to densitometric quantitation J). Data information: the experiments were performed at least three times independently with similar results. All the statistical data are presented as mean ± s.d. of three independent experiments. Statistical analysis was performed using two-tailed unpaired Student's t-tests J), **P < 0.01.


Ubiquitination assays were performed in HEK293T after co - transfection with full HA - ULK1 or indicated truncated constructs ( K ) in presence or absence of Flag - TRIM27 . Data information : , experiments were performed at least three times independently with similar results .

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Ubiquitination assays were performed in HEK293T cells after co-transfection with full length HA-ULK1 or the indicated truncated constructs (K) in the presence or absence of Flag-TRIM27. Data information: , the experiments were performed at least three times independently with similar results.


Ubiquitination assays were performed in HEK293T after co - transfection with double ( K568 / K571R ) or single ( K568R , K571R ) lysine substitution mutants ( L ) in presence or absence of Flag - TRIM27 . Data information : experiments were performed at least three times independently with similar results .

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Ubiquitination assays were performed in HEK293T cells after co-transfection with double (K568/K571R) or single (K568R, K571R) lysine substitution mutants (L) in the presence or absence of Flag-TRIM27. Data information: the experiments were performed at least three times independently with similar results.


M . Ubiquitination of HA - ULK1 in co - expressing Flag - TRIM27 with either His - Ub WT , His - Ub K48 , or His - Ub K63 , respectively . Data information : experiments were performed at least three times independently with similar results .

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M. Ubiquitination of HA-ULK1 in cells co-expressing Flag-TRIM27 with either His-Ub WT, His-Ub K48, or His-Ub K63, respectively. Data information: the experiments were performed at least three times independently with similar results.


N . In vitro ubiquitination assays against affinity - purified HA - ULK1 WT or HA - ULK1 K568 / K571R mutant in presence or absence of GST - TRIM27 . Data information : experiments were performed at least three times independently with similar results .

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N. In vitro ubiquitination assays against affinity-purified HA-ULK1 WT or the HA-ULK1 K568/K571R mutant in the presence or absence of GST-TRIM27. Data information: the experiments were performed at least three times independently with similar results.


O . ULK1 / 2 knockout MEFs stably expressing indicated HA - ULK1 constructs were subjected to Western blot against HA or actin before ( 0h ) after treatment with CHX for 12 hours . Data information : experiments were performed at least three times independently with similar results .

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O. ULK1/2 knockout MEFs stably expressing the indicated HA-ULK1 constructs were subjected to Western blot analysis against HA or actin before (0h) and after treatment with CHX for 12 hours. Data information: the experiments were performed at least three times independently with similar results.


A . Western blot of autophagy marker ( p62 Ser318 phosphorylated ATG13 ) in WT HeLa two independent TRIM27 KO clones . ULK1 TRIM27 were measured , along with actin as a loading control .

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A. Western blot analysis of autophagy marker proteins (p62 and Ser318 phosphorylated ATG13) in WT HeLa cells and two independent TRIM27 KO clones. ULK1 and TRIM27 levels were measured, along with actin as a loading control.


B . TRIM27 KO HeLa were reconstituted with Flag - TRIM27 Western blotting was performed as per ( A ) .

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B. TRIM27 KO HeLa cells were reconstituted with Flag-TRIM27 and Western blotting was performed as per (A).


C . LC3 conversion was measured by Western blot in WT or TRIM27 KO HeLa with or without pretreatment with 200 nM bafilomycin A1 ( BAF A1 ) .

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C. LC3 conversion was measured by Western blot in WT or TRIM27 KO HeLa cells with or without pretreatment with 200 nM bafilomycin A1 (BAF A1).


D . Western blot of indicated in MEFs generated from Trim27 + / + or Trim27 - / - mice .

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D. Western blot analysis of indicated proteins in MEFs generated from Trim27 +/+ or Trim27 -/- mice.


E - H . Immunofluorescence of endogenous LC3 in MEFs generated from Trim27 + / + or Trim27 - / - mice cultured in fed ( E ) or starved conditions ( G ) . of LC3B Fed conditions ( F ) , starved conditions ( H ) ( n = 50 per condition ) . Data are mean ± s . d . in F H . Scale bar , 10 μm . Data information : ( F , H , Statistical was performed using two - unpaired Student ' s t - , * P < 0 . 05 , * * * P < 0 . 001 .

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E-H. Immunofluorescence analysis of endogenous LC3 in MEFs generated from Trim27+/+ or Trim27-/- mice cultured in fed (E) or starved conditions (G). Quantification of LC3B puncta Fed conditions (F), starved conditions (H) (n = 50 cells per condition). Data are mean ± s.d. in F and H. Scale bar, 10 μm. Data information: (F, H, Statistical analysis was performed using two-tailed unpaired Student's t-tests, *P< 0.05,***P<0.001.


I - J . Western blot of autophagy markers in kidneys of three - month - old Trim27 + / + Trim27 - / - mice . Each lane represents a different mouse ( I ) . of p62 , LC3 - II / LC3 - I , LC3 - II / actin in I ( J ) . Data are mean ± s . e . m . for three mice per genotype . Data information : J , Statistical was performed using two - unpaired Student ' s t - , * P < 0 . 05 , * * * P < 0 . 001 .

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I-J. Western blot analysis of autophagy markers in the kidneys of three-month-old Trim27 +/+ and Trim27-/- mice. Each lane represents a different mouse (I). Quantification of p62, LC3-II/LC3-I ratio, and LC3-II/actin ratio in I (J). Data are mean ± s.e.m. for three mice per genotype. Data information: J, Statistical analysis was performed using two-tailed unpaired Student's t-tests, *P< 0.05,***P<0.001.


K . Microscopy of GFP - LC3 in MEFs generated from indicated genotype mice in normal or starvation conditions . Scale bar , 10 μm . L - M . Quantitation of data presented in ( K ) shows mean number of GFP - LC3 per ± s . d . ( n = 50 per condition ) . Data information : L M ) Statistical was performed using two - unpaired Student ' s t - , * P < 0 . 05 , * * * P < 0 . 001 .

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K. Microscopy analysis of GFP-LC3 puncta in MEFs generated from indicated genotype mice in normal or starvation conditions. Scale bar, 10 μm. L-M. Quantitation of data presented in (K) shows mean number of GFP-LC3 puncta per cell ± s.d. (n = 50 cells per condition). Data information: L and M) Statistical analysis was performed using two-tailed unpaired Student's t-tests, *P< 0.05,***P<0.001.


A . Ubiquitination of HA - ULK1 in HeLa derivatives co - expressing His - Ub with or without TRIM27 knockout , were treated with starvation for indicated time .

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A. Ubiquitination analysis of HA-ULK1 in HeLa derivatives co-expressing His-Ub with or without TRIM27 knockout, the cells were treated with starvation for the indicated time.


B . Western blot of ULK1 in HeLa with or without TRIM27 knockout . were treated with starvation for indicated time . lysates were analyzed using anti - ULK1 , anti - TRIM27 , or anti - Actin .

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B. Western blot analysis of ULK1 levels in HeLa cells with or without TRIM27 knockout. The cells were treated with starvation for the indicated time. The lysates were analyzed using anti-ULK1, anti-TRIM27, or anti-Actin.


C . HA - ULK1 , HA - ULK1 K568 / K571R were stably expressed in ULK1 knockout HeLa , were treated with starvation , lysates were analyzed by western blot using anti - HA , or anti - Actin .

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C. HA-ULK1, HA-ULK1 K568/K571R were stably expressed in ULK1 knockout HeLa cells, the cells were treated with starvation, the lysates were analyzed by western blot using anti-HA, or anti-Actin.


E . Co - immunoprecipitation of Flag - TRIM27 with HA - ULK1 in HEK293T with or without STK38L knockdown , were treated with starvation . Flag - TRIM27 was immunoprecipitated using anti - Flag , bound HA - ULK1 was detected using anti - HA .

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E. Co-immunoprecipitation of Flag-TRIM27 with HA-ULK1 in HEK293T cells with or without STK38L knockdown, the cells were treated with starvation. Flag-TRIM27 was immunoprecipitated using anti-Flag, the bound HA-ULK1 was detected using anti-HA.


F . Co - immunoprecipitation of endogenous TRIM27 with endogenous STK38L in HeLa .

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F. Co-immunoprecipitation of endogenous TRIM27 with endogenous STK38L in HeLa cells.


G . Co - immunoprecipitation of Flag - STK38L with ULK1 , or TRIM27 in HEK293T with or without starvation treatment . Flag - STK38L was immunoprecipitated using anti - Flag , immunoprecipitates lysates were analyzed with anti - ULK1 , anti - TRIM27 .

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G. Co-immunoprecipitation of Flag-STK38L with ULK1, or TRIM27 in HEK293T cells with or without starvation treatment. Flag-STK38L was immunoprecipitated using anti-Flag, the immunoprecipitates and lysates were analyzed with anti-ULK1, anti-TRIM27.


H . Co - immunoprecipitation of endogenous TRIM27 with endogenous ULK1 in HeLa with or without STK38L knockout . were treated with starvation before lysis .

