C Immunofluorescence microscopy of HeLa expressing GFP ‐ KSHV ‐ TK ‐ DEAD ( arrowheads ) with indicated phospho antibodies .

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C Immunofluorescence microscopy analysis of HeLa cells expressing GFP‐KSHV‐TK‐DEAD (white arrowheads) with the indicated phospho antibodies.


D Immunofluorescence microscopy of localisation of FAK zyxin in HeLa expressing GFP ‐ KSHV ‐ TK ‐ DEAD ( arrowheads ) .

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D Immunofluorescence microscopy images of the localisation of FAK and zyxin in HeLa cells expressing GFP‐KSHV‐TK‐DEAD (white arrowheads).


E Purified recombinant GST ‐ EBV ‐ TK , GST ‐ MuHV ‐ 4 - TK GST ‐ KSHV ‐ TKimmunoblotted for phosphotyrosine GST following a sequential dephosphorylation kinase reaction .

E Purified recombinant GST‐EBV‐TK, GST‐MuHV‐4-TK and GST‐KSHV‐TKimmunoblotted for phosphotyrosine and GST following a sequential dephosphorylation and kinase reaction.


F Purified recombinant GST ‐ KSHV ‐ TK GST ‐ KSHV ‐ TK ‐ KINASE ‐ DEAD immunoblotted for phosphotyrosine GST .

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F Purified recombinant GST‐KSHV‐TK and GST‐KSHV‐TK‐KINASE‐DEAD immunoblotted for phosphotyrosine and GST.


G Immunoblot demonstrating that N ‐ terminus of KSHV ‐ TK is only auto ‐ phosphorylated by kinase domain in cis .

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G Immunoblot analysis demonstrating that the N‐terminus of KSHV‐TK is only auto‐phosphorylated by the kinase domain in cis.


A Immunoblot of N ‐ terminal deletions of KSHV ‐ TK demonstrates that first 100 are required for tyrosine phosphorylation .

A Immunoblot analysis of N‐terminal deletions of KSHV‐TK demonstrates that the first 100 amino acids are required for tyrosine phosphorylation.


B Immunoblot reveals that mutation of tyrosines 65 , 85 120 ablates tyrosine phosphorylation of KSHV ‐ TK .

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B Immunoblot analysis reveals that mutation of tyrosines 65, 85 and 120 ablates tyrosine phosphorylation of the KSHV‐TK.


C - E Immunofluorescence microscopy with indicated antibodies reveals that mutation of tyrosines 65 , 85 120 to phenylalanine abrogates ability of GFP ‐ KSHV ‐ TK to induce loss of phosphotyrosine epitopes , phospho ‐ paxillin phospho ‐ FAK from focal adhesions .

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C-E Immunofluorescence microscopy analysis with the indicated antibodies reveals that mutation of tyrosines 65, 85 and 120 to phenylalanine abrogates the ability of GFP‐KSHV‐TK to induce loss of phosphotyrosine epitopes, phospho‐paxillin and phospho‐FAK from focal adhesions.


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F Immunoblot of level of GTP ‐ bound total RhoA in expressing indicated GFP ‐ tagged KSHV ‐ TK .

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F Immunoblot analysis of level of GTP‐bound and total RhoA in cells expressing the indicated GFP‐tagged KSHV‐TK proteins.


G Immunofluorescence microscopy reveals that localisation of paxillin at focal adhesions is unaffected by KSHV ‐ TK ‐ Y3F expressed in context of lytic infection .

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G Immunofluorescence microscopy analysis reveals that localisation of paxillin at focal adhesions is unaffected by KSHV‐TK‐Y3F expressed in the context of lytic infection.


A Immunoblot reveals that GFP ‐ KSHV ‐ TK WT , Y2F Y3F but not GFP ‐ KSHV ‐ TK ‐ DEAD associate with FAK .

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A Immunoblot analysis reveals that GFP‐KSHV‐TK WT, Y2F and Y3F but not GFP‐KSHV‐TK‐DEAD associate with FAK.


B GFP ‐ KSHV ‐ TK does not displace paxillin or zyxin from focal adhesions ( arrowheads ) or induce contraction in absence of FAK . Scale bar , 20 μm .

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B GFP‐KSHV‐TK does not displace paxillin or zyxin from focal adhesions (white arrowheads) or induce contraction in the absence of FAK. Scale bar, 20 μm.


C GFP ‐ KSHV ‐ TK does not induce loss of zyxin p ‐ Tyr from focal adhesions or contraction of HeLa depleted of paxillin . Error bars represent from three independent experiments , n = 196 for mock siRNA n = 229 for paxillin siRNA ; * * * P 0 . 001 . Scale bar , 20 μm .

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C GFP‐KSHV‐TK does not induce loss of zyxin and p‐Tyr from focal adhesions or contraction of HeLa cells depleted of paxillin. Error bars represent SEM from three independent experiments, n = 196 for mock siRNA and n = 229 for paxillin siRNA; ***P 0.001. Scale bar, 20 μm.


B Immunoblot of GFP antibody immunoprecipitates reveals that GFP ‐ KSHV ‐ TK but not Y2F or GFP associates with CrkI , II L .

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B Immunoblot analysis of GFP antibody immunoprecipitates reveals that GFP‐KSHV‐TK but not Y2F or GFP associates with CrkI, II and L.


