Figure 1A . LN229 and U87 cells treated with Wnt 3a ( 0 , 10 , 20 , 50 , 100 ng / ml ) for 4 hr . The indicated proteins were analyzed by Western blotting . B . LN229 cells treated with or without Wnt3a ( 50 ng / ml ) were pulsed with [ 35S ] methionine for 30 min and chased for the times indicated . Protein extracts were used for IP with FoxM1 antibodies and subjected to SDS - PAGE and autoradiography . The intensities of the FoxM1 bands in autoradiography were then quantified by using NIH Image software , and the densities of the FoxM1 bands at time 0 were set as 100 % . Values are mean ± SD from two independent experiments . C . Levels of FoxM1 were analyzed by Western blotting in LN229 and U87 cells treated with Wnt inhibitor DKK1 ( 100 ng / ml ) for indicated times . Noted that the basal levels of FoxM1 and β - catenin in ( A ) and ( C ) are similar but look different due to variations on the exposure time of the Western blots . D . FoxM1 expression levels were determined in LN229 cells treated with or without GSK3 inhibitor LiCl ( 10 mM ) and CHX ( 100 µg / ml ) for indicated times . E . GSK3 wild - type ( WT ) and knockout ( KO ) cells were treated with CHX ( 100 µg / ml ) for indicated times , and FoxM1 and GSK3α / β expression levels were determined by Western blotting . F . Flag - FoxM1 and GSK3βKA or GSK3βKD plasmids were co - transfected into 293T cells for 48 hr . The cells were then treated with CHX ( 100 µg / ml ) for indicated times . Exogenous Flag - FoxM1 expression level was determined by Western blotting .

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Figure 1A. LN229 and U87 cells treated with Wnt 3a (0, 10, 20, 50, 100 ng/ml) for 4 hr. The indicated proteins were analyzed by Western blotting.B. LN229 cells treated with or without Wnt3a (50 ng/ml) were pulsed with [35S] methionine for 30 min and chased for the times indicated. Protein extracts were used for IP with FoxM1 antibodies and subjected to SDS-PAGE and autoradiography. The intensities of the FoxM1 bands in autoradiography were then quantified by using NIH Image software, and the densities of the FoxM1 bands at time 0 were set as 100%. Values are mean ± SD from two independent experiments.C. Levels of FoxM1 were analyzed by Western blotting in LN229 and U87 cells treated with Wnt inhibitor DKK1 (100 ng/ml) for indicated times. Noted that the basal levels of FoxM1 and β-catenin in (A) and (C) are similar but look different due to variations on the exposure time of the Western blots.D. FoxM1 expression levels were determined in LN229 cells treated with or without GSK3 inhibitor LiCl (10 mM) and CHX (100 µg/ml) for indicated times.E. GSK3 wild-type (WT) and knockout (KO) cells were treated with CHX (100 µg/ml) for indicated times, and FoxM1 and GSK3α/β expression levels were determined by Western blotting.F. Flag-FoxM1 and GSK3βKA or GSK3βKD plasmids were co-transfected into 293T cells for 48 hr. The cells were then treated with CHX (100 µg/ml) for indicated times. Exogenous Flag-FoxM1 expression level was determined by Western blotting.


Figure 2B . Lysates from 293T cells expressing Flag - FoxM1 or its mutants were subjected to IP using mouse anti - Flag antibody , followed by IB with GSK3α / β antibody ( upper panel ) and rabbit anti - Flag antibody ( middle panel ) . D . HA - ubiquitin and Flag - tagged FoxM1 ( WT ) or mutants FoxM1S228A , FoxM1T309A , or Flag - FoxM1S474A plasmid were co - transfected into 293T cells . After 36 hr , cells were treated with 25 nM MG132 for 6 hr . Cell lysates were subjected to IP with Flag antibody , followed by IB with Flag and HA antibody . E . HA - ubiquitin was co - transfected into 293T cells . After 36 hr , cells were treated with 25 nM MG132 for 6 hr . Cell lysates were subjected to IP with IgG or FoxM1 phospho - S474 antibody , followed by IB with FoxM1 and HA antibody . F . HA - tagged K48 - only ubiquitin or K63 - only ubiquitin construct was transfected into 293T cells . After 36 hr , cells were treated with 25 nM MG132 for 6 hr . Cell lysates were subjected to IP with IgG or FoxM1 antibody , followed by IB with FoxM1 and HA antibody . G . GSK3βCA or vector and Flag - tagged FoxM1 ( WT ) , mutants S474A or S478A were co - transfected into 293T cells . Lysates of the cells were subjected to IP with Flag antibody and followed by IB with FoxM1 phospho - S474 antibody .

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Figure 2B. Lysates from 293T cells expressing Flag-FoxM1 or its mutants were subjected to IP using mouse anti-Flag antibody, followed by IB with GSK3α/β antibody (upper panel) and rabbit anti-Flag antibody (middle panel).D. HA-ubiquitin and Flag-tagged FoxM1 (WT) or mutants FoxM1S228A, FoxM1T309A, or Flag-FoxM1S474A plasmid were co-transfected into 293T cells. After 36 hr, cells were treated with 25 nM MG132 for 6 hr. Cell lysates were subjected to IP with Flag antibody, followed by IB with Flag and HA antibody.E. HA-ubiquitin was co-transfected into 293T cells. After 36 hr, cells were treated with 25 nM MG132 for 6 hr. Cell lysates were subjected to IP with IgG or FoxM1 phospho-S474 antibody, followed by IB with FoxM1 and HA antibody.F. HA-tagged K48-only ubiquitin or K63-only ubiquitin construct was transfected into 293T cells. After 36 hr, cells were treated with 25 nM MG132 for 6 hr. Cell lysates were subjected to IP with IgG or FoxM1 antibody, followed by IB with FoxM1 and HA antibody.G. GSK3βCA or vector and Flag-tagged FoxM1 (WT), mutants S474A or S478A were co-transfected into 293T cells. Lysates of the cells were subjected to IP with Flag antibody and followed by IB with FoxM1 phospho-S474 antibody.


Figure 3A . Cell lysates of LN229 cells were subjected to IP using Axin antibody or control IgG , followed by IB with indicated antibodies . B . Axin siRNA or control siRNA were transfected into LN229 cells , after 36 hours , lysate of the cells was performed with co - IP using FoxM1 antibody followed by IB with indicated antibodies . C . Axin siRNA or control siRNA were transfected into LN229 cells for 36 hours . Lysates of the cells were subjected to IB with FoxM1 phospho - S474 antibody . D . Cell lysates of LN229 cells with or without Wnt - 3a treatment for 4 hours were subjected to IP using Axin antibody or control IgG , followed by IB with FoxM1 and Axin antibodies . . E . Axin siRNA or control siRNA with HA - Ubiquitin were transfected into 293T cells , after 36 hours , cells were treated with MG132 with or without Wnt - 3a ( 50 ng / ml ) for six hours . Then lysate of the cells was performed with IP using FoxM1 antibody followed by IB with indicated antibodies . F . Wild type Flag - FoxM1 or S474A mutant was transfected into 293T cells after 36 hours , cells were treated with MG132 for six hours . Then lysate of the cells was performed with IP using Flag antibody followed by IB with indicated antibodies . G . HA - Ubiquitin and / or FBXW7 , GSK3β CA , Flag - FoxM1 were co - transfected into 293T cells , after 36 hours , cells were treated with MG132 for six hours . Then lysate of the cells was performed with IP using Flag antibody followed by IB with indicated antibodies . H . FBXW7 siRNA or control siRNA and HA - Ubiquitin were transfected into 293T cells , after 36 hours , cells were treated with MG132 with or without Wnt - 3a ( 50 ng / ml ) for six hours . Then lysate of the cells was performed with IP using FoxM1 antibody followed by IB with indicated antibodies .

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Figure 3A. Cell lysates of LN229 cells were subjected to IP using Axin antibody or control IgG, followed by IB with indicated antibodies.B. Axin siRNA or control siRNA were transfected into LN229 cells, after 36 hours, lysate of the cells was performed with co-IP using FoxM1 antibody followed by IB with indicated antibodies.C. Axin siRNA or control siRNA were transfected into LN229 cells for 36 hours. Lysates of the cells were subjected to IB with FoxM1 phospho-S474 antibody.D. Cell lysates of LN229 cells with or without Wnt-3a treatment for 4 hours were subjected to IP using Axin antibody or control IgG, followed by IB with FoxM1 and Axin antibodies..E. Axin siRNA or control siRNA with HA-Ubiquitin were transfected into 293T cells, after 36 hours, cells were treated with MG132 with or without Wnt-3a (50 ng/ml) for six hours. Then lysate of the cells was performed with IP using FoxM1 antibody followed by IB with indicated antibodies.F. Wild type Flag-FoxM1 or S474A mutant was transfected into 293T cells after 36 hours, cells were treated with MG132 for six hours. Then lysate of the cells was performed with IP using Flag antibody followed by IB with indicated antibodies.G. HA-Ubiquitin and/or FBXW7, GSK3β CA, Flag-FoxM1 were co-transfected into 293T cells, after 36 hours, cells were treated with MG132 for six hours. Then lysate of the cells was performed with IP using Flag antibody followed by IB with indicated antibodies.H. FBXW7 siRNA or control siRNA and HA-Ubiquitin were transfected into 293T cells, after 36 hours, cells were treated with MG132 with or without Wnt-3a (50 ng/ml) for six hours. Then lysate of the cells was performed with IP using FoxM1 antibody followed by IB with indicated antibodies.