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H. Co-immunoprecipitation of endogenous TRIM27 with endogenous ULK1 in HeLa cells with or without STK38L knockout. The cells were treated with starvation before lysis.


I . Western blot of ULK1 in starved HeLa expressing either Myc - STK38L , or Flag - TRIM27 , or both . lysates were analyzed using anti - ULK1 , anti - Flag , anti - Myc , or anti - actin .

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I. Western blot analysis of ULK1 levels in starved HeLa cells expressing either Myc-STK38L, or Flag-TRIM27, or both. The lysates were analyzed using anti-ULK1, anti-Flag, anti-Myc, or anti-actin.


J . Ubiquitination of HA - ULK1 in HEK293T co - expressing Flag - TRIM27 His - Ub , or together with Myc - STK38L .

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J. Ubiquitination analysis of HA-ULK1 in HEK293T cells co-expressing Flag-TRIM27 and His-Ub, or together with Myc-STK38L.


K . Western blot of ULK1 in HeLa with or without STK38L knockout , were treated with starvation for indicated time .

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K. Western blot analysis of ULK1 levels in HeLa cells with or without STK38L knockout, the cells were treated with starvation for the indicated time.


L . Western blot of p62 or LC3 in HeLa with or without STK38L knockout , were treated with starvation for indicated time . Bafilomycin A1 ( BAF A1 ) was used to inhibit fusion between autophagosomes lysosomes .

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L. Western blot analysis of p62 or LC3 levels in HeLa cells with or without STK38L knockout, the cells were treated with starvation for the indicated time. Bafilomycin A1 (BAF A1) was used to inhibit the fusion between autophagosomes and lysosomes.


A . Western blot of ULK1 in HeLa stably expressing Flag - TRIM27 , were transfected with either Myc - vector , Myc - STK38L , or Myc - STK38L - K119A . Then were treated with starvation before lysis .

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A. Western blot analysis of ULK1 levels in HeLa cells stably expressing Flag-TRIM27, the cells were transfected with either Myc-vector, Myc-STK38L, or Myc-STK38L-K119A. Then the cells were treated with starvation before lysis.


B . Phosphorylation of HA - ULK1 in HeLa STK38L knockout with or without expression of Flag - STK38L . HA - ULK1 was immunoprecipitated then analyzed by western blot using anti - phosphoserine or anti - phosphothreonine , lysates were analyzed using anti - HA anti - Myc .

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B. Phosphorylation of HA-ULK1 in HeLa STK38L knockout cells with or without expression of Flag-STK38L. HA-ULK1 was immunoprecipitated and then analyzed by western blot using anti-phosphoserine or anti-phosphothreonine, and the cell lysates were analyzed using anti-HA and anti-Myc.


C . Co - immunoprecipitation of Flag - TRIM27 with HA - ULK1 or HA - ULK1 - S495A in HEK293T . HA - ULK1 or HA - ULK1 S495A was immunoprecipitated using anti - HA , immunoprecipitates lysates were analyzed with anti - HA or anti - Flag .

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C. Co-immunoprecipitation of Flag-TRIM27 with HA-ULK1 or HA-ULK1-S495A in HEK293T cells. HA-ULK1 or HA-ULK1 S495A was immunoprecipitated using anti-HA, the immunoprecipitates and lysates were analyzed with anti-HA or anti-Flag.


D . Ubiquitination of HA - ULK1 WT or HA - ULK1 S495A in HEK293T co - expressing Flag - TRIM27 His - Ub .

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D. Ubiquitination analysis of HA-ULK1 WT or HA-ULK1 S495A in HEK293T cells co-expressing Flag-TRIM27 and His-Ub.


E . Phosphorylation of HA - ULK1 in HEK293T co - expressing Myc - STK38L with or without CIP treatment . HA - ULK1 was immunoprecipitated with anti - HA analyzed by western blot using a specific antibody against p - ULK1 - S495 ( Ser495 ) .

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E. Phosphorylation of HA-ULK1 in HEK293T cells co-expressing Myc-STK38L with or without CIP treatment. HA-ULK1 was immunoprecipitated with anti-HA and analyzed by western blot using a specific antibody against p-ULK1-S495 (Ser495).


F . Phosphorylation of HA - ULK1 or HA - ULK1 S495A in HEK293T co - expressing Myc - STK38L . HA - ULK1 or HA - ULK1 S495A was immunoprecipitated with anti - HA analyzed by western blot using a specific antibody against p - ULK1 - S495 ( Ser495 ) .

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F. Phosphorylation of HA-ULK1 or HA-ULK1 S495A in HEK293T cells co-expressing Myc-STK38L. HA-ULK1 or HA-ULK1 S495A was immunoprecipitated with anti-HA and analyzed by western blot using a specific antibody against p-ULK1-S495 (Ser495).


G . In vitro kinase using GST - STK38L , or GST - STK38L K119A ( a kinase - inactive mutant ) HA - ULK1 as substrate . GST - STK38L or GST - STK38L K119A was pulled down using glutathione Sepharose beads . HA - ULK1 was immunoprecipitated from HEK293T using anti - HA treated with lambda phosphatase , reaction product was analyzed by western blot using p - ULK1 - S495 ( Ser495 ) .

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G. In vitro kinase assay using GST-STK38L, or GST-STK38L K119A (a kinase-inactive mutant) and HA-ULK1 as substrate. GST-STK38L or GST-STK38L K119A was pulled down using glutathione Sepharose beads. HA-ULK1 was immunoprecipitated from HEK293T using anti-HA and treated with lambda phosphatase, the reaction product was analyzed by western blot using p-ULK1-S495 (Ser495).


H . Ubiquitination of Myc - STK38L in HEK293T co - expressing Flag - TRIM27 His - Ub .

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H. Ubiquitination analysis of Myc-STK38L in HEK293T cells co-expressing Flag-TRIM27 and His-Ub.


I . Ubiquitination of Flag - STK38L in HEK293T co - expressing His - Ub with or without TRIM27 knockdown , were cultured in fed or starved conditions as indicated .

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I. Ubiquitination analysis of Flag-STK38L in HEK293T cells co-expressing His-Ub with or without TRIM27 knockdown, cells were cultured in fed or starved conditions as indicated.


J . Ubiquitination of Flag - STK38L in HEK293T co - expressing Myc - TRIM27 with either His - Ub ( wild - type ) or His - Ub K6R .

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J. Ubiquitination analysis of Flag-STK38L in HEK293T cells co-expressing Myc-TRIM27 with either His-Ub (wild-type) or His-Ub K6R.


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K. Ubiquitination analysis of Myc-STK38L WT, Myc-STK38L-K432R+K434R or Myc-STK38L-K181R+K215R+K224R+K432R+K434R (Myc-STK38L-5KR) mutant in HEK293T cells co-expressing HA-TRIM27 and His-Ub.


L . Ubiquitination of Myc - STK38L K1 - 38R mutant ( lysines were mutated to arginines ) , or Myc - STK38L - R181 / 215 / 224 / 432 / 434K ( lysines were mutated to arginines except indicated five lysine residues ) mutant in HEK293T co - expressing HA - TRIM27 His - Ub .

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L. Ubiquitination analysis of Myc-STK38L K1-38R mutant (All lysines were mutated to arginines), or Myc-STK38L-R181/215/224/432/434K (All lysines were mutated to arginines except the indicated five lysine residues) mutant in HEK293T cells co-expressing HA-TRIM27 and His-Ub.


M . Phosphorylation of HA - ULK1 S495 ( Ser495 ) in HEK293T co - expressing Myc - STK38L WT , or Myc - STK38L - 5KR mutant in K . were cultured in fed or starved conditions as indicated HA - ULK1 was immunoprecipitated using anti - HA , immunoprecipitates lysates were analyzed with a specific antibody against p - ULK1 - S495 ( Ser495 ) , anti - Myc , anti - HA , or anti - actin .

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M. Phosphorylation of HA-ULK1 S495(Ser495) in HEK293T cells co-expressing Myc-STK38L WT, or Myc-STK38L-5KR mutant in K. The cells were cultured in fed or starved conditions as indicated and HA-ULK1 was immunoprecipitated using anti-HA, the immunoprecipitates and lysates were analyzed with a specific antibody against p-ULK1-S495 (Ser495), anti-Myc, anti-HA, or anti-actin.


N . Phosphorylation of HA - ULK1 in HeLa with or without TRIM27 - knockout , were cultured in fed or starved conditions as indicated , HA - ULK1 was immunoprecipitated using anti - HA , immunoprecipitates lysates were analyzed with a specific antibody against p - ULK1 - S495 ( Ser495 ) , anti - HA , or anti - actin .

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N. Phosphorylation of HA-ULK1 in HeLa cells with or without TRIM27-knockout, cells were cultured in fed or starved conditions as indicated, HA-ULK1 was immunoprecipitated using anti-HA, the immunoprecipitates and lysates were analyzed with a specific antibody against p-ULK1-S495 (Ser495), anti-HA, or anti-actin.