C Immunofluorescence microscopy reveals CrkII associates with GFP ‐ KSHV ‐ TK but not Y2F mutant . Scale bars , 20 μm .

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C Immunofluorescence microscopy analysis reveals CrkII associates with GFP‐KSHV‐TK but not the Y2F mutant. Scale bars, 20 μm.


D Immunoblot of RFP antibody immunoprecipitates reveals that RFP ‐ KSHV ‐ TK specifically associates with SH2 domain of CrkII CrkL .

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D Immunoblot analysis of RFP antibody immunoprecipitates reveals that RFP‐KSHV‐TK specifically associates with the SH2 domain of CrkII and CrkL.


E Immunofluorescence microscopy of phosphotyrosine epitopes in expressing GFP ‐ CrkII SH2 , GFP ‐ CrkL SH2 , RFP ‐ KSHV ‐ TK or a combination of CrkII / LSH2 RFP ‐ KSHV ‐ TK . Arrowheads represent contracted KSHV ‐ TK ‐ expressing . SH2 domains of CrkII or L block KSHV ‐ TK ‐ induced rounding . Scale bars , 20 μm .

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E Immunofluorescence microscopy analysis of phosphotyrosine epitopes in cells expressing GFP‐CrkII SH2, GFP‐CrkL SH2, RFP‐KSHV‐TK or a combination of CrkII/LSH2 and RFP‐KSHV‐TK. Arrowheads represent contracted KSHV‐TK‐expressing cells. The SH2 domains of CrkII or L block KSHV‐TK‐induced cell rounding. Scale bars, 20 μm.


B Immunoblot reveals that GFP ‐ KSHV ‐ TK WT Y2F but not Y120F associate with p85 of PI3 ‐ Kinase .

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B Immunoblot analysis reveals that GFP‐KSHV‐TK WT and Y2F but not Y120F associate with p85 of PI3‐Kinase.


C Immunoblot of p85PI3 ‐ Kinase antibody immunoprecipitates reveals that GFP ‐ KSHV ‐ TK its Y2F mutant but not Y120F associate with p85 of PI3 ‐ Kinase .

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C Immunoblot analysis of p85PI3‐Kinase antibody immunoprecipitates reveals that GFP‐KSHV‐TK and its Y2F mutant but not Y120F associate with p85 of PI3‐Kinase.


D Immunoblot of CrkII CrkL antibody immunoprecipitates reveals that GFP ‐ KSHV ‐ TK p85PI3 ‐ Kinase associate with CrkII CrkL . No association is observed with Y2F , Y3F or kinase ‐ dead TK .

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D Immunoblot analysis of CrkII and CrkL antibody immunoprecipitates reveals that GFP‐KSHV‐TK and the p85PI3‐Kinase associate with CrkII and CrkL. No association is observed with the Y2F, Y3F or kinase‐dead TK.


E Tyrosine phosphorylation of CrkII CrkL is only upregulated in expressing GFP ‐ KSHV ‐ TK .

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E Tyrosine phosphorylation of CrkII and CrkL is only upregulated in cells expressing GFP‐KSHV‐TK.


F Immunoblot reveals that recombinant His ‐ CrkII binds to is phosphorylated by KSHV ‐ TK but not Y2F or Y3F mutants .

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F Immunoblot analysis reveals that recombinant His‐CrkII binds to and is phosphorylated by KSHV‐TK but not the Y2F or Y3F mutants.


( A ) expression pattern of FOXM1 in human cortex at GW15 . VZ , ventricular zone ; ISVZ , inner subventricular zone ; OSVZ , outer subventricular zone . Right panels show higher magnification . Arrows show that FOXM1 + were colocalized with SOX2 + . Scale bars , 100μm ( left ) ; 50μm ( right ) .

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(A) The expression pattern of FOXM1 in fetal human cortex at GW15. VZ, ventricular zone; ISVZ, inner subventricular zone; OSVZ, outer subventricular zone. Right panels show higher magnification images. Arrows show that FOXM1+cells were colocalized with SOX2+ cells. Scale bars, 100μm (left); 50μm (right).


( B ) for NESTIN FOXM1 in human neural progenitor cultured in vitro . Down panels show higher magnification . Scale bars , 50μm ( up ) ; 10μm ( down ) .

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(B) Staining for NESTIN and FOXM1 in human neural progenitor cells cultured in vitro. Down panels show higher magnification images. Scale bars, 50μm(up); 10μm(down).


( D ) for Pax6 in E15 . 5 mice after IUE at E13 . 5 . Arrowheads indicate Pax6 + in enlarged SVZ . Right panels show higher magnification . VZ , ventricular zone ; SVZ , subventricular zone ; IZ , intermediate zone ; CP , cortical plate . Scale bars , 50μm ( left ) ; 20μm ( right ) .

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(D) Staining for Pax6 in E15.5 mice after IUE at E13.5. Arrowheads indicate Pax6+ cell in enlarged SVZ. Right panels show higher magnification images. VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate. Scale bars,50μm(left); 20μm (right).


( G ) for upper neuron marker Cux1 in P3 mice after IUE at E13 . 5 . Arrowhead asterisk indicate deformation of cortical surface at regions containing electroporated . Right panels show higher magnification . Arrows show GFP + Cux1 + double - positive . Scale bars , 100μm ( left ) ; 20μm ( right ) .