Figure 4A . 293T cells were transfected with USPs cDNA or control plasmid . After 36 hr of transfection , cells were treated with 50 ng / ml Wnt - 3a for 4 hr . FoxM1 protein levels in the cells were then determined by Western blotting . B . 293T cells were transfected with 1 µg control plasmid or USP5 cDNA in 6 - well plates . After 36 hr of transfection , cells were treated with 100 µg / ml CHX for 0 , 2 , 4 , or 6 hr . FoxM1 protein levels in the cells were then determined by Western blotting . C . GSK3 - β CA , USP5 , or control plasmid was co - transfected with Flag - FoxM1 into 293T cells . After 36 hr of transfection , cells were treated with 50 ng / ml Wnt - 3a or control PBS for 4 hr . Cell lysates were subjected to IP with USP5 antibody and followed by IB with Flag or USP5 antibody . D . GSK3βCA , USP5 , or control plasmid was co - transfected with Flag - FoxM1 into 293T cells . After 36 hr of transfection , cells were treated with 50 ng / ml Wnt - 3a or control PBS for 6 hr . Cell lysates were subjected to USP5 with Flag antibody and followed by USP5 with Flag or USP5 antibody . E . USP5 and Flag - FoxM1 ( WT ) or mutants Flag - FoxM1S474A or Flag - FoxM1S474E plasmids were co - transfected into 293T cells for 36 hr . Cell lysates were subjected to IP with Flag antibody and followed by IB with Flag or USP5 antibody . F . GSK3β CA , USP5 , or control plasmid was co - transfected with Flag - FoxM1 ( WT ) or mutants Flag - FoxM1S474A into 293T cells for 36 hr . Cell lysates were subjected to IP with Flag antibody and followed by IB with Flag or USP5 antibody . G . HA - ubiquitin and Flag - FoxM1 were co - transfected with or without GSK3β CA into 293T cells . After 36 hr , cells were treated with 25 nM MG132 for 6 hr . Cell lysates were subjected to IP with Flag followed by IB with HA or FoxM1 antibody . H . HA - ubiquitin and Flag - FoxM1 were co - transfected with or without siUSP5 into U87 cells . After 36 hr , cells were treated with 50 ng / ml Wnt - 3a and 25 nM MG132 for 6 hr . Cell lysates were subjected to IP with Flag antibody followed by IB with HA or FoxM1 antibody .

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Figure 4A. 293T cells were transfected with USPs cDNA or control plasmid. After 36 hr of transfection, cells were treated with 50 ng/ml Wnt-3a for 4 hr. FoxM1 protein levels in the cells were then determined by Western blotting.B. 293T cells were transfected with 1 µg control plasmid or USP5 cDNA in 6-well plates. After 36 hr of transfection, cells were treated with 100 µg/ml CHX for 0, 2, 4, or 6 hr. FoxM1 protein levels in the cells were then determined by Western blotting.C. GSK3-β CA, USP5, or control plasmid was co-transfected with Flag-FoxM1 into 293T cells. After 36 hr of transfection, cells were treated with 50 ng/ml Wnt-3a or control PBS for 4 hr. Cell lysates were subjected to IP with USP5 antibody and followed by IB with Flag or USP5 antibody.D. GSK3βCA, USP5, or control plasmid was co-transfected with Flag-FoxM1 into 293T cells. After 36 hr of transfection, cells were treated with 50 ng/ml Wnt-3a or control PBS for 6 hr. Cell lysates were subjected to USP5 with Flag antibody and followed by USP5 with Flag or USP5 antibody.E. USP5 and Flag-FoxM1 (WT) or mutants Flag-FoxM1S474A or Flag-FoxM1S474E plasmids were co-transfected into 293T cells for 36 hr. Cell lysates were subjected to IP with Flag antibody and followed by IB with Flag or USP5 antibody.F. GSK3β CA, USP5, or control plasmid was co-transfected with Flag-FoxM1 (WT) or mutants Flag-FoxM1S474A into 293T cells for 36 hr. Cell lysates were subjected to IP with Flag antibody and followed by IB with Flag or USP5 antibody.G. HA-ubiquitin and Flag-FoxM1 were co-transfected with or without GSK3β CA into 293T cells. After 36 hr, cells were treated with 25 nM MG132 for 6 hr. Cell lysates were subjected to IP with Flag followed by IB with HA or FoxM1 antibody.H. HA-ubiquitin and Flag-FoxM1 were co-transfected with or without siUSP5 into U87 cells. After 36 hr, cells were treated with 50 ng/ml Wnt-3a and 25 nM MG132 for 6 hr. Cell lysates were subjected to IP with Flag antibody followed by IB with HA or FoxM1 antibody.


Figure 5A . Expression of FoxM1 and ICAT was examined via IHC staining in 30 GBM and paired adjacent normal tissues . Representative ICAT and FoxM1 expressions are shown in a section of a GBM tumor with adjacent normal brain . Scale bar : 50 µmB . ICAT stainings were scored as 1 - 12 according to the intensity and percentage in the 30 GBM and paired adjacent normal tissues . Boxes indicate interquartile range . Bars from each box extend to largest and smallest observations . C . FoxM1 stainings were scored as 1 - 12 according to the intensity and percentage in the 30 GBM and paired adjacent normal tissues . Boxes indicate interquartile range . Bars from each box extend to largest and smallest observations . D . Increased amounts of FoxM1 plasmids were transfected into 293T cells . After 36 hr , cells were treated with 50 ng / ml Wnt - 3a for 6 hr . Cell lysates were then subjected to IP with β - catenin antibody , followed by IB with β - catenin , ICAT , FoxM1 or IgG antibodies . E . Increased amounts of ICAT plasmids were transfected into 293T cells . After 36 hr , cells were treated with 50 ng / ml Wnt - 3a for 6 hr . Cell lysates were then subjected to IP with β - catenin antibody , followed by IB with β - catenin , ICAT , FoxM1 or IgG antibodies . F . FoxM1 siRNA or control siRNA was transfected into U87 cells , lysates were subjected to IP with β - catenin antibody , followed by IB with β - catenin , ICAT , FoxM1 or IgG antibodies . G . ICAT siRNA or control siRNA was transfected into LN229 cells , lysates were subjected to IP with β - catenin antibody , followed by IB with β - catenin , ICAT , FoxM1 or IgG antibodies . H . ChIP analyses of endogenous β - catenin binding at the TCF - binding site of the Axin2 promoter were performed in vector - , FoxM1 - or / and ICAT plasmid - transfected U87 cells . Fold was calculated relative to that in cells transfected with the vector , which was set as 1 . I . ChIP analyses of endogenous β - catenin binding at the TCF - binding site of the Axin2 promoter were performed in FoxM1 siRNA - , ICAT siRNA - , control siRNA - or combination of FoxM1 siRNA and ICAT siRNA - transfected U87 cells as described in ( H ) . J . ChIP analyses of endogenous β - catenin binding at the TCF - binding site of the Axin2 promoter were performed in vector - , β - catenin - NLS - , sh - FoxM1 - , FoxM1 - shR - and / or ICAT plasmid - transfected U87 cells as described in ( 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 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 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 5A. Expression of FoxM1 and ICAT was examined via IHC staining in 30 GBM and paired adjacent normal tissues. Representative ICAT and FoxM1 expressions are shown in a section of a GBM tumor with adjacent normal brain. Scale bar: 50 µmB. ICAT stainings were scored as 1-12 according to the intensity and percentage in the 30 GBM and paired adjacent normal tissues. Boxes indicate interquartile range. Bars from each box extend to largest and smallest observations.C. FoxM1 stainings were scored as 1-12 according to the intensity and percentage in the 30 GBM and paired adjacent normal tissues. Boxes indicate interquartile range. Bars from each box extend to largest and smallest observations.D. Increased amounts of FoxM1 plasmids were transfected into 293T cells. After 36 hr, cells were treated with 50 ng/ml Wnt-3a for 6 hr. Cell lysates were then subjected to IP with β-catenin antibody, followed by IB with β-catenin, ICAT, FoxM1 or IgG antibodies.E. Increased amounts of ICAT plasmids were transfected into 293T cells. After 36 hr, cells were treated with 50 ng/ml Wnt-3a for 6 hr. Cell lysates were then subjected to IP with β-catenin antibody, followed by IB with β-catenin, ICAT, FoxM1 or IgG antibodies.F. FoxM1 siRNA or control siRNA was transfected into U87 cells, lysates were subjected to IP with β-catenin antibody, followed by IB with β-catenin, ICAT, FoxM1 or IgG antibodies.G. ICAT siRNA or control siRNA was transfected into LN229 cells, lysates were subjected to IP with β-catenin antibody, followed by IB with β-catenin, ICAT, FoxM1 or IgG antibodies.H. ChIP analyses of endogenous β-catenin binding at the TCF-binding site of the Axin2 promoter were performed in vector-, FoxM1- or/and ICAT plasmid-transfected U87 cells. Fold was calculated relative to that in cells transfected with the vector, which was set as 1.I. ChIP analyses of endogenous β-catenin binding at the TCF-binding site of the Axin2 promoter were performed in FoxM1 siRNA-, ICAT siRNA-, control siRNA- or combination of FoxM1 siRNA and ICAT siRNA-transfected U87 cells as described in (H).J. ChIP analyses of endogenous β-catenin binding at the TCF-binding site of the Axin2 promoter were performed in vector-, β-catenin-NLS-, sh-FoxM1-, FoxM1-shR- and/or ICAT plasmid-transfected U87 cells as described in (H).


Figure 6A . FoxM1 or / and ICAT plasmids were co - transfected with Axin - 2 promoter luciferase and TK - Renilla plasmid into U87 cells . Luciferase activity was measured with the Dural Luciferase reporter assay , and relative luciferase activity was normalized to that of the vector group . Values are mean ± SD for triplicate samples . B . FoxM1 siRNA # 1 , # 2 or control siRNA were co - transfected to U87 cells with Axin - 2 promoter reporter . Luciferase activity was measured with the Dural Luciferase reporter assay , and relative luciferase activity was normalized to that of the control siRNA group . Values are mean ± SD for triplicate samples . C . ICAT siRNA or control siRNA were co - transfected into LN229 cells with Axin - 2 promoter reporter . Luciferase activity was measured as in ( C ) . D . FoxM1 or / and ICAT plasmids were co - transfected with TOP - Flash reporter and TK - Renilla plasmids into U87 cells . Luciferase activity of TOP - Flash in the cells was measured as in A . Values are mean ± SD for triplicate samples . E . ICAT , FoxM1 or FoxM1 - ∆ FB ( FoxM1 mutant lacking the forkhead box domain ) plasmid or their combinations were co - transfected with TOP - Flash reporter and TK - Renilla plasmids into LN229 cells . Luciferase activity of TOP - Flash in the cells was measured as in A . Values are mean ± SD for triplicate samples . F . FoxM1 siRNA and / or ICAT siRNA were co - transfected with TOP - Flash reporter and TK - Renilla plasmids into LN229 cells , and the cells were treated with Wnt3a ( 50 ng / ml ) or control PBS . Luciferase activity of TOP - Flash in the cells was measured as in A . Values are mean ± SD for triplicate samples . G . Vector - , β - catenin - NLS - , sh - FoxM1 - , FoxM1 - shR - and / or ICAT plasmids were co - transfected with TOP - Flash reporter and TK - Renilla plasmids into U87 cells . Luciferase activity of TOP - Flash in the cells was measured as in ( A ) . Values are mean ± SD triplicate samples . H . Levels of Axin - 2 and cyclin D1 protein in LN229 cells transfected with FoxM1 siRNA # 1 or # 2 were determined by Western blotting . I . Levels of Axin - 2 and cyclin D1 protein in LN229 cells transfected with ICAT siRNA were determined by Western blotting . J . Levels of Axin - 2 and cyclin D1 protein in LN229 cells transfected with FoxM1 or / and ICAT plasmids were determined by Western blotting .

