A ) Kinetics of S . Typhimurium replication in Tbk1 - / - MEFs stably expressing the indicated TBK1 alleles . Intracellular bacteria were enumerated by their ability to form colonies on agar plates following cell lysis at the indicated time points . Statistical analysis comparing Tbk1 - / - MEFs and cells expressing TBK1 alleles . Western blot for Flag - tagged TBK1 variants in post nuclear cell lysates .

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

A) Kinetics of S. Typhimurium replication in Tbk1-/- MEFs stably expressing the indicated TBK1 alleles. Intracellular bacteria were enumerated by their ability to form colonies on agar plates following cell lysis at the indicated time points. Statistical analysis comparing Tbk1-/- MEFs and cells expressing TBK1 alleles. Western blot for Flag-tagged TBK1 variants in post nuclear cell lysates.


B ) Replication of S . Typhimurium in Atg5 - / - MEFs cells complemented with ATG5 or mock and treated with the TBK1 kinase inhibitor MRT68843 ( 10 nM ) or DMSO . Fold replication was determined by counting bacterial colonies on agar plates at 2 and 8 h post inoculation ( p . i . ) following cell lysis .

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

B) Replication of S. Typhimurium in Atg5-/- MEFs cells complemented with ATG5 or mock and treated with the TBK1 kinase inhibitor MRT68843 (10 nM) or DMSO. Fold replication was determined by counting bacterial colonies on agar plates at 2 and 8 h post inoculation (p.i.) following cell lysis.


C ) Analysis of Tbk1 - / - MEFs stably expressing the indicated TBK1 alleles and infected with S . Typhimurium . Percentage of S . Typhimurium coated with ubiquitin ( detected by FK2 antibody ) at 1 or 4 h p . i . Western blot for Flag - tagged TBK1 variants in post nuclear cell lysates .

O O 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 B-MEASURED_VAR 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

C) Analysis of Tbk1-/- MEFs stably expressing the indicated TBK1 alleles and infected with S. Typhimurium. Percentage of S. Typhimurium coated with ubiquitin (detected by FK2 antibody) at 1 or 4 h p.i. Western blot for Flag-tagged TBK1 variants in post nuclear cell lysates.


A - H ) Analysis of Tbk1 - / - MEFs stably expressing the indicated TBK1 alleles and infected with S . Typhimurium at 1 h p . i . Percentage of S . Typhimurium coated with GFP : LC3B ( A ) . Where indicated , Wortmannin ( Wort ) was added at 100 nM .

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

A-H) Analysis of Tbk1-/- MEFs stably expressing the indicated TBK1 alleles and infected with S. Typhimurium at 1 h p.i. Percentage of S. Typhimurium coated with GFP:LC3B (A). Where indicated, Wortmannin (Wort) was added at 100 nM.


A - H ) Analysis of Tbk1 - / - MEFs stably expressing the indicated TBK1 alleles and infected with S . Typhimurium at 1 h p . i . Percentage of S . Typhimurium coated with the indicated GFP : WIPI alleles ( B ) . Where indicated , Wortmannin ( Wort ) was added at 100 nM .

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

A-H) Analysis of Tbk1-/- MEFs stably expressing the indicated TBK1 alleles and infected with S. Typhimurium at 1 h p.i. Percentage of S. Typhimurium coated with the indicated GFP:WIPI alleles (B). Where indicated, Wortmannin (Wort) was added at 100 nM.


A - H ) Analysis of Tbk1 - / - MEFs stably expressing the indicated TBK1 alleles and infected with S . Typhimurium at 1 h p . i . Confocal microscopy of MEFs expressing the indicated GFP fusion proteins or immunolabeled for WIPI2 and infected with mCherry expressing S . Typhimurium ( C - H ) .

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

A-H) Analysis of Tbk1-/- MEFs stably expressing the indicated TBK1 alleles and infected with S. Typhimurium at 1 h p.i. Confocal microscopy of MEFs expressing the indicated GFP fusion proteins or immunolabeled for WIPI2 and infected with mCherry expressing S. Typhimurium (C-H).


A - H ) Analysis of Tbk1 - / - MEFs stably expressing the indicated TBK1 alleles and infected with S . Typhimurium at 1 h p . i . Percentage of S . Typhimurium coated with GFP : DFCP1 ( FYVE * denotes a PI ( 3 ) P - binding mutant of DFCP1 ) ( D - E ) . Where indicated , Wortmannin ( Wort ) was added at 100 nM .

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

A-H) Analysis of Tbk1-/- MEFs stably expressing the indicated TBK1 alleles and infected with S. Typhimurium at 1 h p.i. Percentage of S. Typhimurium coated with GFP:DFCP1 (FYVE* denotes a PI(3)P-binding mutant of DFCP1) (D-E). Where indicated, Wortmannin (Wort) was added at 100 nM.


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


A - H ) Analysis of Tbk1 - / - MEFs stably expressing the indicated TBK1 alleles and infected with S . Typhimurium at 1 h p . i . Percentage of S . Typhimurium coated with GFP : ML1N * 2 , a probe for PI ( 3 , 5 ) P2 ( G ) . Where indicated , Wortmannin ( Wort ) was added at 100 nM .

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

A-H) Analysis of Tbk1-/- MEFs stably expressing the indicated TBK1 alleles and infected with S. Typhimurium at 1 h p.i. Percentage of S. Typhimurium coated with GFP:ML1N*2, a probe for PI(3,5)P2 (G). Where indicated, Wortmannin (Wort) was added at 100 nM.


O O O O O O B-CONTROLLED_VAR 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


A - B ) Kinetics of S . Typhimurium replication in MEFs depleted of WIPI1 ( A ) . Intracellular bacteria were enumerated by their ability to form colonies on agar plates . Western blot for GFP : WIPI1 upon the indicated siRNA treatments .

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A-B) Kinetics of S. Typhimurium replication in MEFs depleted of WIPI1 (A). Intracellular bacteria were enumerated by their ability to form colonies on agar plates. Western blot for GFP:WIPI1 upon the indicated siRNA treatments.


A - B ) Kinetics of S . Typhimurium replication in MEFs depleted of WIPI2 ( B ) . Intracellular bacteria were enumerated by their ability to form colonies on agar plates . quantitative RT - PCR for WIPI2 upon the indicated siRNA treatments .

A-B) Kinetics of S. Typhimurium replication in MEFs depleted of WIPI2 (B). Intracellular bacteria were enumerated by their ability to form colonies on agar plates. quantitative RT-PCR for WIPI2 upon the indicated siRNA treatments.


A - D ) Analysis of Tbk1 - / - MEFs complemented with the indicated Flag - tagged TBK1ΔC - adaptor fusion proteins . S . Typhimurium replication kinetics ( A , D ) . Infected cells were lysed at the indicated time points post inoculation ( p . i . ) and bacteria were enumerated by their ability to form colonies on agar plates . Mean and s . d . of triplicate MEF cultures and duplicate colony counts . Data are representative of at least two repeats . Statistical significance to Tbk1 - / - MEFs expressing TBK1ΔC is indicated . Western blot for Flag - tagged TBK1 variants in post nuclear cell lysates .

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

A-D) Analysis of Tbk1-/- MEFs complemented with the indicated Flag-tagged TBK1ΔC-adaptor fusion proteins. S. Typhimurium replication kinetics (A,D). Infected cells were lysed at the indicated time points post inoculation (p.i.) and bacteria were enumerated by their ability to form colonies on agar plates. Mean and s.d. of triplicate MEF cultures and duplicate colony counts. Data are representative of at least two repeats. Statistical significance to Tbk1-/- MEFs expressing TBK1ΔC is indicated. Western blot for Flag-tagged TBK1 variants in post nuclear cell lysates.


A - D ) Analysis of Tbk1 - / - MEFs complemented with the indicated Flag - tagged TBK1ΔC - adaptor fusion proteins . ( B ) Percentage of GFP : WIPI1 positive S . Typhimurium at 1 h p . i . in the indicated complemented MEFs . Mean and s . e . m . of four independent experiments . > 200 bacteria counted per coverslip . * p < 0 . 05 , * * * p < 0 . 001 , one - way ANOVA with Dunnett ' s multiple comparisons test .

O O O O O O B-CONTROLLED_VAR 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 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

A-D) Analysis of Tbk1-/- MEFs complemented with the indicated Flag-tagged TBK1ΔC-adaptor fusion proteins. (B) Percentage of GFP:WIPI1 positive S. Typhimurium at 1 h p.i. in the indicated complemented MEFs. Mean and s.e.m. of four independent experiments. >200 bacteria counted per coverslip. *p<0.05, ***p<0.001, one-way ANOVA with Dunnett's multiple comparisons test.


A - D ) Analysis of Tbk1 - / - MEFs complemented with the indicated Flag - tagged TBK1ΔC - adaptor fusion proteins . S . Typhimurium replication kinetics ( A , D ) . Infected cells were lysed at the indicated time points post inoculation ( p . i . ) and bacteria were enumerated by their ability to form colonies on agar plates . Mean and s . d . of triplicate MEF cultures and duplicate colony counts . Data are representative of at least two repeats . Statistical significance to Tbk1 - / - MEFs expressing TBK1ΔC is indicated . Western blot for Flag - tagged TBK1 variants in post nuclearcell lysates .

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

A-D) Analysis of Tbk1-/- MEFs complemented with the indicated Flag-tagged TBK1ΔC-adaptor fusion proteins. S. Typhimurium replication kinetics (A,D). Infected cells were lysed at the indicated time points post inoculation (p.i.) and bacteria were enumerated by their ability to form colonies on agar plates. Mean and s.d. of triplicate MEF cultures and duplicate colony counts. Data are representative of at least two repeats. Statistical significance to Tbk1-/- MEFs expressing TBK1ΔC is indicated. Western blot for Flag-tagged TBK1 variants in post nuclearcell lysates.


E ) Lumier binding assay . Binding of the indicated luciferase - tagged NDP52 variants to beads coated with GST , GST : galectin - 8 , or GST : ubiquitin . Coomassie stain of purified GST proteins . Western blot for luciferase - tagged NDP52 alleles in total cell lysates .