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(G) Staining for upper neuron marker Cux1 in P3 mice after IUE at E13.5. Arrowhead and asterisk indicate deformation of the cortical surface at regions containing electroporated cells. Right panels show higher magnification images. Arrows show GFP+ Cux1+ double-positive cells. Scale bars,100μm(left); 20μm (right).


( A ) Representative of whole brains from wild - type ( WT ) hFOXM1f / + - NesCre ( hFOXM1 - cKI ) mice pups at P0 . Scale bar , 100μm . ( B ) of cortical width at P0 ( WT n = 7 ; hFOXM1 - cKI n = 7 ; two - unpaired t - , P = 0 . 0361for width , P = 0 . 2866 for ) . ( C ) of brain / at P0 ( WT n = 9 ; hFOXM1 - cKI n = 9 ; two - unpaired t - , P = 0 . 0004 ) . ( D ) of cortex thickness at P0 ( WT n = 7 ; hFOXM1 - cKI n = 11 ; two - unpaired t - , P = 0 . 0002 ) . ( E )

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(A) Representative images of whole brains from wild-type (WT) and hFOXM1f/+-NesCre (hFOXM1-cKI) mice pups at P0. Scale bar,100μm. (B) Quantification of cortical width and length at P0 (WT n=7; hFOXM1-cKI n=7; two-tailed unpaired t-test, P= 0.0361for width, P= 0.2866 for length). (C) Quantification of brain/body weight at P0 (WT n=9; hFOXM1-cKI n=9; two-tailed unpaired t-test, P= 0.0004). (D) Quantification of cortex thickness at P0 (WT n=7; hFOXM1-cKI n=11; two-tailed unpaired t-test, P= 0.0002). (E)


( E ) for Satb2 Ctip2 in P0 WT hFOXM1 - cKI mice folding structure . Asterisks indicate gyrus arrows indicate sulcus structures . A1 , A2 , B1and B2 ( DAPI ) are high - magnification . Scale bars , 200μm ( left ) ; 100μm ( right ) . ( F ) of GI of WT hFOXM1 - cKI mice pups at P0 . of GI was analyzed in pre - selected hFOXM1 - cKI mice with folding . ( n = 6 each group ; two - u unpaired t - , P = 0 . 0010 ) .

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(E) Staining for Satb2 and Ctip2 in P0 WT and hFOXM1-cKI mice folding structure. Asterisks indicate gyrus and arrows indicate sulcus structures. A1, A2, B1and B2 (DAPI) are high-magnification images. Scale bars, 200μm(left); 100μm(right). (F) Quantification of GI of WT and hFOXM1-cKI mice pups at P0. The quantification of the GI was analyzed in pre-selected hFOXM1-cKI mice with folding. (n = 6 each group; two-tailed u unpaired t-test, P= 0.0010).


( H ) Graph shows number of Satb2 + in 4 × 104µm2 at P0 ( WT n = 5 ; hFOXM1 - cKI n = 5 ; two - unpaired t - , P = 0 . 0086 ) . ( I ) Graph shows number of Ctip2 + in 4 × 104µm2 at P0 ( WT n = 5 ; hFOXM1 - cKI n = 5 ; two - unpaired t - , P = 0 . 0025 ) . (

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(H) Graph shows the number of Satb2+cells in 4×104µm2 at P0 (WT n=5; hFOXM1-cKI n=5; two-tailed unpaired t-test, P= 0.0086). (I) Graph shows the number of Ctip2 +cells in 4×104µm2 at P0 (WT n=5; hFOXM1-cKI n=5; two-tailed unpaired t-test, P= 0.0025). (


( K ) for Cux1 in P0 WT hFOXM1 - cKI mice . Scale bar , 100μm . ( I ) Graph shows number of Cux1 + in 4 × 104µm2 at P0 ( WT n = 3 ; hFOXM1 - cKI n = 3 ; two - unpaired t - , P = 0 . 0451 ) .

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(K) Staining for Cux1 in P0 WT and hFOXM1-cKI mice. Scale bar,100μm. (I) Graph shows the number of Cux1+cells in 4×104µm2 at P0 (WT n=3; hFOXM1-cKI n=3; two-tailed unpaired t-test, P= 0.0451). D


( A ) for Pax6 p - Vim in E15 WT hFOXM1 - cKI mice . right A B are high - magnification . Arrows indicate double positive of Pax6 + p - Vim + with with a basal process ( arrowheads ) . Scale bars , 50μm ( left ) ; 5μm ( right ) . ( B ) Graph shows of Pax6 + in enlarged SVZ ( n = 6 each group ; two - unpaired t - , P = 0 . 0002 ) . ( C ) Graph shows number of Pax6 + p - Vim + in 4x10 ^ 4 μm2 in enlarged SVZ ( n = 6 each group ; two - unpaired t - , P = 0 . 0003 ) . (

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(A) Staining for Pax6 and p-Vim in E15 WT and hFOXM1-cKI mice. The right A and B are high-magnification images. Arrows indicate double positive of Pax6+ p-Vim+ cells with cells with a basal process (arrowheads). Scale bars, 50μm (left); 5μm (right). (B) Graph shows the percentage of Pax6+ cells in enlarged SVZ (n = 6 each group; two-tailed unpaired t-test, P=0.0002). (C) Graph shows the number of Pax6+ p-Vim+ cells in 4x10^4 μm2 in enlarged SVZ (n = 6 each group; two-tailed unpaired t-test, P=0.0003). (D