A . Comparison of relative mRNA of TRIM27 in normal breast tissues ( Normal , n = 113 ) indicated breast tumors ( Basal ， n = 143 ； Her2 ， n = 67 ； LumA ， n = 427 ； LumB ， n = 190 ) in TCGA database . number of biological replicates are shown as indicated . P < 0 . 0001 ( one - way ANOVA ) .

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A. Comparison of the relative mRNA levels of TRIM27 in normal breast tissues (Normal, n=113) and indicated breast tumors (Basal，n=143；Her2，n=67；LumA，n=427；LumB，n=190) in the TCGA database. The number of biological replicates are shown as indicated. P < 0.0001 (one-way ANOVA).


B . of correlation between ULK1 TRIM27 expression in CPTAC proteomics database . P = 0 . 0027 ( Pearson ' s correlation method ) .

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B. Analysis of the correlation between ULK1 and TRIM27 expression in the CPTAC proteomics database. P = 0.0027 (Pearson's correlation method).


Western blot of ULK1 TRIM27 from tissues ( T ) , Pericarcinomatous tissues ( P ) , their matched surrounding normal tissues ( N ) . GAPDH was used as a loading control ( C ) .

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Western blot analysis of ULK1 and TRIM27 levels from tumor tissues (T), Pericarcinomatous tissues (P), and their matched surrounding normal tissues (N). GAPDH was used as a loading control (C).


F . Representative tumors in MMTV - PyMT mice of indicated Trim27 genotypes . Scale bar , 5mm .

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F. Representative tumors in MMTV-PyMT mice of the indicated Trim27 genotypes. Scale bar, 5mm.


G . Western blot of indicated in Trim27 - / - PyMT or Trim27 + / - PyMT mice in breast tumors . Actin was used as a loading control .

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G. Western blot analysis of indicated proteins in Trim27-/- PyMT or Trim27+/- PyMT mice in primary breast tumors. Actin was used as a loading control.


H . Kaplan - Meier - free survival curves of Trim27 + / - PyMT ( median = 75 d , number = 20 ) , or Trim27 - / - PyMT mice ( median = 95 d , number = 16 ) . P < 0 . 0001 ( Mantel - Cox ) .

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H. Kaplan-Meier tumor-free survival curves of Trim27+/- PyMT (median = 75 d, number = 20), or Trim27 -/- PyMT mice (median = 95 d, number = 16). P < 0.0001 (Mantel-Cox test).


Pulmonary surface nodules in Trim27 + / - PyMT mice Trim27 - / - PyMT mice ( I Scale bar , 1mm .

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Pulmonary surface nodules in Trim27+/- PyMT mice and Trim27-/- PyMT mice (I Scale bar, 1mm.


Pulmonary surface nodules hematoxylin eosin ( H & E ) in Trim27 + / - PyMT mice Trim27 - / - PyMT mice J ) . Scale bar , 1mm . ( K ) . Bar chart showing for number of metastatic nodules per lung lope ( Trim27 + / - , n = 6 mice ; Trim27 - / - n = 6 mice ) , when compared at same final timepoint . Each symbol represents a mouse ; Data are presented as mean ± s . d . from three independent experiments ; * * * P < 0 . 001 ( unpaired student ' s t - ) .

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Pulmonary surface nodules and hematoxylin and eosin (H&E) staining in Trim27+/- PyMT mice and Trim27-/- PyMT mice J). Scale bar, 1mm. (K). Bar chart showing quantification for the number of metastatic nodules per lung lope (Trim27+/-, n=6 mice; Trim27-/- n=6 mice), when compared at the same final timepoint. Each symbol represents a mouse; Data are presented as mean ± s.d. from three independent experiments; ***P<0.001 (unpaired student's t-test).


Western blot of a dCas9 immunoprecipitation experiment in promoter - targeted at ring stage . of dCas9 histone H3 in ( Cyt ) , ( excluding , Nuc ) , ( Ch ) fractions are revealed with anti - HA anti - H3 antibodies , respectively . dCas9 is enriched in immunoprecipitated fraction ( IP ) compared to unbound supernatant ( SN ) input ( i . e . fraction ) of IP . Molecular weights are shown to right .

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Western blot analysis of a dCas9 immunoprecipitation experiment in the promoter-targeted strain at ring stage. Levels of dCas9 and histone H3 in the cytoplasmic (Cyt), nuclear (excluding chromatin, Nuc), and chromatin (Ch) fractions are revealed with anti-HA and anti-H3 antibodies, respectively. dCas9 is enriched in the immunoprecipitated fraction (IP) compared to the unbound supernatant (SN) and input (i.e. chromatin fraction) of the IP. Molecular weights are shown to the right.


Circos plots of ChIP - seq data showing genome - wide enrichment of dCas9 in ring stage . 14 chromosomes are represented circularly by outer grey bars , with chromosome number indicated in roman numerals chromosome distances indicated in Arabic numerals ( Mbp ) . Enrichment for intron - or promoter - targeted dCas9 ( normalized to non - targeted dCas9 ) is shown as average reads per million ( RPM ) over bins of 1 , 000 nt . maximum y - axis value is 1 , 200 RPM for promoter - targeted dCas9 ( rings represent increments of 200 ) 400 RPM for intron - targeted dCas9 ( rings represent increments of 66 . 7 ) . var genes are represented by red bars . An asterisk indicates an unintended binding event .

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Circos plots of ChIP-seq data showing genome-wide enrichment of dCas9 in ring stage parasites. The 14 chromosomes are represented circularly by the outer grey bars, with chromosome number indicated in roman numerals and chromosome distances indicated in Arabic numerals (Mbp). Enrichment for intron- or promoter-targeted dCas9 (normalized to non-targeted dCas9) is shown as average reads per million (RPM) over bins of 1,000 nt. The maximum y-axis value is 1,200 RPM for the promoter-targeted dCas9 (rings represent increments of 200) and 400 RPM for the intron-targeted dCas9 (rings represent increments of 66.7). var genes are represented by red bars. An asterisk indicates an unintended binding event.


ChIP - seq data show enrichment of dCas9 in strains at ring stage expressing non - targeted " Control " ( grey ) , var intron - targeted ( blue ) , or var promoter - targeted ( red ) sgRNAs . Genome location is indicated at top of each panel . x - axis is , with genes represented by black boxes indented to delineate introns labeled with arrowheads to indicate transcription direction . y - axis is input - subtracted ChIP enrichment . q - values for promoter - targeted dCas9 peaks shown are 2 . 22x10 - 289 for PF3D7 _ 0413100 7 . 07x10 - 257 for PF3D7 _ 1240400 . q - values for intron - targeted dCas9 peaks shown are 2 . 03x10 - 76 for PF3D7 _ 412700 , 6 . 81x10 - 46 for PF3D7 _ 0412900 , 1 . 53x10 - 62 for PF3D7 _ 0413100 , 8 . 37x10 - 4 for PF3D7 _ 1240900 .

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ChIP-seq data show enrichment of dCas9 in strains at ring stage expressing non-targeted "Control" (grey), var intron-targeted (blue), or var promoter-targeted (red) sgRNAs. Genome location is indicated at the top of each panel. The x-axis is DNA sequence, with genes represented by black boxes indented to delineate introns and labeled with white arrowheads to indicate transcription direction. The y-axis is input-subtracted ChIP enrichment. q-values for promoter-targeted dCas9 peaks shown are 2.22x10-289 for PF3D7_0413100 and 7.07x10-257 for PF3D7_1240400. q-values for intron-targeted dCas9 peaks shown are 2.03x10-76 for PF3D7_412700, 6.81x10-46 for PF3D7_0412900, 1.53x10-62 for PF3D7_0413100, and 8.37x10-4 for PF3D7_1240900.


ChIP - seq ( red ) RIP - seq ( green ) data show enrichment of , respectively , from var genes in promoter - targeted non - targeted " Control " dCas9 immunoprecipitation from non - clonal bulk population of ring stage . Genome location is indicated at top of each panel . x - axis is , with genes represented by black boxes indented to delineate introns labeled with arrowheads to indicate transcription direction . y - axis is input - subtracted dCas9 ChIP enrichment in var gene promoter - targeted dCas9 ( red ) , dCas9 RIP enrichment normalized to IgG control in var gene promoter - targeted dCas9 or non - targeted " Control " dCas9 ( green ) , or var gene transcript in promoter - targeted dCas9 ( RPKM , grey ) . One replicate was performed for ChIP - seq - seq one replicate was performed for HA IgG control RIP - seq .

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ChIP-seq (red) and RIP-seq (green) data show enrichment of DNA and RNA, respectively, from var genes in the promoter-targeted and non-targeted "Control" dCas9 immunoprecipitation from non-clonal bulk population of ring stage parasites. Genome location is indicated at the top of each panel. The x-axis is DNA sequence, with genes represented by black boxes indented to delineate introns and labeled with white arrowheads to indicate transcription direction. The y-axis is input-subtracted dCas9 ChIP enrichment in the var gene promoter-targeted dCas9 strain (red), dCas9 RIP enrichment normalized to IgG control in the var gene promoter-targeted dCas9 strain or non-targeted "Control" dCas9 strain (green), or var gene transcript levels in the promoter-targeted dCas9 strain (RPKM, grey). One replicate was performed for ChIP-seq and RNA-seq and one replicate was performed for HA and IgG control RIP-seq.