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

E) Lumier binding assay. Binding of the indicated luciferase-tagged NDP52 variants to beads coated with GST, GST:galectin-8, or GST:ubiquitin. Coomassie stain of purified GST proteins. Western blot for luciferase-tagged NDP52 alleles in total cell lysates.


F ) Kinetics of S . Typhimurium replication in Tbk1 - / - MEFs complemented with the indicated Flag - tagged TBK1ΔC - NDP52 fusion proteins . Infected cells were lysed at the indicated times post inoculation ( p . i . ) and bacteria were enumerated by their ability to form colonies on agar plates . Mean and s . d . of triplicate MEF cultures and duplicate colony counts . Data are representative of at least two repeats . Statistical comparison with Tbk1 - / - MEFs expressing TBK1ΔC is indicated . * * * p < 0 . 001 , one - way ANOVA with Dunnett ' s multiple comparisons test . Western blot for Flag - tagged TBK1 variants in post nuclear cell lysates .

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

F) Kinetics of S. Typhimurium replication in Tbk1-/- MEFs complemented with the indicated Flag-tagged TBK1ΔC-NDP52 fusion proteins. Infected cells were lysed at the indicated times post inoculation (p.i.) and bacteria were enumerated by their ability to form colonies on agar plates. Mean and s.d. of triplicate MEF cultures and duplicate colony counts. Data are representative of at least two repeats. Statistical comparison with Tbk1-/- MEFs expressing TBK1ΔC is indicated. ***p<0.001, one-way ANOVA with Dunnett's multiple comparisons test. Western blot for Flag-tagged TBK1 variants in post nuclear cell lysates.


A - B ) Analysis of Tbk1 - / - MEFs complemented with the indicated Flag - tagged TBK1ΔC - galectin fusion proteins . S . Typhimurium replication kinetics ( A ) . Infected cells were lysed at the indicated times post inoculation ( p . i . ) . and bacteria were enumerated by their ability to form colonies on agar plates . Mean and s . d . of triplicate MEF cultures and duplicate colony counts . Data are representative of at least two repeats . Statistical differences to Tbk1 - / - MEFs expressing TBK1ΔC are shown . * p < 0 . 05 , * * * p < 0 . 001 , one - way ANOVA with Dunnett ' s multiple comparisons test . Western blot for Flag - tagged TBK1 variants in post nuclear cell lysates .

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

A-B) Analysis of Tbk1-/- MEFs complemented with the indicated Flag-tagged TBK1ΔC-galectin fusion proteins. S. Typhimurium replication kinetics (A). Infected cells were lysed at the indicated times post inoculation (p.i.). and bacteria were enumerated by their ability to form colonies on agar plates. Mean and s.d. of triplicate MEF cultures and duplicate colony counts. Data are representative of at least two repeats. Statistical differences to Tbk1-/- MEFs expressing TBK1ΔC are shown. *p<0.05, ***p<0.001, one-way ANOVA with Dunnett's multiple comparisons test. Western blot for Flag-tagged TBK1 variants in post nuclear cell lysates.


A - B ) Analysis of Tbk1 - / - MEFs complemented with the indicated Flag - tagged TBK1ΔC - galectin fusion proteins . ( B ) Percentage of GFP : WIPI1 positive S . Typhimurium at 1 h p . i . in the indicated complemented MEFs . Mean and s . e . m . of three independent experiments . > 200 bacteria counted per coverslip . * p < 0 . 05 , one - way ANOVA with Dunnett ' s multiple comparisons test .

O O O O O O B-CONTROLLED_VAR 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 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

A-B) Analysis of Tbk1-/- MEFs complemented with the indicated Flag-tagged TBK1ΔC-galectin fusion proteins. (B) Percentage of GFP:WIPI1 positive S. Typhimurium at 1 h p.i. in the indicated complemented MEFs. Mean and s.e.m. of three independent experiments. >200 bacteria counted per coverslip. *p<0.05, one-way ANOVA with Dunnett's multiple comparisons test.


A ) Binding of NDP52 to ubiquitin tetramers of different linkage types . Beads coated with the indicated GST - tagged proteins were incubated with pooled M1 - , K63 - , and K48 - linked tetra - ubiquitin chains . Anti - ubiquitin Western blot of input and proteins bound to beads .

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

A) Binding of NDP52 to ubiquitin tetramers of different linkage types. Beads coated with the indicated GST-tagged proteins were incubated with pooled M1-, K63-, and K48-linked tetra-ubiquitin chains. Anti-ubiquitin Western blot of input and proteins bound to beads.


B - D ) Kinetics of S . Typhimurium replication in Tbk1 - / - MEFs complemented with the indicated Flag - tagged TBK1ΔC fusion proteins . Infected cells were lysed at the indicated time points post inoculation ( p . i . ) and bacteria were enumerated by their ability to form colonies on agar plates . Mean and s . d . of triplicate MEF cultures and duplicate colony counts . Data are representative of at least two repeats . Statistical differences to Tbk1 - / - MEFs expressing TBK1ΔC are shown . * p < 0 . 5 , * * p < 0 . 01 , * * * p < 0 . 001 one - way ANOVA with Dunnett ' s multiple comparisons test . Western blot for Flag - tagged TBK1 variants in post nuclear cell lysates .

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 O O O O O O O O O O O O O O O O O O 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 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-MEASURED_VAR O O O O O O O

B-D) Kinetics of S. Typhimurium replication in Tbk1-/- MEFs complemented with the indicated Flag-tagged TBK1ΔC fusion proteins. Infected cells were lysed at the indicated time points post inoculation (p.i.) and bacteria were enumerated by their ability to form colonies on agar plates. Mean and s.d. of triplicate MEF cultures and duplicate colony counts. Data are representative of at least two repeats. Statistical differences to Tbk1-/- MEFs expressing TBK1ΔC are shown. *p<0.5, **p<0.01, ***p<0.001 one-way ANOVA with Dunnett's multiple comparisons test. Western blot for Flag-tagged TBK1 variants in post nuclear cell lysates.


B - D ) Kinetics of S . Typhimurium replication in Tbk1 - / - MEFs complemented with the indicated Flag - tagged TBK1ΔC fusion proteins . Infected cells were lysed at the indicated time points post inoculation ( p . i . ) and bacteria were enumerated by their ability to form colonies on agar plates . Mean and s . d . of triplicate MEF cultures and duplicate colony counts . Data are representative of at least two repeats . Statistical differences to Tbk1 - / - MEFs expressing TBK1ΔC are shown . * p < 0 . 5 , * * p < 0 . 01 , * * * p < 0 . 001 one - way ANOVA with Dunnett ' s multiple comparisons test . Western blot for Flag - tagged TBK1 variants in post nuclearcell lysates .

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 O O O O O O O O O O O O O O O O O O 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 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-MEASURED_VAR O O O O O O

B-D) Kinetics of S. Typhimurium replication in Tbk1-/- MEFs complemented with the indicated Flag-tagged TBK1ΔC fusion proteins. Infected cells were lysed at the indicated time points post inoculation (p.i.) and bacteria were enumerated by their ability to form colonies on agar plates. Mean and s.d. of triplicate MEF cultures and duplicate colony counts. Data are representative of at least two repeats. Statistical differences to Tbk1-/- MEFs expressing TBK1ΔC are shown. *p<0.5, **p<0.01, ***p<0.001 one-way ANOVA with Dunnett's multiple comparisons test. Western blot for Flag-tagged TBK1 variants in post nuclearcell lysates.


( A m6A dot blot of the P493 - 6 cells treated with Tet for 0h or 72h ( A ) equal mRNA loading was verified by methylene blue staining . The shown data are representative of at least three independent experiments .

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(A m6A dot blot of the P493-6 cells treated with Tet for 0h or 72h (A) equal mRNA loading was verified by methylene blue staining. The shown data are representative of at least three independent experiments.


B ) m6A dot blot of Raji cells that expressed NTC or MYC shRNAs ( B ) ; equal mRNA loading was verified by methylene blue staining . The shown data are representative of at least three independent experiments .

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B) m6A dot blot of Raji cells that expressed NTC or MYC shRNAs (B); equal mRNA loading was verified by methylene blue staining. The shown data are representative of at least three independent experiments.


( C ) Quantification of m6A abundance by HPLC - MS . * * * P < 0 . 001 as compared to corresponding high MYC group ( mean ± SD , n = 3 biological replicates , Student ' s t ‐ test ) .

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

(C) Quantification of m6A abundance by HPLC-MS. *** P<0.001 as compared to corresponding high MYC group (mean ± SD, n = 3 biological replicates, Student's t‐test).


( D ) Metagene profiles of m6A peak distribution along a normalized transcript composed of three rescaled non - overlapping segments : 5 ′ UTR , CDS , and 3 ′ UTR in P493 - 6 cells treated with Tet for 0h or 72h .

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

(D) Metagene profiles of m6A peak distribution along a normalized transcript composed of three rescaled non-overlapping segments: 5′UTR, CDS, and 3′UTR in P493-6 cells treated with Tet for 0h or 72h.


( F ) IGV graph showing location of m6A peaks on representative genes .

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(F) IGV graph showing location of m6A peaks on representative genes.


( G ) m6A - RIP assay in P493 - 6 cells treated with Tet for 0h or 72h . HPRT1 serves as negative control . * * * P < 0 . 001 as compared to corresponding high MYC group , ns , not significant ( mean ± SD , n = 3 biological replicates , Student ' s t ‐ test ) .

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-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

(G) m6A-RIP assay in P493-6 cells treated with Tet for 0h or 72h. HPRT1 serves as negative control. *** P<0.001 as compared to corresponding high MYC group, ns, not significant (mean ± SD, n = 3 biological replicates, Student's t‐test).


( H Western blot analysis for protein levels in P493 - 6 cells treated with Tet for 0h , 24h , 48h and 72h ( H ) HPRT1 and β - actin serve as negative and loading controls , respectively . Data are representative of at least three independent experiments .