( D ) for Sox2 in E15 WT hFOXM1 - cKI mice . Scale bar , 50μm . ( E ) Graph shows number of Sox2 + in 4x104 μm2 in enlarged SVZ ( WT n = 3 ; hFOXM1 - cKI n = 4 ; two - unpaired t - , P = 0 . 0292 ) . (

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(D) Staining for Sox2 in E15 WT and hFOXM1-cKI mice. Scale bar, 50μm. (E) Graph shows the number of Sox2+ cells in 4x104 μm2 in enlarged SVZ (WT n=3; hFOXM1-cKI n=4; two-tailed unpaired t-test, P=0.0292). (


( F ) for Tbr2 in E15 WT hFOXM1 - cKI mice . Scale bar , 50μm . ( G ) Graph shows number of Tbr2 + in 4x104 μm2 in enlarged SVZ ( WT n = 6 ; hFOXM1 - cKI n = 9 ; two - unpaired t - , P = 0 . 0003 ) . (

(F) Staining for Tbr2 in E15 WT and hFOXM1-cKI mice. Scale bar, 50μm. (G) Graph shows the number of Tbr2+ cells in 4x104 μm2 in enlarged SVZ (WT n=6; hFOXM1-cKI n=9; two-tailed unpaired t-test, P=0.0003). (


( H ) for HOPX in E15 WT hFOXM1 - cKI mice . Scale bar , 50μm . ( I ) Graph shows number of HOPX + in 4 × 104 μm2 in enlarged SVZ ( n = 4 each group ; two - unpaired t - , P = 0 . 0044 ) . (

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(H) Staining for HOPX in E15 WT and hFOXM1-cKI mice. Scale bar, 50μm. (I) Graph shows the number of HOPX+ cells in 4×104 μm2 in enlarged SVZ (n = 4 each group; two-tailed unpaired t-test, P= 0.0044). (


( J ) for Ki67 in E15 WT hFOXM1 - cKI mice . Scale bar , 50μm .

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(J) Staining for Ki67 in E15 WT and hFOXM1-cKI mice. Scale bar, 50μm.


( L ) experimental scheme for BrdU EdU in E15 WT hFOXM1 - cKI mice . arrows represent BrdU EdU double - positive . Scale bars , 50μm ( left ) ; 20μm ( right ) . ( M ) Graph shows of BrdU EdU double - positive in E15 WT hFOXM1 - cKI mice ( n = 6 each group ; two - unpaired t - , P = 0 . 0413 ) .

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(L) The experimental scheme and staining for BrdU and EdU in E15 WT and hFOXM1-cKI mice. White arrows represent BrdU and EdU double-positive cells. Scale bars, 50μm(left);20μm(right). (M) Graph shows the percentage of BrdU and EdU double-positive cells in E15 WT and hFOXM1-cKI mice (n = 6 each group; two-tailed unpaired t-test, P=0.0413). D


( B ) immunostaining for HOPX in control , hFOXM1 - FL mutants in mice neural progenitors 3 days following lentiviral infection . arrows represent GFP HOPX double - positive . Scale bar , 50μm . ( C ) Graph shows of HOPX + among GFP + ( Control n = 3 ; hFOXM1 - FL n = 6 ; hFLΔexon9 n = 5 ; one - way ANOVA , F ( 2 , 12 ) = 29 . 13 , control vs . hFOXM1 , P = 0 . 0002 ; control vs . hFOXM1Δexon9 , P = 0 . 9080 ) . ( D ) immunostaining for HOPX in control , mFoxm1 - FL mutants in mice neural progenitors 3 days following lentiviral infection . arrows represent GFP HOPX double - positive . Scale bar , 50μm . ( E ) ( E ) Graph shows of HOPX + among GFP + ( Control n = 4 ; mFoxm1 - FL n = 6 ; mFL + h exon9 n = 4 ; one - way ANOVA , F ( 2 , 11 ) = 8 . 163 , control vs . mFoxm1 , P = 0 . 4747 , control vs . mFoxm1 + h exon9 , P = 0 . 0066 ) . (

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(B) Cell immunostaining for HOPX in control, hFOXM1-FL and mutants in mice primary neural progenitors 3 days following lentiviral infection. White arrows represent GFP HOPX double-positive cells. Scale bar, 50μm. (C) Graph shows the percentage of HOPX+ cells among GFP+ cells (Control n=3; hFOXM1-FL n=6; hFLΔexon9 n=5; one-way ANOVA, F (2, 12) = 29.13, control vs. hFOXM1, P=0.0002; control vs. hFOXM1Δexon9, P=0.9080). (D) Cell immunostaining for HOPX in control, mFoxm1-FL and mutants in mice primary neural progenitors 3 days following lentiviral infection. White arrows represent GFP HOPX double-positive cells. Scale bar, 50μm. (E) (E) Graph shows the percentage of HOPX+ cells among GFP+ cells (Control n=4; mFoxm1-FL n=6; mFL+h exon9 n=4; one-way ANOVA, F (2, 11) = 8.163, control vs. mFoxm1, P= 0.4747, control vs. mFoxm1+h exon9, P= 0.0066). (


( J ) for Tbr2 in E15 . 5 mice after IUE at E13 . 5 . Down panels show higher magnification . arrows represent GFP TBR2 double - positive . Scale bars , 50μm . ( K ) Graph shows of GFP + Tbr2 + / GFP + in enlarged SVZ ( n = 3 each group ; one - way ANOVA , F ( 3 , 8 ) = 8 . 787 , P = 0 . 0065 ) .

