A . A total of 1 × 104 LN229 were transfected with control vector , FoxM1 , / or ICAT plasmids treated with Wnt3a ( 50 ng / ml ) were then plated counted 48 hr after seeding . proliferation was calculated relative to that in transfected with control vector , which was set as 100 % . Data represent mean ± SD of three independent experiments .

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A. 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 were transfected with vector control , FoxM1 , FoxM1 plus sh - β - catenin or FoxM1 plus ICAT plasmid . proliferation was determined as described in ( A ) .

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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 were transfected with FoxM1 - shR ( siRNA - resistant FoxM1 ) , or FoxM1 - shR plus ICAT plasmid . were then treated with Wnt3a ( 50 ng / ml ) . proliferation was determined as described in ( A ) .

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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 were transfected with USP5 / or FoxM1 siRNA . proliferation was determined as described in ( A ) .

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D. LN229 cells were transfected with USP5 and/or FoxM1 siRNA. The proliferation percentage was determined as described in (A).


E . LN229 were transfected with USP5 siRNA / or FoxM1 plasmid . were then treated with Wnt3a ( 50 ng / ml ) . proliferation was determined as described in ( A ) . Data shown in ( A ) - ( E ) represent mean ± SD of three independent experiments .

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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 USP5 was examined via in 50 GBM specimens . of FoxM1 , Cyclin D1 , or USP5 was scored as 1 - 12 according to . significance of correlations was determined by Pearson ' s correlation .

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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 in above 50 GBM specimens were examined through RNAscope technology . was scored as 1 - 12 according to . significance of correlations was determined by Pearson ' s correlation .

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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.


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

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A. 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.


A . A close - up view of sucrose - binding pocket . Residues hydrogen - bonding with sucrose are shown as yellow sticks , sucrose molecule is shown as pink stick .

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A. 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 . 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 of 200 μg / ml is shown in Appendix Table S1 . two kinds of Dln1 - binding glycan structures ( No . 214 316 ) are illustrated in schematic .

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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 Manα1 - 3Man binding sites . mannobiose molecules are shown as cyan gray sticks , respectively . Fo - Fc electron density maps of disaccharides disaccharide - binding sites are shown as gray mesh countered at 3 . 0 . hydrogen - bonding residues are shown as yellow sticks . Polar interactions with mannobiose are shown as dashed lines .

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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.


A & amp ; amp ; B . ELISA of Dln1 towards glycosylated gp120 or yeast mannan . curve represents average of triplicate detections , using BSA as control . Points are averages ( ± standard error of mean ) of triplicate determinations .

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A&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 D135A mutant to saccharomyces cerevisiae . scale bar represents 5 μm .

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C. Binding of FITC-labeled Dln1 and D135A mutant to saccharomyces cerevisiae cells. The scale bar represents 5 μm.


D . Immunoblot of Dln1 D135A mutant after incubating with S . cerevisiae . seeing bands represent - bound after extensive washing yeast . monomer oligomer bands are highlighted by black arrow .

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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.


A . Oligomerization of Dln1 in presence of S . cerevisiae mannan or liposomes at different pH values . Histograms are averages ( ± standard error of mean ) of triplicate

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A. 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 determinations


B & amp ; amp ; C . Dye release velocity from liposomes in presence of Dln1 at different concentrations , various pH values . Liposomes were incubated with Dln1 of different concentrations at pH 5 . 5 , 1 μM Dln1 at various pH values , respectively .

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B&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 . samples for electrophoresis were prepared with boiling at 95 ° C for 5 min , comparative gels were processed in parallel . oligomer band was quantified by Image J software with average of triplicate detections , using marker band as a sample processing control . SDS - PAGE profiles of Dln1 oligomerization were shown in Appendix Fig S4 . Histograms are averages ( ± standard error of mean ) of triplicate determinations .

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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 .

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E. Liposomes were treated with Dln1M5 or Dln1 of different concentrations at a fixed pH value of 5.5.


F . inhibition of dye release in presence of 0 . 5 M Dln1M5 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 to maximal release upon addition of Triton X - 100 . Carboxyfluorescein was loaded in liposomes for detection of dye efflux .

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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.


A . Negative - stain electron microscopy image of Dln1M5 in presence of liposomes . B . Side views of Dln1M5 oligomers attached to of liposomes . inset represents respective class averages .

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A. 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 . inset represents a few 2D class averages showing octamer pattern from some rings .

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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 lipid monolayer . inset represents computed diffraction pattern Fourier transform of image . F . Projection map with a P4 symmetry .

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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 . top , side cut - open views of 3D reconstruction of Dln1M5 octamer at 20 Å resolution are shown as gray surface representation . surface of is indicated by dashed lines . scale bar is 10 nm .

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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.