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

(H Western blot analysis for protein levels in P493-6 cells treated with Tet for 0h, 24h, 48h and 72h (H) HPRT1 and β-actin serve as negative and loading controls, respectively. Data are representative of at least three independent experiments.


I ) Western blot analysis for protein levels in Raji cells that expressed NTC or MYC shRNAs ( I ) . HPRT1 and β - actin serve as negative and loading controls , respectively . Data are representative of at least three independent experiments .

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

I) Western blot analysis for protein levels in Raji cells that expressed NTC or MYC shRNAs (I). HPRT1 and β-actin serve as negative and loading controls, respectively. Data are representative of at least three independent experiments.


( A Western blot analysis for protein levels of m6A methyltransferases ( METTL3 and METTL14 ) and demethylases ( ALKBH5 and FTO ) in P493 - 6 cells treated with Tet for 0h , 24h , 48h and 72h ( A ) β - actin serves as loading controls . Data are representative of at least three independent experiments .

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

(A Western blot analysis for protein levels of m6A methyltransferases (METTL3 and METTL14) and demethylases (ALKBH5 and FTO) in P493-6 cells treated with Tet for 0h, 24h, 48h and 72h (A) β-actin serves as loading controls. Data are representative of at least three independent experiments.


B ) Western blot analysis for protein levels of m6A methyltransferases ( METTL3 and METTL14 ) and demethylases ( ALKBH5 and FTO ) in Raji cells that expressed NTC or MYC shRNAs ( B ) . β - actin serves as loading controls . Data are representative of at least three independent experiments .

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B) Western blot analysis for protein levels of m6A methyltransferases (METTL3 and METTL14) and demethylases (ALKBH5 and FTO) in Raji cells that expressed NTC or MYC shRNAs (B). β-actin serves as loading controls. Data are representative of at least three independent experiments.


( C ) RT - qPCR analysis of the mRNA levels of methyltransferases and demethylases in P493 - 6 cells treated with Tet for 0h or 24h . Data were presented as mean ( ± SD ) , n = 3 biological replicates . * P < 0 . 05 relative to corresponding - Tet group ( Student ' s t ‐ test ) .

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(C) RT-qPCR analysis of the mRNA levels of methyltransferases and demethylases in P493-6 cells treated with Tet for 0h or 24h. Data were presented as mean (±SD), n = 3 biological replicates. * P<0.05 relative to corresponding -Tet group (Student's t‐test).


( D ) IGV graph showing location of MYC binding peaks on ALKBH5 and FTO from published ChIP - seq datasets ( Walz et al , 2014 ; Data ref : Eilers M , 2014 ) . In this dataset , high levels of MYC were induced with 1µg / ml doxycycline for 30h and ChIPed by MYC antibody . Red triangle indicates the E - box .

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(D) IGV graph showing location of MYC binding peaks on ALKBH5 and FTO from published ChIP-seq datasets (Walz et al, 2014; Data ref: Eilers M, 2014). In this dataset, high levels of MYC were induced with 1µg/ml doxycycline for 30h and ChIPed by MYC antibody. Red triangle indicates the E-box.


( E ) RIP assay , using ALKBH5 , FTO or IgG antibody to detect the binding to MRGs ( SPI1 and PHF12 ) in P493 - 6 cells treated with Tet or not . HPRT1 serves as negative control . * * * P < 0 . 001 as compared to corresponding IgG group ( mean ± SD , n = 3 biological replicates , Student ' s t ‐ test ) .

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(E) RIP assay, using ALKBH5, FTO or IgG antibody to detect the binding to MRGs (SPI1 and PHF12) in P493-6 cells treated with Tet or not. HPRT1 serves as negative control. *** P<0.001 as compared to corresponding IgG group (mean ± SD, n = 3 biological replicates, Student's t‐test).


( F Western blot analysis for protein levels in P493 - 6 cells that overexpressed empty vector ( EV ) , ALKBH5 , or FTO and were then treated with Tet or not ( F ) HPRT1 and β - actin served as negative and loading controls , respectively . Data are representative of at least three independent experiments .

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(F Western blot analysis for protein levels in P493-6 cells that overexpressed empty vector (EV), ALKBH5, or FTO and were then treated with Tet or not (F) HPRT1 and β-actin served as negative and loading controls, respectively. Data are representative of at least three independent experiments.


G ) Western blot analysis for protein levels in Raji cells expressed NTC or MYC shRNAs ( G ) . HPRT1 and β - actin served as negative and loading controls , respectively . Data are representative of at least three independent experiments .

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G) Western blot analysis for protein levels in Raji cells expressed NTC or MYC shRNAs (G). HPRT1 and β-actin served as negative and loading controls, respectively. Data are representative of at least three independent experiments.


( H ) m6A RIP assay in P493 - 6 cells that overexpressed EV , ALKBH5 , or FTO and were then treated with Tet or not . HPRT1 serves as negative control . * * * P < 0 . 001 as compared between indicated groups . ns , not significant ( mean ± SD , n = 3 biological replicates , Student ' s t ‐ test ) .

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(H) m6A RIP assay in P493-6 cells that overexpressed EV, ALKBH5, or FTO and were then treated with Tet or not. HPRT1 serves as negative control. *** P<0.001 as compared between indicated groups. ns, not significant (mean ± SD, n = 3 biological replicates, Student's t‐test).


( I , Western blot analysis for protein levels in P493 - 6 cells that expressed NTC or ALKBH5 shRNAs and were then treated with Tet or not ( I ) HPRT1 and β - actin serve as negative and loading controls , respectively . Data are representative of at least three independent experiments .

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(I, Western blot analysis for protein levels in P493-6 cells that expressed NTC or ALKBH5 shRNAs and were then treated with Tet or not (I) HPRT1 and β-actin serve as negative and loading controls, respectively. Data are representative of at least three independent experiments.


J ) Western blot analysis for protein levels in Raji cells expressed NTC or ALKBH5 shRNAs and knocked down MYC or not ( J ) . HPRT1 and β - actin serve as negative and loading controls , respectively . Data are representative of at least three independent experiments .

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J) Western blot analysis for protein levels in Raji cells expressed NTC or ALKBH5 shRNAs and knocked down MYC or not (J). HPRT1 and β-actin serve as negative and loading controls, respectively. Data are representative of at least three independent experiments.


( K ) m6A RIP assay HPRT1 serves as negative control . * * * P < 0 . 001 as compared between indicated groups ( mean ± SD , n = 3 biological replicates , Student ' s t ‐ test ) .

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(K) m6A RIP assay HPRT1 serves as negative control. *** P<0.001 as compared between indicated groups (mean ± SD, n = 3 biological replicates, Student's t‐test).


( A ) RIP assay in P493 - 6 cells treated with Tet for 0h or 72h . HPRT1 serves as negative control . * * * P < 0 . 001 relative to indicating groups ( mean ± SD , n = 3 biological replicates , Student ' s t ‐ test ) .

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(A) RIP assay in P493-6 cells treated with Tet for 0h or 72h. HPRT1 serves as negative control. *** P<0.001 relative to indicating groups (mean ± SD, n = 3 biological replicates, Student's t‐test).


( B , Western blot analysis for protein levels in P493 - 6 cells that expressed NTC or YTHDF3 shRNAs and were then treated with Tet or not ( B ) HPRT1 and β - actin serve as negative and loading controls , respectively . Data are representative of at least three independent experiments .

(B, Western blot analysis for protein levels in P493-6 cells that expressed NTC or YTHDF3 shRNAs and were then treated with Tet or not (B) HPRT1 and β-actin serve as negative and loading controls, respectively. Data are representative of at least three independent experiments.


C ) Western blot analysis for protein levels in Raji cells that expressed NTC or YTHDF3 shRNAs and knocked down MYC or not ( C ) . HPRT1 and β - actin serve as negative and loading controls , respectively . Data are representative of at least three independent experiments .

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C) Western blot analysis for protein levels in Raji cells that expressed NTC or YTHDF3 shRNAs and knocked down MYC or not (C). HPRT1 and β-actin serve as negative and loading controls, respectively. Data are representative of at least three independent experiments.


( D ) Polysomes profiling assay in P493 - 6 cells that expressed NTC or YTHDF3 shRNA .

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(D) Polysomes profiling assay in P493-6 cells that expressed NTC or YTHDF3 shRNA.


( E ) RT - qPCR assay for mRNA levels of MRGs ( SPI1 and PHF12 ) in different fractions in ( D ) . HPRT1 serves as negative control . Data were presented as mean ( ± SD ) , n = 3 biological replicates . * * P < 0 . 01 and * * * P < 0 . 001 as compared to NTC group ( Student ' s t ‐ test ) .

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(E) RT-qPCR assay for mRNA levels of MRGs (SPI1 and PHF12) in different fractions in (D). HPRT1 serves as negative control. Data were presented as mean (±SD), n = 3 biological replicates. ** P<0.01 and *** P<0.001 as compared to NTC group (Student's t‐test).


( F ) Click - iT AHA ( L - azidohomoalaine ) experiments were performed using IgG , anti - SPI1 or anti - PHF12 antibody . P493 - 6 cells expressing NTC or YTHDF3 shRNAs were incubated for 1 hour in medium containing 100 ug / mL AHA . The translated proteins were detected by Western blot . Arrow indicates translated MRGs .

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(F) Click-iT AHA (L-azidohomoalaine) experiments were performed using IgG, anti-SPI1 or anti-PHF12 antibody. P493-6 cells expressing NTC or YTHDF3 shRNAs were incubated for 1 hour in medium containing 100 ug/mL AHA. The translated proteins were detected by Western blot. Arrow indicates translated MRGs.


( G Western blot analysis for protein levels in P493 - 6 cells ( G ) β - actin serve as negative and loading controls , respectively . Data are representative of at least three independent experiments .

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(G Western blot analysis for protein levels in P493-6 cells (G) β-actin serve as negative and loading controls, respectively. Data are representative of at least three independent experiments.


H ) Western blot analysis for protein levels in Raji cells ( H ) that expressed NTC , or ALKBH5 shRNA , or YTHDF3 shRNA . β - actin serve as negative and loading controls , respectively . Data are representative of at least three independent experiments .

