Figure 4 ( A ) WT , and Irgm2 - / - BMMs were stimulated overnight with IFNγ or left untreated and cell lysates were collected . Lysates were assessed for Casp11 , Gbp2 , and actin protein levels via immunoblotting . ( B ) IFNγ - primed and unprimed WT , Irgm2 - / - , Gbpchr3 - / - and Aim2 - / - BMMs were infected with Francisella novicida ( MOI 10 ) and LDH release measured at 4 hpi . ( C ) WT , Irgm2 - / - , Gbpchr3 - / - and Irgm2 - / - Gbpchr3 - / - BMMs were treated with LPS ( 1 µg / ml ) . IL - 1β , IL - 18 , and LDH release were assessed at 24 hpt . ( D ) IFNγ - primed WT , Irgm2 - / - , Gbpchr3 - / - and Irgm2 - / - Gbpchr3 - / - BMMs were treated with LPS ( 1 µg / ml ) for 24 hours and cell lysates and supernatants collected . Protein levels in cell lysates ( Caspase - 1 , and actin ) and supernatants ( Caspase - 1 p20 ) were visualized via immunoblotting .

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Figure 4 (A) WT, and Irgm2-/- BMMs were stimulated overnight with IFNγ or left untreated and cell lysates were collected. Lysates were assessed for Casp11, Gbp2, and actin protein levels via immunoblotting.  (B) IFNγ-primed and unprimed WT, Irgm2-/-, Gbpchr3-/- and Aim2-/- BMMs were infected with Francisella novicida (MOI 10) and LDH release measured at 4 hpi.  (C) WT, Irgm2-/-, Gbpchr3-/- and Irgm2-/- Gbpchr3-/- BMMs were treated with LPS (1 µg/ml). IL-1β, IL-18, and LDH release were assessed at 24 hpt.  (D) IFNγ-primed WT, Irgm2-/-, Gbpchr3-/- and Irgm2-/- Gbpchr3-/- BMMs were treated with LPS (1 µg/ml) for 24 hours and cell lysates and supernatants collected. Protein levels in cell lysates (Caspase-1, and actin) and supernatants (Caspase-1 p20) were visualized via immunoblotting.


Figure 5 ( A ) WT , Irgm2 - / - , and Nlrp3 - / - BMMs were treated with LPS ( 0 . 1 µg / ml ) for 3 h followed by nigericin for 1 h and IL - 1β / LDH release was measured . ( B ) WT , Irgm2 - / - , Casp11 - / - and Irgm2 - / - Casp11 - / - BMMs were treated with LPS ( 0 . 1 µg / ml ) for 3 h followed by nigericin and / or MCC950 for 1 h and IL - 1β / LDH release was measured . ( C ) WT , Irgm2 - / - , Casp11 - / - and Irgm2 - / - Casp11 - / - BMMs were treated with LPS ( 1 µg / ml ) and / or MCC950 for 24 h and IL - 1β / LDH release was measured . ( D ) IFNγ - primed WT , Irgm2 - / - , Casp11 - / - and Irgm2 - / - Casp11 - / - BMMs were transfected with LPS using lipofectamine LTX and LDH release was measured at 2 hpt . ( E ) IFNγ - primed WT , Irgm2 - / - , Casp11 - / - and Irgm2 - / - Casp11 - / - BMMs were co - treated with LPS ( indicated doses ) and Listeria monocytogenes ( MOI 10 ) and LDH release measured at 4 hpt .

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Figure 5 (A) WT, Irgm2-/-, and Nlrp3-/- BMMs were treated with LPS (0.1 µg/ml) for 3 h followed by nigericin for 1 h and IL-1β/LDH release was measured.  (B) WT, Irgm2-/-, Casp11-/- and Irgm2-/-Casp11-/- BMMs were treated with LPS (0.1 µg/ml) for 3 h followed by nigericin and/or MCC950 for 1 h and IL-1β/LDH release was measured.  (C) WT, Irgm2-/-, Casp11-/- and Irgm2-/-Casp11-/- BMMs were treated with LPS (1 µg/ml) and/or MCC950 for 24 h and IL-1β/LDH release was measured.  (D) IFNγ-primed WT, Irgm2-/-, Casp11-/- and Irgm2-/- Casp11-/- BMMs were transfected with LPS using lipofectamine LTX and LDH release was measured at 2 hpt.  (E) IFNγ-primed WT, Irgm2-/-, Casp11-/- and Irgm2-/- Casp11-/- BMMs were co-treated with LPS (indicated doses) and Listeria monocytogenes (MOI 10) and LDH release measured at 4 hpt.