A . Top side views of eight lectin modules docked to 20 Å EM map . Due to moderate resolution of EM map , flexible aerolysin module cannot be fit in map . It is important to notice that this model is only a hypothetical fit . Each pair of lectin modules are colored in yellow orange , respectively . β - barrel of γ - hemolysin ( PDB 3B07 ) was used to mimic β - barrel backbone of Dln1 . position of is indicated by dashed lines .

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A. 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 . mobility is represented by coil thickness from thin ( low ) to thick ( high ) , color from blue ( low ) to red ( high ) . Notably , pre - hairpin distal moiety of aerolysin module are particularly mobile .

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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 ) pre - hairpin extraction form ( blue ) are shown with lectin modules superimposed ( 200 structures for each ) .

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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).


( A ) Bacterially purified 6xHis - rRv2626c analyzed by Coomassie blue ( left ) or immuno blotting ( IB ) with αHis ( right ) .

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(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 indicated times then viability measured with MTT .

(B) BMDMs were incubated with rRv2626c for the indicated times and then cell viability measured with MTT assay.


( C ) confocal showing in BMDMs treated with Rv2626c ( 2 . 5 μg / ml ) for 30 min , fixed , immunostained with antibodies for His ( Alexa 488 ) DAPI . Scale bar , 10 μm .

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(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 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 , of TNF - α , IL - 6 , IL - 12p40 , IL - 10 were measured by ELISA .

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(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 then subjected to IB for αIκBα His . αActin was used as a loading control .

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(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.


BMDMs were pretreated with 2 . 5 μg / ml rRv2626c or rVector for 1 h , stimulated with 100 ng / ml LPS ( A for indicated times . ( A Culture supernatants were harvested , of TNF - α , IL - 6 , IL - 12p40 , IL - 10 were measured by ELISA .

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BMDMs 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 , stimulated with 100 ng / ml Pam2CSK4 ( B ) B ) Culture supernatants were harvested , of TNF - α , IL - 6 , IL - 12p40 , IL - 10 were measured by ELISA .

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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 ) were harvested then subjected to IB for phosphorylated forms of AKT , MAPK ( ERK , p38 , JNK ) , IκBα , total forms of IκBα . αActin was used as a loading control ( left ) . densitometric values were calculated from ratios of relative to actin ( right ) .

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(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).


( A ) Identification of TLR2 , IRAK1 , RIP1 , TRAF6 , MAPK11 TOLLIP as endogenous binding partners of rRv2626c . THP - 1 lysates were incubated with His tagged - rRv2626c or rVector then IP with His - agarose beads . Binding partners were confirmed by silver mass spectrometric . red - colored letters indicate peptides identified from mass spectrometry .

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(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 were co - transfected with Flag - TRAF6 Myc - Rv2626c IP with αFlag or αMyc . WCLs were used for IB with αFlag , αMyc or αActin .

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(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 BMDMs were stimulated with rRv2626c ( 2 . 5 μg / ml ) for indicated times , followed by IP with αHis ( up ) or αTRAF6 ( down ) IB with αTRAF6 , αHis , αActin .

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(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 . then immunolabeled with antibody to His - rRv2626c ( Alexa Fluor 488 ) or TRAF6 ( Alexa Fluor 568 ) . DAPI ( blue ) stained nuclei .

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(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.


( A ) Schematic diagram of structures of Rv2626c ( up ) . 293T were co - transfected with Flag - TRAF6 together with GST - vector or GST - Rv2626c its mutant constructs , subjected to GST pulldown , followed by IB with αFlag . WCLs were used for IB with αGST , αFlag or αActin ( down ) .

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(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 293T 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 .

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(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 structures of TRAF6 ( up ) . 293T were co - transfected with Myc - Rv2626c together with GST - vector or GST - TRAF6 its mutant constructs , subjected to GST pulldown , followed by IB with αMyc . WCLs were used for IB with αGST , αMyc or αActin ( down ) .

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(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 were co - transfected with Flag - TRAF6 , Myc - Rv2626c as indicated doses HA - ubiquitin ( left ) , HA - K48 - linked ubiquitin ( middle ) , HA - K63 - linked ubiquitin ( right ) then IP with αFlag , followed by IB with αHA . WCLs were used for IB with αFlag , αMyc , or αActin .

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(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 , 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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(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.


( B ) Bacterially purified 6xHis - rRv2626c analyzed by Coomassie blue ( left ) or immuno blotting ( IB ) with αHis ( right ) .

(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 indicated times then viability measured with MTT .

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(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 , stimulated with 100 ng / ml LPS for 18 h . Culture supernatants were harvested , of TNF - α , IL - 6 , IL - 12p40 , IL - 10 were measured by ELISA .

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(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 were pretreated with rRv2626c - WT ( 2 . 5 μg / ml ) , rRv2626c - CA ( 10 ng / ml ) , rRv2626c - DN ( 2 . 5 μg / ml ) , for 1 h , stimulated with 100 ng / ml LPS for 30 min , followed by IP with αMyc or αTRAF6 , IB with αMyc ubiquitin . WCLs were used for IB with αMyc , αTRAF6 , αHis or αActin .