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H) Western blot analysis for protein levels in Raji cells (H) that expressed NTC, or ALKBH5 shRNA, or YTHDF3 shRNA. β-actin serve as negative and loading controls, respectively. Data are representative of at least three independent experiments.


( I ) m6A RIP assay HPRT1 serves as negative control . * * * P < 0 . 001 as compared between indicated groups , ns , not significant ( mean ± SD , n = 3 biological replicates , Student ' s t ‐ test ) .

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(I) m6A RIP assay HPRT1 serves as negative control. *** P<0.001 as compared between indicated groups, ns, not significant (mean ± SD, n = 3 biological replicates, Student's t‐test).


( A ) Trypan blue counting was used to analyze growth curves for P493 - 6 cells that expressed NTC or SPI1 shRNA or PHF12 shRNA . * * P < 0 . 01 as compared between indicated groups . ( mean ± SD , n = 4 biological replicates , Student ' s t ‐ test )

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(A) Trypan blue counting was used to analyze growth curves for P493-6 cells that expressed NTC or SPI1 shRNA or PHF12 shRNA. ** P<0.01 as compared between indicated groups. (mean ± SD, n = 4 biological replicates, Student's t‐test)


( B , C ) Trypan blue counting was used to analyze growth curves for P493 - 6 cells overexpressing ALKBH5 and further infected with viruses expressing SPI1 ( B ) or PHF12 ( C ) . * * P < 0 . 01 as compared between indicated groups ( mean ± SD , n = 4 biological replicates , Student ' s t ‐ test ) .

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(B, C) Trypan blue counting was used to analyze growth curves for P493-6 cells overexpressing ALKBH5 and further infected with viruses expressing SPI1 (B) or PHF12 (C). ** P<0.01 as compared between indicated groups (mean ± SD, n = 4 biological replicates, Student's t‐test).


( D , E ) Raji cells stably expressing EV or MYC were infected with viruses expressing shALKBH5 . Cells were injected subcutaneously into nude mice ( n = 5 for each group ) . Tumor growth curves were measured starting from 12 days post injection ( D ) . Photo of tumors collected at the end of the experiment ( day 27 ) ( E ) . Data are presented as mean ( ± SEM ) . * * P < 0 . 01 or * * * P < 0 . 001 as compared between indicated groups ( Student ' s t ‐ test ) .

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(D, E) Raji cells stably expressing EV or MYC were infected with viruses expressing shALKBH5. Cells were injected subcutaneously into nude mice (n=5 for each group). Tumor growth curves were measured starting from 12 days post injection (D). Photo of tumors collected at the end of the experiment (day 27) (E). Data are presented as mean (±SEM). ** P<0.01 or *** P<0.001 as compared between indicated groups (Student's t‐test).


( F P493 - 6 cells stably expressing NTC or shSPI1 or shPHF12 were injected subcutaneously into nude mice ( n = 5 for each group ) . Tumor growth curves were measured starting from 12 days post injection ( F ) . Data are presented as mean ( ± SEM ) . * * P < 0 . 01 as compared between indicated groups ( Student ' s t ‐ test ) .

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(F P493-6 cells stably expressing NTC or shSPI1 or shPHF12 were injected subcutaneously into nude mice (n=5 for each group). Tumor growth curves were measured starting from 12 days post injection (F). Data are presented as mean (±SEM). ** P<0.01 as compared between indicated groups (Student's t‐test).


G ) P493 - 6 cells stably expressing NTC or shSPI1 or shPHF12 were injected subcutaneously into nude mice ( n = 5 for each group ) . Photo ( G ) of tumors collected at the end of the experiment ( day 25 ) .

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G) P493-6 cells stably expressing NTC or shSPI1 or shPHF12 were injected subcutaneously into nude mice (n=5 for each group). Photo (G) of tumors collected at the end of the experiment (day 25).


( H , I ) P493 - 6 cells stably expressing EV or ALKBH5 were infected with viruses expressing SPI1 or PHF12 . Cells were injected subcutaneously into nude mice ( n = 5 for each group ) . Tumor growth curves were measured starting from 17 days post injection ( H ) . Photo ( I ) of tumors collected at the end of the experiment ( day 32 ) . Data are presented as mean ( ± SEM ) . * P < 0 . 05 as compared between indicated groups ( Student ' s t ‐ test ) .

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(H, I) P493-6 cells stably expressing EV or ALKBH5 were infected with viruses expressing SPI1 or PHF12. Cells were injected subcutaneously into nude mice (n=5 for each group). Tumor growth curves were measured starting from 17 days post injection (H). Photo (I) of tumors collected at the end of the experiment (day 32). Data are presented as mean (±SEM). * P<0.05 as compared between indicated groups (Student's t‐test).


( J , K ) Representative IHC images ( J ) , and analysis ( K ) of MYC , ALKBH5 , SPI1 and PHF12 expressions in normal lymphocyte tissue ( normal ) and lymphoma specimens . Scale bar : 50 um . Data are presented as mean ( ± SEM ) , clinical samples n = 30 for normal group , n = 28 for low MYC expression group , n = 27 for high MYC expression group , P value was presented between indicated groups ( Student ' s t ‐ test ) .

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(J, K) Representative IHC images (J), and analysis (K) of MYC, ALKBH5, SPI1 and PHF12 expressions in normal lymphocyte tissue (normal) and lymphoma specimens. Scale bar: 50 um. Data are presented as mean (±SEM), clinical samples n = 30 for normal group, n = 28 for low MYC expression group, n = 27 for high MYC expression group, P value was presented between indicated groups (Student's t‐test).


A Western blot analysis of the localization of endogenous PHGDH in PLC , HepG2 , and THLE3 cells . Lamin B1 , Tubulin , and Tom20 were used as markers for nuclear ( Nuc ) , cytosolic ( Cyto ) , and mitochondrial ( Mito ) proteins , respectively .

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A Western blot analysis of the localization of endogenous PHGDH in PLC, HepG2, and THLE3 cells. Lamin B1, Tubulin, and Tom20 were used as markers for nuclear (Nuc), cytosolic (Cyto), and mitochondrial (Mito) proteins, respectively.


B Western blot analysis of the localization of endogenous PHGDH in tumour tissues and adjacent normal tissues . Lamin B1 , GAPDH , and Tom20 were used as markers for Nuc , Cyto , and Mito proteins , respectively .

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B Western blot analysis of the localization of endogenous PHGDH in tumour tissues and adjacent normal tissues. Lamin B1, GAPDH, and Tom20 were used as markers for Nuc, Cyto, and Mito proteins, respectively.


C Western blot analysis of the localization of endogenous and exogenous Flag - tagged PHGDH in PLC cells . Lamin B1 , Tubulin , and Tom20 were used as markers for Nuc , Cyto , and Mito proteins , respectively .

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C Western blot analysis of the localization of endogenous and exogenous Flag-tagged PHGDH in PLC cells. Lamin B1, Tubulin, and Tom20 were used as markers for Nuc, Cyto, and Mito proteins, respectively.


D Immunofluorescence images showing colocalization of Flag - PHGDH ( green ) and mitochondria ( red ) acquired with a laser confocal microscope system in PLC cells . Nuclei were stained with DAPI ( blue ) , and mitochondria were stained with MitoTracker Red . Scale bar , 10 μm ( left panel ) , 2 μm ( right panel ) .

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D Immunofluorescence images showing colocalization of Flag-PHGDH (green) and mitochondria (red) acquired with a laser confocal microscope system in PLC cells. Nuclei were stained with DAPI (blue), and mitochondria were stained with MitoTracker Red. Scale bar, 10 μm (left panel), 2 μm (right panel).


F Subcellular localization of PHGDH was determined by western blot in PLC cells overexpressing of Flag - tagged wild - type PHGDH , PHGDH ∆ SBD1 , or PHGDH ∆ RD mutants . WCL : whole - cell lysates , Mito : mitochondria . Tom20 was used as loading control .

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F Subcellular localization of PHGDH was determined by western blot in PLC cells overexpressing of Flag-tagged wild-type PHGDH, PHGDH∆SBD1, or PHGDH∆RD mutants. WCL: whole-cell lysates, Mito: mitochondria. Tom20 was used as loading control.


G Proteinase K protection assays were performed on purified mitochondria isolated from PLC and HepG2 cells . The outer mitochondrial membrane ( OMM ) protein Tom20 , the inner mitochondrial membrane ( IMM ) protein ANT2 , and the mitochondrial matrix ( MM ) protein TFAM were used as markers of the OMM , IMM , and MM , respectively .

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G Proteinase K protection assays were performed on purified mitochondria isolated from PLC and HepG2 cells. The outer mitochondrial membrane (OMM) protein Tom20, the inner mitochondrial membrane (IMM) protein ANT2, and the mitochondrial matrix (MM) protein TFAM were used as markers of the OMM, IMM, and MM, respectively.


H Purified mitochondria of PLC or HepG2 cells were incubated with Na2CO3 ( pH 11 . 5 ) for 30 min at 4 ℃ . The soluble component ( Supernatant / Sup . ) and membrane component ( Pellet ) were separated by centrifugation prior to western blot analysis . COX2 , ATP8 , and ANT2 are markers for the membrane compotent ; CYTC and TFAM are markers for the soluble component .

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H Purified mitochondria of PLC or HepG2 cells were incubated with Na2CO3 (pH 11.5) for 30 min at 4 ℃. The soluble component (Supernatant/Sup.) and membrane component (Pellet) were separated by centrifugation prior to western blot analysis. COX2, ATP8, and ANT2 are markers for the membrane compotent; CYTC and TFAM are markers for the soluble component.


A The oxygen consumption rate ( OCR ) was measured by successive injections of oligomycin ( oligo ) , FCCP , and antimycin A / rotenone ( AA / Rot ) in PLC cells infected with viruses expressing nontargeting control ( NTC ) shRNA or shPHGDH . Basal respiration , ATP - linked respiration , and maximal respiration were analysed with the OCR curve .