Figure 6 ( A ) WT ( n = 9 ) , Irgm2 - / - ( n = 10 ) , Casp11 - / - ( n = 7 ) and Irgm2 - / - Casp11 - / - ( n = 8 ) mice were injected i . p . with LPS ( 8 mg / kg ) . Morbidity and mortality were observed for 48 h at 3 hour intervals . ( B ) WT ( n = 9 ) , Irgm2 - / - ( n = 8 ) , Casp11 - / - ( n = 7 ) and Irgm2 - / - Casp11 - / - ( n = 10 ) mice were injected i . p . with LPS ( 8 mg / kg ) . Serum was collected 4 hpi and concentration of IL - 1β , and IL - 18 was measured by ELISA . Data shown are from 2 pooled experiments .

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Figure 6 (A) WT (n = 9), Irgm2-/- (n = 10), Casp11-/- (n = 7) and Irgm2-/-Casp11-/- (n = 8) mice were injected i.p. with LPS (8 mg/kg). Morbidity and mortality were observed for 48 h at 3 hour intervals.  (B) WT (n = 9), Irgm2-/- (n = 8), Casp11-/- (n = 7) and Irgm2-/-Casp11-/- (n = 10) mice were injected i.p. with LPS (8 mg/kg). Serum was collected 4 hpi and concentration of IL-1β, and IL-18 was measured by ELISA. Data shown are from 2 pooled experiments.


Figure 7 ( A ) WT , Irgm2 - / - , Irgm3 - / - , Irgm2 - / - Irgm3 - / - , panIrgm - / - , and Casp11 - / - BMMs were treated with LPS ( 1 µg / ml ) or infected with E . coli K - 12 ( MOI 25 ) and IL - 1β , and LDH release were assessed at 24 hpt ( n = 3 independent experiments ) . ( B ) IFNγ - primed WT , Irgm2 - / - , Irgm3 - / - , Irgm2 - / - Irgm3 - / - , panIrgm - / - , and Casp11 - / - BMMs were treated with LPS ( 1 µg / ml ) or infected with E . coli K - 12 ( MOI 25 ) and IL - 1β , and LDH release were assessed at 8 hpt / hpi ( n = 3 independent experiments ) . ( C ) WT ( n = 15 ) , Irgm2 - / - ( n = 12 ) , and panIrgm - / - ( n = 13 ) mice were injected i . p . with LPS ( 8 mg / kg ) . Serum was collected 4 hpi and concentration of IL - 1β and TNFα was measured via ELISA . Data shown are from 3 pooled experiments . ( D ) WT , Irgm2 - / - , panIrgm - / - and Casp11 - / - mice ( n = 8 mice / genotype ) were injected i . p . with LPS ( 8 mg / kg body weight ) . Morbidity and mortality were observed for 48 h at 3 - h intervals .

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Figure 7 (A) WT, Irgm2-/-, Irgm3-/-, Irgm2-/-Irgm3-/-, panIrgm-/-, and Casp11-/- BMMs were treated with LPS (1 µg/ml) or infected with E. coli K-12 (MOI 25) and IL-1β, and LDH release were assessed at 24 hpt (n = 3 independent experiments). (B) IFNγ-primed WT, Irgm2-/-, Irgm3-/-, Irgm2-/-Irgm3-/-, panIrgm-/-, and Casp11-/- BMMs were treated with LPS (1 µg/ml) or infected with E. coli K-12 (MOI 25) and IL-1β, and LDH release were assessed at 8 hpt/ hpi (n = 3 independent experiments).  (C) WT (n = 15), Irgm2-/- (n = 12), and panIrgm-/- (n = 13) mice were injected i.p. with LPS (8 mg/kg). Serum was collected 4 hpi and concentration of IL-1β and TNFα was measured via ELISA. Data shown are from 3 pooled experiments.  (D) WT, Irgm2-/-, panIrgm-/- and Casp11-/- mice (n = 8 mice/genotype) were injected i.p. with LPS (8 mg/kg body weight). Morbidity and mortality were observed for 48 h at 3-h intervals.