Volcano plot representation of label - free quantitative proteomic of P . falciparum present in intron - ( left ) promoter - targeted ( right ) dCas9 immunoprecipitations . Each dot represents a , its corresponds to sum of peptides from both conditions used to quantify of enrichment . For main volcano plot , x - axis = log2 ( fold - change ) , y - axis = - log10 ( p - value ) , horizontal yellow indicates adjusted p - value = 0 . 05 , vertical yellow lines indicate absolute fold - change = 1 . 5 . Side panels indicate uniquely identified in either sample ( y - axis = number of peptides per 100 ) . dCas9 is highlighted in green for reference . Two replicates were used for promoter - targeted dCas9 four replicates were used for intron - targeted dCas9 . Fold enrichment adjusted p - values for each highlighted in red ( promoter ) blue ( intron ) can be found in Tables EV5 EV6 , respectively .

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Volcano plot representation of label-free quantitative proteomic analysis of P. falciparum proteins present in intron- (left) and promoter-targeted (right) dCas9 immunoprecipitations. Each dot represents a protein, and its size corresponds to the sum of peptides from both conditions used to quantify the ratio of enrichment. For the main volcano plot, x-axis = log2(fold-change), y-axis = -log10(p-value), horizontal yellow line indicates adjusted p-value = 0.05, and vertical yellow lines indicate absolute fold-change = 1.5. Side panels indicate proteins uniquely identified in either sample (y-axis = number of peptides per 100 amino acids). dCas9 protein is highlighted in green for reference. Two replicates were used for the promoter-targeted dCas9 and four replicates were used for the intron-targeted dCas9. Fold enrichment and adjusted p-values for each protein highlighted in red (promoter) and blue (intron) can be found in Tables EV5 and EV6, respectively.


Correlation plot of label - free quantitative proteomic of P . falciparum enriched in promoter - targeted ( top right quadrant in red ) versus non - targeted control ( top left quadrant in grey ) or intron - ( bottom right quadrant in blue ) dCas9 immunoprecipitations . Each dot represents a , its corresponds to sum of peptides from both conditions used to quantify of enrichment . x - axis = log2 [ fold - change ( promoter / non - targeted control ) ] y - axis = log2 [ fold - change ( promoter / intron ) ] . dCas9 is highlighted in green for reference . Two replicates were used for promoter - targeted dCas9 four replicates were used for intron - targeted non - targeted dCas9 . Fold enrichment adjusted p - values can be found for each highlighted in red ( promoter ) in Tables EV5 ( intron comparison ) EV7 ( control comparison ) for each highlighted in blue ( intron ) in Tables EV6 ( promoter comparison ) EV8 ( control comparison ) .

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Correlation plot of label-free quantitative proteomic analysis of P. falciparum proteins enriched in promoter-targeted (top right quadrant in red) versus non-targeted control (top left quadrant in grey) or intron- (bottom right quadrant in blue) dCas9 immunoprecipitations. Each dot represents a protein, and its size corresponds to the sum of peptides from both conditions used to quantify the ratio of enrichment. The x-axis = log2[fold-change(promoter/non-targeted control)] and y-axis = log2[fold-change(promoter/intron)]. dCas9 protein is highlighted in green for reference. Two replicates were used for the promoter-targeted dCas9 and four replicates were used for the intron-targeted and non-targeted dCas9. Fold enrichment and adjusted p-values can be found for each protein highlighted in red (promoter) in Tables EV5 (intron comparison) and EV7 (control comparison) and for each protein highlighted in blue (intron) in Tables EV6 (promoter comparison) and EV8 (control comparison).


Western blot of ( Cty ) extracts from a bulk population of ISWI - 3HA - ribo in absence ( - ) or presence ( + ) of glucosamine ( GlcN ) . ISWI - 3HA is detected with an anti - HA antibody . Antibodies against aldolase ( Ald . ) histone H3 are controls for extracts , respectively . Molecular weights are shown to right .

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Western blot analysis of cytoplasmic (Cty) and nuclear extracts from a bulk population of ISWI-3HA-ribo parasites in the absence (-) or presence (+) of glucosamine (GlcN). ISWI-3HA is detected with an anti-HA antibody. Antibodies against aldolase (Ald.) and histone H3 are controls for the cytoplasmic and nuclear extracts, respectively. Molecular weights are shown to the right.


MA plot of log2 [ glucosamine - treated / untreated , M ] plotted over mean abundance of each gene ( A ) at 12 hpi . Transcripts with a significantly higher ( above x - axis ) or lower ( below x - axis ) abundance in presence of glucosamine are highlighted in red ( q ≤ 0 . 05 ) . iswi is highlighted in blue ( q = 6 . 71x10 - 10 ) active var gene is highlighted in green ( q = 2 . 53x10 - 5 ) . Two three replicates were used for untreated glucosamine - treated , respectively . p - values were calculated with a Wald for significance of coefficients in a negative binomial generalized linear model as implemented in DESeq2 ( Love et al , 2014 ) . q = Bonferroni corrected p - value .

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MA plot of log2[glucosamine-treated/untreated, M] plotted over the mean abundance of each gene (A) at 12 hpi. Transcripts with a significantly higher (above x-axis) or lower (below x-axis) abundance in the presence of glucosamine are highlighted in red (q ≤ 0.05). iswi is highlighted in blue (q = 6.71x10-10) and the active var gene is highlighted in green (q = 2.53x10-5). Two and three replicates were used for untreated and glucosamine-treated parasites, respectively. p-values were calculated with a Wald test for significance of coefficients in a negative binomial generalized linear model as implemented in DESeq2 (Love et al, 2014). q = Bonferroni corrected p-value.


Frequency plot showing time in IDC of peak transcript level [ comparison to microarray time course in ( Bozdech et al , 2003 ; Data ref : Bozdech et al , 2003 ) ] for genes that are significantly down - regulated ( blue ) or up - regulated ( yellow ) following ISWI knockdown .

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Frequency plot showing the time in the IDC of peak transcript level [comparison to microarray time course in (Bozdech et al, 2003; Data ref: Bozdech et al, 2003)] for genes that are significantly down-regulated (blue) or up-regulated (yellow) following ISWI knockdown.


Meta - gene plot showing average ISWI enrichment ( y - axis = ChIP / Input ) in clonal ISWI - 3HA at 12 hpi from 1 . 5 kb upstream of translation start site ( " Start " ) to 1 . 5 kb downstream of translation stop site ( " Stop " ) for genes that are down - ( blue ) or up - regulated ( yellow ) upon ISWI knockdown . One replicate was used for ISWI ChIP - seq .

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Meta-gene plot showing average ISWI enrichment (y-axis = ChIP/Input) in clonal ISWI-3HA parasites at 12 hpi from 1.5 kb upstream of the translation start site ("Start") to 1.5 kb downstream of the translation stop site ("Stop") for genes that are down- (blue) or up-regulated (yellow) upon ISWI knockdown. One replicate was used for the ISWI ChIP-seq.


ChIP - seq data show enrichment of ISWI ( blue ) in clonal ISWI - 3HA at 12 hpi relative to regions of accessible ( " ATAC , " red ) . accessibility ( ATAC - seq ) data were taken from 15 hpi time point in ( Toenhake et al , 2018 ; Data ref : Toenhake et al , 2018 ) . Genome location is indicated at top of panel . x - axis is , with genes represented by black boxes indented to delineate introns labeled with arrowheads to indicate transcription direction . y - axis is enrichment ( ChIP / Input or ATAC - seq / genomic ) . One replicate was used for ISWI ChIP - seq .

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ChIP-seq data show enrichment of ISWI (blue) in clonal ISWI-3HA parasites at 12 hpi relative to regions of accessible chromatin ("ATAC," red). Chromatin accessibility (ATAC-seq) data were taken from the 15 hpi time point in (Toenhake et al, 2018; Data ref: Toenhake et al, 2018). Genome location is indicated at the top of the panel. The x-axis is DNA sequence, with genes represented by black boxes indented to delineate introns and labeled with white arrowheads to indicate transcription direction. The y-axis is enrichment (ChIP/Input or ATAC-seq/genomic DNA). One replicate was used for the ISWI ChIP-seq.


Meta - gene plot showing average ISWI enrichment ( y - axis = ChIP / Input ) in clonal ISWI - 3HA at 12 hpi from 1 . 5 kb upstream of translation start site ( " Start " ) to 1 . 5 kb downstream of translation stop site ( " Stop " ) for genes , which are grouped into quartiles on their transcript ( RPKM ) at 12 hpi . red represents genes with highest transcript ( " 1st " ) , red represents genes with second highest transcript ( " 2nd " ) , orange represents genes with third highest transcript ( " 3rd " ) , yellow represents genes with lowest transcript ( " 4th " ) . One replicate was used for ISWI ChIP - seq transcription data are an average from two replicates of untreated ISWI - 3HA clone used for differential expression .