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A The oxygen consumption rate (OCR) was measured by successive injections of oligomycin (oligo), FCCP, and antimycin A/rotenone (AA/Rot) in PLC cells infected with viruses expressing nontargeting control (NTC) shRNA or shPHGDH. Basal respiration, ATP-linked respiration, and maximal respiration were analysed with the OCR curve.


B Western blot analysis of mtDNA - encoded proteins in PLC cells stably expressing NTC or shPHGDH and empty vector ( EV ) or PLVX - PHGDH . β - Actin was used as the loading control .

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B Western blot analysis of mtDNA-encoded proteins in PLC cells stably expressing NTC or shPHGDH and empty vector (EV) or PLVX-PHGDH. β-Actin was used as the loading control.


C PLC cells with endogenous PHGDH knockdown were further infected with viruses expressing EV or PLVX - PHGDH prior to western blot analysis of mtDNA - encoded proteins . β - Actin was used as the loading control .

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C PLC cells with endogenous PHGDH knockdown were further infected with viruses expressing EV or PLVX-PHGDH prior to western blot analysis of mtDNA-encoded proteins. β-Actin was used as the loading control.


D PLC cells expressing NTC or shPHGDH were treated with 100 μg / ml cycloheximide ( CHX ) for 20 min prior to puromycin treatment for 15 min . Purified mitochondrial lysates were subjected to a SUnSET assay with an anti - puromycin antibody to measure the actively translated polypeptide chains . Western blot analysis of PHGDH confirmed the knockdown efficiency of the PHGDH shRNAs . β - Actin was used as the loading control .

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D PLC cells expressing NTC or shPHGDH were treated with 100 μg/ml cycloheximide (CHX) for 20 min prior to puromycin treatment for 15 min. Purified mitochondrial lysates were subjected to a SUnSET assay with an anti-puromycin antibody to measure the actively translated polypeptide chains. Western blot analysis of PHGDH confirmed the knockdown efficiency of the PHGDH shRNAs. β-Actin was used as the loading control.


E SUnSET assays were performed in the indicated cell lines using whole - cell lysates .

E SUnSET assays were performed in the indicated cell lines using whole-cell lysates.


F PLC cells with or without PLVX - Flag - PHGDH overexpression were treated with DMSO or 50 μM chloramphenicol ( CAP ) in combination with 50 μM thiamphenicol ( TAP ) for 24 h prior to SUnSET assays . The assays were performed using whole - cell lysate .

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F PLC cells with or without PLVX-Flag-PHGDH overexpression were treated with DMSO or 50 μM chloramphenicol (CAP) in combination with 50 μM thiamphenicol (TAP) for 24 h prior to SUnSET assays. The assays were performed using whole-cell lysate.


G PLC cells with or without PLVX - PHGDH overexpression were treated with DMSO or 50 μM CAP in combination with 50 μM TAP for 24 h prior to western blot analysis of mtDNA - encoded proteins . β - Actin was used as the loading control .

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

G PLC cells with or without PLVX-PHGDH overexpression were treated with DMSO or 50 μM CAP in combination with 50 μM TAP for 24 h prior to western blot analysis of mtDNA-encoded proteins. β-Actin was used as the loading control.


H The translation efficiency of mtDNA - encoded proteins was determined by a SUnSET assay in PLC cells with endogenous PHGDH knockdown that were further infected with viruses expressing EV , wild - type PHGDH , or the catalytically inactive PHGDH mutant PHGDHR236E as indicated . β - Actin was used as the loading control .

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

H The translation efficiency of mtDNA-encoded proteins was determined by a SUnSET assay in PLC cells with endogenous PHGDH knockdown that were further infected with viruses expressing EV, wild-type PHGDH, or the catalytically inactive PHGDH mutant PHGDHR236E as indicated. β-Actin was used as the loading control.


I mtDNA - encoded proteins were analysed by western blotting in PLC cells overexpressing EV , wild - type PHGDH , or PHGDHR236E . β - Actin was used as the loading control .

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

I mtDNA-encoded proteins were analysed by western blotting in PLC cells overexpressing EV, wild-type PHGDH, or PHGDHR236E. β-Actin was used as the loading control.


A HEK293T cells were transfected with Flag - tagged PHGDH in combination with HA - tagged EV , mtIF2 , mtIF3 , mtEFTu , mtEFG1 , mtEFG2 , or mtRRF . Co - immunoprecipitation ( Co - IP ) was performed with an anti - Flag antibody prior to western blot analysis .

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A HEK293T cells were transfected with Flag-tagged PHGDH in combination with HA-tagged EV, mtIF2, mtIF3, mtEFTu, mtEFG1, mtEFG2, or mtRRF. Co-immunoprecipitation (Co-IP) was performed with an anti-Flag antibody prior to western blot analysis.


B HEK293T cells were transfected with HA - tagged PHGDHWT or PHGDHR236E in combination with Flag - EV or Flag - mtEFG2 , and Co - IP was performed with an anti - Flag antibody prior to western blot analysis .

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B HEK293T cells were transfected with HA-tagged PHGDHWT or PHGDHR236E in combination with Flag-EV or Flag-mtEFG2, and Co-IP was performed with an anti-Flag antibody prior to western blot analysis.


C HEK293T cells were transfected with HA - tagged PHGDHWT or PHGDHR236E in combination with Flag - EV or Flag - ANT2 , and Co - IP was performed with an anti - Flag antibody prior to western blot analysis .

C HEK293T cells were transfected with HA-tagged PHGDHWT or PHGDHR236E in combination with Flag-EV or Flag-ANT2, and Co-IP was performed with an anti-Flag antibody prior to western blot analysis.


D HEK293T cells were transfected with HA - mtEFG2 in combination with Flag - EV or Flag - ANT2 , and Co - IP was performed with an anti - Flag antibody prior to western blot analysis .

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

D HEK293T cells were transfected with HA-mtEFG2 in combination with Flag-EV or Flag-ANT2, and Co-IP was performed with an anti-Flag antibody prior to western blot analysis.


E GST pull - down of His - PHGDHWT and His - PHGDHR236E by GST - ANT2 using proteins purified in E . coli , followed by western blot analysis with anti - His and anti - GST antibodies .

O O O O O O O O B-CONTROLLED_VAR 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

E GST pull-down of His-PHGDHWT and His-PHGDHR236E by GST-ANT2 using proteins purified in E. coli, followed by western blot analysis with anti-His and anti-GST antibodies.


F GST pull - down of His - mtEFG2 by GST - ANT2 using proteins purified in E . coli , followed by western blot analysis with anti - His and anti - GST antibodies .

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

F GST pull-down of His-mtEFG2 by GST-ANT2 using proteins purified in E. coli, followed by western blot analysis with anti-His and anti-GST antibodies.


G PLC cells stably expressing Flag - mtEFG2 and HA - PHGDH were further infected with viruses expressing NTC or shANT2 , and then cell lysates were immunoprecipitated with an anti - Flag antibody or IgG prior to western blot analysis .

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

G PLC cells stably expressing Flag-mtEFG2 and HA-PHGDH were further infected with viruses expressing NTC or shANT2, and then cell lysates were immunoprecipitated with an anti-Flag antibody or IgG prior to western blot analysis.


H PLC cells overexpressing PHGDH were further infected with viruses expressing NTC or shANT2 . The translation efficiency was measured by a SUnSET assay .

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

H PLC cells overexpressing PHGDH were further infected with viruses expressing NTC or shANT2. The translation efficiency was measured by a SUnSET assay.


I PLC cells overexpressing PLVX - PHGDH were further infected with viruses expressing NTC or shANT2 . The expression of mtDNA - encoded proteins was determined by western blotting . β - Actin was used as the loading control .

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

I PLC cells overexpressing PLVX-PHGDH were further infected with viruses expressing NTC or shANT2. The expression of mtDNA-encoded proteins was determined by western blotting. β-Actin was used as the loading control.


PLC cells overexpressing PHGDH were further infected with viruses expressing NTC or shmtEFG2 . The translation efficiency was measured by a SUnSET assay .

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

PLC cells overexpressing PHGDH were further infected with viruses expressing NTC or shmtEFG2. The translation efficiency was measured by a SUnSET assay.


K PLC cells overexpressing pSin - 3xFlag - PHGDH were further infected with viruses expressing NTC or shmtEFG2 . The expression of mtDNA - encoded proteins was determined by western blotting . β - Actin was used as the loading control .

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

K PLC cells overexpressing pSin-3xFlag-PHGDH were further infected with viruses expressing NTC or shmtEFG2. The expression of mtDNA-encoded proteins was determined by western blotting. β-Actin was used as the loading control.


A HEK293T cells were transfected with HA - mtEFG2 in combination with Flag - EV or Flag - mtRRF , and Co - IP was performed with an anti - Flag antibody prior to western blot analysis .

O O O O O O O O B-CONTROLLED_VAR 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

A HEK293T cells were transfected with HA-mtEFG2 in combination with Flag-EV or Flag-mtRRF, and Co-IP was performed with an anti-Flag antibody prior to western blot analysis.


B PLC cells stably expressing Flag - mtEFG2 and HA - mtRRF were further infected with viruses expressing NTC or shPHGDH , and then cell lysates were immunoprecipitated with anti - Flag antibody or IgG prior to western blot analysis .

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

B PLC cells stably expressing Flag-mtEFG2 and HA-mtRRF were further infected with viruses expressing NTC or shPHGDH, and then cell lysates were immunoprecipitated with anti-Flag antibody or IgG prior to western blot analysis.


C PLC cells stably overexpressing mtEFG2 were further infected with viruses expressing NTC or shPHGDH . The translation efficiency was measured by a SUnSET assay .

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

C PLC cells stably overexpressing mtEFG2 were further infected with viruses expressing NTC or shPHGDH. The translation efficiency was measured by a SUnSET assay.


D PLC cells stably overexpressing mtEFG2 were further infected with viruses expressing NTC or shPHGDH . mtDNA - encoded proteins were analysed by western blot . β - Actin was used as the loading control .

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

D PLC cells stably overexpressing mtEFG2 were further infected with viruses expressing NTC or shPHGDH. mtDNA-encoded proteins were analysed by western blot. β-Actin was used as the loading control.