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Figure 6A. FoxM1 or/and ICAT plasmids were co-transfected with Axin-2 promoter luciferase and TK-Renilla plasmid into U87 cells. Luciferase activity was measured with the Dural Luciferase reporter assay, and relative luciferase activity was normalized to that of the vector group. Values are mean ± SD for triplicate samples.B. FoxM1 siRNA#1, #2 or control siRNA were co-transfected to U87 cells with Axin-2 promoter reporter. Luciferase activity was measured with the Dural Luciferase reporter assay, and relative luciferase activity was normalized to that of the control siRNA group. Values are mean ± SD for triplicate samples.C. ICAT siRNA or control siRNA were co-transfected into LN229 cells with Axin-2 promoter reporter. Luciferase activity was measured as in (C).D. FoxM1 or/and ICAT plasmids were co-transfected with TOP-Flash reporter and TK-Renilla plasmids into U87 cells. Luciferase activity of TOP-Flash in the cells was measured as in A. Values are mean ± SD for triplicate samples.E. ICAT, FoxM1 or FoxM1-∆FB (FoxM1 mutant lacking the forkhead box domain) plasmid or their combinations were co-transfected with TOP-Flash reporter and TK-Renilla plasmids into LN229 cells. Luciferase activity of TOP-Flash in the cells was measured as in A. Values are mean ± SD for triplicate samples.F. FoxM1 siRNA and/or ICAT siRNA were co-transfected with TOP-Flash reporter and TK-Renilla plasmids into LN229 cells, and the cells were treated with Wnt3a (50 ng/ml) or control PBS. Luciferase activity of TOP-Flash in the cells was measured as in A. Values are mean ± SD for triplicate samples.G. Vector-, β-catenin-NLS-, sh-FoxM1-, FoxM1-shR- and/or ICAT plasmids were co-transfected with TOP-Flash reporter and TK-Renilla plasmids into U87 cells. Luciferase activity of TOP-Flash in the cells was measured as in (A). Values are mean ± SD triplicate samples.H. Levels of Axin-2 and cyclin D1 protein in LN229 cells transfected with FoxM1 siRNA#1 or #2 were determined by Western blotting.I. Levels of Axin-2 and cyclin D1 protein in LN229 cells transfected with ICAT siRNA were determined by Western blotting.J. Levels of Axin-2 and cyclin D1 protein in LN229 cells transfected with FoxM1 or/and ICAT plasmids were determined by Western blotting.


Figure 7A . A total of 1 × 104 LN229 cells were transfected with control vector , FoxM1 , and / or ICAT plasmids and treated with Wnt3a ( 50 ng / ml ) and were then plated and counted 48 hr after seeding . The proliferation percentage was calculated relative to that in cells transfected with the control vector , which was set as 100 % . Data represent the mean ± SD of three independent experiments . B . LN229 cells were transfected with vector control , FoxM1 , FoxM1 plus sh - β - catenin or FoxM1 plus ICAT plasmid . The proliferation percentage was determined as described in ( A ) . C . LN229 - shFoxM1 cells were transfected with FoxM1 - shR ( siRNA - resistant FoxM1 ) , or FoxM1 - shR plus ICAT plasmid . The cells were then treated with Wnt3a ( 50 ng / ml ) . The proliferation percentage was determined as described in ( A ) . D . LN229 cells were transfected with USP5 and / or FoxM1 siRNA . The proliferation percentage was determined as described in ( A ) . E . LN229 cells were transfected with USP5 siRNA and / or FoxM1 plasmid . The cells were then treated with Wnt3a ( 50 ng / ml ) . The proliferation percentage was determined as described in ( A ) . Data shown in ( A ) - ( E ) represent the mean ± SD of three independent experiments . F - H . Expression of FoxM1 , Cyclin D1 and USP5 proteins was examined via IHC staining in 50 GBM specimens . Staining of FoxM1 , Cyclin D1 , or USP5 was scored as 1 - 12 according to the intensity and percentage . The significance of the correlations was determined by Pearson ' s correlation test . I - J . Axin2 mRNA levels in the above 50 GBM specimens were examined through RNAscope technology . The staining was scored as 1 - 12 according to the intensity and percentage . The significance of the correlations was determined by Pearson ' s correlation 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 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 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 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 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

Figure 7A. A total of 1 × 104 LN229 cells were transfected with control vector, FoxM1, and/or ICAT plasmids and treated with Wnt3a (50 ng/ml) and were then plated and counted 48 hr after seeding. The proliferation percentage was calculated relative to that in cells transfected with the control vector, which was set as 100%. Data represent the mean ± SD of three independent experiments.B. LN229 cells were transfected with vector control, FoxM1, FoxM1 plus sh-β-catenin or FoxM1 plus ICAT plasmid. The proliferation percentage was determined as described in (A).C. LN229-shFoxM1 cells were transfected with FoxM1-shR (siRNA-resistant FoxM1), or FoxM1-shR plus ICAT plasmid. The cells were then treated with Wnt3a (50 ng/ml). The proliferation percentage was determined as described in (A).D. LN229 cells were transfected with USP5 and/or FoxM1 siRNA. The proliferation percentage was determined as described in (A).E. LN229 cells were transfected with USP5 siRNA and/or FoxM1 plasmid. The cells were then treated with Wnt3a (50 ng/ml). The proliferation percentage was determined as described in (A). Data shown in (A)-(E) represent the mean ± SD of three independent experiments.F-H. Expression of FoxM1, Cyclin D1 and USP5 proteins was examined via IHC staining in 50 GBM specimens. Staining of FoxM1, Cyclin D1, or USP5 was scored as 1-12 according to the intensity and percentage. The significance of the correlations was determined by Pearson's correlation test.I-J. Axin2 mRNA levels in the above 50 GBM specimens were examined through RNAscope technology. The staining was scored as 1-12 according to the intensity and percentage. The significance of the correlations was determined by Pearson's correlation test.


Figure 1A . Cartoon representation of the Dln1 dimer ( PDB 4ZNO ) . Subunit A is colored in gray . The lectin module of subunit B is colored in yellow , whereas the middle and C - terminal moieties of the aerolysin module are colored in hot pink and green , respectively . The pre - stem hairpin is colored in blue . The bound sucrose molecules are shown as light pink sticks . B . The dimeric interface . Residues involved in dimeric interactions are shown as sticks .

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Figure 1A. Cartoon representation of the Dln1 dimer (PDB 4ZNO). Subunit A is colored in gray. The lectin module of subunit B is colored in yellow, whereas the middle and C-terminal moieties of the aerolysin module are colored in hot pink and green, respectively. The pre-stem hairpin is colored in blue. The bound sucrose molecules are shown as light pink sticks.B. The dimeric interface. Residues involved in dimeric interactions are shown as sticks.


Figure 2A . A close - up view of the sucrose - binding pocket . Residues hydrogen - bonding with sucrose are shown as yellow sticks , and the sucrose molecule is shown as light pink stick . B . The glycan binding profile of Dln1 . This profile represents FITC - labeled Dln1 at 200 μg / ml screened against 609 printed glycans on Mammalian Printed Array version 5 . 2 in replicates of six . A list of top 8 hits reacted with a concentration of 200 μg / ml is shown in Appendix Table S1 . The two kinds of Dln1 - binding glycan structures ( No . 214 and 316 ) are illustrated in schematic . C & amp ; amp ; D . Close - up views of Manα1 - 2Man and Manα1 - 3Man binding sites . The mannobiose molecules are shown as cyan and gray sticks , respectively . The Fo - Fc electron density maps of disaccharides and the disaccharide - binding sites are shown as gray mesh and countered at 3 . 0 . The hydrogen - bonding residues are shown as yellow sticks . Polar interactions with the mannobiose are shown as dashed lines .

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Figure 2A. A close-up view of the sucrose-binding pocket. Residues hydrogen-bonding with sucrose are shown as yellow sticks, and the sucrose molecule is shown as light pink stick.B. The glycan binding profile of Dln1. This profile represents FITC-labeled Dln1 at 200 μg/ml screened against 609 printed glycans on Mammalian Printed Array version 5.2 in replicates of six. A list of top 8 hits reacted with a concentration of 200 μg/ml is shown in Appendix Table S1. The two kinds of Dln1-binding glycan structures (No. 214 and 316) are illustrated in schematic.C&amp;D. Close-up views of Manα1-2Man and Manα1-3Man binding sites. The mannobiose molecules are shown as cyan and gray sticks, respectively. The Fo-Fc electron density maps of disaccharides and the disaccharide-binding sites are shown as gray mesh and countered at 3.0 . The hydrogen-bonding residues are shown as yellow sticks. Polar interactions with the mannobiose are shown as dashed lines.


Figure 3A & amp ; amp ; B . ELISA of Dln1 towards glycosylated gp120 or yeast mannan . The curve represents the average of triplicate detections , using BSA as the control . Points are averages ( ± standard error of the mean ) of triplicate determinations . C . Binding of FITC - labeled Dln1 and D135A mutant to saccharomyces cerevisiae cells . The scale bar represents 5 μm . D . Immunoblot analysis of Dln1 and D135A mutant after incubating with S . cerevisiae cells . The seeing bands represent the cell - bound protein after extensive washing the yeast cells . The monomer and oligomer bands are highlighted by the black arrow .

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Figure 3A&amp;B. ELISA of Dln1 towards glycosylated gp120 or yeast mannan. The curve represents the average of triplicate detections, using BSA as the control. Points are averages (±standard error of the mean) of triplicate determinations.C. Binding of FITC-labeled Dln1 and D135A mutant to saccharomyces cerevisiae cells. The scale bar represents 5 μm.D. Immunoblot analysis of Dln1 and D135A mutant after incubating with S. cerevisiae cells. The seeing bands represent the cell-bound protein after extensive washing the yeast cells. The monomer and oligomer bands are highlighted by the black arrow.