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(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.


( A - C ) Schematic of CLP model treated with rRv2626c - CA or its mutants ( upper ) . survival of mice was monitored for 10 days ; mortality was measured for n = 25 mice per group ( lower ) . Statistical differences compared with rVector - treated mice are indicated ( log - rank ) .

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(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).


( E ) representative hematoxylin eosin ( H & E ) of lung , liver 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 .

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(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 .

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(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 , 18 h in a CLP - induced sepsis model . Pharmacokinetics biodistribution were observed in spleen , liver , lung , kidney tissues .

(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 ) of F4 / 80 + macrophages rRv2626c - CA - His were found in spleen , liver lung using - activated sorting in background of CLP - induced sepsis .

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(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.


( A ) burden was evaluated 20 h after treatment of CLP mice with rRv2626c - CA or its mutants ( n = 10 mice per group ) .

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(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 ) of F4 / 80 + macrophages rRv2626c - CA - His or its mutants were found in spleen or lung using - activated sorting in background of CLP - induced sepsis .

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(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 Arg1 mRNA were determined by real - time PCR .

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(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 .

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(D) Expression of SR-A, FcR, TLR2, TLR4, NRP1 or CXCR2 were measured by flow cytometry analysis.


Figure 1 - origin of N - terminal proteoforms . A . 2 , 578 proteoforms were identified including 2 , 135 dbTIS 443 aTIS . For majority of we found a unique translation initiation site ( grey ) . Additionally , 335 N - terminal peptides pointed to 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 transcript level using Ribo - Seq data obtained in Jurkat HCT 116 ( data unpublished ) . Although in this study , degree of in vivo Nt - acetylation could not be directly determined , retained N - termini were compliant with rules of N - terminal processing from previous experiments ( Van Damme et al , 2014 ) . In case N - termini were found to be in vivo Nt - acetylated ( previous MS / MS - evidence ( Van Damme et al , 2014 ) ) this information is indicated . dbTIS aTIS peptides were further mapped to N - termini annotated in Swiss - Prot Isoform , UniProt TrEMBL Ensembl databases neo - N - termini ( proteolytic products ) of knowledgebase TopFind 3 . 0 . number of matching N - termini is indicated in 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 metadata we assigned most likely origin of aTIS peptides confirmed that alternative translation initiation within same transcript ( leaky scanning ) contributes to majority of alternative TIS identified .

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Figure 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 2 - Examples of translation initiation events confirmed by Ribo - Seq in Jurkat . A - B . Alternative translation initiation due to leaky scanning . C . Alternative splicing . D . 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 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.


A . Jurkat were pre - labeled with or medium L - Arg isotopes . A label swap was performed of growing in medium Arg , whereas were cultured in same medium . were harvested at different time points equal proteome amounts were mixed , followed by N - terminal COFRADIC fractionation LC - MS / MS .

A. 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 . distribution of turnover times ( N = 1 , 894 ) in our experiment was not unimodal , with a sizable number of quickly turned over , with median turnover rate being 21 . 6 h . doubling time of Jurkat ( 24 h ) is indicated in red .

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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.


A . Degradation profiles of AIMP2 , MARE2 AN32E dbTIS aTIS proteoforms as representative examples of N - terminal proteoforms that differ in stability .

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A. 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 N - termini of two proteoforms originating from aminoacyl tRNA synthase - interacting multifunctional 2 ( AIMP2 ) : a dbTIS indicative N - terminus with processed iMet starting at second acid Pro , an aTIS indicative peptide starting at position 3 retaining its iMet . As evident from MS spectra shown , these N - terminal proteoforms displayed largely different turnover rates .

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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.


A . Jurkat were treated with 100 µg / ml CHX for 0 , 0 . 5 , 1 . 5 , 4 , 8 , 12 or 24 hours . degradation was monitored by Western blotting stabilities of several short - lived endogenous were confirmed using antibodies ( including lamin B , securin , β - catenin , GCIP interacting p29 ) compared to stable ( such as GAPDH actin ) . Proteoform - specific bands were used to calculate half - lives which were in a good agreement with turnover times obtained from

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A. 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 pSILAC


B - C . Validation of differential turnover time of dbTIS aTIS derived proteoforms of MARE2 AN32E . Selective C - terminal V5 - tagged proteoforms were overexpressed in HCT116 for 24 hours CHXpulse - chase experiments were performed as described for Jurkat . Degradation of overexpressed proteoforms was monitored by an anti - V5 antibody compared to turnover of stable such as actin tubulin .

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B-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.


H , I WT 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 total expression were analyzed by Western blot with α - tubulin β - Actin as loading controls .

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H, 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.


B Western blot for verification of pulldown - MS results with β - Actin as a negative loading control .