Figure 1Immunostaining of H3K27ac in mouse germinal vesicle ( GV ) oocytes ( n = 8 ) , metaphase II ( MII ) oocytes ( n = 7 ) , zygotes ( n = 7 ) , early 2 - cell ( 2C ) ( n = 7 ) and late 2C ( n = 9 ) embryos . Right panel is the quantification of H3K27ac immunostaining relative signal intensities at different stages . Scale bar : 20 μm . Data information : For boxplots in A the central band represents the median . The lower and upper edges of the box represent the first and third quartiles , respectively . The whiskers of the boxplot extend to 1 . 5 times inter - quartile range ( IQR ) .

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Figure 1Immunostaining of H3K27ac in mouse germinal vesicle (GV) oocytes (n = 8), metaphase II (MII) oocytes (n = 7), zygotes (n = 7), early 2-cell (2C) (n = 7) and late 2C (n = 9) embryos. Right panel is the quantification of H3K27ac immunostaining relative signal intensities at different stages. Scale bar: 20 μm. Data information: For boxplots in A the central band represents the median. The lower and upper edges of the box represent the first and third quartiles, respectively. The whiskers of the boxplot extend to 1.5 times inter-quartile range (IQR).


Figure 4Immunostaining of H3K27ac in A - 485 treated embryos versus control ( DMSO treated ) at different time points . Scale bar : 20 μm . Developmental rate of embryos treated with A - 485 versus control .

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Figure 4Immunostaining of H3K27ac in A-485 treated embryos versus control (DMSO treated) at different time points. Scale bar: 20 μm.Developmental rate of embryos treated with A-485 versus control.


Figure 6Immunostaining of H3K27ac in TSA treated embryos under different concentrations versus control ( DMSO treated ) at 28 hpi . Scale bar : 20 μm . Developmental rate of embryos treated with TSA at different time points ( 0 - 20 hpi and 8 - 28 hpi ) versus control .

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Figure 6Immunostaining of H3K27ac in TSA treated embryos under different concentrations versus control (DMSO treated) at 28 hpi. Scale bar: 20 μm.Developmental rate of embryos treated with TSA at different time points (0-20 hpi and 8-28 hpi) versus control.


Figure 1A Live cell imaging reveals that expression of GFP ‐ KSHV ‐ TK , but not GFP induces contraction of HeLa cells ( see Supplementary Movies S1 and S2 ) . The time from the start of imaging is indicated above each panel , and the scale bars represent 18 μm . Quantification of GFP ‐ KSHV ‐ TK ‐ induced cell contraction . The graphs show cell area ( μm2 ) of cells expressing the indicated protein , and error bars represent SEM from three independent experiments in which n = 224 for GFP and n = 203 for GFP ‐ KSHV ‐ TK . * * * P 0 . 001 . B Phase images taken from Supplementary Movie S3 illustrating GFP ‐ KSHV ‐ TK induced blebbing of contracted cells . The time from the start of imaging is indicated above each panel , and the scale bars represent 18 μm . C Scanning electron microscopy of HeLa cells expressing GFP or GFP ‐ KSHV ‐ TK . D Immunofluorescence microscopy images illustrating the association of GFP ‐ KSHV ‐ TK , but not GFP , with actinstress fibres . Scale bar , 10 μm .