Figure 4A . Oligomerization of Dln1 in the presence of S . cerevisiae mannan or liposomes at different pH values . Histograms are averages ( ± standard error of the mean ) of triplicate determinationsB & amp ; amp ; C . Dye release velocity from the liposomes in the presence of Dln1 at different concentrations , and various pH values . Liposomes were incubated with Dln1 of different concentrations at pH 5 . 5 , and 1 μM Dln1 at various pH values , respectively . D . Oligomerization of Dln1M5 with or without liposomes at different pH values . All samples for electrophoresis were prepared with boiling at 95 ° C for 5 min , and the comparative gels were processed in parallel . The oligomer band was quantified by Image J software with the average of triplicate detections , using the marker band as a sample processing control . The SDS - PAGE profiles of Dln1 oligomerization were shown in Appendix Fig S4 . Histograms are averages ( ± standard error of the mean ) of triplicate determinations . E . Liposomes were treated with Dln1M5 or Dln1 of different concentrations at a fixed pH value of 5 . 5 . F . The inhibition of dye release in presence of 0 . 5 M Dln1M5 and 25 mM polyethylene glycols ( PEG ) of different molecular weights at a fixed pH value of 5 . 5 . Dye efflux was monitored in a period of 1400 sec . Triton X - 100 at 0 . 1 % was added to completely disrupt liposomes . Dye efflux was measured as a percentage to the maximal release upon addition of Triton X - 100 . Carboxyfluorescein was loaded in the liposomes for the detection of dye efflux .

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Figure 4A. Oligomerization of Dln1 in the presence of S. cerevisiae mannan or liposomes at different pH values. Histograms are averages (±standard error of the mean) of triplicate determinationsB&amp;C. Dye release velocity from the liposomes in the presence of Dln1 at different concentrations, and various pH values. Liposomes were incubated with Dln1 of different concentrations at pH 5.5, and 1 μM Dln1 at various pH values, respectively.D. Oligomerization of Dln1M5 with or without liposomes at different pH values. All samples for electrophoresis were prepared with boiling at 95°C for 5 min, and the comparative gels were processed in parallel. The oligomer band was quantified by Image J software with the average of triplicate detections, using the marker band as a sample processing control. The SDS-PAGE profiles of Dln1 oligomerization were shown in Appendix Fig S4. Histograms are averages (±standard error of the mean) of triplicate determinations.E. Liposomes were treated with Dln1M5 or Dln1 of different concentrations at a fixed pH value of 5.5.F. The inhibition of dye release in presence of 0.5 M Dln1M5 and 25 mM polyethylene glycols (PEG) of different molecular weights at a fixed pH value of 5.5. Dye efflux was monitored in a period of 1400 sec. Triton X-100 at 0.1% was added to completely disrupt liposomes. Dye efflux was measured as a percentage to the maximal release upon addition of Triton X-100. Carboxyfluorescein was loaded in the liposomes for the detection of dye efflux.


Figure 5A . Negative - stain electron microscopy image of Dln1M5 in presence of liposomes . B . Side views of Dln1M5 oligomers attached to the membrane of liposomes . The inset represents the respective class averages . C & amp ; amp ; D . Transmission electron microscope micrograph of dispersed ring - like structures of Dln1M5 or Dln1D135A oligomers on lipid monolayer . The inset represents a few 2D class averages showing the octamer pattern from some individual rings . C & amp ; amp ; D . Transmission electron microscope micrograph of dispersed ring - like structures of Dln1M5 or Dln1D135A oligomers on lipid monolayer . The inset represents a few 2D class averages showing the octamer pattern from some individual rings . E . EM picture of a negatively stained 2D crystal of Dln1M5 on the lipid monolayer . The inset represents the computed diffraction pattern Fourier transform of the image . F . Projection map with a P4 symmetry . G . The top , side and cut - open views of the 3D reconstruction of Dln1M5 octamer at 20 Å resolution are shown as gray surface representation . The surface of membrane is indicated by dashed lines . The scale bar is 10 nm .

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 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 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

Figure 5A. Negative-stain electron microscopy image of Dln1M5 in presence of liposomes.B. Side views of Dln1M5 oligomers attached to the membrane of liposomes. The inset represents the respective class averages.C&amp;D. Transmission electron microscope micrograph of dispersed ring-like structures of Dln1M5 or Dln1D135A oligomers on lipid monolayer. The inset represents a few 2D class averages showing the octamer pattern from some individual rings.C&amp;D. Transmission electron microscope micrograph of dispersed ring-like structures of Dln1M5 or Dln1D135A oligomers on lipid monolayer. The inset represents a few 2D class averages showing the octamer pattern from some individual rings.E. EM picture of a negatively stained 2D crystal of Dln1M5 on the lipid monolayer. The inset represents the computed diffraction pattern Fourier transform of the image.F. Projection map with a P4 symmetry.G. The top, side and cut-open views of the 3D reconstruction of Dln1M5 octamer at 20 Å resolution are shown as gray surface representation. The surface of membrane is indicated by dashed lines. The scale bar is 10 nm.


Figure 6A . Top and side views of eight lectin modules docked to the 20 Å EM map . Due to the moderate resolution of the EM map , the flexible aerolysin module cannot be fit in the map . It is important to notice that this model is only a hypothetical fit . Each pair of the lectin modules are colored in yellow and orange , respectively . The β - barrel of γ - hemolysin ( PDB 3B07 ) was used to mimic the β - barrel backbone of Dln1 . The position of cell membrane is indicated by dashed lines . B . B - factor of Dln1 subunit B . The mobility is represented by coil thickness from thin ( low ) to thick ( high ) , and color from blue ( low ) to red ( high ) . Notably , the pre - stem hairpin and distal moiety of aerolysin module are particularly mobile . C . Position of Cα atoms along MD simulations of apo - form ( red ) and the pre - stem hairpin extraction form ( blue ) are shown with the lectin modules superimposed ( 200 structures for each ) .

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 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

Figure 6A. Top and side views of eight lectin modules docked to the 20 Å EM map. Due to the moderate resolution of the EM map, the flexible aerolysin module cannot be fit in the map. It is important to notice that this model is only a hypothetical fit. Each pair of the lectin modules are colored in yellow and orange, respectively. The β-barrel of γ-hemolysin (PDB 3B07) was used to mimic the β-barrel backbone of Dln1. The position of cell membrane is indicated by dashed lines.B. B-factor of Dln1 subunit B. The mobility is represented by coil thickness from thin (low) to thick (high), and color from blue (low) to red (high). Notably, the pre-stem hairpin and distal moiety of aerolysin module are particularly mobile.C. Position of Cα atoms along MD simulations of apo-form (red) and the pre-stem hairpin extraction form (blue) are shown with the lectin modules superimposed (200 structures for each).


Figure 1 ( A ) Bacterially purified 6xHis - rRv2626c and analyzed by Coomassie blue staining ( left ) or immuno blotting ( IB ) with αHis ( right ) . ( B ) BMDMs were incubated with rRv2626c for the indicated times and then cell viability measured with MTT assay . ( C ) Fluorescence confocal images showing in BMDMs treated with Rv2626c ( 2 . 5 μg / ml ) for 30 min , fixed , immunostained with antibodies for His ( Alexa 488 ) and DAPI . Scale bar , 10 μm . ( D and E ) BMDMs from WT mice were treated with LPS ( 100 ng / ml ) or rRv2626c for indicated times ( D ) . BMDMs from WT , TLR2 - / - , TLR4 - / - , MyD88 - / - , TRIF - / - , IRAK1 - / - , TRAF6 - / - , TBK1 - / - mice were treated with 2 . 5 μg / ml rRv2626c or rVector for 18 h ( E ) . Culture supernatants were harvested , and the levels of TNF - α , IL - 6 , IL - 12p40 , and IL - 10 were measured by ELISA . ( F ) BMDMs were treated with 2 . 5 μg / ml rRv2626c for 1 h , harvested and then subjected to IB for αIκBα and His . αActin was used as a loading control .

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Figure 1(A) Bacterially purified 6xHis-rRv2626c and analyzed by Coomassie blue staining (left) or immuno blotting (IB) with αHis (right).(B) BMDMs were incubated with rRv2626c for the indicated times and then cell viability measured with MTT assay.(C) Fluorescence confocal images showing in BMDMs treated with Rv2626c (2.5 μg/ml) for 30 min, fixed, immunostained with antibodies for His (Alexa 488) and DAPI. Scale bar, 10 μm.(D and E) BMDMs from WT mice were treated with LPS (100 ng/ml) or rRv2626c for indicated times (D). BMDMs from WT, TLR2-/-, TLR4-/-, MyD88-/-, TRIF-/-, IRAK1-/-, TRAF6-/-, TBK1-/- mice were treated with 2.5 μg/ml rRv2626c or rVector for 18 h (E). Culture supernatants were harvested, and the levels of TNF-α, IL-6, IL-12p40, and IL-10 were measured by ELISA.(F) BMDMs were treated with 2.5 μg/ml rRv2626c for 1 h, harvested and then subjected to IB for αIκBα and His. αActin was used as a loading control.


Figure 2BMDMs were pretreated with 2 . 5 μg / ml rRv2626c or rVector for 1 h , and stimulated with 100 ng / ml LPS ( A for indicated times . ( A Culture supernatants were harvested , and the levels of TNF - α , IL - 6 , IL - 12p40 , and IL - 10 were measured by ELISA . BMDMs were pretreated with 2 . 5 μg / ml rRv2626c or rVector for 1 h , and stimulated with 100 ng / ml Pam2CSK4 ( B ) B ) Culture supernatants were harvested , and the levels of TNF - α , IL - 6 , IL - 12p40 , and IL - 10 were measured by ELISA . ( C ) Cells were harvested and then subjected to IB for the phosphorylated forms of AKT , MAPK ( ERK , p38 , JNK ) , IκBα , total forms of IκBα . αActin was used as a loading control ( left ) . The densitometric values were calculated from the ratios of protein levels relative to actin ( right ) .