B 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 ) PKR were transfected into iBMMs for 48 h , mRNA expression level of PKR were tested by RT - qPCR Western blot , respectively .

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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 VSV - G mRNA were transcribed from particles for - pulldown , interactions between VSV with PKR was checked by Western blot , β - Actin was shown as a negative control .

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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 pulled down from iBMMs infected with VSV for 4 h , PKR was detected by Western blot GAPDH was shown as a loading control .

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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 - pulldown using biotinylated lincRNA - EPS from lysates of polyI : C ( 1 2 . 5 μg / mL ) transfected iBMMs for 4 h , PKR was detected by Western blot β - Actin was shown as a loading control .

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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.


A 20 nM siRNA for negative control ( NC ) PKR were transfected into iBMMs following infected with VSV ( MOI 0 . 1 ) for 4 h or 8 h , phosphorylation total of STAT1 , eIF2α PKR were detected by Western blot β - Actin was shown as a loading control .

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A 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 for 24 h following stimulated with IFN - β ( 50 ng / mL ) for 1 h to STAT1 phosphorylation by Western blot β - Actin was shown as a loading control .

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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.


( A ) HT29 were infected with Shigella WT , S325 , or ospD deletion mutants , incubated for 8 h . Aliquots of supernatants were subjected to cytotoxicity assays . * P < 0 . 05 ( one - way ANOVA ) .

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(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 were infected with Shigella WT or ∆ ospD3 incubated for 8 h . Infected were fixed subjected to TUNEL PI . Percentages of positive ( TUNEL , green ; PI , red ) are shown in graph at right . nuclei were stained with DAPI ( blue ) . Scale bar : 100 μm . n . s . , not significant ; * P < 0 . 05 ( unpaired two - Student ' s t - ) .

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(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 were infected with Shigella WT , S325 , or ∆ ospD3 incubated for 8 h . Infected were subjected to Giemsa . Arrows indicate in which cytoplasm disappeared . Scale bar : 20 μm .

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(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.


( A ) HT29 were infected with Shigella WT , S325 , or ospD deletion mutants , incubated for 8 h . lysates were subjected to immunoblotting .

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(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 were infected with indicated Shigella strains in presence or absence of RIPK1 inhibitor , RIPK3 inhibitor , or caspase inhibitor ( z - VAD ) , then incubated for 8 h . lysates aliquots of supernatants were subjected to

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(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 immunoblotting


C ) HT29 were infected with indicated Shigella strains in presence or absence of RIPK1 inhibitor , RIPK3 inhibitor , or caspase inhibitor ( z - VAD ) , then incubated for 8 h . lysates aliquots of supernatants were subjected to cytotoxicity

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C) 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 treated with indicated siRNAs were infected with Shigella WT or ∆ ospD3 . lysates aliquots of supernatants were subjected to immunoblotting ( top ) cytotoxicity assays ( bottom ) , respectively . knockdown efficiency of indicated siRNAs was assessed by immunoblotting ( inset ) . n . s . , not significant ; * P < 0 . 05 ( unpaired two - Student ' s t - ) .

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(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 treated with indicated siRNAs were infected with Shigella WT or ∆ ospD3 . lysates aliquots of supernatants were subjected to immunoblotting ( top ) cytotoxicity assays ( bottom ) , respectively . knockdown efficiency of indicated siRNAs was assessed by immunoblotting ( inset ) . n . s . , not significant ; * P < 0 . 05 ( unpaired two - Student ' s t - ) .

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).


( A ) HT29 were infected with indicated Shigella strains incubated for 8 h , then lysates were subjected to immunoblotting .

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(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 were transfected with Myc - tagged ospD expression plasmids . After 24 h , were harvested subjected to immunoblotting . Arrows indicate RIPK1 .

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(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 were transfected with a series of plasmids expressing ospD3 point mutants . After 24 h , were harvested subjected to immunoblotting . Arrows indicate RIPK1 .

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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 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 cysteine residue at position 64 was replaced by serine ) strains incubated for 8 h . lysates aliquots of supernatants were subjected to

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(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 immunoblotting


F ) HT29 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 cysteine residue at position 64 was replaced by serine ) strains incubated for 8 h . lysates aliquots of supernatants were subjected to cytotoxicity

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F) 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


( A ) HT29 were infected with indicated Shigella strains incubated for 8 h . were harvested subjected to measurement of caspase - 8 activation . Caspase - 8 activity is reported as relative units ( RLU ) of infected samples , minus value in uninfected samples . * P < 0 . 05 ( one - way ANOVA ) .

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(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 were infected with indicated Shigella strains incubated for 8 h . lysates were subjected to immunoblotting . Arrows indicate forms of caspase - 8 , PARP , GSDMD , IL - 18 , respectively .