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Meta-gene plot showing average ISWI enrichment (y-axis = ChIP/Input) in clonal ISWI-3HA parasites at 12 hpi from 1.5 kb upstream of the translation start site ("Start") to 1.5 kb downstream of the translation stop site ("Stop") for all genes, which are grouped into quartiles based on their transcript levels (RPKM) at 12 hpi. Dark red represents genes with the highest transcript levels ("1st"), light red represents genes with the second highest transcript levels ("2nd"), orange represents genes with the third highest transcript levels ("3rd"), and yellow represents genes with the lowest transcript levels ("4th"). One replicate was used for the ISWI ChIP-seq and the transcription data are an average from the two replicates of the untreated ISWI-3HA clone used for the differential expression analysis.


ChIP - seq data show enrichment of ISWI ( blue ) H3K9ac ( green ) in clonal ISWI - 3HA at 12 hpi at active var gene ( PF3D7 _ 1240600 ) . - seq data from this clone at 12 hpi show transcript for this gene ( grey ) . Genome location is indicated at top of panel . x - axis is , with genes represented by black boxes indented to delineate introns labeled with arrowheads to indicate transcription direction . y - axis is ChIP / Input for ChIP data RPKM for - seq data . One replicate was used for each ChIP - seq - seq data is from a single replicate from untreated ISWI - 3HA clone used for differential expression .

O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

ChIP-seq data show enrichment of ISWI (blue) and H3K9ac (green) in clonal ISWI-3HA parasites at 12 hpi at the active var gene (PF3D7_1240600). RNA-seq data from this clone at 12 hpi show transcript levels for this gene (grey). Genome location is indicated at the top of the panel. The x-axis is DNA sequence, with genes represented by black boxes indented to delineate introns and labeled with white arrowheads to indicate transcription direction. The y-axis is ChIP/Input for ChIP data and RPKM for RNA-seq data. One replicate was used for each ChIP-seq and the RNA-seq data is from a single replicate from the untreated ISWI-3HA clone used for the differential expression analysis.


Meta - gene plot showing average ISWI enrichment ( y - axis = ChIP / Input ) in clonal ISWI - 3HA at 12 hpi from 1 . 5 kb upstream of translation start site ( " Start " ) to 1 . 5 kb downstream of translation stop site ( " Stop " ) for active var gene ( PF3D7 _ 1240600 , blue ) or silent var genes ( yellow ) . One replicate was used for ISWI ChIP - seq .

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Meta-gene plot showing average ISWI enrichment (y-axis = ChIP/Input) in clonal ISWI-3HA parasites at 12 hpi from 1.5 kb upstream of the translation start site ("Start") to 1.5 kb downstream of the translation stop site ("Stop") for the active var gene (PF3D7_1240600, blue) or silent var genes (yellow). One replicate was used for the ISWI ChIP-seq.


( B ) Anti - GA response in vaccinated mice measured by ELISA using GST - ( GA ) 15 antigen . Tukey - style box plot shows 25th , 50th 75th percentile whiskers extend to ± 1 . 5 interquartile range . Outliers depicted as dots . Two - way repeated - measure ANOVA ( group x time ) revealed a significant main effect of treatment ( F ( 5 , 57 ) = 478 . 2 , P < 0 . 0001 ) time ( F ( 6 , 342 ) = 296 . 1 , P < 0 . 0001 ) a significant interaction between factors ( F ( 30 , 342 ) = 119 . 5 , P < 0 . 0001 ) , followed by Tukey ' s post hoc .

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(B) Anti-GA response in vaccinated mice measured by ELISA using GST-(GA)15 antigen. Tukey-style box plot shows 25th, 50th and 75th percentile and whiskers extend to ±1.5 interquartile range. Outliers depicted as dots. Two-way repeated-measure ANOVA (group x time) revealed a significant main effect of treatment (F(5, 57) = 478.2, P <0.0001) and time (F(6,342) = 296.1, P <0.0001) and a significant interaction between factors (F(30,342) = 119.5, P <0.0001), followed by Tukey's post hoc test.


( C ) Immunohistochemistry of sections from a C9orf72 a healthy control . Monoclonal anti - GA antiserum from Ova - GA vaccinated mice detect neuronal inclusions ( arrows ) in molecular layer of cerebellum . Note that crude antiserum shows higher background than monoclonal antibody . Scale bar indicates 20 μm . ( D ) Quantitative for GA - positive inclusions per 100 neurons in ( C ) . One - way ANOVA , Tukey ' s post hoc , F ( 3 , 15 ) = 2 . 676 , p = 0 . 0846 , C9 : Anti - GA 1A12 vs C9 : TG - Ova - ( GA ) 10 serum p = 0 . 9483 .

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(C) Immunohistochemistry of sections from a C9orf72 patient and a healthy control. Monoclonal anti-GA and antiserum from Ova-GA vaccinated mice detect neuronal cytoplasmic inclusions (arrows) in the molecular layer of the cerebellum. Note that crude antiserum shows higher background than the monoclonal antibody. Scale bar indicates 20 μm. (D) Quantitative analysis for GA-positive inclusions per 100 neurons in (C). One-way ANOVA, Tukey's post hoc test, F(3,15)=2.676, p=0.0846, C9: Anti-GA 1A12 vs C9: TG-Ova-(GA)10 serum p=0.9483.


( E ) Immunohistochemistry of sections from occipital cortex of a C9orf72 a healthy control with monoclonal anti - GA antiserum preincubated with 0 . 1 mg / ml recombinant GST - ( GA ) 15 or GST . Scale bar indicates 20 μm .

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(E) Immunohistochemistry of sections from occipital cortex of a C9orf72 patient and a healthy control with monoclonal anti-GA and antiserum preincubated with 0.1 mg/ml recombinant GST-(GA)15 or GST. Scale bar indicates 20 μm.


( A ) of motor function in vaccinated GA - CFP mice wild - type in a beam walk . Average time to cross beam from duplicate repeat measurements in consecutive . Pairwise Wilcoxon rank sum with Benjamini - Hochberg correction . comparisons with WT - PBS TG - Ova - ( GA ) 10 mice vs . TG - PBS comparison are depicted with * p < 0 . 05 , * * , p < 0 . 01 .

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(A) Analysis of motor function in vaccinated GA-CFP mice and wild-type littermates in a beam walk assay. Average time to cross the beam from duplicate repeat measurements in consecutive weeks. Pairwise Wilcoxon rank sum test with Benjamini-Hochberg correction. All comparisons with WT-PBS and the TG-Ova-(GA)10 mice vs. TG-PBS comparison are depicted with * p<0.05, **, p<0.01.


( B ) Average number of drop - downs per mouse from aggregated runs . Kruskal - Wallis with Dunn ' s multiple comparisons . * p < 0 . 05 . TG - Ova - ( GA ) 10 vs . TG - PBS p = 0 . 0188 .

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(B) Average number of drop-downs per mouse from all aggregated runs. Kruskal-Wallis test with Dunn's multiple comparisons. * p<0.05. TG-Ova-(GA)10 vs. TG-PBS p=0.0188.


( C ) Representative image of poly - GA in anterior horn in spinal cord using immunohistochemistry .

(C) Representative image of poly-GA aggregates in the anterior horn in spinal cord using immunohistochemistry.


( D ) Quantitative of poly - GA in spinal cord using immunohistochemistry . from complete spinal cord sections at 1 mm intervals . One - way ANOVA , Tukey ' s post hoc , F ( 5 , 12 ) = 1 . 625 , p = 0 . 2270 . * * * p < 0 . 001 , ns not significant . TG - Ova - ( GA ) 10 vs . TG - PBS p < 0 . 0001 ; TG - ( GA ) 15 vs . TG - PBS p = 0 . 2460 . Scale bar indicates 100 μm .

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(D) Quantitative analysis of poly-GA aggregates in the spinal cord using immunohistochemistry. Images from complete spinal cord sections at 1 mm intervals. One-way ANOVA, Tukey's post hoc test, F(5,12)=1.625, p=0.2270. *** p<0.001, ns not significant. TG-Ova-(GA)10 vs. TG-PBS p<0.0001; TG-(GA)15 vs. TG-PBS p=0.2460. Scale bar indicates 100 μm.


( E ) Immunoassay of poly - GA from insoluble fraction . One - way ANOVA , Tukey ' s post hoc , F ( 5 , 24 ) = 3 . 098 , p = 0 . 0269 . * * p < 0 . 001 , ns not significant . TG - Ova - ( GA ) 10 vs . TG - PBS p < 0 . 0001 ; TG - ( GA ) 15 vs . TG - PBS p = 0 . 1975 .

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(E) Immunoassay of poly-GA from the insoluble fraction. One-way ANOVA, Tukey's post hoc test, F(5,24)=3.098, p=0.0269. ** p<0.001, ns not significant. TG-Ova-(GA)10 vs. TG-PBS p<0.0001; TG-(GA)15 vs. TG-PBS p=0.1975.