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Figure 2BMDMs were pretreated with 2.5 μg/ml rRv2626c or rVector for 1 h, and stimulated with 100 ng/ml LPS (A for indicated times. (A Culture supernatants were harvested, and the levels of TNF-α, IL-6, IL-12p40, and IL-10 were measured by ELISA.BMDMs were pretreated with 2.5 μg/ml rRv2626c or rVector for 1 h, and stimulated with 100 ng/ml Pam2CSK4 (B) B) Culture supernatants were harvested, and the levels of TNF-α, IL-6, IL-12p40, and IL-10 were measured by ELISA.(C) Cells were harvested and then subjected to IB for the phosphorylated forms of AKT, MAPK (ERK, p38, JNK), IκBα, total forms of IκBα. αActin was used as a loading control (left). The densitometric values were calculated from the ratios of protein levels relative to actin (right).


Figure 3 ( A ) Identification of TLR2 , IRAK1 , RIP1 , TRAF6 , MAPK11 and TOLLIP as endogenous binding partners of rRv2626c . THP - 1 cell lysates were incubated with His tagged - rRv2626c or rVector and then IP with His - agarose beads . Binding partners were confirmed by silver staining and mass spectrometric analysis . The red - colored letters indicate the peptides identified from mass spectrometry analysis . ( B ) 293T cells were co - transfected with Flag - TRAF6 and Myc - Rv2626c and IP with αFlag or αMyc . WCLs were used for IB with αFlag , αMyc or αActin . ( C ) THP - 1 and BMDMs were stimulated with rRv2626c ( 2 . 5 μg / ml ) for the indicated times , followed by IP with αHis ( up ) or αTRAF6 ( down ) and IB with αTRAF6 , αHis , and αActin . ( D ) Immunostaining of BMDMs treated with 2 . 5 μg / ml rRv2626c for 30 min . and then immunolabeled with antibody to His - rRv2626c ( Alexa Fluor 488 ) or TRAF6 ( Alexa Fluor 568 ) . DAPI ( blue ) stained nuclei .

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Figure 3(A) Identification of TLR2, IRAK1, RIP1, TRAF6, MAPK11 and TOLLIP as endogenous binding partners of rRv2626c. THP-1 cell lysates were incubated with His tagged-rRv2626c or rVector and then IP with His-agarose beads. Binding partners were confirmed by silver staining and mass spectrometric analysis. The red-colored letters indicate the peptides identified from mass spectrometry analysis.(B) 293T cells were co-transfected with Flag-TRAF6 and Myc-Rv2626c and IP with αFlag or αMyc. WCLs were used for IB with αFlag, αMyc or αActin.(C) THP-1 and BMDMs were stimulated with rRv2626c (2.5 μg/ml) for the indicated times, followed by IP with αHis (up) or αTRAF6 (down) and IB with αTRAF6, αHis, and αActin.(D) Immunostaining of BMDMs treated with 2.5 μg/ml rRv2626c for 30 min. and then immunolabeled with antibody to His-rRv2626c (Alexa Fluor 488) or TRAF6 (Alexa Fluor 568). DAPI (blue) stained nuclei.


Figure 4 ( A ) Schematic diagram of the structures of Rv2626c ( up ) . 293T cells were co - transfected with Flag - TRAF6 together with GST - vector or GST - Rv2626c and its mutant constructs , subjected to GST pulldown , followed by IB with αFlag . WCLs were used for IB with αGST , αFlag or αActin ( down ) . ( B ) At 12 hr post - transfection with mammalian Myc - Rv2626c constructs together with Flag - TRAF6 and 293T cells treated with several Tat - CBS2 peptide for 12 h ( 10 µM ) for 6h , subjected to IP with αMyc , followed by IB with αFlag . WCLs were used for IB with αMyc , αFlag or αActin . ( C ) Schematic diagram of the structures of TRAF6 ( up ) . 293T cells were co - transfected with Myc - Rv2626c together with GST - vector or GST - TRAF6 and its mutant constructs , subjected to GST pulldown , followed by IB with αMyc . WCLs were used for IB with αGST , αMyc or αActin ( down ) . ( D ) 293T cells were co - transfected with Flag - TRAF6 , Myc - Rv2626c as indicated doses and HA - ubiquitin ( left ) , HA - K48 - linked ubiquitin ( middle ) , HA - K63 - linked ubiquitin ( right ) and then IP with αFlag , followed by IB with αHA . WCLs were used for IB with αFlag , αMyc , or αActin . ( E ) BMDMs were pretreated with 2 . 5 μg / ml rRv2626c for 1 h , and stimulated with 100 ng / ml LPS for 30 min , followed by IP with αTRAF6 , IB with ubiquitin , K48 - linked ubiquitin , or K63 - linked ubiquitin . WCLs were used for IB with αHis , αTRAF6 , or αActin .

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Figure 4(A) Schematic diagram of the structures of Rv2626c (up). 293T cells were co-transfected with Flag-TRAF6 together with GST-vector or GST-Rv2626c and its mutant constructs, subjected to GST pulldown, followed by IB with αFlag. WCLs were used for IB with αGST, αFlag or αActin (down).(B) At 12 hr post-transfection with mammalian Myc-Rv2626c constructs together with Flag-TRAF6 and 293T cells treated with several Tat-CBS2 peptide for 12 h (10 µM) for 6h, subjected to IP with αMyc, followed by IB with αFlag. WCLs were used for IB with αMyc, αFlag or αActin.(C) Schematic diagram of the structures of TRAF6 (up). 293T cells were co-transfected with Myc-Rv2626c together with GST-vector or GST-TRAF6 and its mutant constructs, subjected to GST pulldown, followed by IB with αMyc. WCLs were used for IB with αGST, αMyc or αActin (down).(D) 293T cells were co-transfected with Flag-TRAF6, Myc-Rv2626c as indicated doses and HA-ubiquitin (left), HA-K48-linked ubiquitin (middle), HA-K63-linked ubiquitin (right) and then IP with αFlag, followed by IB with αHA. WCLs were used for IB with αFlag , αMyc, or αActin.(E) BMDMs were pretreated with 2.5 μg/ml rRv2626c for 1 h, and stimulated with 100 ng/ml LPS for 30 min, followed by IP with αTRAF6, IB with ubiquitin, K48-linked ubiquitin, or K63-linked ubiquitin. WCLs were used for IB with αHis, αTRAF6, or αActin.


Figure 5 ( B ) Bacterially purified 6xHis - rRv2626c and analyzed by Coomassie blue staining ( left ) or immuno blotting ( IB ) with αHis ( right ) . ( C ) BMDMs were incubated with rRv2626c - CA , rRv2626c - DN , or rVehicle for the indicated times and then cell viability measured with MTT assay . ( D ) BMDMs were pretreated with rRv2626c - WT , rRv2626c - CA , or rRv2626c - DN for 1 h , and stimulated with 100 ng / ml LPS for 18 h . Culture supernatants were harvested , and the levels of TNF - α , IL - 6 , IL - 12p40 , and IL - 10 were measured by ELISA . ( E ) Myc - Rv2626c - expressing Raw264 . 7 or THP - 1 cells were pretreated with rRv2626c - WT ( 2 . 5 μg / ml ) , rRv2626c - CA ( 10 ng / ml ) , rRv2626c - DN ( 2 . 5 μg / ml ) , for 1 h , and stimulated with 100 ng / ml LPS for 30 min , followed by IP with αMyc or αTRAF6 , IB with αMyc and ubiquitin . WCLs were used for IB with αMyc , αTRAF6 , αHis or αActin .

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 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 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 O O O O O 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 5(B) Bacterially purified 6xHis-rRv2626c and analyzed by Coomassie blue staining (left) or immuno blotting (IB) with αHis (right).(C) BMDMs were incubated with rRv2626c-CA, rRv2626c-DN, or rVehicle for the indicated times and then cell viability measured with MTT assay.(D) BMDMs were pretreated with rRv2626c-WT, rRv2626c-CA, or rRv2626c-DN for 1 h, and stimulated with 100 ng/ml LPS for 18 h. Culture supernatants were harvested, and the levels of TNF-α, IL-6, IL-12p40, and IL-10 were measured by ELISA.(E) Myc-Rv2626c-expressing Raw264.7 or THP-1 cells were pretreated with rRv2626c-WT (2.5 μg/ml), rRv2626c-CA (10 ng/ml), rRv2626c-DN (2.5 μg/ml), for 1 h, and stimulated with 100 ng/ml LPS for 30 min, followed by IP with αMyc or αTRAF6, IB with αMyc and ubiquitin. WCLs were used for IB with αMyc, αTRAF6, αHis or αActin.


Figure 6 ( A - C ) Schematic of the CLP model treated with rRv2626c - CA or its mutants ( upper ) . The survival of mice was monitored for 10 days ; mortality was measured for n = 25 mice per group ( lower ) . Statistical differences compared with the rVector - treated mice are indicated ( log - rank test ) . ( D ) Serum cytokine levels ( E ) representative hematoxylin and eosin ( H & E ) staining of the lung , liver and spleen ( left ) from 10 mice per group . Histopathology scores were obtained from H & E stained were determined at 20 h in CLP mice were treated with rRv2626c - CA or its mutants . Scale bar , 100 μm . ( F ) Splenocytes were used for IP with αHis or αTRAF6 , followed by IB with αHis or αUb . WCLs were used for IB with αTRAF6 , αHis or αActin . ( G ) In vivo imaging using IVIS spectrum - chromatography CT system . Intraperitoneal administration of rRv2626c - CA / Cy5 . 5 was carried out at intervals of 0 , 6 , 12 , and 18 h in a CLP - induced sepsis model . Pharmacokinetics and biodistribution were observed in spleen , liver , lung , and kidney tissues . ( H ) The percentage of F4 / 80 + macrophages cells and rRv2626c - CA - His were found in the spleen , liver and lung using Fluorescence - activated cell sorting analysis in the background of CLP - induced sepsis .

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Figure 6(A-C) Schematic of the CLP model treated with rRv2626c-CA or its mutants (upper). The survival of mice was monitored for 10 days; mortality was measured for n=25 mice per group (lower). Statistical differences compared with the rVector-treated mice are indicated (log-rank test).(D) Serum cytokine levels(E) representative hematoxylin and eosin (H&E) staining of the lung, liver and spleen (left) from 10 mice per group. Histopathology scores were obtained from H&E stained were determined at 20 h in CLP mice were treated with rRv2626c-CA or its mutants. Scale bar, 100 μm.(F) Splenocytes were used for IP with αHis or αTRAF6, followed by IB with αHis or αUb. WCLs were used for IB with αTRAF6, αHis or αActin.(G) In vivo imaging using IVIS spectrum-chromatography CT system. Intraperitoneal administration of rRv2626c-CA/Cy5.5 was carried out at intervals of 0, 6, 12, and 18 h in a CLP-induced sepsis model. Pharmacokinetics and biodistribution were observed in spleen, liver, lung, and kidney tissues.(H) The percentage of F4/80+ macrophages cells and rRv2626c-CA-His were found in the spleen, liver and lung using Fluorescence-activated cell sorting analysis in the background of CLP-induced sepsis.