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(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 were infected with indicated Shigella strains incubated for 8 h . Aliquots of supernatants were subjected to cytotoxicity . * P < 0 . 05 ; n . s . , not significant ( one - way ANOVA ) .

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(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 were infected with Shigella WT or ∆ ospC1 mutant incubated for 8 h . Infected were then fixed stained with caspase - 8 ( green ) , rhodamine - phalloidin ( red ) , DAPI ( blue ) . Percentages of positive are shown in graph at right ( * P < 0 . 05 ; unpaired two - Student ' s t - ) . Scale bar : 100 μm .

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(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 were infected with indicated Shigella strains incubated for 8 h . were harvested subjected to measurement of caspase - 3 / 7 activation . Caspase - 3 / 7 activity is reported as relative units ( RLU ) of infected samples minus uninfected samples . * P < 0 . 05 ( one - way ANOVA ) .

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(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 were infected with Shigella WT , S325 , or ∆ ospC1 strains , incubated for up to 8 h . After incubation for indicated times , were subjected to real - time Annexin V apoptosis . Annexin V binding is reported as relative units ( RLU ) in infected samples minus value in uninfected samples . * P < 0 . 05 ( one - way ANOVA ) .

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(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).


( A HT29 were infected with Shigella WT , ∆ ospC1 , ∆ ospD3 , ∆ ospC1 ∆ ospD3 , or ∆ ospC1 ∆ ospD3 / ospC1 ( ∆ ospC1 ∆ ospD3 complemented with ospC1 ) strains incubated for 8 h . lysates aliquots of supernatants were subjected to

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(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 immunoblotting


B ) HT29 were infected with Shigella WT , ∆ ospC1 , ∆ ospD3 , ∆ ospC1 ∆ ospD3 , or ∆ ospC1 ∆ ospD3 / ospC1 ( ∆ ospC1 ∆ ospD3 complemented with ospC1 ) strains incubated for 8 h . lysates aliquots of supernatants were subjected to cytotoxicity

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B) 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 were infected with Shigella WT , ∆ ospD3 , or ∆ ospC1 . lysates obtained at indicated time points were subjected to immunoblotting .

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(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 treated with DMSO or caspase - 8 inhibitor were infected with Shigella WT , ∆ ospC1 , ∆ ospD3 , or ∆ ospC1 ∆ ospD3 strains incubated for 8 h . lysates aliquots of supernatants were subjected to

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(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 immunoblotting


E ) HT29 treated with DMSO or caspase - 8 inhibitor were infected with Shigella WT , ∆ ospC1 , ∆ ospD3 , or ∆ ospC1 ∆ ospD3 strains incubated for 8 h . lysates aliquots of supernatants were subjected to cytotoxicity

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E) 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 treated with control or caspase - 8 siRNAs were infected with indicated Shigella strains incubated for 8 h . lysates were subjected to immunoblotting . knockdown efficiency of indicated siRNAs was assessed by immunoblotting .

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(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.


A , B histones were prepared from NSPCs treated with various concentrations of crotonate ( A ) 1 μM MS - 275 ( B ) for 24 h subjected to western blotting using anti - Kcr antibody , experiments were repeated three times .

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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 10 mM acetate ( Ac + C646 ) , 10 mM crotonate ( Cr ) , 20 μM C646 10 mM crotonate ( Cr + C646 ) for 24 h , then histones were extracted subjected to western blotting using anti - Kcr antibody , experiments were repeated three times .

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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.


E of changes in histone Kcr in NSPCs treated with DMSO ( Control ) , 5 μM triptolide ( FP ) , 5 μM flavopiridol 10 mM crotonate ( FP + CR ) , 10 mM crotonate ( CR ) for 1 h . Data are presented as mean ± of three independent biological replicates , n = 3 . One - way ANOVA with post hoc Tukey ' s was used to analyze statistical significance ; NS : no significance , P < 0 . 01 , P < 0 . 001 .

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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 indicating RNAP2 binding in H3K9cr enriched regions in NSPCs . H3 was used as a loading control for input .

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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 of changes in openness at Tgfb1 Notum promoter regions by FAIRE - qPCR among different groups of NSPCs . Data are presented as mean ± of three independent biological replicates , n = 3 . Two - unpaired Student ' s t - was used to analyze statistical significance ; P < 0 . 05 , P < 0 . 01 .

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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.


A , B Representative ( A ) ( B ) of changes in proliferation ability measured by BrdU incorporation rate among different groups of NSPCs . Scale bar , 50 μm . Data are presented as mean ± of three independent biological replicates , n = 3 . Two - unpaired Student ' s t - was used to analyze statistical significance ; P < 0 . 01 .

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A, B Representative images (A) and quantification analysis (B) of changes in cell proliferation ability measured by BrdU incorporation rate among different groups of NSPCs. Scale bar, 50 μm. 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 < 0.01.


