E ) IP assays of FTSJ1 - Asn221DIZPK . The empty vector was transfected as a negative control . ( F ) A list of top FTSJ1 - Asn221DIZPK putative interacting proteins identified by IP - MS in HEK293T cells .

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E) IP assays of FTSJ1-Asn221DIZPK. The empty vector was transfected as a negative control. (F) A list of top FTSJ1-Asn221DIZPK putative interacting proteins identified by IP-MS in HEK293T cells.


Mass chromatograms of the nucleosides , Cm ( Q1 / Q3 = 258 . 1 / 112 . 1 ) , Gm ( Q1 / Q3 = 298 . 1 / 152 . 1 ) and A ( Q1 / Q3 = 268 . 1 / 136 . 2 ) of tRNAPhe ( GAA ) isolated from WT , ftsj1 KO and wdr6 KO cells . Target peaks are indicated by black triangles . Cm , 2 ' - O - methylcytidine ; Gm , 2 ' - O - methylguanosine ; Q1 / Q3 : the mass of the precursor ion and the mass of the product ion .

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Mass chromatograms of the nucleosides, Cm (Q1/Q3=258.1/112.1), Gm (Q1/Q3=298.1/152.1) and A (Q1/Q3=268.1/136.2) of tRNAPhe(GAA) isolated from WT, ftsj1 KO and wdr6 KO cells. Target peaks are indicated by black triangles. Cm, 2'-O-methylcytidine; Gm, 2'-O-methylguanosine; Q1/Q3: the mass of the precursor ion and the mass of the product ion.


Mass chromatograms of the nucleosides m1G ( Q1 / Q3 = 298 . 1 / 166 . 1 ) , m7G ( Q1 / Q3 = 298 . 1 / 166 . 1 ) and m2G ( Q1 / Q3 = 298 . 1 / 166 . 1 ) , o2yW ( Q1 / Q3 = 541 . 2 / 409 . 0 ) , m5C ( Q1 / Q3 = 258 . 1 / 126 . 1 ) and A ( Q1 / Q3 = 268 . 1 / 136 . 2 ) of tRNAPhe ( GAA ) isolated from WT , ftsj1 KO and wdr6 KO cells . Target peaks are indicated by black triangles . m2G , 2 - methylguanosine ; D , dihydrouridine ; m22G , N2 , N2 - dimethylguanosine ; m5C , 5 - methylcytidine ; m7G , 7 - methylguanosine ; m5U , 5 - methyluridine ; m1A : 1 - methyladenosine ; ψ : pseudouridine ; o2yW , peroxywybutosine . Q1 / Q3 : the mass of the precursor ion and the mass of the product ion . Combined the retention time of standard product , we marked the o2yW in Figure 3G . Considering the change of this peak area was consistent with that of o2yW , we speculated the other peak was generated by the intermediate product of o2yW with nature isotope labelled .

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Mass chromatograms of the nucleosides m1G (Q1/Q3=298.1/166.1), m7G (Q1/Q3=298.1/166.1) and m2G (Q1/Q3=298.1/166.1), o2yW (Q1/Q3=541.2/409.0), m5C (Q1/Q3=258.1/126.1) and A (Q1/Q3=268.1/136.2) of tRNAPhe(GAA) isolated from WT, ftsj1 KO and wdr6 KO cells. Target peaks are indicated by black triangles. m2G, 2-methylguanosine; D, dihydrouridine; m22G, N2,N2-dimethylguanosine; m5C, 5-methylcytidine; m7G, 7-methylguanosine; m5U, 5-methyluridine; m1A: 1-methyladenosine; ψ: pseudouridine; o2yW, peroxywybutosine. Q1/Q3: the mass of the precursor ion and the mass of the product ion. Combined the retention time of standard product, we marked the o2yW in Figure 3G. Considering the change of this peak area was consistent with that of o2yW, we speculated the other peak was generated by the intermediate product of o2yW with nature isotope labelled.


( B , C ) Quantification of the Cm / A and Gm / A of tRNAPhe ( GAA ) , △ ftsj1 _ tRNAPhe ( GAA ) , and m1G37 _ tRNAPhe ( GAA ) after incubation with FTSJ1 , WDR6 , and FTSJ1 - WDR6 by UPLC - MS / MS analysis , respectively . Error bars represent the standard deviation of three independent experiments . n . d . , not detected .