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(J) Staining for Tbr2 in E15.5 mice after IUE at E13.5. Down panels show higher magnification images. White arrows represent GFP and TBR2 double-positive cells. Scale bars, 50μm. (K) Graph shows the percentage of GFP+Tbr2+/GFP+ in enlarged SVZ (n = 3 each group; one-way ANOVA, F (3,8) =8.787, P=0.0065). D


( C ) Volcano plots shows gene expression states . yellow dots are up - regulated genes , blue dots are down - regulated genes . Lin28a gene ( red dot ) is significantly up - regulated .

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(C) Volcano plots shows the gene expression states. The yellow dots are the up-regulated genes, and the blue dots are the down-regulated genes. The Lin28a gene (red dot) is significantly up-regulated.


( D ) Western blot for Lin28a in cortex of E15 WT hFOXM1 - cKI mice . β - actin was detected as loading control .

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(D) Western blot analysis for Lin28a in cortex of E15 WT and hFOXM1-cKI mice. β-actin was detected as loading control.


( F ) for Sox2 in E15 . 5 mice after IUE at E13 . 5 . Arrowheads indicate Sox2 + in enlarged SVZ . Scale bar , 50μm . ( G ) Graph shows of Sox2 + among GFP + in enlarged SVZ ( n = 3 each group ; two - unpaired t - , P = 0 . 0154 ) . ( H ) Graph shows of Sox2 + among GFP + ( n = 3 each group ; two - unpaired t - , P = 0 . 0018 ) .

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(F) Staining for Sox2 in E15.5 mice after IUE at E13.5. Arrowheads indicate Sox2+ cell in enlarged SVZ. Scale bar, 50μm. (G) Graph shows the percentage of Sox2+ cells among GFP+ cells in enlarged SVZ (n = 3 each group; two-tailed unpaired t-test, P=0.0154). (H) Graph shows the percentage of Sox2+ cells among GFP+ cells (n = 3 each group; two-tailed unpaired t-test, P=0.0018).


( I ) LIN28A - shRNA1 rescues neuronal progenitor increase in hFOXM1 - cKI mice . Brain slice for Sox2 two days after IUE at E13 . Scale bar , 50μm . ( J ) Graph shows of Sox2 + among GFP + ( n = 3 each group ; one - way

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(I) LIN28A-shRNA1 rescues the neuronal progenitor cell increase in hFOXM1-cKI mice. Brain slice staining for Sox2 two days after IUE at E13. Scale bar, 50μm. (J) Graph shows the percentage of Sox2+ cells among GFP+ cells (n=3 each group; one-way A


( B ) Representative cases of FOXM1 + / + FOXM1 - / - NPCs clones . Scale bar , 20μm . ( C ) Graph shows number of per clone in FOXM1 + / + FOXM1 - / - human NPCs ( FOXM1 + / + n = 12 ; FOXM1 - / - n = 13 ; at least three biological replicates . two - unpaired t - , P = 0 . 0014 ) . (

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(B) Representative cases of FOXM1+/+ and FOXM1-/- NPCs clones. Scale bar, 20μm. (C) Graph shows the number of cells per clone in FOXM1+/+ and FOXM1-/- human NPCs (FOXM1+/+ n=12; FOXM1-/- n=13; at least three biological replicates. two-tailed unpaired t-test, P= 0.0014). (


( D ) Overexpression of FOXM1 or LIN28A rescues human neural progenitor reduction in FOXM1 - / - lines . Western blot for SOX2 , PAX6 HOPX . β - actin was detected as loading control . ( E ) Statistics of relative of SOX2 , PAX6 HOPX ( PAX6 : n = 3 biological replicates ; one - way ANOVA , F ( 2 , 6 ) = 8 . 716 , FOXM1 - / - ( Control ) vs . FOXM1 - / - ( hFOXM1 ) , P = 0 . 0173 , FOXM1 + / + ( Control ) vs . FOXM1 - / - ( hFOXM1 ) , P = 0 . 0476 . SOX2 : n = 5 biological replicates ; one - way ANOVA , F ( 2 , 12 ) = 14 . 72 , FOXM1 + / + ( Control ) vs . FOXM1 - / - ( Control ) , P = 0 . 0015 , FOXM1 + / + ( Control ) vs . FOXM1 - / - ( hFOXM1 ) , P = 0 . 9906 . HOPX : n = 4 biological replicates , one - way ANOVA F ( 2 , 9 ) = 25 . 28 , FOXM1 + / + ( Control ) vs . FOXM1 - / - ( Control ) , P = 0 . 0018 , FOXM1 + / + ( Control ) vs . FOXM1 - / - ( hFOXM1 ) , P = 0 . 2211 ) . (