E PLC cell lysates were immunoprecipitated with an anti - MRPL44 antibody or IgG prior to western blot analysis of MRPL44 , MRPL46 , MRPL48 , MRPS15 , MRPS16 , and MRPS35 .

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E PLC cell lysates were immunoprecipitated with an anti-MRPL44 antibody or IgG prior to western blot analysis of MRPL44, MRPL46, MRPL48, MRPS15, MRPS16, and MRPS35.


F Binding of mtDNA - encoded mRNA by endogenous MRPL44 ( representing mitochondrial ribosomes ) was evaluated by RNA immunoprecipitation ( RIP ) in PLC cells with PHGDH knockdown .

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 B-CONTROLLED_VAR O O

F Binding of mtDNA-encoded mRNA by endogenous MRPL44 (representing mitochondrial ribosomes) was evaluated by RNA immunoprecipitation (RIP) in PLC cells with PHGDH knockdown.


G The same amount of mitochondria from PLC cells expressing NTC or shPHGDH treated with CAP and TAP were loaded onto the sucrose density gradient . After centrifugation , all fractions were subjected to western blot analysis of MRPS35 , MRPL44 , and MRPL48 proteins . RNA concentrations of ND6 were measured .

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

G The same amount of mitochondria from PLC cells expressing NTC or shPHGDH treated with CAP and TAP were loaded onto the sucrose density gradient. After centrifugation, all fractions were subjected to western blot analysis of MRPS35, MRPL44, and MRPL48 proteins. RNA concentrations of ND6 were measured.


A PLC cells overexpressing EV , PHGDHWT , or PHGDHR236E were further infected with viruses expressing NTC or shmtEFG2 . The OCR was measured after successive injections of oligomycin ( oligo ) , FCCP , and antimycin A / rotenone ( AA / Rot ) . Basal respiration , ATP - linked respiration , and maximal respiration were analysed with the OCR curve .

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

A PLC cells overexpressing EV, PHGDHWT, or PHGDHR236E were further infected with viruses expressing NTC or shmtEFG2. The OCR was measured after successive injections of oligomycin (oligo), FCCP, and antimycin A/rotenone (AA/Rot). Basal respiration, ATP-linked respiration, and maximal respiration were analysed with the OCR curve.


B PLC cells overexpressing EV , PHGDHWT , or PHGDHR236E were further infected with viruses expressing NTC or shmtEFG2 prior to western blot analysis . β - Actin was used as the loading control ( left ) . Cell growth curves were constructed by cell counting with trypan blue exclusion ( right ) .

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 O O O O O O O O

B PLC cells overexpressing EV, PHGDHWT, or PHGDHR236E were further infected with viruses expressing NTC or shmtEFG2 prior to western blot analysis. β-Actin was used as the loading control (left). Cell growth curves were constructed by cell counting with trypan blue exclusion (right).


C PLC cells overexpressing EV , PHGDHWT , or PHGDHR236E were further treated with H2O or tigecycline prior to western blot analysis . β - Actin was used as the loading control ( left ) . Cell growth curves were determined by cell counting with trypan blue exclusion ( right ) .

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

C PLC cells overexpressing EV, PHGDHWT, or PHGDHR236E were further treated with H2O or tigecycline prior to western blot analysis. β-Actin was used as the loading control (left). Cell growth curves were determined by cell counting with trypan blue exclusion (right).


D - F PLC cells overexpressing EV or PHGDH were further infected with viruses expressing NTC or shmtEFG2 . Then , these cells were subcutaneously injected into male nude mice ( n = 5 in each group ) . Tumour sizes were measured beginning 14 days after inoculation ( D ) . At the end of the experiment , the tumours were excised ( E ) , and PHGDH and mtEFG2 expression in tumour lysates was analysed by western blotting ( F ) .

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

D-F PLC cells overexpressing EV or PHGDH were further infected with viruses expressing NTC or shmtEFG2. Then, these cells were subcutaneously injected into male nude mice (n = 5 in each group). Tumour sizes were measured beginning 14 days after inoculation (D). At the end of the experiment, the tumours were excised (E), and PHGDH and mtEFG2 expression in tumour lysates was analysed by western blotting (F).


G - I PLC cells overexpressing EV or PHGDH were subcutaneously injected into male nude mice ( n = 5 in each group ) . Mice were intraperitoneally injected with 100 mg / kg tigecycline daily beginning 14 days after inoculation . Tumour sizes were measured beginning 14 days after inoculation ( G ) . At the end of the experiment , the tumours were excised ( H ) , and PHGDH expression in tumour lysates was analysed by western blotting ( I ) .

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

G-I PLC cells overexpressing EV or PHGDH were subcutaneously injected into male nude mice (n = 5 in each group). Mice were intraperitoneally injected with 100 mg/kg tigecycline daily beginning 14 days after inoculation. Tumour sizes were measured beginning 14 days after inoculation (G). At the end of the experiment, the tumours were excised (H), and PHGDH expression in tumour lysates was analysed by western blotting (I).


( A - F ) TNF - α , Cxcl1 and Cxcl2 ( A , C , and E respectively ) were measured at 24 h after stimulation of bone - marrow - derived neutrophils ( 5 x 105 cells ) with increasing concentrations of bacterial nucleic acids ( 1 , 5 , 10 and 20 µg of DNA + RNA in equal proportions ) , HK - bacteria ( 1 , 5 , 10 or 20 μg / ml ) or live bacteria ( MOIs of 2 , 5 , 10 and 20 ) . LPS ( 1 , 10 , 100 and 1000 ng / ml ) was used as a positive control stimulus . ( B , D and F ) , Effect of FPR antagonist Boc - 2 on GBS - induced TNF - α , Cxcl1 and Cxcl2 production , respectively . Bone - marrow - derived neutrophils were exposed to Boc - 2 ( 50 µM ) for 1 hour and then stimulated with HK - GBS ( 10 µg / ml ) or live GBS ( MOI of 5 ) . Data are expressed as means + standard deviations of measurements from three independent experiments conducted in duplicate . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , as determined by unpaired t - test ; ns , not significant .

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

(A-F) TNF-α, Cxcl1 and Cxcl2 (A, C, and E respectively) were measured at 24 h after stimulation of bone-marrow-derived neutrophils (5 x 105 cells) with increasing concentrations of bacterial nucleic acids (1, 5, 10 and 20 µg of DNA + RNA in equal proportions), HK-bacteria (1, 5, 10 or 20 μg/ml) or live bacteria (MOIs of 2, 5, 10 and 20). LPS (1, 10, 100 and 1000 ng/ml) was used as a positive control stimulus. (B, D and F), Effect of FPR antagonist Boc-2 on GBS-induced TNF-α, Cxcl1 and Cxcl2 production, respectively. Bone-marrow-derived neutrophils were exposed to Boc-2 (50 µM) for 1 hour and then stimulated with HK-GBS (10 µg/ml) or live GBS (MOI of 5). Data are expressed as means + standard deviations of measurements from three independent experiments conducted in duplicate. *P < 0.05, **P < 0.01, ***P < 0.001, as determined by unpaired t-test; ns, not significant.


Bone - marrow - derived neutrophils were exposed to Boc - 2 ( 10 µM ) or WRW4 ( 1 , 5 and 10 µM ) for 1 hour and then stimulated with HK - GBS ( 10 µg / ml ) or live GBS ( MOI of 5 ) . Data are expressed as means + standard deviations of measurements from three independent experiments conducted in duplicate . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , as determined by unpaired t - test ; ns , not significant .

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

Bone-marrow-derived neutrophils were exposed to Boc-2 (10 µM) or WRW4 (1, 5 and 10 µM) for 1 hour and then stimulated with HK-GBS (10 µg/ml) or live GBS (MOI of 5). Data are expressed as means + standard deviations of measurements from three independent experiments conducted in duplicate. *P < 0.05, **P < 0.01, ***P < 0.001, as determined by unpaired t-test; ns, not significant.


( A - F ) Cxcl1 and Cxcl2 concentrations in 24 - h culture supernatants of neutrophils ( A and B ) , CSF - polarized ( C and D ) or GM - CSF - polarized ( E and F ) BMDMs ( ΜΦ ) from WT or Fpr - deficient mice stimulated with live GBS ( MOI of 2 , 5 , and 10 ) or HK - GBS ( 10 µg / ml ) . LPS ( 100 ng / ml ) was used as a positive control stimulus . Means + SD of data from three independent experiments conducted in duplicate . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 vs WT mice , determined by unpaired t - test .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F) Cxcl1 and Cxcl2 concentrations in 24-h culture supernatants of neutrophils (A and B), CSF-polarized (C and D) or GM-CSF-polarized (E and F) BMDMs (ΜΦ) from WT or Fpr-deficient mice stimulated with live GBS (MOI of 2, 5, and 10) or HK-GBS (10 µg/ml). LPS (100 ng/ml) was used as a positive control stimulus. Means + SD of data from three independent experiments conducted in duplicate. *P < 0.05, **P < 0.01, ***P < 0.001 vs WT mice, determined by unpaired t-test.


( E ) , Kaplan - Meier analysis of survival rates in WT , Fpr1 and Fpr2 KO mice after i . p . challenge with 2x105 bacteria . Data are the cumulative results from two experiments , each involving 8 animals per group . * * P < 0 . 01 , * * * * P < 0 . 0001 vs WT mice , as determined by Kaplan - Meier analysis . Data information : means + SDs of three duplicate determinations , each conducted in a different animal .

O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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), Kaplan-Meier analysis of survival rates in WT, Fpr1 and Fpr2 KO mice after i.p. challenge with 2x105 bacteria. Data are the cumulative results from two experiments, each involving 8 animals per group. **P < 0.01, ****P < 0.0001 vs WT mice, as determined by Kaplan-Meier analysis. Data information: means + SDs of three duplicate determinations, each conducted in a different animal.