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Figure 1A Live cell imaging reveals that expression of GFP‐KSHV‐TK, but not GFP induces contraction of HeLa cells (see Supplementary Movies S1 and S2). The time from the start of imaging is indicated above each panel, and the scale bars represent 18 μm. Quantification of GFP‐KSHV‐TK‐induced cell contraction. The graphs show cell area (μm2) of cells expressing the indicated protein, and error bars represent SEM from three independent experiments in which n = 224 for GFP and n = 203 for GFP‐KSHV‐TK. ***P 0.001.B Phase images taken from Supplementary Movie S3 illustrating GFP‐KSHV‐TK induced blebbing of contracted cells. The time from the start of imaging is indicated above each panel, and the scale bars represent 18 μm.C Scanning electron microscopy of HeLa cells expressing GFP or GFP‐KSHV‐TK.D Immunofluorescence microscopy images illustrating the association of GFP‐KSHV‐TK, but not GFP, with actinstress fibres. Scale bar, 10 μm.


Figure 2A Co ‐ expression of GFP ‐ RhoN19 but not GFP inhibits RFP ‐ KSHV ‐ TK ‐ induced cell contraction . Graphs represent cell area ( μm2 ) of KSHV ‐ TK , RhoN19 or KSHV ‐ TK and RhoN19 expressing cells . Error bars represent SEM from 3 independent experiments , n = 204 for KSHV ‐ TK , n = 223 for RhoN19 and n = 189 for KSHV ‐ TK and RhoN19 . B GFP ‐ KSHV ‐ TK ‐ induced cell contraction does not occur in cells treated with the ROCK and myosin II inhibitors Y ‐ 27632 and blebbistatin , respectively . Error bars represent SEM from three independent experiments , n = 213 for DMSO , n = 189 for Y ‐ 27632 and n = 245 . * * * P 0 . 001 . C Immunoblot analysis of rhotekinpull ‐ down ( P . D . ) assays reveals that GFP ‐ KSHV ‐ TK increases the level of GTP ‐ bound RhoA ( GTP ‐ RhoA ) . Actin is included as a loading control , and the input lysates are indicated . D GFP ‐ KSHV ‐ TK but not GFP induces the loss of phosphotyrosine , phospho ‐ FAK Y925 and phospho ‐ paxillin Y31 epitopes from focal adhesions .

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Figure 2A Co‐expression of GFP‐RhoN19 but not GFP inhibits RFP‐KSHV‐TK‐induced cell contraction. Graphs represent cell area (μm2) of KSHV‐TK, RhoN19 or KSHV‐TK and RhoN19 expressing cells. Error bars represent SEM from 3 independent experiments, n = 204 for KSHV‐TK, n = 223 for RhoN19 and n = 189 for KSHV‐TK and RhoN19.B GFP‐KSHV‐TK‐induced cell contraction does not occur in cells treated with the ROCK and myosin II inhibitors Y‐27632 and blebbistatin, respectively. Error bars represent SEM from three independent experiments, n = 213 for DMSO, n = 189 for Y‐27632 and n = 245. ***P 0.001.C Immunoblot analysis of rhotekinpull‐down (P.D.) assays reveals that GFP‐KSHV‐TK increases the level of GTP‐bound RhoA (GTP‐RhoA). Actin is included as a loading control, and the input lysates are indicated.D GFP‐KSHV‐TK but not GFP induces the loss of phosphotyrosine, phospho‐FAK Y925 and phospho‐paxillin Y31 epitopes from focal adhesions.


Figure 3B Immunoblot analysis of BHK cells infected with MuHV ‐ 4 and MuHV ‐ 4 [ g ‐ KSHV ‐ TK ] or uninfected cells using antibodies against phosphotyrosine ( p ‐ Tyr ) , GFP , MuHV ‐ 4 TK , ORF17 ( capsid ) and glycoprotein N . C Immunofluorescence microscopy analysis of MuHV ‐ 4 ‐ and MuHV ‐ 4 [ g ‐ KSHV ‐ TK ] ‐ infected cells with the indicated antibodies reveals GFP ‐ KSHV ‐ TK expressed in the context of lytic infection ( MuHV ‐ 4 [ g ‐ KSHV ‐ TK ] ) induces the loss of phospho ‐ FAK Y925 and paxillin Y31 from focal adhesions . White arrowheads indicate examples of infected cells expressing GFP ‐ KSHV ‐ TK or GFP ( indicating WT MuHV ‐ 4 infection , BAC + virus ) . D Immunofluorescence microscopy analysis reveals that GFP ‐ KSHV ‐ TK expressed in the context of lytic infection ( MuHV ‐ 4 [ g ‐ KSHV ‐ TK ] ) does not alter focal adhesion integrity ( paxillin ) in the presence of the ROCK inhibitor Y ‐ 27632 . E Immunofluorescence microscopy analysis of paxillin in the lytically induced rKSHV . 219 / Vero cell line in the presence or absence of Y ‐ 27632 .