( C , D ) of microglia / macrophage activation using Iba1 immunohistochemistry from complete spinal cord sections at 1 mm interval . One - way ANOVA , Tukey ' s post hoc . * * p < 0 . 01 , * p < 0 . 05 , ns not significant . Scale bar indicates 100 μm . F ( 5 , 12 ) = 0 . 6974 , p = 0 . 6357 ; TG - Ova - ( GA ) 10 vs . TG - PBS p = 0 . 0131 ; TG - ( GA ) 15 vs . TG - PBS p = 0 . 7650 . TG - PBS vs . WT - PBS p = 0 . 0018 .

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(C, D) Analysis of microglia/macrophage activation using Iba1 immunohistochemistry from complete spinal cord sections at 1 mm interval. One-way ANOVA, Tukey's post hoc test. ** p<0.01, * p<0.05, ns not significant. Scale bar indicates 100 μm. F(5,12)=0.6974, p=0.6357; TG-Ova-(GA)10 vs. TG-PBS p= 0.0131; TG-(GA)15 vs. TG-PBS p=0.7650. TG-PBS vs. WT-PBS p=0.0018.


( E ) of with partial mislocalization of TDP - 43 was quantified from six from spinal cord sections at 1 mm intervals were analyzed . One - way ANOVA , Tukey ' s post hoc . * p < 0 . 05 , ns not significant . F ( 5 , 12 ) = 0 . 6533 , p = 0 . 6650 ; TG - Ova - ( GA ) 10 vs . TG - PBS p = 0 . 0096 ; TG - ( GA ) 15 vs . TG - PBS p = 0 . 4215 .

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(E) The percentage of cells with partial cytoplasmic mislocalization of TDP-43 was quantified from six images from spinal cord sections at 1 mm intervals were analyzed. One-way ANOVA, Tukey's post hoc test. * p<0.05, ns not significant. F(5,12)=0.6533, p=0.6650; TG-Ova-(GA)10 vs. TG-PBS p= 0.0096; TG-(GA)15 vs. TG-PBS p=0.4215.


( F ) Immunoassay of NFL level in cerebrospinal fluid . One - way ANOVA , Tukey ' s post hoc , F ( 3 , 11 ) = 1 . 911 , p = 0 . 1862 . * * p < 0 . 01 . TG - Ova - ( GA ) 10 vs . TG - PBS p = 0 . 0081 .

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(F) Immunoassay of NFL level in cerebrospinal fluid. One-way ANOVA, Tukey's post hoc test, F(3,11)=1.911, p=0.1862. ** p<0.01. TG-Ova-(GA)10 vs. TG-PBS p=0.0081.


B , C measured by lactase dehydrogenase ( LDH ) ( B ) viability measured by 3 - ( 4 , 5 - dimethyl - thiazol - . 2 - yl ) - 2 , 5 - diphenyl tetrazolium bromide ( MTT ) ( C ) in T98 42 hrs post - transfection of pure interrupted SCA8 repeat tracts ; LDH n = 8 , MTT n = 12 , n = independent experiments , * p < 0 . 05 , NT not transfected , EV : empty vector ; unpaired t ; mean ± .

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B, C Cell death measured by lactase dehydrogenase (LDH) (B) and cell viability measured by 3-(4,5- dimethyl-thiazol-. 2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (C) in T98 cells 42 hrs post-transfection of pure and interrupted SCA8 repeat tracts; LDH n=8, MTT n=12, n=independent experiments, * p < 0.05, NT not transfected, EV: empty vector; unpaired t test; mean ± SEM.


D , E qRT - PCR showing transcript of Pure 96 Int . 95 ( D , n = 6 ; p = 0 . 0187 ) , Pure 104 Int . 102 ( E , n = 6 ; * * * * p < 0 . 0001 ) repeats ; n = independent experiments ; unpaired t ; mean ± .

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D, E qRT-PCR showing transcript levels of Pure 96 and Int.95 (D, n=6; p=0.0187), and Pure 104 and Int.102 (E, n=6; **** p<0.0001) repeats; n=independent experiments; unpaired t test; mean ± SEM.


B , C ( B ; LDH , n = 8 ) viability ( C ; MTT , n = 6 ) assays in T98 42 - hrs post - transfection . NT : not transfected ; EV : empty vector ; n = independent experiments ; * p < 0 . 05 ; unpaired t ; mean ± .

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B, C Cell death (B; LDH, n=8) and viability (C; MTT, n=6) assays in T98 cells 42-hrs post-transfection. NT: not transfected; EV: empty vector; n=independent experiments; * p < 0.05; unpaired t test; mean ± SEM.


blots of polyGln expressed in transfected HEK293T with interrupted or pure polyGln repeats ; EV : empty vector ; n = 3

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Protein blots of polyGln proteins expressed in transfected HEK293T cells with interrupted or pure polyGln repeats; EV: empty vector; n=3 transfections


Immunofluorescence of intranuclear inclusions for polyGln expressed from Alt . polyGln Alt . Int . polyGln constructs in HEK293T ; scale bar : 50μm ; green arrow heads indicate positive for Alt . Int . polyGln intranuclear inclusions ; dashed box in merge panels indicates region show in orthogonal ( Ortho ) zoomed which show cross section localization of diffuse polyGln polyGln ( Arg ) inclusions with alpha tubulin as a marker ; n = 4 experiments with n > 55 per

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Immunofluorescence of intranuclear inclusions for polyGln expressed from Alt. polyGln and Alt. Int. polyGln constructs in HEK293T cells; scale bar: 50μm; green arrow heads indicate cells positive for Alt. Int. polyGln intranuclear inclusions; dashed white box in merge panels indicates the region show in the orthogonal (Ortho) zoomed images which show cross section and localization of diffuse polyGln staining and nuclear polyGln(Arg) inclusions with alpha tubulin as a cytoplasmic marker; n=4 experiments with n>55 cells per experiment


K PolyGln ( Arg ) but not pure polyGln colocalize with nucleophosmin in HEK293T ; scale bars : 20µm ; dashed box in merge panels indicates region shown as an orthogonal projection in ortho zoom panels which show co - localization of polyGln inclusions nucleophosmin in three dimensions .

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K PolyGln(Arg) proteins but not pure polyGln proteins colocalize with nucleophosmin in HEK293T cells; scale bars: 20µm; dashed white box in merge panels indicates the region shown as an orthogonal projection in the ortho zoom panels which show the co-localization of polyGln inclusions and nucleophosmin in three dimensions.


A - C blotting ( A ) ( B , C ) of polySer RAN in HEK293T from interrupted ( Int . 95 ; Int . 102 ) or pure ( Pure 96 ; Pure 104 ) CAG repeats . EV : empty vector , n = 3 transfections , * * p < 0 . 01 , * p < 0 . 05 , unpaired t , mean ± .

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A-C Protein blotting (A) and quantification (B, C) of polySer RAN proteins in HEK293T cells from interrupted (Int.95; Int.102) or pure (Pure 96; Pure 104) CAG repeats. EV: empty vector, n=3 transfections, ** p<0.01, *p<0.05, unpaired t test, mean ± SEM.


D , Immunofluorescence of RAN polySer from CGG interrupted pure CAG repeat tracts in HEK293T ; scale bar : 10μm . n > 20 per construct for each experiment ( Exp . )

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D, Immunofluorescence of RAN polySer protein aggregates from CGG interrupted and pure CAG repeat tracts in HEK293T cells; scale bar: 10μm. n>20 cells per construct for each experiment (Exp.)


F , blot of polyAla RAN expressed from pure or interrupted constructs ; n = 3

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F, Protein blot of polyAla RAN proteins expressed from pure or interrupted constructs; n=3 transfections


I , J blotting ( I ) ( J ) of phospho eIF2α ( p - eIF2α ) relative to total eIF2α ; n = 4 transfections , * p < 0 . 05 , unpaired t , mean ± SD .

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I, J Protein blotting (I) and quantification (J) of phospho eIF2α (p-eIF2α) relative to total eIF2α; n=4 transfections, * p<0.05, unpaired t test, mean ± SD.


Representative Southern blots showing DSB repair products in WT , rad59 ∆ , rad52 - R70A rad59 ∆ rad52 - R70A . was digested with Bsp1286I probed with a MAT - distal ( yellow box in panel B ) .

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Representative Southern blots showing DSB repair products in WT, rad59∆, rad52-R70A and rad59∆ rad52-R70A cells. DNA was digested with Bsp1286I and probed with a MAT-distal sequence (yellow box in panel B).


Viability of indicated strains ( mean ± SD ; n = 3 ) . Data information : Welch ' s unpaired t - was used to determine p -

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Viability of indicated strains (mean ± SD; n = 3). Data information: Welch's unpaired t-test was used to determine the p-value


Representative Southern blots showing DSB repair products in WT , rad59 ∆ , rad52 - R70A . was digested with XhoI EcoRI probed with a Z ( yellow box ) .