Figure 7 ( A ) The bacterial burden was evaluated 20 h after treatment of CLP mice with rRv2626c - CA or its mutants ( n = 10 mice per group ) . ( B ) The percentage of F4 / 80 + macrophages cells and rRv2626c - CA - His or its mutants were found in the spleen or lung using Fluorescence - activated cell sorting analysis in the background of CLP - induced sepsis . ( C ) CD86 , iNOS , CD163 and Arg1 mRNA levels were determined by real - time PCR . ( D ) Expression of SR - A , FcR , TLR2 , TLR4 , NRP1 or CXCR2 were measured by flow cytometry analysis .

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Figure 7(A) The bacterial burden was evaluated 20 h after treatment of CLP mice with rRv2626c-CA or its mutants (n=10 mice per group).(B) The percentage of F4/80+ macrophages cells and rRv2626c-CA-His or its mutants were found in the spleen or lung using Fluorescence-activated cell sorting analysis in the background of CLP-induced sepsis.(C) CD86, iNOS, CD163 and Arg1 mRNA levels were determined by real-time PCR.(D) Expression of SR-A, FcR, TLR2, TLR4, NRP1 or CXCR2 were measured by flow cytometry analysis.


Figure 1Figure 1 - The origin of N - terminal proteoforms . A . 2 , 578 proteoforms were identified including 2 , 135 dbTIS and 443 aTIS . For the majority of proteins we found a unique translation initiation site ( grey ) . Additionally , 335 N - terminal peptides pointed to proteins with multiple N - termini created by alternative translation initiation ( blue ) , whereas 81 N - termini were generated by alternative iMet processing ( green ) . B - C . Proteomics - derived translation initiation events were verified at the transcript level using Ribo - Seq data obtained in Jurkat cells and HCT 116 cells ( data unpublished ) . Although in this study , the degree of in vivo Nt - acetylation could not be directly determined , all retained N - termini were compliant with the rules of N - terminal processing from previous experiments ( Van Damme et al , 2014 ) . In the case N - termini were found to be in vivo Nt - acetylated ( previous MS / MS - based evidence ( Van Damme et al , 2014 ) ) this information is indicated . dbTIS and aTIS peptides were further mapped to protein N - termini annotated in Swiss - Prot Isoform , UniProt TrEMBL and Ensembl databases and neo - N - termini ( proteolytic products ) of the knowledgebase TopFind 3 . 0 . The number of matching N - termini is indicated in dark grey . N - terminal peptides supported by at least one source of metadata pointing to translation ( Nt - acetylation , Ribo - Seq , Swiss - Prot Isoforms , TrEMBL or Ensembl ) were classified as highly confident TIS ( indicated in green ) . D . Using experimental and metadata we assigned the most likely origin of aTIS peptides and confirmed that alternative translation initiation within the same transcript ( leaky scanning ) contributes to the majority of alternative TIS identified .

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Figure 1Figure 1 - The origin of N-terminal proteoforms. A. 2,578 proteoforms were identified including 2,135 dbTIS and 443 aTIS. For the majority of proteins we found a unique translation initiation site (grey). Additionally, 335 N-terminal peptides pointed to proteins with multiple N-termini created by alternative translation initiation (blue), whereas 81 N-termini were generated by alternative iMet processing (green). B-C. Proteomics-derived translation initiation events were verified at the transcript level using Ribo-Seq data obtained in Jurkat cells and HCT 116 cells (data unpublished). Although in this study, the degree of in vivo Nt-acetylation could not be directly determined, all retained N-termini were compliant with the rules of N-terminal processing from previous experiments (Van Damme et al, 2014). In the case N-termini were found to be in vivo Nt-acetylated (previous MS/MS-based evidence (Van Damme et al, 2014)) this information is indicated. dbTIS and aTIS peptides were further mapped to protein N-termini annotated in Swiss-Prot Isoform, UniProt TrEMBL and Ensembl databases and neo-N-termini (proteolytic products) of the knowledgebase TopFind 3.0. The number of matching N-termini is indicated in dark grey. N-terminal peptides supported by at least one source of metadata pointing to translation (Nt-acetylation, Ribo-Seq, Swiss-Prot Isoforms, TrEMBL or Ensembl) were classified as highly confident TIS (indicated in green). D. Using experimental and metadata we assigned the most likely origin of aTIS peptides and confirmed that alternative translation initiation within the same transcript (leaky scanning) contributes to the majority of alternative TIS identified.


Figure 2Figure 2 - Examples of translation initiation events confirmed by Ribo - Seq in Jurkat cells . A - B . Alternative translation initiation due to leaky scanning . C . Alternative splicing . D . The preferential expression of an N - terminally truncated proteoform confirmed by Ribo - Seq data . E - F . Evidence for alternative translation initiation occurring at two consecutive AUG codons .

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Figure 2Figure 2 - Examples of translation initiation events confirmed by Ribo-Seq in Jurkat cells. A-B. Alternative translation initiation due to leaky scanning. C. Alternative splicing. D. The preferential expression of an N-terminally truncated proteoform confirmed by Ribo-Seq data. E-F. Evidence for alternative translation initiation occurring at two consecutive AUG codons.


Figure 3A . Jurkat cells were pre - labeled with light or medium L - Arg isotopes . A label swap was performed of cells growing in medium Arg , whereas light cells were cultured in the same medium . Cells were harvested at different time points and equal proteome amounts were mixed , followed by N - terminal COFRADIC fractionation and LC - MS / MS analysis . C . The distribution of turnover times ( N = 1 , 894 ) in our experiment was not unimodal , with a sizable number of quickly turned over proteins , with the median protein turnover rate being 21 . 6 h . The doubling time of Jurkat cells ( 24 h ) is indicated in red .

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Figure 3A. Jurkat cells were pre-labeled with light or medium L-Arg isotopes. A label swap was performed of cells growing in medium Arg, whereas light cells were cultured in the same medium. Cells were harvested at different time points and equal proteome amounts were mixed, followed by N-terminal COFRADIC fractionation and LC-MS/MS analysis.C. The distribution of turnover times (N=1,894) in our experiment was not unimodal, with a sizable number of quickly turned over proteins, with the median protein turnover rate being 21.6 h. The doubling time of Jurkat cells (24 h) is indicated in red.


Figure 6A . Degradation profiles of AIMP2 , MARE2 and AN32E dbTIS and aTIS proteoforms as representative examples of N - terminal proteoforms that differ in stability . B . MS spectra of the N - termini of two proteoforms originating from the aminoacyl tRNA synthase complex - interacting multifunctional protein 2 ( AIMP2 ) : a dbTIS indicative N - terminus with processed iMet and starting at the second amino acid Pro , an aTIS indicative peptide starting at position 3 and retaining its iMet . As evident from the MS spectra shown , these N - terminal proteoforms displayed largely different turnover rates .

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Figure 6A. Degradation profiles of AIMP2, MARE2 and AN32E dbTIS and aTIS proteoforms as representative examples of N-terminal proteoforms that differ in stability.B. MS spectra of the N-termini of two proteoforms originating from the aminoacyl tRNA synthase complex-interacting multifunctional protein 2 (AIMP2): a dbTIS indicative N-terminus with processed iMet and starting at the second amino acid Pro, an aTIS indicative peptide starting at position 3 and retaining its iMet. As evident from the MS spectra shown, these N-terminal proteoforms displayed largely different turnover rates.


Figure 8A . Jurkat cells were treated with 100 µg / ml CHX for 0 , 0 . 5 , 1 . 5 , 4 , 8 , 12 or 24 hours . Protein degradation was monitored by Western blotting and stabilities of several short - lived endogenous proteins were confirmed using antibodies ( including lamin B , securin , β - catenin , GCIP interacting protein p29 ) and compared to stable proteins ( such as GAPDH and actin ) . Proteoform - specific bands were used to calculate protein half - lives which were in a good agreement with turnover times obtained from pSILACB - C . Validation of the differential turnover time of the dbTIS and aTIS derived proteoforms of MARE2 and AN32E . Selective C - terminal V5 - tagged proteoforms were overexpressed in HCT116 cells for 24 hours and CHXpulse - chase experiments were performed as described for Jurkat cells . Degradation of overexpressed proteoforms was monitored by an anti - V5 antibody and compared to the turnover of stable proteins such as actin and tubulin .

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Figure 8A. Jurkat cells were treated with 100 µg/ml CHX for 0, 0.5, 1.5, 4, 8, 12 or 24 hours. Protein degradation was monitored by Western blotting and stabilities of several short-lived endogenous proteins were confirmed using antibodies (including lamin B, securin, β-catenin, GCIP interacting protein p29) and compared to stable proteins (such as GAPDH and actin). Proteoform-specific bands were used to calculate protein half-lives which were in a good agreement with turnover times obtained from pSILACB-C. Validation of the differential turnover time of the dbTIS and aTIS derived proteoforms of MARE2 and AN32E. Selective C-terminal V5-tagged proteoforms were overexpressed in HCT116 cells for 24 hours and CHXpulse-chase experiments were performed as described for Jurkat cells. Degradation of overexpressed proteoforms was monitored by an anti-V5 antibody and compared to the turnover of stable proteins such as actin and tubulin.


Figure 5H , I WT and lincRNA - EPS - / - iBMMs were infected with VSV ( MOI 0 . 1 ) for 6 h or 10 h ( H ) or transfected with polyI : C ( 1 μg / mL ) for 4 h or 8 h ( I ) , phosphorylation and total protein expression were analyzed by Western blot with α - tubulin and β - Actin as loading controls .

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Figure 5H, I WT and lincRNA-EPS-/- iBMMs were infected with VSV (MOI 0.1) for 6 h or 10 h (H) or transfected with polyI:C (1 μg/mL) for 4 h or 8 h (I), phosphorylation and total protein expression were analyzed by Western blot with α-tubulin and β-Actin as loading controls.