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

(D) Overexpression of FOXM1 or LIN28A rescues the human neural progenitor cell reduction in FOXM1-/- cell lines. Western blot analysis for SOX2, PAX6 and HOPX. β-actin was detected as loading control. (E) Statistics of relative intensity of SOX2, PAX6 and HOPX (PAX6: n=3 biological replicates; one-way ANOVA, F (2,6) = 8.716, FOXM1-/-(Control) vs. FOXM1-/-(hFOXM1), P= 0.0173, FOXM1+/+(Control) vs. FOXM1-/-(hFOXM1), P= 0.0476. SOX2: n=5 biological replicates; one-way ANOVA, F (2, 12) = 14.72, FOXM1+/+(Control) vs. FOXM1-/-(Control), P= 0.0015, FOXM1+/+(Control) vs. FOXM1-/-(hFOXM1), P=0.9906. HOPX: n=4 biological replicates, one-way ANOVA F (2, 9) = 25.28, FOXM1+/+(Control) vs. FOXM1-/- (Control), P= 0.0018, FOXM1+/+(Control) vs. FOXM1-/-(hFOXM1), P= 0.2211). (


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

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 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 B-MEASURED_VAR 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 complemented with ATG5 or mock treated with TBK1 kinase inhibitor MRT68843 ( 10 nM ) or DMSO . Fold replication was determined by counting colonies on agar plates at 2 8 h post inoculation ( p . i . ) following lysis .

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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 ) of Tbk1 - / - MEFs stably expressing indicated TBK1 alleles infected with S . Typhimurium . 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 lysates .

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

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

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

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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 ) of Tbk1 - / - MEFs stably expressing indicated TBK1 alleles infected with S . Typhimurium at 1 h p . i . 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 .

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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.


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A - H ) of Tbk1 - / - MEFs stably expressing indicated TBK1 alleles infected with S . Typhimurium at 1 h p . i . 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 .

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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.


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A - B ) Kinetics of S . Typhimurium replication in MEFs depleted of WIPI1 ( A ) . Intracellular were enumerated by their ability to form colonies on agar plates . Western blot for GFP : WIPI1 upon 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 were enumerated by their ability to form colonies on agar plates . quantitative RT - PCR for WIPI2 upon 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 ) of Tbk1 - / - MEFs complemented with indicated Flag - tagged TBK1ΔC - adaptor fusion . S . Typhimurium replication kinetics ( A , D ) . Infected were lysed at indicated time points post inoculation ( p . i . ) were enumerated by their ability to form colonies on agar plates . Mean s . d . of triplicate MEF cultures duplicate colony . 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 lysates .

O O O O O B-CONTROLLED_VAR 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 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

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

O O O O 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 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

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 ) of Tbk1 - / - MEFs complemented with indicated Flag - tagged TBK1ΔC - adaptor fusion . S . Typhimurium replication kinetics ( A , D ) . Infected were lysed at indicated time points post inoculation ( p . i . ) were enumerated by their ability to form colonies on agar plates . Mean s . d . of triplicate MEF cultures duplicate colony . 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 B-CONTROLLED_VAR 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 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 . Binding of indicated luciferase - tagged NDP52 variants to beads coated with GST , GST : galectin - 8 , or GST : ubiquitin . Coomassie stain of purified GST . Western blot for luciferase - tagged NDP52 alleles in total lysates .

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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 indicated Flag - tagged TBK1ΔC - NDP52 fusion . Infected were lysed at indicated times post inoculation ( p . i . ) were enumerated by their ability to form colonies on agar plates . Mean s . d . of triplicate MEF cultures duplicate colony . 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 . Western blot for Flag - tagged TBK1 variants in post lysates .

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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 ) of Tbk1 - / - MEFs complemented with indicated Flag - tagged TBK1ΔC - galectin fusion . S . Typhimurium replication kinetics ( A ) . Infected were lysed at indicated times post inoculation ( p . i . ) . were enumerated by their ability to form colonies on agar plates . Mean s . d . of triplicate MEF cultures duplicate colony . 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 . Western blot for Flag - tagged TBK1 variants in post lysates .

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

O O O O 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 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

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 indicated GST - tagged were incubated with pooled M1 - , K63 - , K48 - linked tetra - ubiquitin chains . Anti - ubiquitin Western blot of input bound to beads .

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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 indicated Flag - tagged TBK1ΔC fusion . Infected were lysed at indicated time points post inoculation ( p . i . ) were enumerated by their ability to form colonies on agar plates . Mean s . d . of triplicate MEF cultures duplicate colony . 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 . Western blot for Flag - tagged TBK1 variants in post lysates .

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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 indicated Flag - tagged TBK1ΔC fusion . Infected were lysed at indicated time points post inoculation ( p . i . ) were enumerated by their ability to form colonies on agar plates . Mean s . d . of triplicate MEF cultures duplicate colony . 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 . 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 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 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 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 P493 - 6 treated with Tet for 0h or 72h ( A ) equal mRNA loading was verified by methylene blue . 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 that expressed NTC or MYC shRNAs ( B ) ; equal mRNA loading was verified by methylene blue . 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 ) 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 ‐ ) .

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(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 , 3 ′ UTR in P493 - 6 treated with Tet for 0h or 72h .

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(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 .

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


( G ) m6A - RIP in P493 - 6 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 ‐ ) .