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Figure 3B Immunoblot analysis of BHK cells infected with MuHV‐4 and MuHV‐4 [g‐KSHV‐TK] or uninfected cells using antibodies against phosphotyrosine (p‐Tyr), GFP, MuHV‐4 TK, ORF17 (capsid) and glycoprotein N.C Immunofluorescence microscopy analysis of MuHV‐4‐ and MuHV‐4 [g‐KSHV‐TK]‐infected cells with the indicated antibodies reveals GFP‐KSHV‐TK expressed in the context of lytic infection (MuHV‐4 [g‐KSHV‐TK]) induces the loss of phospho‐FAK Y925 and paxillin Y31 from focal adhesions. White arrowheads indicate examples of infected cells expressing GFP‐KSHV‐TK or GFP (indicating WT MuHV‐4 infection, BAC+ virus).D Immunofluorescence microscopy analysis reveals that GFP‐KSHV‐TK expressed in the context of lytic infection (MuHV‐4 [g‐KSHV‐TK]) does not alter focal adhesion integrity (paxillin) in the presence of the ROCK inhibitor Y‐27632.E Immunofluorescence microscopy analysis of paxillin in the lytically induced rKSHV.219/Vero cell line in the presence or absence of Y‐27632.


Figure 4A Cellular localisation and immunoblot analysis of phosphotyrosine ( upper panel ) and GFP ( lower panel ) in cells expressing wild ‐ type or kinase ‐ dead GFP ‐ KSHV ‐ TK . B Scanning electron microscopy of HeLa cells expressing GFP or GFP ‐ KSHV ‐ TK ‐ DEAD . C Immunofluorescence microscopy analysis of HeLa cells expressing GFP ‐ KSHV ‐ TK ‐ DEAD ( white arrowheads ) with the indicated phospho antibodies . 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 and GST ‐ KSHV ‐ TKimmunoblotted for phosphotyrosine and GST following a sequential dephosphorylation and kinase reaction . F Purified recombinant GST ‐ KSHV ‐ TK and GST ‐ KSHV ‐ TK ‐ KINASE ‐ DEAD immunoblotted for phosphotyrosine and GST . G Immunoblot analysis demonstrating that the N ‐ terminus of KSHV ‐ TK is only auto ‐ phosphorylated by the kinase domain in cis .

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Figure 4A Cellular localisation and immunoblot analysis of phosphotyrosine (upper panel) and GFP (lower panel) in cells expressing wild‐type or kinase‐dead GFP‐KSHV‐TK.B Scanning electron microscopy of HeLa cells expressing GFP or GFP‐KSHV‐TK‐DEAD.C Immunofluorescence microscopy analysis of HeLa cells expressing GFP‐KSHV‐TK‐DEAD (white arrowheads) with the indicated phospho antibodies.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 and GST‐KSHV‐TKimmunoblotted for phosphotyrosine and GST following a sequential dephosphorylation and kinase reaction.F Purified recombinant GST‐KSHV‐TK and GST‐KSHV‐TK‐KINASE‐DEAD immunoblotted for phosphotyrosine and GST.G Immunoblot analysis demonstrating that the N‐terminus of KSHV‐TK is only auto‐phosphorylated by the kinase domain in cis.


Figure 5A Immunoblot analysis of N ‐ terminal deletions of KSHV ‐ TK demonstrates that the first 100 amino acids are required for tyrosine phosphorylation . B Immunoblot analysis reveals that mutation of tyrosines 65 , 85 and 120 ablates tyrosine phosphorylation of the KSHV ‐ TK . 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 . 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 . F Immunoblot analysis of level of GTP ‐ bound and total RhoA in cells expressing the indicated GFP ‐ tagged KSHV ‐ TK proteins . 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 .