C , D Representative ( C ) ( D ) of changes in neuronal differentiation potential defined by TUJ1 positivity among different groups of NSPCs . Scale bar , 20 μm . Data are presented as mean ± of three independent biological replicates , n = 4 . Two - unpaired Student ' s t - was used to analyze statistical significance ; P < 0 . 001 .

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C, D Representative images (C) and quantification analysis (D) of changes in neuronal differentiation potential defined by TUJ1 positivity among different groups of NSPCs. Scale bar, 20 μm. Data are presented as mean ± SEM of three independent biological replicates, n = 4. Two-tailed unpaired Student's t-test was used to analyze statistical significance; P < 0.001.


I , J of expression changes of Mir - 203 ( I ) Bmi1 ( J ) by qRT - PCR among different groups of NSPCs . Data are presented as mean ± of three independent biological replicates , n = 3 . Two - unpaired Student ' s t - was used to analyze statistical significance ; P < 0 . 05 , P < 0 . 01 .

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I, J Quantification analysis of expression changes of Mir-203 (I) and Bmi1 (J) by qRT-PCR 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.


K Representative blots ( top panel ) ( bottom panel ) of changes in BMI1 among different groups of NSPCs . Data are presented as mean ± of three independent biological replicates , n = 3 . Two - unpaired Student ' s t - was used to analyze statistical significance ; P < 0 . 05 .

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K Representative blots (top panel) and quantification analysis (bottom panel) of changes in BMI1 protein levels 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.


A , B histones were prepared from E13 . 5 forebrain explants treated with various concentrations of crotonate for 3 h subjected to western blotting using anti - H3K9cr ( A ) anti - Kcr ( B ) antibodies . Naïve : isolated forebrains without subsequent culture treatment . C , D of changes in H3K9cr ( C ) H3 - Kcr ( D ) by western blotting among different groups of explants . Data are presented as mean ± of three independent biological replicates , n = 3 . Two - unpaired Student ' s t - was used to analyze statistical significance ; NS : no significance , P < 0 . 01 , P < 0 . 001 .

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A, B Core histones were prepared from E13.5 forebrain explants treated with various concentrations of crotonate for 3 h and subjected to western blotting analysis using anti-H3K9cr (A) and anti-Kcr (B) antibodies. Naïve: isolated forebrains without subsequent culture and treatment. C, D Quantification analysis of changes in H3K9cr (C) and H3-Kcr (D) levels by western blotting among different groups of explants. 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; NS: no significance, P < 0.01, P < 0.001.


A , B histones were prepared from E13 . 5 forebrain explants treated with various concentrations of crotonate for 3 h subjected to western blotting using anti - H3K9cr ( A ) anti - Kcr ( B ) antibodies . Naïve : isolated forebrains without subsequent culture treatment .

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A, B Core histones were prepared from E13.5 forebrain explants treated with various concentrations of crotonate for 3 h and subjected to western blotting analysis using anti-H3K9cr (A) and anti-Kcr (B) antibodies. Naïve: isolated forebrains without subsequent culture and treatment.


G , H Quantitative of changes in H3K9cr ( G , left panel of H ) H3K14la ( right panel of H ) by western blotting among different groups of embryonic forebrain . Data are presented as mean ± of three independent biological replicates , n = 3 . Two - unpaired Student ' s t - was used to analyze statistical significance ; NS : no significance , P < 0 . 05 , P < 0 . 001 .

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G, H Quantitative analysis of changes in H3K9cr (G, left panel of H) and H3K14la (right panel of H) levels by western blotting among different groups of the embryonic forebrain. 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; NS: no significance, P < 0.05, P <0.001.


Two top - ranked SplashRNA - designed shRNAmirs targeting human Nurd components a control shRNAmir targeting human CD4 were cloned into both miR - AB miR - E lentiviral vectors with Venus as reporter . lentiviruses packaged by these constructs were used for transduction of multiple human lines . RNAi efficiency was determined by western blot ( WB ) of target in lysates of transduced . - actin served as loading control .

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Two top-ranked SplashRNA-designed shRNAmirs targeting human Nurd complex components and a control shRNAmir targeting human CD4 were cloned into both miR-AB and miR-E lentiviral vectors with Venus as reporter. The lentiviruses packaged by these constructs were used for transduction of multiple human cell lines. RNAi efficiency was determined by western blot (WB) analysis of target protein levels in the lysates of the transduced cells. -actin served as loading control.


RNAi efficiency of Pten . 1524 Pten . 932 shRNAmir mediated by miR - AB vs . miR - E was tested in NIH - 3T3 in context of < 30 % transduction efficiency ( GFP as reporter ) . Representative western blots of Pten expression of FACS - sorted GFP + from two biological replicates were shown . β - actin served as loading control .

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The RNAi efficiency of Pten.1524 and Pten.932 shRNAmir mediated by miR-AB vs. miR-E was tested in NIH-3T3 cells in the context of <30% transduction efficiency (GFP as reporter). Representative western blots of Pten protein expression of the FACS-sorted GFP+ cells from two biological replicates were shown. β-actin served as loading control.