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(B, C) Quantification of the Cm/A and Gm/A of tRNAPhe(GAA), △ftsj1_tRNAPhe(GAA), and m1G37_tRNAPhe(GAA) after incubation with FTSJ1, WDR6, and FTSJ1-WDR6 by UPLC-MS/MS analysis, respectively. Error bars represent the standard deviation of three independent experiments. n.d., not detected.


( D ) UPLC - MS / MS analysis of 2 ' - O - methylation of m1G37 _ tRNAPhe ( GAA ) , - G34A , - G34C and G34U after incubation with FTSJ1 - WDR6 . 2 μL of Gm ( 1 ng / mL ) , Am ( 1 ng / mL ) , Cm ( 1 ng / mL ) and Um ( 5 ng / mL ) standards were injected to UPLC - MS / MS as control . cps , counts per second .

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(D) UPLC-MS/MS analysis of 2'-O-methylation of m1G37_tRNAPhe(GAA), -G34A, -G34C and G34U after incubation with FTSJ1-WDR6. 2 μL of Gm (1 ng/mL), Am (1 ng/mL), Cm (1 ng/mL) and Um (5 ng/mL) standards were injected to UPLC-MS/MS as control. cps, counts per second.


A ) The SAM binding affinity of FTSJ1 as measured by ITC .

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A) The SAM binding affinity of FTSJ1 as measured by ITC.


( B The binding affinity of FTSJ1 alone or FTSJ1 - WDR6 for tRNA analyzed by the gel mobility shift assay .

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(B The binding affinity of FTSJ1 alone or FTSJ1-WDR6 for tRNA analyzed by the gel mobility shift assay.


C ) For the reaction in figure C , 0 . 2 μΜ GST , 0 . 2 μΜ GST - WDR6 , 1 μM FTSJ1 , the mixture of 1 μM FTSJ1 with 0 . 2 μΜ GST - WDR6 or 0 . 2 μΜ GST , 0 . 5 mM SAM and 250 nM tRNAPhe ( GAA ) transcript were incubated .

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C) For the reaction in figure C, 0.2 μΜ GST, 0.2 μΜ GST-WDR6, 1 μM FTSJ1, the mixture of 1 μM FTSJ1 with 0.2 μΜ GST-WDR6 or 0.2 μΜ GST, 0.5 mM SAM and 250 nM tRNAPhe(GAA) transcript were incubated.


The representative graphs for quantification of the expected Nm32 / A % and Nm34 / A % ratios of G37 - tRNAPhe ( GAA ) , tRNALeu ( UAA ) , tRNATrp ( CCA ) , tRNAArg ( ACG ) , tRNAArg ( UCG ) , and A37 - tRNAMet ( CAU ) , which were isolated from WT , ftsj1 KO , and wdr6 KO cells by UPLC - MS / MS analysis . Error bars represent the standard deviation of three independent experiments . p values were determined using two - tailed Student ' s t - test for paired samples . * * * p < 0 . 001 . n . s . , no significance . n . d . , not detected .

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

The representative graphs for quantification of the expected Nm32/A% and Nm34/A% ratios of G37-tRNAPhe(GAA), tRNALeu(UAA), tRNATrp(CCA), tRNAArg(ACG), tRNAArg(UCG), and A37-tRNAMet(CAU), which were isolated from WT, ftsj1 KO, and wdr6 KO cells by UPLC-MS/MS analysis. Error bars represent the standard deviation of three independent experiments. p values were determined using two-tailed Student's t-test for paired samples. ***p < 0.001. n.s., no significance. n.d., not detected.


( A , B ) The growth curve of WT and two ftsj1 KO HEK293T cell lines under normal culture or in the culture condition with 0 . 2 mg / mL paromomycin assayed by cell counting kit - 8 proliferation analysis .

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(A, B) The growth curve of WT and two ftsj1 KO HEK293T cell lines under normal culture or in the culture condition with 0.2 mg/mL paromomycin assayed by cell counting kit-8 proliferation analysis.


( C , D ) The growth curve of WT with or without overexpressing tRNAPhe ( GAA ) under normal culture or with 0 . 2 mg / mL paromomycin .

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(C, D) The growth curve of WT with or without overexpressing tRNAPhe(GAA) under normal culture or with 0.2 mg/mL paromomycin.


( E , F ) The growth curve of ftsj1 KO cells with or without overexpressing tRNAPhe ( GAA ) under normal culture or with 0 . 2 mg / mL paromomycin .