( F - G ) Neutrophil influx ( F ) and bacterial burden ( G ) in peritoneal lavage fluid samples collected from Fpr2 - / - mice at 3 h after i . p . challenge with live GBS ( 2x107 CFU ) . At 1h post - challenge mice were inoculated i . p . with 1 μg of recombinant Cxcl1 or Cxcl2 or with vehicle . Data information : , individual data points and means . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 versus vehicle treated mice determined by unpaired t test or Mann - Whitney U

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

(F-G) Neutrophil influx (F) and bacterial burden (G) in peritoneal lavage fluid samples collected from Fpr2-/- mice at 3 h after i.p. challenge with live GBS (2x107 CFU). At 1h post-challenge mice were inoculated i.p. with 1 μg of recombinant Cxcl1 or Cxcl2 or with vehicle. Data information: , individual data points and means. *P < 0.05, **P < 0.01, ***P < 0.001 versus vehicle treated mice determined by unpaired t test or Mann-Whitney U test


( C and D ) , Cxcl2 release in neutrophil cultures stimulated with combinations of HK - bacteria , GBS fMet peptides or TLR agonists . Neutrophils were exposed to a 50 µM concentration of Fpr agonists ( fMIFL , WKYMVM or fPep8 and fPep10 ) for 1 h before the addition of the indicated TLR agonists , which included HK - GBS ( 10 μg / ml ) , LPS ( 1 µg / ml ) and RNA ( 1 µg / ml ) . In some cases , neutrophils were exposed to graded doses ( 10 , 20 and 50 µM ) of the indicated Fpr agonists alone . Data information : Data are expressed as means + SD from three independent experiments , each conducted in duplicate . * * P < 0 . 01 , * * * P < 0 . 001 , as determined by unpaired t - test .

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 B-CONTROLLED_VAR O 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 B-CONTROLLED_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 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 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

(C and D), Cxcl2 release in neutrophil cultures stimulated with combinations of HK-bacteria, GBS fMet peptides or TLR agonists. Neutrophils were exposed to a 50 µM concentration of Fpr agonists (fMIFL, WKYMVM or fPep8 and fPep10) for 1 h before the addition of the indicated TLR agonists, which included HK-GBS (10 μg/ml), LPS (1 µg/ml) and RNA (1 µg /ml). In some cases, neutrophils were exposed to graded doses (10, 20 and 50 µM) of the indicated Fpr agonists alone. Data information: Data are expressed as means + SD from three independent experiments, each conducted in duplicate. **P < 0.01, ***P < 0.001, as determined by unpaired t-test.


( A ) , Effect of actinonin on Cxcl2 production by neutrophils . Neutrophils were stimulated with live GBS ( MOI 5 ) in the presence or absence of the formyl peptidase inhibitor actinonin ( 32µg / ml ) . Data information : Data are means + SD from three independent experiments conducted in duplicate . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , as determined by unpaired t - test .

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(A), Effect of actinonin on Cxcl2 production by neutrophils. Neutrophils were stimulated with live GBS (MOI 5) in the presence or absence of the formyl peptidase inhibitor actinonin (32µg/ml). Data information: Data are means + SD from three independent experiments conducted in duplicate. *P < 0.05, **P < 0.01, ***P < 0.001, as determined by unpaired t-test.


( D ) , Effect of treatment with ROS scavengers / inhibitors on Cxcl2 production . Neutrophils were treated with the singlet oxygen scavenger MnTBAP ( 100 μM ) , the hydrogen peroxide scavenger DMTU ( 10 mM ) , the NADPH oxidase inhibitor DPI ( 20 μM ) or the ASK1 inhibitor GS - 444217 ( 1 μM ) for 1 h before the addition of live GBS ( MOI of 5 ) . Data information : Data are means + SD from three independent experiments conducted in duplicate . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , as determined by unpaired t - test .

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

(D), Effect of treatment with ROS scavengers/inhibitors on Cxcl2 production. Neutrophils were treated with the singlet oxygen scavenger MnTBAP (100 μM), the hydrogen peroxide scavenger DMTU (10 mM), the NADPH oxidase inhibitor DPI (20 μM) or the ASK1 inhibitor GS-444217 (1 μM) for 1 h before the addition of live GBS (MOI of 5). Data information: Data are means + SD from three independent experiments conducted in duplicate. *P < 0.05, **P < 0.01, ***P < 0.001, as determined by unpaired t-test.


( A - B ) Bone - marrow - derived neutrophils were exposed to different concentrations of signaling molecule ( A ) or transcription factors inhibitors ( B ) for 1 hour and then stimulated with live GBS ( MOI 5 ) . Target molecules are indicated in parentheses . Data are means + SD from three independent experiments conducted in duplicate . * * P < 0 . 01 , * * * P < 0 . 001 , as determined by unpaired t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Bone-marrow-derived neutrophils were exposed to different concentrations of signaling molecule (A) or transcription factors inhibitors (B) for 1 hour and then stimulated with live GBS (MOI 5). Target molecules are indicated in parentheses. Data are means + SD from three independent experiments conducted in duplicate. **P < 0.01, ***P < 0.001, as determined by unpaired t-test.


( C ) , Effect of p38MAPK inhibition on the activation of AP - 1 family members in neutrophils stimulated with live GBS . Neutrophils were pretreated with SB202190 ( 5μM ) , stimulated for 90min with live GBS ( MOI 5 ) , lysed and assayed for AP - 1 activation . Data are means + SD from three independent experiments conducted in duplicate . * P < 0 . 05 , * * * P < 0 . 001 , as determined by unpaired t - test .

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(C), Effect of p38MAPK inhibition on the activation of AP-1 family members in neutrophils stimulated with live GBS. Neutrophils were pretreated with SB202190 (5μM), stimulated for 90min with live GBS (MOI 5), lysed and assayed for AP-1 activation. Data are means + SD from three independent experiments conducted in duplicate. *P < 0.05, ***P < 0.001, as determined by unpaired t-test.


( D ) , Effect of the absence of Fpr1 , Fpr2 or the TLR adaptor MyD88 on the activation of AP - 1 family members in neutrophils . Neutrophils from WT , Fpr - or MyD88 - deficient mice were stimulated for 90 min with live GBS ( MOI 5 ) , lysed and assayed for AP - 1 activation . Data are means + SD from three independent experiments conducted in duplicate . * P < 0 . 05 , * * * P < 0 . 001 vs WT mice , as determined by unpaired t - test .

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

(D), Effect of the absence of Fpr1, Fpr2 or the TLR adaptor MyD88 on the activation of AP-1 family members in neutrophils. Neutrophils from WT, Fpr- or MyD88-deficient mice were stimulated for 90 min with live GBS (MOI 5), lysed and assayed for AP-1 activation. Data are means + SD from three independent experiments conducted in duplicate. *P < 0.05, ***P < 0.001 vs WT mice, as determined by unpaired t-test.


( E ) , Effect of TAK1 inhibition on p38 MAPK activation in neutrophils stimulated with live GBS ( MOI 5 ) .

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(E), Effect of TAK1 inhibition on p38 MAPK activation in neutrophils stimulated with live GBS (MOI 5).


( F ) , Effect of the absence of Fpr1 , Fpr2 or the TLR adaptor MyD88 on p38MAPK activation in neutrophils stimulated with live GBS ( MOI 5 ) . Shown is a blot representative of three separate experiments .

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(F), Effect of the absence of Fpr1, Fpr2 or the TLR adaptor MyD88 on p38MAPK activation in neutrophils stimulated with live GBS (MOI 5). Shown is a blot representative of three separate experiments.


A ) ELISA quantification of PD - L1 , pTau 181 and Aβx - 42 in the CSF of control and AD patients ( n = 10 + / - SEM , Student ' s t - test , * p < 0 . 05 , * * * p < 0 . 001 , PD - L1 : t = 2 . 47 df = 18 , Aβx - 42 : t = 4 . 83 df = 18 , pTau 181 : t = 5 , 768 df = 18 ) .

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

A) ELISA quantification of PD-L1, pTau 181 and Aβx-42 in the CSF of control and AD patients (n=10 +/-SEM, Student's t-test, * p<0.05, *** p<0.001, PD-L1: t=2.47 df = 18, Aβx-42: t=4.83 df=18, pTau 181: t=5,768 df=18).


B , C ) ( B ) Colocalization of PD - L1 and GFAP in AD and ( C ) in control patients ( MX - O4 = amyloid deposits , upper picture : bar = 100 µm , lower picture : bar = 30 µm ) . D ) Immunohistochemical detection of PD - L1 in 9 - month - old female APP / PS1 mice ( bar = 50 µM ) . E ) Immunohistochemical detection of PD - L1 in 4 - month - old female APP / PS1 mice ( bar = 20 µm ) .

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

B, C) (B) Colocalization of PD-L1 and GFAP in AD and (C) in control patients (MX-O4 = amyloid deposits, upper picture: bar = 100 µm, lower picture: bar=30 µm). D) Immunohistochemical detection of PD-L1 in 9-month-old female APP/PS1 mice (bar = 50 µM). E) Immunohistochemical detection of PD-L1 in 4-month-old female APP/PS1 mice (bar = 20 µm).


A ) C6 cells expressing PD - L1 - myc treated with the γ - secretase inhibitor DAPT for 18 h . PD - L1 was detected using a myc - antibody .

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A) C6 cells expressing PD-L1-myc treated with the γ-secretase inhibitor DAPT for 18 h. PD-L1 was detected using a myc-antibody.


B ) Immunoblot of HEK293 cells expressing PD - L1 - myc and presenilin 1 ( hPS1 ) or a presenilin 1 dominant negative mutant ( hPS1 D246A ) .

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B) Immunoblot of HEK293 cells expressing PD-L1-myc and presenilin 1 (hPS1) or a presenilin 1 dominant negative mutant (hPS1 D246A).


D ) 18 h 1 μM PMA treatment of C6 cells expressing PD - L1 - myc . Secretion of PD - L1 in the conditioned medium ( CM ) was detected using antibody AF1019 .

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D) 18 h 1 μM PMA treatment of C6 cells expressing PD-L1-myc. Secretion of PD-L1 in the conditioned medium (CM) was detected using antibody AF1019.


E ) Mouse astrocytes were incubated for 48 h with 1 μM PMA and 10 μg / ml TNFα / 100 U / μl IFNγ and lysates and the CM were immunoblotted using antibody AF1019 .