Figure 6B Western blot for verification of RNA pulldown - MS results with β - Actin as a negative loading control . D 20 nM siRNA targeting negative control ( NC ) and PKR were transfected into iBMMs for 48 h , the protein and mRNA expression level of PKR were tested by RT - qPCR and Western blot , respectively . G VSV - N and VSV - G mRNA were transcribed from virus particles for RNA - pulldown , the interactions between VSV RNA with PKR protein was checked by Western blot , β - Actin was shown as a negative control . H LincRNA - EPS was transcribed in vitro and pulled down proteins from iBMMs infected with VSV for 4 h , PKR protein was detected by Western blot and GAPDH was shown as a loading control . I RNA - pulldown using biotinylated lincRNA - EPS from cell lysates of polyI : C ( 1 and 2 . 5 μg / mL ) transfected iBMMs for 4 h , PKR protein was detected by Western blot and β - Actin was shown as a loading control .

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Figure 6B Western blot for verification of RNA pulldown-MS results with β-Actin as a negative loading control.D 20 nM siRNA targeting negative control (NC) and PKR were transfected into iBMMs for 48 h, the protein and mRNA expression level of PKR were tested by RT-qPCR and Western blot, respectively.G VSV-N and VSV-G mRNA were transcribed from virus particles for RNA-pulldown, the interactions between VSV RNA with PKR protein was checked by Western blot, β-Actin was shown as a negative control.H LincRNA-EPS was transcribed in vitro and pulled down proteins from iBMMs infected with VSV for 4 h, PKR protein was detected by Western blot and GAPDH was shown as a loading control.I RNA-pulldown using biotinylated lincRNA-EPS from cell lysates of polyI:C (1 and 2.5 μg/mL) transfected iBMMs for 4 h, PKR protein was detected by Western blot and β-Actin was shown as a loading control.


Figure 7A 20 nM siRNA for negative control ( NC ) and PKR were transfected into iBMMs following infected with VSV ( MOI 0 . 1 ) for 4 h or 8 h , phosphorylation and total levels of STAT1 , eIF2α and PKR were detected by Western blot and β - Actin was shown as a loading control . B PKR was transfected into A549 cells for 24 h following stimulated with IFN - β ( 50 ng / mL ) for 1 h to test STAT1 phosphorylation by Western blot and β - Actin was shown as a loading control .

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Figure 7A 20 nM siRNA for negative control (NC) and PKR were transfected into iBMMs following infected with VSV (MOI 0.1) for 4 h or 8 h, phosphorylation and total levels of STAT1, eIF2α and PKR were detected by Western blot and β-Actin was shown as a loading control.B PKR was transfected into A549 cells for 24 h following stimulated with IFN-β (50 ng/mL) for 1 h to test STAT1 phosphorylation by Western blot and β-Actin was shown as a loading control.


Figure 1 ( A ) HT29 cells were infected with Shigella WT , S325 , or ospD deletion mutants , and incubated for 8 h . Aliquots of cellular supernatants were subjected to cytotoxicity assays . * P < 0 . 05 ( one - way ANOVA ) . ( B ) HT29 cells were infected with Shigella WT or ∆ ospD3 and incubated for 8 h . Infected cells were fixed and subjected to TUNEL and PI staining . Percentages of positive cells ( TUNEL , green ; PI , red ) are shown in graph at right . The nuclei were stained with DAPI ( blue ) . Scale bar : 100 μm . n . s . , not significant ; * P < 0 . 05 ( unpaired two - tailed Student ' s t - test ) . ( C ) HT29 cells were infected with Shigella WT , S325 , or ∆ ospD3 and incubated for 8 h . Infected cells were subjected to Giemsa staining . Arrows indicate cells in which the cytoplasm disappeared . Scale bar : 20 μm .

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Figure 1(A) HT29 cells were infected with Shigella WT, S325, or ospD deletion mutants, and incubated for 8 h. Aliquots of cellular supernatants were subjected to cytotoxicity assays. *P<0.05 (one-way ANOVA).(B) HT29 cells were infected with Shigella WT or ∆ospD3 and incubated for 8 h. Infected cells were fixed and subjected to TUNEL and PI staining. Percentages of positive cells (TUNEL, green; PI, red) are shown in graph at right. The nuclei were stained with DAPI (blue). Scale bar: 100 μm. n.s., not significant; *P<0.05 (unpaired two-tailed Student's t-test).(C) HT29 cells were infected with Shigella WT, S325, or ∆ospD3 and incubated for 8 h. Infected cells were subjected to Giemsa staining. Arrows indicate cells in which the cytoplasm disappeared. Scale bar: 20 μm.


Figure 2 ( A ) HT29 cells were infected with Shigella WT , S325 , or ospD deletion mutants , and incubated for 8 h . Cell lysates were subjected to immunoblotting . ( B HT29 cells were infected with the indicated Shigella strains in the presence or absence of RIPK1 inhibitor , RIPK3 inhibitor , or caspase inhibitor ( z - VAD ) , and then incubated for 8 h . Cell lysates and aliquots of cellular supernatants were subjected to immunoblottingC ) HT29 cells were infected with the indicated Shigella strains in the presence or absence of RIPK1 inhibitor , RIPK3 inhibitor , or caspase inhibitor ( z - VAD ) , and then incubated for 8 h . Cell lysates and aliquots of cellular supernatants were subjected to cytotoxicity assay ( D HT29 cells treated with the indicated siRNAs were infected with Shigella WT or ∆ ospD3 . Cell lysates and aliquots of cellular supernatants were subjected to immunoblotting ( top ) and cytotoxicity assays ( bottom ) , respectively . The knockdown efficiency of the indicated siRNAs was assessed by immunoblotting ( inset ) . n . s . , not significant ; * P < 0 . 05 ( unpaired two - tailed Student ' s t - test ) . E ) HT29 cells treated with the indicated siRNAs were infected with Shigella WT or ∆ ospD3 . Cell lysates and aliquots of cellular supernatants were subjected to immunoblotting ( top ) and cytotoxicity assays ( bottom ) , respectively . The knockdown efficiency of the indicated siRNAs was assessed by immunoblotting ( inset ) . n . s . , not significant ; * P < 0 . 05 ( unpaired two - tailed Student ' s t - test ) .

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Figure 2(A) HT29 cells were infected with Shigella WT, S325, or ospD deletion mutants, and incubated for 8 h. Cell lysates were subjected to immunoblotting.(B HT29 cells were infected with the indicated Shigella strains in the presence or absence of RIPK1 inhibitor, RIPK3 inhibitor, or caspase inhibitor (z-VAD), and then incubated for 8 h. Cell lysates and aliquots of cellular supernatants were subjected to immunoblottingC) HT29 cells were infected with the indicated Shigella strains in the presence or absence of RIPK1 inhibitor, RIPK3 inhibitor, or caspase inhibitor (z-VAD), and then incubated for 8 h. Cell lysates and aliquots of cellular supernatants were subjected to cytotoxicity assay(D HT29 cells treated with the indicated siRNAs were infected with Shigella WT or ∆ospD3. Cell lysates and aliquots of cellular supernatants were subjected to immunoblotting (top) and cytotoxicity assays (bottom), respectively. The knockdown efficiency of the indicated siRNAs was assessed by immunoblotting (inset). n.s., not significant; *P<0.05 (unpaired two-tailed Student's t-test).E) HT29 cells treated with the indicated siRNAs were infected with Shigella WT or ∆ospD3. Cell lysates and aliquots of cellular supernatants were subjected to immunoblotting (top) and cytotoxicity assays (bottom), respectively. The knockdown efficiency of the indicated siRNAs was assessed by immunoblotting (inset). n.s., not significant; *P<0.05 (unpaired two-tailed Student's t-test).


Figure 3 ( A ) HT29 cells were infected with the indicated Shigella strains and incubated for 8 h , and then cell lysates were subjected to immunoblotting . ( B ) 293T cells were transfected with Myc - tagged ospD expression plasmids . After 24 h , cells were harvested and subjected to immunoblotting . Arrows indicate cleaved RIPK1 . D ) 293T cells were transfected with a series of plasmids expressing ospD3 point mutants . After 24 h , cells were harvested and subjected to immunoblotting . Arrows indicate cleaved RIPK1 . ( E HT29 cells were infected with Shigella WT , S325 , ∆ ospD3 , ∆ ospD3 / D3 ( ∆ ospD3 complemented with wild - type ospD3 ) , or ∆ ospD3 / D3CS ( ∆ ospD3 complemented with a protease activity - deficient mutant , in which the cysteine residue at position 64 was replaced by serine ) strains and incubated for 8 h . Cell lysates and aliquots of cellular supernatants were subjected to immunoblottingF ) HT29 cells were infected with Shigella WT , S325 , ∆ ospD3 , ∆ ospD3 / D3 ( ∆ ospD3 complemented with wild - type ospD3 ) , or ∆ ospD3 / D3CS ( ∆ ospD3 complemented with a protease activity - deficient mutant , in which the cysteine residue at position 64 was replaced by serine ) strains and incubated for 8 h . Cell lysates and aliquots of cellular supernatants were subjected to cytotoxicity

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Figure 3(A) HT29 cells were infected with the indicated Shigella strains and incubated for 8 h, and then cell lysates were subjected to immunoblotting.(B) 293T cells were transfected with Myc-tagged ospD expression plasmids. After 24 h, cells were harvested and subjected to immunoblotting. Arrows indicate cleaved RIPK1.D) 293T cells were transfected with a series of plasmids expressing ospD3 point mutants. After 24 h, cells were harvested and subjected to immunoblotting. Arrows indicate cleaved RIPK1.(E HT29 cells were infected with Shigella WT, S325, ∆ospD3, ∆ospD3/D3 (∆ospD3 complemented with wild-type ospD3), or ∆ospD3/D3CS (∆ospD3 complemented with a protease activity-deficient mutant, in which the cysteine residue at position 64 was replaced by serine) strains and incubated for 8 h. Cell lysates and aliquots of cellular supernatants were subjected to immunoblottingF) HT29 cells were infected with Shigella WT, S325, ∆ospD3, ∆ospD3/D3 (∆ospD3 complemented with wild-type ospD3), or ∆ospD3/D3CS (∆ospD3 complemented with a protease activity-deficient mutant, in which the cysteine residue at position 64 was replaced by serine) strains and incubated for 8 h. Cell lysates and aliquots of cellular supernatants were subjected to cytotoxicity assays