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

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

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

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(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 for of m6A methyltransferases ( METTL3 METTL14 ) demethylases ( ALKBH5 FTO ) in Raji 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 of mRNA of methyltransferases demethylases in P493 - 6 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 ‐ ) .

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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 FTO from published ChIP - seq datasets ( Walz et al , 2014 ; Data ref : Eilers M , 2014 ) . In this dataset , high of MYC were induced with 1µg / ml doxycycline for 30h ChIPed by MYC antibody . Red triangle indicates 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 , using ALKBH5 , FTO or IgG antibody to detect binding to MRGs ( SPI1 PHF12 ) in P493 - 6 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 ‐ ) .

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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 for in P493 - 6 that overexpressed empty vector ( EV ) , ALKBH5 , or FTO were then treated with Tet or not ( F ) HPRT1 β - actin served as negative 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 for in Raji expressed NTC or MYC shRNAs ( G ) . HPRT1 β - actin served as negative 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 in P493 - 6 that overexpressed EV , ALKBH5 , or FTO 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 ‐ ) .

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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 for in P493 - 6 that expressed NTC or ALKBH5 shRNAs were then treated with Tet or not ( I ) HPRT1 β - actin serve as negative 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 for in Raji expressed NTC or ALKBH5 shRNAs knocked down MYC or not ( J ) . HPRT1 β - actin serve as negative 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 HPRT1 serves as negative control . * * * P < 0 . 001 as compared between indicated groups ( mean ± SD , n = 3 biological replicates , Student ' s t ‐ ) .

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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 in P493 - 6 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 ‐ ) .

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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 for in P493 - 6 that expressed NTC or YTHDF3 shRNAs were then treated with Tet or not ( B ) HPRT1 β - actin serve as negative 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 for in Raji that expressed NTC or YTHDF3 shRNAs knocked down MYC or not ( C ) . HPRT1 β - actin serve as negative 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 in P493 - 6 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 for mRNA of MRGs ( SPI1 PHF12 ) in different fractions in ( D ) . HPRT1 serves as negative control . Data were presented as mean ( ± SD ) , n = 3 biological replicates . * * P < 0 . 01 * * * P < 0 . 001 as compared to NTC group ( Student ' s t ‐ ) .

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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 expressing NTC or YTHDF3 shRNAs were incubated for 1 hour in medium containing 100 ug / mL AHA . translated 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 for in P493 - 6 ( G ) β - actin serve as negative 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 for in Raji ( H ) that expressed NTC , or ALKBH5 shRNA , or YTHDF3 shRNA . β - actin serve as negative 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 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 ‐ ) .

O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 ‐ )

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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 overexpressing ALKBH5 further infected with expressing SPI1 ( B ) or PHF12 ( C ) . * * P < 0 . 01 as compared between indicated groups ( mean ± SD , n = 4 biological replicates , Student ' s t ‐ ) .

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

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

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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 stably expressing NTC or shSPI1 or shPHF12 were injected subcutaneously into nude mice ( n = 5 for each group ) . Photo ( G ) of tumors collected at end of 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 stably expressing EV or ALKBH5 were infected with expressing SPI1 or PHF12 . were injected subcutaneously into nude mice ( n = 5 for each group ) . growth curves were measured starting from 17 days post injection ( H ) . Photo ( I ) of tumors collected at end of experiment ( day 32 ) . Data are presented as mean ( ± ) . * P < 0 . 05 as compared between indicated groups ( Student ' s t ‐ ) .

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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 ( J ) , ( K ) of MYC , ALKBH5 , SPI1 PHF12 expressions in normal lymphocyte tissue ( normal ) lymphoma specimens . Scale bar : 50 um . Data are presented as mean ( ± ) , 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 ‐ ) .

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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 of localization of endogenous PHGDH in PLC , HepG2 , THLE3 . Lamin B1 , Tubulin , Tom20 were used as markers for ( Nuc ) , ( Cyto ) , mitochondrial ( Mito ) , 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 of localization of endogenous PHGDH in tumour tissues adjacent normal tissues . Lamin B1 , GAPDH , Tom20 were used as markers for Nuc , Cyto , Mito , 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 of localization of endogenous exogenous Flag - tagged PHGDH in PLC . Lamin B1 , Tubulin , Tom20 were used as markers for Nuc , Cyto , Mito , 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 showing colocalization of Flag - PHGDH ( green ) mitochondria ( red ) acquired with a confocal microscope system in PLC . Nuclei were stained with DAPI ( blue ) , 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 overexpressing of Flag - tagged wild - type PHGDH , PHGDH ∆ SBD1 , or PHGDH ∆ RD mutants . WCL : whole - 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 HepG2 . outer mitochondrial ( OMM ) Tom20 , inner mitochondrial ( IMM ) ANT2 , mitochondrial matrix ( MM ) TFAM were used as markers of OMM , IMM , 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 were incubated with Na2CO3 ( pH 11 . 5 ) for 30 min at 4 ℃ . soluble component ( Supernatant / Sup . ) component ( Pellet ) were separated by centrifugation prior to western blot . COX2 , ATP8 , ANT2 are markers for compotent ; CYTC TFAM are markers for 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 oxygen consumption rate ( OCR ) was measured by successive injections of oligomycin ( oligo ) , FCCP , antimycin A / rotenone ( AA / Rot ) in PLC infected with expressing nontargeting control ( NTC ) shRNA or shPHGDH . Basal respiration , ATP - linked respiration , maximal respiration were analysed with 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 of mtDNA - encoded in PLC stably expressing NTC or shPHGDH empty vector ( EV ) or PLVX - PHGDH . β - Actin was used as loading control .