Two top - ranked SplashRNA - designed shRNAmirs targeting human or mouse surface FAS were cloned into Venus - expressing miR - AB lentiviral vectors which harbor different promoters to drive shRNAmir expression . packaged lentiviruses were used to infect indicated human or mouse lines . A shRNAmir targeting human or mouse CD4 driven under hCMV promoter was used as control . After > 96h of infection , FAS in transduced Venus - positive were determined by surface shown in histograms ( top panels ) quantified by MFI of APC ( bottom panels ) .

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Two top-ranked SplashRNA-designed shRNAmirs targeting human or mouse surface protein FAS were cloned into Venus-expressing miR-AB lentiviral vectors which harbor different promoters to drive the shRNAmir expression. The packaged lentiviruses were used to infect the indicated human or mouse cell lines. A shRNAmir targeting human or mouse CD4 driven under hCMV promoter was used as control. After >96h of infection, FAS protein levels in the transduced Venus-positive cells were determined by surface staining and shown in histograms (top panels) and quantified by MFI of APC (bottom panels).


293T were co - transduced with four miR - AB lentiviruses at 1 : 1 , which carries a neutral shRNAmir targeting CD4 or CD19 , with Azurite , GFP , Ametrine or mOrange as reporter respectively . were cultured for 22 days positive were quantified at time points as indicated . Representative contour FACS plots on day 4 day 22 shown ( top panels ) . numbers of untransduced , single positive quadruple positive were quantified normalized to untransduced on day 4 , plotted over time ( bottom panel ) . Data are shown as mean ± s . d . n = 4 . NS indicates no significant difference ( P > 0 . 05 , two - unpaired Student ' s t - ) .

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

293T cells were co-transduced with four miR-AB lentiviruses at 1:1 ratio, which carries a neutral shRNAmir targeting CD4 or CD19, with Azurite, GFP, Ametrine or mOrange as reporter respectively. Cells were cultured for 22 days and fluorescence positive cells were quantified at the time points as indicated. Representative contour FACS plots cells on day 4 and day 22 shown (top panels). Cell numbers of untransduced, single fluorescence positive cells and quadruple positive cells were quantified and normalized to untransduced cells on day 4, and plotted over time (bottom panel). Data are shown as mean ± s.d. and n=4. NS indicates no significant difference (P>0.05, two-tailed unpaired Student's t-test).


CD8 + T were activated for 16h then co - transduced with miR - AB retroviruses at 1 : 1 , which carries a SplashRNA - designed shRNAmir , targeting surface CD127 , CD90 , CD44 or CD8 , with Azurite , GFP , Ametrine or mCherry2 as reporters respectively for 4h . miR - AB retroviruses expressing CD19 - specific shRNAmirs with same reporters were used as controls . Surface was performed 72h after transduction . FACS data were shown as overlaid histograms of control shRNAmirs ( shCD19s ) target shRNAmirs ( shCD127 , shCD90 , shCD44 or shCD8 ) . were categorized as sixteen populations expressing single , double , triple or quadruple or not ( none ) reporter - positive plots were highlighted with thick frames background . Each population ' s surface profile was shown on right . data was representative of two biologically independent experiments .

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

CD8+ T cells were activated for 16h and then co-transduced with miR-AB retroviruses at 1:1 ratio, which carries a SplashRNA-designed shRNAmir, targeting surface protein CD127, CD90, CD44 or CD8, with Azurite, GFP, Ametrine or mCherry2 as fluorescent reporters respectively for 4h. miR-AB retroviruses expressing CD19-specific shRNAmirs with same fluorescent reporters were used as controls. Surface staining was performed 72h after transduction. FACS data were shown as overlaid histograms of control shRNAmirs (shCD19s) and target shRNAmirs (shCD127, shCD90, shCD44 or shCD8). Cells were categorized as sixteen populations expressing single, double, triple or quadruple fluorescent proteins or not (none) and fluorescent reporter-positive plots were highlighted with thick frames and white background. Each population's surface staining profile was shown on the right. The data was representative of two biologically independent experiments.


( C ) qRT - PCR expression of endogenous repeats following shRNA - mediated KAP1 depletion in NTERA - 2 . Results were normalized to β2 microglobulin ( B2M ) . KAP1 expression were verified by qRT - PCR Western blot . A representative experiment of 2 experiments is shown . Two - unpaired t were done ( HERVK14C _ 2 p - value = 0 . 002300 ) .

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(C) qRT-PCR expression of endogenous repeats following shRNA-mediated KAP1 depletion in NTERA-2 cells. Results were normalized to β2 microglobulin (B2M). KAP1 expression levels were verified by qRT-PCR and Western blot. A representative experiment of 2 experiments is shown. Two-tailed unpaired t tests were done (HERVK14C_2 p-value = 0.002300).