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Representative Southern blots showing DSB repair products in WT, rad59∆, and rad52-R70A. DNA was digested with XhoI and EcoRI and probed with a Z sequence (yellow box).


of chromosome loss of indicated mutants . Data information : χ2 is used to determine p - value number of colonies tested per mutant is indicated in Appendix Table S1 .

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Percentage of chromosome loss of indicated mutants. Data information: χ2 test is used to determine the p-value and number of colonies tested per mutant is indicated in Appendix Table S1.


Schematic of mutation during allelic recombination ( left ) . Mutation reporter cassette is inserted within repair template 16 kb away from strand invasion site . Mutation rate ( right ) of WT , rad59 ∆ , rad52 - R70A was calculated as previously described ( Saini et . al . , 2013 ) . Data information : χ Mutation rates are reported as median value , statistical comparisons between median mutation rates were performed using Mann - Whitney U - .

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Schematic of mutation analysis during allelic recombination (left). Mutation reporter cassette is inserted within the repair template 16 kb away from strand invasion site. Mutation rate (right) of WT, rad59∆, rad52-R70A was calculated as previously described (Saini et. al., 2013). Data information: χ Mutation rates are reported as the median value, and statistical comparisons between median mutation rates were performed using the Mann-Whitney U-test.


Representative Southern blots showing DSB repair products of indicated mutants in regular mating type switching ( H - 1400 ) .

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Representative Southern blots showing DSB repair products of indicated mutants in regular mating type switching (H-1400 assay).


Viability of indicated mutants are shown . ( mean ± SD ; n = 3 ) . Data information : Welch ' s unpaired t - was used to determine p -

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

Viability of indicated mutants are shown. (mean ± SD; n = 3). Data information: Welch's unpaired t-test was used to determine the p-value


( A - C ) Representative Southern blots showing DSB repair products ( top ) , of BIR among repair products ( bottom left ) viability ( bottom right ) of WT sir2 ∆ in H - 150 ( A ) , H - - 150 no - gap ( B ) or native mating type switching H - 1400 ( C ) . ( mean ± SD ; n = 3 ) . Welch ' s unpaired t - was used to determine p - value in panels A - C .

O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C) Representative Southern blots showing DSB repair products (top), percentage of BIR among repair products (bottom left) and viability (bottom right) of WT and sir2∆ in the H-150 assay (A), H--150 no-gap assay (B) or native mating type switching H-1400 assay (C). (mean ± SD; n = 3). Welch's unpaired t-test was used to determine the p-value in panels A-C.


( A - B ) Representative Southern blots showing DSB repair products ( top ) , of BIR among repair products ( bottom left ) viability ( bottom right ) of indicated mutants in H - 150 no - gap ( A ) or H - 150 ( B ) . ( mean ± SD ; n ≥ 3 ) . Welch ' s unpaired t - was used to determine p - value in panels A B .

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(A-B) Representative Southern blots showing DSB repair products (top), percentage of BIR among repair products (bottom left) and viability (bottom right) of indicated mutants in the H-150 no-gap assay (A) or H-150 assay (B). (mean ± SD; n ≥3). Welch's unpaired t-test was used to determine the p-value in panels A and B.


Representative microscopy of total , CD142 - CD142 + ASPCs isolated with use of respective anti - CD142 antibodies : SinoBiological ( SB ) , SinoBiological - LL ( SB - LL ) , BiOrbyt ( BO ) R & D Systems ( RnD ) ( Appendix Fig . S2B , Appendix Fig . S5A , Methods ) , after in vitro adipogenic differentiation ;

O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative fluorescence microscopy images of total, CD142- and CD142+ ASPCs isolated with the use of the respective anti-CD142 antibodies: SinoBiological (SB), SinoBiological-LL (SB-LL), BiOrbyt (BO) and R&D Systems (RnD) (Appendix Fig. S2B, Appendix Fig. S5A, Methods), after in vitro adipogenic differentiation;


Fraction of differentiated per ASPC type shown in E , as quantified by " adiposcore " ; marker shapes correspond to different anti - CD142 antibodies used for isolation as indicated , n = 9 - 15 , 3 - 4 biological replicates , 3 - 5 independent wells for each ;

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Fraction of differentiated cells per ASPC type shown in E, as quantified by the "adiposcore"; marker shapes correspond to different anti-CD142 antibodies used for isolation as indicated, n=9-15, 3-4 biological replicates, 3-5 independent wells for each;


Representative microscopy of CD142 - , 20 % CD142 + ( 5 - 7 % ) CD142 + ASPCs isolated following sorting strategy shown in Appendix Fig . S7A , after in vitro adipogenic differentiation with indicated adipogenic differentiation cocktails ( Methods ) ;

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Representative fluorescence microscopy images of CD142-, 20% CD142+ and (5-7%) CD142+ ASPCs isolated following the sorting strategy shown in Appendix Fig. S7A, after in vitro adipogenic differentiation with the indicated white adipogenic differentiation cocktails (Methods);


Fraction of differentiated per ASPC type differentiation cocktail shown in J , as quantified by " adipo - score " ; marker shapes correspond to different biological replicates , n = 8 - 17 , 3 - 5 biological replicates , 2 - 5 independent wells for each ;

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Fraction of differentiated cells per ASPC type and differentiation cocktail shown in J, as quantified by the "adipo- score"; marker shapes correspond to different biological replicates, n=8-17, 3-5 biological replicates, 2-5 independent wells for each;


Representative microscopy of - - derived total , CD142 - CD142 + ASPCs , isolated following sorting strategy shown in Appendix Fig . S8A after in vitro adipogenic differentiation ; Fraction of differentiated per ASPC type per sex shown in L , as quantified by " adiposcore " ; bar colour shading corresponds to - - derived as indicated ; marker shapes correspond to different biological replicates , n = 9 - 10 , 4 biological replicates , 2 - 3 independent wells for each ;

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Representative fluorescence microscopy images of male- and female-derived total, CD142- and CD142+ ASPCs, isolated following the sorting strategy shown in Appendix Fig. S8A after in vitro adipogenic differentiation;   Fraction of differentiated cells per ASPC type and per sex shown in L, as quantified by the "adiposcore"; bar colour shading corresponds to male- and female-derived cells as indicated; marker shapes correspond to different biological replicates, n=9-10, 4 biological replicates, 2-3 independent wells for each;


Representative microscopy of P0 - , P12 - 17 - , 4wo - , 7wo - 11wo - derived total , CD142 - CD142 + APSCs after in vitro adipogenic differentiation ; Fraction of differentiated per ASPC type shown in D , as quantified by " adiposcore " ; bar colour shading corresponds to pre - post - weaning - derived as indicated ; marker shapes correspond to different biological replicates , n = 14 - 65 , 12 - 13 biological replicates , 2 - 6 independent wells for each

O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative fluorescence microscopy images of P0-, P12-17-, 4wo-, 7wo- and 11wo-derived total, CD142- and CD142+ APSCs after in vitro adipogenic differentiation;   Fraction of differentiated cells per ASPC type shown in D, as quantified by the "adiposcore"; bar colour shading corresponds to pre- and post-weaning-derived cells as indicated; marker shapes correspond to different biological replicates, n=14-65, 12-13 biological replicates, 2-6 independent wells for each


Representative microscopy of CD142 - ASPCs co - cultured with an empty transwell ( T - well ) or with wild - type ( WT ) CD142 + ASPCs after in vitro adipogenic differentiation ; Fraction of differentiated CD142 - ASPCs co - cultured with an empty transwell ( T - well ) or with wild - type CD142 + ASPCs ( non - treated or treated with control scrambled siRNA ) after in vitro adipogenic differentiation as quantified by " adiposcore " ; marker shapes correspond to different biological replicates , n = 10 , 4 biological replicates , 2 - 3 independent wells for each

O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative fluorescence microscopy images of CD142- ASPCs co-cultured with an empty transwell (T-well) or with wild-type (WT) CD142+ ASPCs after in vitro adipogenic differentiation;   Fraction of differentiated CD142- ASPCs co-cultured with an empty transwell (T-well) or with wild-type CD142+ ASPCs (non-treated or treated with control scrambled siRNA) after in vitro adipogenic differentiation and as quantified by the "adiposcore"; marker shapes correspond to different biological replicates, n=10, 4 biological replicates, 2-3 independent wells for each


Representative microscopy imaged of human - derived stromal vascular fraction ( SVF ) , containing ASPCs , after in vitro adipogenic differentiation when co - cultured with total , CD142 - or CD142 + mouse ASPCs ; Fraction of differentiated human - derived SVF , containing ASPCs co - cultured with total , CD142 - or CD142 + mouse ASPCs after in vitro adipogenic differentiation ( Methods ) shown in E ; marker shapes correspond to different biological replicates , n = 9 , 3 biological replicates , 3 independent wells for each

O O O O O 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-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative fluorescence microscopy imaged of human-derived stromal vascular fraction (SVF), containing the ASPCs, after in vitro adipogenic differentiation when co-cultured with total, CD142- or CD142+ mouse ASPCs;   Fraction of differentiated human-derived SVF, containing the ASPCs co-cultured with total, CD142- or CD142+ mouse ASPCs after in vitro adipogenic differentiation (Methods) shown in E; marker shapes correspond to different biological replicates, n=9, 3 biological replicates, 3 independent wells for each