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

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 with endogenous PHGDH knockdown were further infected with expressing EV or PLVX - PHGDH prior to western blot of mtDNA - encoded . β - Actin was used as loading control .

O O O 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 O O O O

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 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 with an anti - puromycin antibody to measure actively translated polypeptide chains . Western blot of PHGDH confirmed knockdown efficiency of PHGDH shRNAs . β - Actin was used as 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 indicated lines using whole - lysates .

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


F PLC 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 . assays were performed using whole - 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 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 of mtDNA - encoded . β - Actin was used as loading control .

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

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

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

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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 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 .

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

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

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

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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 His - PHGDHR236E by GST - ANT2 using purified in E . coli , followed by western blot with anti - His anti - GST antibodies .

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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 purified in E . coli , followed by western blot with anti - His anti - GST antibodies .

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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 stably expressing Flag - mtEFG2 HA - PHGDH were further infected with expressing NTC or shANT2 , then lysates were immunoprecipitated with an anti - Flag antibody or IgG prior to western blot .

O O O O O O O O O O O O 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 overexpressing PHGDH were further infected with expressing NTC or shANT2 . translation efficiency was measured by a SUnSET .

O O O B-CONTROLLED_VAR 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 overexpressing PLVX - PHGDH were further infected with expressing NTC or shANT2 . expression of mtDNA - encoded was determined by western blotting . β - Actin was used as 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

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 overexpressing PHGDH were further infected with expressing NTC or shmtEFG2 . translation efficiency was measured by a SUnSET .

O O B-CONTROLLED_VAR 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 overexpressing pSin - 3xFlag - PHGDH were further infected with expressing NTC or shmtEFG2 . expression of mtDNA - encoded was determined by western blotting . β - Actin was used as loading control .

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

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

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

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 stably expressing Flag - mtEFG2 HA - mtRRF were further infected with expressing NTC or shPHGDH , then lysates were immunoprecipitated with anti - Flag antibody or IgG prior to western blot .

O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O 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 stably overexpressing mtEFG2 were further infected with expressing NTC or shPHGDH . translation efficiency was measured by a SUnSET .

O O O O B-CONTROLLED_VAR 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 stably overexpressing mtEFG2 were further infected with expressing NTC or shPHGDH . mtDNA - encoded were analysed by western blot . β - Actin was used as loading control .

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

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 lysates were immunoprecipitated with an anti - MRPL44 antibody or IgG prior to western blot of MRPL44 , MRPL46 , MRPL48 , MRPS15 , MRPS16 , 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 immunoprecipitation ( RIP ) in PLC 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 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 same amount of mitochondria from PLC expressing NTC or shPHGDH treated with CAP TAP were loaded onto sucrose density gradient . After centrifugation , fractions were subjected to western blot of MRPS35 , MRPL44 , MRPL48 . concentrations of ND6 were measured .

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

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

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

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

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

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

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 B-MEASURED_VAR I-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 overexpressing EV or PHGDH were subcutaneously injected into 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 end of experiment , tumours were excised ( H ) , PHGDH expression in tumour lysates was analysed by western blotting ( I ) .

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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 Cxcl2 ( A , C , E respectively ) were measured at 24 h after stimulation of bone - marrow - derived neutrophils ( 5 x 105 ) with increasing concentrations of nucleic ( 1 , 5 , 10 20 µg of + in equal proportions ) , HK - ( 1 , 5 , 10 or 20 μg / ml ) or live ( MOIs of 2 , 5 , 10 20 ) . LPS ( 1 , 10 , 100 1000 ng / ml ) was used as a positive control stimulus . ( B , D F ) , Effect of FPR antagonist Boc - 2 on GBS - induced TNF - α , Cxcl1 Cxcl2 production , respectively . Bone - marrow - derived neutrophils were exposed to Boc - 2 ( 50 µM ) for 1 hour 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 - ; ns , not significant .

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(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 10 µM ) for 1 hour 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 - ; ns , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Cxcl2 concentrations in 24 - h culture supernatants of neutrophils ( A B ) , CSF - polarized ( C D ) or GM - CSF - polarized ( E F ) BMDMs ( ΜΦ ) from WT or Fpr - deficient mice stimulated with live GBS ( MOI of 2 , 5 , 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 - .

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

O O O O O O O O O O O O O B-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

(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 ) 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 : , data points means . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 versus vehicle treated mice determined by unpaired t 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 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

(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 D ) , Cxcl2 release in neutrophil cultures stimulated with combinations of HK - , GBS fMet peptides or TLR agonists . Neutrophils were exposed to a 50 µM of Fpr agonists ( fMIFL , WKYMVM or fPep8 fPep10 ) for 1 h before addition of indicated TLR agonists , which included HK - GBS ( 10 μg / ml ) , LPS ( 1 µg / ml ) ( 1 µg / ml ) . In some cases , neutrophils were exposed to graded doses ( 10 , 20 50 µM ) of 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 - .

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

(C 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.