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Figure 5A Immunoblot analysis of N‐terminal deletions of KSHV‐TK demonstrates that the first 100 amino acids are required for tyrosine phosphorylation.B Immunoblot analysis reveals that mutation of tyrosines 65, 85 and 120 ablates tyrosine phosphorylation of the KSHV‐TK.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.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.F Immunoblot analysis of level of GTP‐bound and total RhoA in cells expressing the indicated GFP‐tagged KSHV‐TK proteins.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.


Figure 6A 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 ( white arrowheads ) or induce contraction in the absence of FAK . Scale bar , 20 μm . 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 .

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Figure 6A 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 (white arrowheads) or induce contraction in the absence of FAK. Scale bar, 20 μm.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.


Figure 7B 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 analysis reveals CrkII associates with GFP ‐ KSHV ‐ TK but not the Y2F mutant . Scale bars , 20 μm . 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 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 .

O B-PANEL_START O O 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-PANEL_START 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-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7B 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 analysis reveals CrkII associates with GFP‐KSHV‐TK but not the Y2F mutant. Scale bars, 20 μm.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 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.


Figure 8B Immunoblot analysis reveals that GFP ‐ KSHV ‐ TK WT and Y2F but not Y120F associate with p85 of PI3 ‐ Kinase . 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 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 and CrkL is only upregulated in cells expressing GFP ‐ KSHV ‐ TK . F Immunoblot analysis reveals that recombinant His ‐ CrkII binds to and is phosphorylated by KSHV ‐ TK but not the Y2F or Y3F mutants .

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Figure 8B Immunoblot analysis reveals that GFP‐KSHV‐TK WT and Y2F but not Y120F associate with p85 of PI3‐Kinase.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 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 and CrkL is only upregulated in cells expressing GFP‐KSHV‐TK.F Immunoblot analysis reveals that recombinant His‐CrkII binds to and is phosphorylated by KSHV‐TK but not the Y2F or Y3F mutants.


Figure 1 ( 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 ) 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 ) 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 ) 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 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(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) 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) 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) 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).


Figure 2 ( 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 ) 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 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 ) 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 ) .

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Figure 2(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) 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 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) 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


Figure 3 ( 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 ) 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 ) 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 ) 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 ) Staining for Ki67 in E15 WT and hFOXM1 - cKI mice . Scale bar , 50μm . ( 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 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(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) 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) 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) 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) Staining for Ki67 in E15 WT and hFOXM1-cKI mice. Scale bar, 50μm.(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


Figure 4 ( 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 ) 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 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(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) 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


Figure 5 ( 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 analysis for Lin28a in cortex of E15 WT and hFOXM1 - cKI mice . β - actin was detected as loading control . ( 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 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

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Figure 5(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 analysis for Lin28a in cortex of E15 WT and hFOXM1-cKI mice. β-actin was detected as loading control.(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 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


Figure 6 ( 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 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 ) . (

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(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 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). (


Figure 1A ) 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A) 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.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.


Figure 2A - 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 . 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 . 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 : ML1N * 2 , a probe for PI ( 3 , 5 ) P2 ( G ) . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A-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.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.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:ML1N*2, a probe for PI(3,5)P2 (G). 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).


Figure 3A - 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A-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.


Figure 4A - 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A-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.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.


Figure 5E ) 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5E) 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.


Figure 6A - 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A-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.


Figure 7A ) 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 . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A) 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.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.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.


Figure 1 ( 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 . ( 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 . ( 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 ) . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(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.(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.(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).(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.


Figure 2 ( 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 . ( 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 . ( 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 . 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 ) . ( 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 . ( 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 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(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.(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.(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.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).(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.(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).


Figure 3 ( 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 . 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 . ( 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 . ( 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 . ( 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 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(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.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.(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.(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.(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).


Figure 4 ( 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 ) . ( 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 ) . 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 ) . ( 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 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(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).(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).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).(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).


Figure 1A 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 . 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 ) . 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 . 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A 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.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).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.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.