Fraction of differentiated adult - derived CD142 - ASPCs , as quantified by " adiposcore " , treated with indicated recombinant shown in J ; marker shapes correspond to different biological replicates , n = 11 - 59 , 2 - 10 biological replicates , 3 - 9 independent wells for

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Fraction of differentiated adult-derived CD142- ASPCs, as quantified by the "adiposcore", treated with the indicated recombinant proteins shown in J; marker shapes correspond to different biological replicates, n=11-59, 2-10 biological replicates, 3-9 independent wells for each


Representative microscopy of adult - derived CD142 - ASPCs after in vitro adipogenic differentiation ; induction cocktail was supplemented with recombinant corresponding to selected Areg - specific candidates : CD142 , MGP , GDF10 , CLEC11A , CPE BGN at 100 ng /

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Representative fluorescence microscopy images of adult-derived CD142- ASPCs after in vitro adipogenic differentiation; the induction cocktail was supplemented with recombinant proteins corresponding to the selected Areg-specific candidates: CD142, MGP, GDF10, CLEC11A, CPE and BGN at 100 ng/m


Representative microscopy of CD142 - ASPCs , co - cultured with CD142 + ASPCs ( Aregs ) carrying control ( scr ) or siRNA - mediated knockdowns of selected CD142 + ASPC ( Areg ) - specific candidate genes : F3 , Mgp , Gdf10 after adipogenic differentiation ( i . e . , after exposure to an adipogenic cocktail , Methods )

O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O

Representative fluorescence microscopy images of CD142- ASPCs, co-cultured with CD142+ ASPCs (Aregs) carrying control (scr) or siRNA-mediated knockdowns of selected CD142+ ASPC (Areg)-specific candidate genes: F3, Mgp, and Gdf10 after adipogenic differentiation (i.e., after exposure to an adipogenic cocktail, Methods)


Fraction of differentiated CD142 - ASPCs , as quantified by " adiposcore " , co - cultured with CD142 + ASPCs ( Aregs ) subjected to specific knockdowns ( or scrambled ( scr ) siRNA control ) , as indicated in A ; marker shapes correspond to different biological replicates , n = 10 , 4 biological replicates , 2 - 3 independent wells for each ;

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Fraction of differentiated CD142- ASPCs, as quantified by the "adiposcore", co-cultured with CD142+ ASPCs (Aregs) subjected to specific knockdowns (or scrambled (scr) siRNA control), as indicated in A; marker shapes correspond to different biological replicates, n=10, 4 biological replicates, 2-3 independent wells for each;


Representative microscopy of CD142 - ASPCs , co - cultured with CD142 + ASPCs ( Aregs ) ( in top well ) subjected to indicated compounds at indicated ; AGN BMS are antagonists of RAR , DEAB is an antagonist of RALDH enzymes which convert retinaldehyde to RA , HX is an antagonist of RXR ; Fraction of differentiated CD142 - ASPCs indicated in G , as quantified by " adiposcore " ; marker shapes correspond to different biological replicates , n = 4 - 15 , 2 - 5 biological replicates , 2 - 3 independent wells for each ;

O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative fluorescence microscopy images of CD142- ASPCs, co-cultured with CD142+ ASPCs (Aregs) (in the top well) subjected to the indicated compounds at the indicated concentration; AGN and BMS are antagonists of RAR, DEAB is an antagonist of RALDH enzymes which convert retinaldehyde to RA, and HX is an antagonist of RXR;   Fraction of differentiated CD142- ASPCs indicated in G, as quantified by the "adiposcore"; marker shapes correspond to different biological replicates, n=4-15, 2-5 biological replicates, 2-3 independent wells for each;


Representative microscopy of CD142 - ASPCs , co - cultured with CD142 + ASPCs ( Aregs ) subjected to control ( scr ) or siRNA - mediated knockdowns of selected RA signalling pathway genes : Rara , Rarb , Rarg , Rbp1 , Rxrg after adipogenic differentiation ( i . e . , after exposure to an adipogenic cocktail , Methods ) ; Fraction of differentiated CD142 - ASPCs indicated in I , as quantified by " adiposcore " ; marker shapes correspond to different biological replicates , n = 24 - 29 , 4 biological replicates , 5 - 8 independent wells for each .

O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative fluorescence microscopy images of CD142- ASPCs, co-cultured with CD142+ ASPCs (Aregs) subjected to control (scr) or siRNA-mediated knockdowns of selected RA signalling pathway genes: Rara, Rarb, Rarg, Rbp1, and Rxrg after adipogenic differentiation (i.e., after exposure to an adipogenic cocktail, Methods);   Fraction of differentiated CD142- ASPCs indicated in I, as quantified by the "adiposcore"; marker shapes correspond to different biological replicates, n=24-29, 4 biological replicates, 5-8 independent wells for each.


A Representative image of - bearing mice tumors after subcutaneous injection of Hepa1 - 6 hepatoma into WT Tet2 - / - ( KO ) mice .

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A Representative image of tumor-bearing mice and tumors after subcutaneous injection of Hepa1-6 hepatoma cells into WT and Tet2-/- (KO) mice.


B Mean volumes in WT Tet2 - / - mice ( n = 6 ) .

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B Mean tumor volumes in WT and Tet2-/- mice (n=6).


C weights 18 days after injection into WT KO mice ( n = 6 ) .

C Tumor weights 18 days after tumor cell injection into WT and KO mice (n=6).


D Kaplan - Meier survival curves of - bearing - free WT KO mice ( n = 15 ) .

D Kaplan-Meier survival curves of tumor-bearing and tumor-free WT and KO mice (n=15).


E Representative of lungs with metastasis from WT KO mice injected with Hepa1 - 6 via tail vein , as well as of number of lung metastasis ( n = 4 ) .

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E Representative images of lungs with metastasis from WT and KO mice injected with Hepa1-6 cells via tail vein, as well as quantification of the number of lung metastasis (n=4).


F Representative of tumors from WT KO mice subcutaneously injected with Py8119 breast cancer , as well as of weights ( n = 5 ) .

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F Representative images of tumors from WT and KO mice subcutaneously injected with Py8119 breast cancer cells, as well as quantification of tumor weights (n=5).


A , B Immunohistochemistry of CD8a , CD4 ( A ) , PCNA , Foxp3 ( B ) in tumors from WT Tet2 - / - ( KO ) mice subcutaneously injected with Hepa1 - 6 hepatoma . Corresponding bar charts display positive , quantified using Image Pro Plus 6 . 0 software ( n = 3 ) . Pictures were captured at 400X magnification . Scale bar = 50 μm .

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A, B Immunohistochemistry analysis of CD8a, CD4 (A), PCNA, and Foxp3 (B) in tumors from WT and Tet2-/- (KO) mice subcutaneously injected with Hepa1-6 hepatoma cells. Corresponding bar charts display positive staining, quantified using Image Pro Plus 6.0 software (n=3). Pictures were captured at 400X magnification. Scale bar = 50 μm.


D growth curves in WT KO mice treated with PBS or with anti - PD - 1 Ab ( n = 3 ) .

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D Tumor growth curves in WT and KO mice treated with PBS or with anti-PD-1 Ab (n =3).


E Representatives of day - 19 tumors from WT KO from panel D .

E Representatives images of day-19 tumors from WT and KO from panel D.


F Tumor weights from WT and KO mice at day 19.


G Flow cytometry of splenic CD8 + T in WT KO mice with or without anti - PD - 1 treatment after challenge .

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G Flow cytometry analysis of splenic CD8+ T cells in WT and KO mice with or without anti-PD-1 treatment after tumor challenge.


H - K GSEA showing significant down - regulation of genes in GO biological processes : chemokine signaling pathway ( H ) , T receptor signaling pathway ( I ) , innate immune response ( J ) cytokine receptor interaction ( K ) . x - axis represents that mouse genes are ranked from most up - regulated ( left end ) to most down - regulated ( right end ) in comparison of syngeneic from Hepa1 - 6 - bearing KO to WT mice ( n = 4 ) . y - axis is an enrichment score ( ES ) , which goes up ( > 0 ) down ( < 0 ) representing enrichment of up - regulated genes down - regulated genes , respectively . NES is normalized enrichment score . p - value was calculated by permutation .

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H-K GSEA analysis showing the significant down-regulation of genes in GO biological processes: chemokine signaling pathway (H), T cell receptor signaling pathway (I), innate immune response (J) and cytokine receptor interaction (K). The x-axis represents that the mouse genes are ranked from the most up-regulated (left end) to the most down-regulated (right end) in comparison of syngeneic tumor from Hepa1-6 tumor-bearing KO to WT mice (n=4). The y-axis is an enrichment score (ES), which goes up (>0) and down (<0) representing the enrichment of up-regulated genes and down-regulated genes, respectively. NES is the normalized enrichment score. p-value was calculated by permutation.