Figure 4 ( A ) HT29 cells were infected with the indicated Shigella strains and incubated for 8 h . Cells were harvested and subjected to measurement of caspase - 8 activation . Caspase - 8 activity is reported as relative light units ( RLU ) of infected samples , minus the value in uninfected samples . * P < 0 . 05 ( one - way ANOVA ) . ( B ) HT29 cells were infected with the indicated Shigella strains and incubated for 8 h . Cell lysates were subjected to immunoblotting . Arrows indicate cleaved forms of caspase - 8 , PARP , GSDMD , and IL - 18 , respectively . ( C ) HT29 cells were infected with the indicated Shigella strains and incubated for 8 h . Aliquots of cellular supernatants were subjected to cytotoxicity assay . * P < 0 . 05 ; n . s . , not significant ( one - way ANOVA ) . ( D ) HT29 cells were infected with Shigella WT or ∆ ospC1 mutant and incubated for 8 h . Infected cells were then fixed and stained with cleaved caspase - 8 ( green ) , rhodamine - phalloidin ( red ) , and DAPI ( blue ) . Percentages of positive cells are shown in the graph at right ( * P < 0 . 05 ; unpaired two - tailed Student ' s t - test ) . Scale bar : 100 μm . ( E ) HT29 cells were infected with the indicated Shigella strains and incubated for 8 h . Cells were harvested and subjected to measurement of caspase - 3 / 7 activation . Caspase - 3 / 7 activity is reported as relative light units ( RLU ) of infected samples minus uninfected samples . * P < 0 . 05 ( one - way ANOVA ) . ( F ) HT29 cells were infected with Shigella WT , S325 , or ∆ ospC1 strains , and incubated for up to 8 h . After incubation for the indicated times , cells were subjected to real - time Annexin V apoptosis assay . Annexin V binding is reported as relative light units ( RLU ) in infected samples minus the value in uninfected samples . * P < 0 . 05 ( one - way ANOVA ) .

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Figure 4(A) HT29 cells were infected with the indicated Shigella strains and incubated for 8 h. Cells were harvested and subjected to measurement of caspase-8 activation. Caspase-8 activity is reported as relative light units (RLU) of infected samples, minus the value in uninfected samples. *P<0.05 (one-way ANOVA).(B) HT29 cells were infected with the indicated Shigella strains and incubated for 8 h. Cell lysates were subjected to immunoblotting. Arrows indicate cleaved forms of caspase-8, PARP, GSDMD, and IL-18, respectively.(C) HT29 cells were infected with the indicated Shigella strains and incubated for 8 h. Aliquots of cellular supernatants were subjected to cytotoxicity assay. *P<0.05; n.s., not significant (one-way ANOVA).(D) HT29 cells were infected with Shigella WT or ∆ospC1 mutant and incubated for 8 h. Infected cells were then fixed and stained with cleaved caspase-8 (green), rhodamine-phalloidin (red), and DAPI (blue). Percentages of positive cells are shown in the graph at right (*P<0.05; unpaired two-tailed Student's t-test). Scale bar: 100 μm.(E) HT29 cells were infected with the indicated Shigella strains and incubated for 8 h. Cells were harvested and subjected to measurement of caspase-3/7 activation. Caspase-3/7 activity is reported as relative light units (RLU) of infected samples minus uninfected samples. *P<0.05 (one-way ANOVA).(F) HT29 cells were infected with Shigella WT, S325, or ∆ospC1 strains, and incubated for up to 8 h. After incubation for the indicated times, cells were subjected to real-time Annexin V apoptosis assay. Annexin V binding is reported as relative light units (RLU) in infected samples minus the value in uninfected samples. *P<0.05 (one-way ANOVA).


Figure 5 ( A HT29 cells were infected with Shigella WT , ∆ ospC1 , ∆ ospD3 , ∆ ospC1 ∆ ospD3 , or ∆ ospC1 ∆ ospD3 / ospC1 ( ∆ ospC1 ∆ ospD3 complemented with ospC1 ) strains and incubated for 8 h . Cell lysates and aliquots of cellular supernatants were subjected to immunoblottingB ) HT29 cells were infected with Shigella WT , ∆ ospC1 , ∆ ospD3 , ∆ ospC1 ∆ ospD3 , or ∆ ospC1 ∆ ospD3 / ospC1 ( ∆ ospC1 ∆ ospD3 complemented with ospC1 ) strains and incubated for 8 h . Cell lysates and aliquots of cellular supernatants were subjected to cytotoxicity assay ( C ) HT29 cells were infected with Shigella WT , ∆ ospD3 , or ∆ ospC1 . Cell lysates obtained at the indicated time points were subjected to immunoblotting . ( D HT29 cells treated with DMSO or caspase - 8 inhibitor were infected with Shigella WT , ∆ ospC1 , ∆ ospD3 , or ∆ ospC1 ∆ ospD3 strains and incubated for 8 h . Cell lysates and aliquots of cellular supernatants were subjected to immunoblottingE ) HT29 cells treated with DMSO or caspase - 8 inhibitor were infected with Shigella WT , ∆ ospC1 , ∆ ospD3 , or ∆ ospC1 ∆ ospD3 strains and incubated for 8 h . Cell lysates and aliquots of cellular supernatants were subjected to cytotoxicity assay ( F ) HT29 cells treated with the control or caspase - 8 siRNAs were infected with the indicated Shigella strains and incubated for 8 h . Cell lysates were subjected to immunoblotting . The knockdown efficiency of the indicated siRNAs was assessed by immunoblotting .

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Figure 5(A HT29 cells were infected with Shigella WT, ∆ospC1, ∆ospD3, ∆ospC1∆ospD3, or ∆ospC1∆ospD3/ospC1 (∆ospC1∆ospD3 complemented with ospC1) strains and incubated for 8 h. Cell lysates and aliquots of cellular supernatants were subjected to immunoblottingB) HT29 cells were infected with Shigella WT, ∆ospC1, ∆ospD3, ∆ospC1∆ospD3, or ∆ospC1∆ospD3/ospC1 (∆ospC1∆ospD3 complemented with ospC1) strains and incubated for 8 h. Cell lysates and aliquots of cellular supernatants were subjected to cytotoxicity assay(C) HT29 cells were infected with Shigella WT, ∆ospD3, or ∆ospC1. Cell lysates obtained at the indicated time points were subjected to immunoblotting.(D HT29 cells treated with DMSO or caspase-8 inhibitor were infected with Shigella WT, ∆ospC1, ∆ospD3, or ∆ospC1∆ospD3 strains and incubated for 8 h. Cell lysates and aliquots of cellular supernatants were subjected to immunoblottingE) HT29 cells treated with DMSO or caspase-8 inhibitor were infected with Shigella WT, ∆ospC1, ∆ospD3, or ∆ospC1∆ospD3 strains and incubated for 8 h. Cell lysates and aliquots of cellular supernatants were subjected to cytotoxicity assay(F) HT29 cells treated with the control or caspase-8 siRNAs were infected with the indicated Shigella strains and incubated for 8 h. Cell lysates were subjected to immunoblotting. The knockdown efficiency of the indicated siRNAs was assessed by immunoblotting.


Figure 3 A , B Core histones were prepared from NSPCs treated with various concentrations of crotonate ( A ) and 1 μM MS - 275 ( B ) for 24 h and subjected to western blotting analysis using anti - Kcr antibody , and the experiments were repeated three times . C NSPCs were treated with 20 μM C646 , 10 mM acetate ( Ac ) , 20 μM C646 and 10 mM acetate ( Ac + C646 ) , 10 mM crotonate ( Cr ) , and 20 μM C646 and 10 mM crotonate ( Cr + C646 ) for 24 h , then core histones were extracted and subjected to western blotting analysis using anti - Kcr antibody , the experiments were repeated three times .

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Figure 3 A, B Core histones were prepared from NSPCs treated with various concentrations of crotonate (A) and 1 μM MS-275 (B) for 24 h and subjected to western blotting analysis using anti-Kcr antibody, and the experiments were repeated three times.  C NSPCs were treated with 20 μM C646, 10 mM acetate (Ac), 20 μM C646 and 10 mM acetate (Ac+C646), 10 mM crotonate (Cr), and 20 μM C646 and 10 mM crotonate (Cr+C646) for 24 h, then core histones were extracted and subjected to western blotting analysis using anti-Kcr antibody, the experiments were repeated three times.


Figure 4 E Quantification analysis of changes in histone Kcr levels in NSPCs treated with DMSO ( Control ) , 5 μM triptolide ( FP ) , 5 μM flavopiridol and 10 mM crotonate ( FP + CR ) , and 10 mM crotonate ( CR ) for 1 h . Data are presented as mean ± SEM of three independent biological replicates , n = 3 . One - way ANOVA with post hoc Tukey ' s test was used to analyze statistical significance ; NS : no significance , P < 0 . 01 , P < 0 . 001 . F Co - IP analysis indicating RNAP2 binding in H3K9cr enriched regions in NSPCs . H3 was used as a loading control for the input nuclear proteins . G Quantification analysis of changes in chromatin openness at Tgfb1 and Notum promoter regions by FAIRE - qPCR among different groups of NSPCs . Data are presented as mean ± SEM of three independent biological replicates , n = 3 . Two - tailed unpaired Student ' s t - test was used to analyze statistical significance ; P < 0 . 05 , P < 0 . 01 .

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Figure 4 E Quantification analysis of changes in histone Kcr levels in NSPCs treated with DMSO (Control), 5 μM triptolide (FP), 5 μM flavopiridol and 10 mM crotonate (FP+CR), and 10 mM crotonate (CR) for 1 h. Data are presented as mean ± SEM of three independent biological replicates, n = 3. One-way ANOVA with post hoc Tukey's test was used to analyze statistical significance; NS: no significance, P < 0.01, P < 0.001.  F Co-IP analysis indicating RNAP2 binding in H3K9cr enriched regions in NSPCs. H3 was used as a loading control for the input nuclear proteins.  G Quantification analysis of changes in chromatin openness at Tgfb1 and Notum promoter regions by FAIRE-qPCR among different groups of NSPCs. Data are presented as mean ± SEM of three independent biological replicates, n = 3. Two-tailed unpaired Student's t-test was used to analyze statistical significance; P < 0.05, P < 0.01.