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(E, F) The growth curve of ftsj1 KO cells with or without overexpressing tRNAPhe(GAA) under normal culture or with 0.2 mg/mL paromomycin.


( H , I ) The translation efficiency of F - luc with the 6 × UUC codons or 6 × UUU codons .

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(H, I) The translation efficiency of F-luc with the 6×UUC codons or 6×UUU codons.


( J , K ) The translation efficiency of F - luc with all UUC codons or all UUU codons .

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(J, K) The translation efficiency of F-luc with all UUC codons or all UUU codons.


Co - immunoprecipitation assays in HEK293T cells after co - transfection with Flag - TRIM27 and the indicated combinations of empty HA - vector , HA - tagged FIP200 , ULK1 , or ATG13 before immunoprecipitation with anti - HA antibodies . Immunoprecipitates ( top panel ) and input samples ( bottom panel ) were then subjected to Western blotting against anti - HA or Flag . Data information : the experiments were performed at least three times independently with similar results .

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

Co-immunoprecipitation assays in HEK293T cells after co-transfection with Flag-TRIM27 and the indicated combinations of empty HA-vector, HA-tagged FIP200, ULK1, or ATG13 before immunoprecipitation with anti-HA antibodies. Immunoprecipitates (top panel) and input samples (bottom panel) were then subjected to Western blotting against anti-HA or Flag. Data information: the experiments were performed at least three times independently with similar results.


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 .

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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 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 .

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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 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 .

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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 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 .

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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 and bound ULK1 analyzed by Western blotting . Data information : the 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 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 .

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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 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 .

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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 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 .

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

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 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 .

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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 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 .

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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 . 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 .

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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 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 .

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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 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 .

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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 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 .

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

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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 cells were reconstituted with Flag - TRIM27 and 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 cells 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 analysis of indicated proteins 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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 cells , the cells were treated with starvation , the 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 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 .

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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 cells .

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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 cells with or without starvation treatment . Flag - STK38L was immunoprecipitated using anti - Flag , the immunoprecipitates and 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 cells with or without STK38L knockout . The cells 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 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 .

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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 analysis of HA - ULK1 in HEK293T cells co - expressing Flag - TRIM27 and 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 analysis of ULK1 levels in HeLa cells with or without STK38L knockout , the cells were treated with starvation for the 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 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 .

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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 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 .

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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 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 .

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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 cells . HA - ULK1 or HA - ULK1 S495A was immunoprecipitated using anti - HA , the immunoprecipitates and 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 analysis of HA - ULK1 WT or HA - ULK1 S495A in HEK293T cells co - expressing Flag - TRIM27 and 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 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 ) .

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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 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 ) .

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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 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 ) .

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

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


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 .

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


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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 ) .

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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 . Analysis of the correlation between ULK1 and TRIM27 expression in the 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 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 ) .

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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 the 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 analysis of indicated proteins in Trim27 - / - PyMT or Trim27 + / - PyMT mice in primary 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 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 ) .

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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 and 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 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 ) 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 .

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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 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 .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 ) 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 ) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O 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) 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 all aggregated runs . Kruskal - Wallis test 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 aggregates in the 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O 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) 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 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 .

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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 ) 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 .

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 O O O O 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 O O O O O O O O O O O O O O O O O O O O O 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, 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 ) 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 .

O O O O O O O O O O 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 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

(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 test , F ( 3 , 11 ) = 1 . 911 , p = 0 . 1862 . * * p < 0 . 01 . TG - Ova - ( GA ) 10 vs . TG - PBS p = 0 . 0081 .

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

(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 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 .

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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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 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 .

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 O O O O O O O O O O

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.


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

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

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 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

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

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 ) 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 .

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

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 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 .

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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 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 . )

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

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

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

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


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 .

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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 and rad59 ∆ rad52 - R70A cells . DNA was digested with Bsp1286I and probed with a MAT - distal sequence ( 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 - test was used to determine the 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 ∆ , and rad52 - R70A . DNA was digested with XhoI and EcoRI and probed with a Z sequence ( 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).


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 .

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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 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 .

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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 assay ) .

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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 - test was used to determine the 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 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 ) , 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 .

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 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 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 ) , 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 .

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 O O O O O 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-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 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 ;

O O O O O O O B-MEASURED_VAR O O B-MEASURED_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 O O O O O 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 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 ;

O O 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-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

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 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 ) ;

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

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 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 ;

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

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;