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E) Mouse astrocytes were incubated for 48 h with 1 μM PMA and 10 μg/ml TNFα / 100 U/μl IFNγ and lysates and the CM were immunoblotted using antibody AF1019.


A ) Colocalization of PD - 1 and CD11b ( MX - 04 = methoxy - XO4 , stains amyloid deposits ) in AD by immunohistochemistry ( bar = 50 µm ) . B ) Colocalization of PD - 1 and Iba1 or PD - 1 and Aβ in a female APP / PS1 mouse at 9 months of age by immunohistochemistry ( bar = 20 µm ) .

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A) Colocalization of PD-1 and CD11b (MX-04=methoxy-XO4, stains amyloid deposits) in AD by immunohistochemistry (bar=50 µm). B) Colocalization of PD-1 and Iba1 or PD-1 and Aβ in a female APP/PS1 mouse at 9 months of age by immunohistochemistry (bar=20 µm).


E ) ELISA analysis of TNFα secretion by wild type ( wt ) and PD - 1 - / - microglia induced by 0 . 5 µM Aβ1 - 42 for 6 h in vitro ( n = 3 biological replicates , one - way ANOVA ( DF = 1 , F = 8 . 29 , p = 0 . 021 ) , Tukey ' s HSD , * p < 0 . 05 ) . Data information : central bands represent median , boxes show interquartile range ( IQR ) and whiskers define the + / - 1 . 5xIQR .

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

E) ELISA analysis of TNFα secretion by wild type (wt) and PD-1-/- microglia induced by 0.5 µM Aβ1-42 for 6 h in vitro (n=3 biological replicates, one-way ANOVA (DF = 1, F = 8.29, p= 0.021), Tukey's HSD, * p<0.05). Data information: central bands represent median, boxes show interquartile range (IQR) and whiskers define the +/-1.5xIQR.


F - H ) Microglia from methoxy - XO4 - injected , 8 - month - old female , wild type ( wt ) and APP / PS1 mice were analyzed ( F ) for PD - 1 ( n = 5 biological replicates per group , one - way ANOVA ( DF = 2 , F = 13 . 99 , p = 0 . 0013 ) , Tukey ' s HSD , * * p < 0 . 01 ) , ( G ) for CD11b ( n = 5 biological replicates per group , one - way ANOVA ( DF = 2 , F = 30 . 06 , p = 5 . 47x10 - 05 ) , Tukey ' s HSD , * * * p < 0 . 001 ) and ( H ) CD45 by flow cytometry ( n = 5 biological replicates per group , one - way ANOVA ( DF = 1 , F = 24 . 96 , p = 0 . 00013 ) , Tukey ' s HSD , * * * p < 0 . 001 ) and normalized to the expression of wild type mice ( represented by dashed lines ) . Data information : central bands represent median , boxes show interquartile range ( IQR ) and whiskers define the + / - 1 . 5xIQR .

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

F-H) Microglia from methoxy-XO4-injected, 8-month-old female, wild type (wt) and APP/PS1 mice were analyzed (F) for PD-1 (n=5 biological replicates per group, one-way ANOVA (DF = 2, F = 13.99, p=0.0013), Tukey's HSD, ** p<0.01), (G) for CD11b (n=5 biological replicates per group, one- way ANOVA (DF = 2, F = 30.06, p=5.47x10-05), Tukey's HSD, *** p<0.001) and (H) CD45 by flow cytometry (n=5 biological replicates per group, one-way ANOVA (DF = 1, F = 24.96, p=0.00013), Tukey's HSD, *** p<0.001) and normalized to the expression of wild type mice (represented by dashed lines). Data information: central bands represent median, boxes show interquartile range (IQR) and whiskers define the +/-1.5xIQR.


A , B ) ELISA of the ( A ) RIPA soluble fraction of female APP / PS1 and APP / PS1 PD - 1 - / - mice for Aβx - 40 ( biological replicates with n = 6 for APP / PS1 and n = 5 for APP / PS1 PD - 1 - / - , mean + / - SEM , one - way ANOVA ( DF = 1 , F = 0 . 6 , p = 0 . 45 ) , Tukey ' s HSD , * * p < 0 . 01 , * * * p < 0 . 001 ) and ( B ) of the SDS soluble fraction ( biological replicates with n = 6 for APP / PS1 and n = 5 for APP / PS1 PD - 1 - / - , mean + / - SEM , one - way ANOVA ( DF = 1 , F = 32 . 8 , p = 2x10 - 5 ) , Tukey ' s HSD , * * p < 0 . 01 , * * * p < 0 . 001 ) .

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

A, B) ELISA of the (A) RIPA soluble fraction of female APP/PS1 and APP/PS1 PD-1-/- mice for Aβx-40 (biological replicates with n=6 for APP/PS1 and n=5 for APP/PS1 PD-1-/-, mean+/-SEM, one-way ANOVA (DF = 1, F = 0.6, p=0.45), Tukey's HSD, ** p<0.01, *** p<0.001) and (B) of the SDS soluble fraction (biological replicates with n=6 for APP/PS1 and n=5 for APP/PS1 PD-1-/-, mean+/-SEM, one-way ANOVA(DF=1, F= 32.8, p=2x10-5), Tukey's HSD, ** p<0.01, *** p<0.001).


D , E ) Mice were analyzed for ( D ) percentage of the covered areas ( 5 sections per mouse analyzed with the mean representing an individual data point ; biological replicates with n = 6 for APP / PS1 and n = 9 for APP / PS1 PD - 1 - / - , mean + / - SEM , one - way ANOVA ( DF = 1 , F = 0 . 905 , p = 0 . 90 ) , Tukey ' s HSD , * * p < 0 . 01 , * * * p < 0 . 001 ) and ( E ) the number of plaques per mm2 ( 5 sections per mouse analyzed with the mean representing an individual data point ; biological replicates with n = 6 for APP / PS1 and n = 9 for APP / PS1 PD - 1 - / - , mean + / - SEM , one - way ANOVA ( DF = 1 , F = 4 . 08 , p = 0 . 054 ) , Tukey ' s HSD , * * p < 0 . 01 , * * * p < 0 . 001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O 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 I-CONTROLLED_VAR I-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 O O O O O O O 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 I-CONTROLLED_VAR I-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

D, E) Mice were analyzed for (D) percentage of the covered areas (5 sections per mouse analyzed with the mean representing an individual data point; biological replicates with n=6 for APP/PS1 and n=9 for APP/PS1 PD-1-/-, mean +/-SEM, one-way ANOVA (DF=1, F=0.905, p=0.90), Tukey's HSD, ** p<0.01, *** p<0.001) and (E) the number of plaques per mm2 (5 sections per mouse analyzed with the mean representing an individual data point; biological replicates with n=6 for APP/PS1 and n=9 for APP/PS1 PD-1-/-, mean +/-SEM, one-way ANOVA (DF=1, F=4.08, p=0.054), Tukey's HSD, ** p<0.01, *** p<0.001).


G ) Time needed to reach the hidden platform ( latency in seconds ) in the Morris water maze test ( mean + / - SEM of biological replicates with n = 12 for wt , n = 5 for PD - 1 - / - , n = 7 for APP / PS1 , and n = 8 for APP / PS1 PD - 1 - / - , one - way ANOVA ( DF = 3 , F = 17 . 72 , p = 1x10 - 9 ) , Tukey ' s HSD , * p < 0 . 05 , * * * p < 0 . 001 ) .

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

G) Time needed to reach the hidden platform (latency in seconds) in the Morris water maze test (mean +/-SEM of biological replicates with n=12 for wt, n=5 for PD-1-/-, n=7 for APP/PS1, and n=8 for APP/PS1 PD-1-/-, one-way ANOVA (DF=3, F=17.72, p=1x10-9), Tukey's HSD, * p<0.05, *** p<0.001).


A ) In vitro phagocytosis of FAM - Aβ1 - 42 by wild type or PD - 1 - / - microglia for up to 6 h ( mean + / - SEM of a technical quadruplicate , Student ' s t - test , * * p < 0 . 01 , * * * p < 0 . 001 , representative result of an n = 2 biological replicate ) . B ) In vitro phagocytosis of FAM - Aβ1 - 42 by wild type microglia cultured in astrocyte - conditioned medium ( ACM ) from wild type or PD - L1 - / - astroglial cultures ( mean + / - SEM of a technical quadruplicate , Student ' s t - test , * p < 0 . 05 , * * * p < 0 . 001 , representative result of an n = 2 biological replicate )

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A) In vitro phagocytosis of FAM-Aβ1-42 by wild type or PD-1-/- microglia for up to 6 h (mean +/-SEM of a technical quadruplicate, Student's t-test, ** p<0.01, *** p<0.001, representative result of an n=2 biological replicate). B) In vitro phagocytosis of FAM-Aβ1-42 by wild type microglia cultured in astrocyte-conditioned medium (ACM) from wild type or PD-L1-/- astroglial cultures (mean +/-SEM of a technical quadruplicate, Student's t-test, * p<0.05, *** p<0.001, representative result of an n=2 biological replicate)


D ) Quantification of microglial in vivo phagocytosis in female APP / PS1 and APP / PS1 PD - 1 - / - mice at 8 . 5 month of age after intraperitoneal injection of methoxy - XO4 ( biological replicates with n = 5 for APP / PS1 and n = 4 for APP / PS1 PD - 1 - / - mice , Student ' s t - test ( t = 2 . 1808 , df = 6 . 9829 ) , * p < 0 . 05 ) . Data information : central bands represent median , boxes show interquartile range ( IQR ) and whiskers define the + / - 1 . 5xIQR .

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D) Quantification of microglial in vivo phagocytosis in female APP/PS1 and APP/PS1 PD-1-/- mice at 8.5 month of age after intraperitoneal injection of methoxy-XO4 (biological replicates with n=5 for APP/PS1 and n=4 for APP/PS1 PD-1-/- mice, Student's t-test (t = 2.1808, df = 6.9829), * p<0.05). Data information: central bands represent median, boxes show interquartile range (IQR) and whiskers define the +/-1.5xIQR.