( D qRT - PCR expression of endogenous repeats following KAP1 knockout in HeLa Results were normalized to B2M . KAP1 expression were verified by qRT - PCR Western blot . Two - unpaired t were done p - values are : HERVK14C _ 1 : 0 . 0017 , HERVK14C _ 2 : 0 . 0044 , SVA D VNTR : 0 . 0243 Clones 8 , 12 15 ( from D ) were selected for mRNA - sequencing .

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(D qRT-PCR expression of endogenous repeats following KAP1 knockout in HeLa Results were normalized to B2M. KAP1 expression levels were verified by qRT-PCR and Western blot. Two-tailed unpaired t tests were done and p-values are: HERVK14C_1: 0.0017, HERVK14C_2: 0.0044, SVA D VNTR: 0.0243 Clones 8, 12 and 15 (from D) were selected for mRNA-sequencing.


E ) qRT - PCR expression of endogenous repeats following KAP1 knockout in 293T Results were normalized to B2M . KAP1 expression were verified by qRT - PCR Western blot . For ( E ) , we found that clone I ( also treated with KAP1 sgRNA ) retained KAP1 expression ( E , right panel ) so we only used clones II , III IV to explore phenotype ( E , left panel ) , which represented validated KAP1 knockouts . Two - unpaired t were done p - values are : ( E ) HERVK14C _ 2 : < 0 . 0001 .

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

E) qRT-PCR expression of endogenous repeats following KAP1 knockout in 293T cells Results were normalized to B2M. KAP1 expression levels were verified by qRT-PCR and Western blot. For (E), we found that clone I (also treated with KAP1 sgRNA) retained KAP1 protein expression (E, right panel) so we only used clones II, III and IV to explore phenotype (E, left panel), which represented validated KAP1 knockouts. Two-tailed unpaired t tests were done and p-values are: (E) HERVK14C_2: <0.0001.


( F ) qRT - PCR expression of endogenous repeats following shRNA - mediated KAP1 depletion in PBMCs ( day 6 post - transduction ) . Results were normalized to B2M . error bars show standard deviation ( SD ) . numbers above bars depict fold changes compared to control ( to one decimal place ) .

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

(F) qRT-PCR expression of endogenous repeats following shRNA-mediated KAP1 depletion in PBMCs (day 6 post-transduction). Results were normalized to B2M. All error bars show standard deviation (SD). All numbers above bars depict fold changes compared to control cells (to one decimal place).


( A ) Boxplots showing repeats significantly upregulated ( > 2 fold where p = < 0 . 05 using DESeq2 ) in knockout ( N = 3 ) compared to wildtype ( N = 3 ) HeLa on mRNA - sequencing data . P values = 0 . 0174 ( HERV - S ) , 0 . 0047 ( HERV - K14C ) , 0 . 00013 ( HERV - T LTR6B ) 1 . 90E - 06 ( HERV - T HERVS71 ) . HERV - T HERV - S but not HERVK14C also reached significance when only adjusted p - values were considered , where differences are compared to whole of repBase . KAP1 binding ( ENCODE data ) is shown in Fig S1A . numbers above bars depict fold changes compared to control ( to one decimal place ) .

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

(A) Boxplots showing repeats significantly upregulated (>2 fold where p=<0.05 using DESeq2) in knockout (N=3) compared to wildtype (N=3) HeLa cells based on mRNA-sequencing data. P values = 0.0174 (HERV-S), 0.0047 (HERV-K14C), 0.00013 (HERV-T LTR6B) and 1.90E-06 (HERV-T HERVS71). HERV-T and HERV-S but not HERVK14C also reached significance when only adjusted p-values were considered, where differences are compared to the whole of repBase. KAP1 binding (ENCODE data) is shown in Fig S1A. All numbers above bars depict fold changes compared to control cells (to one decimal place).


( F ) Genomic coordinates of common KAP1 sites identified in Fig EV3D were subjected to ChIP - seq correlation analyses using ChIP - Cor software ( see methods ) . Each plot shows duplicate ChIP - seq experiments from ENCODE . See Fig EV3B for complete data .

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(F) Genomic coordinates of the common KAP1 sites identified in Fig EV3D were subjected to ChIP-seq correlation analyses using ChIP-Cor software (see methods). Each plot shows duplicate ChIP-seq experiments from ENCODE. See Fig EV3B for complete data.


( B ) NTERA - 2 were transduced with either an empty vector ( WT ) or same vector containing an shRNA against KAP1 ( KD ) prior to puromycin selection transduction with increasing doses of GFP reporter vectors . GFP expression was analysed 72 hours post reporter transduction . A representative experiment of 2 experiments is shown . Two - unpaired t p - values : PBS - LTR - GFP : 0 . 0148 ; PBSChr15 - LTR - GFP : 0 .

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

(B) NTERA-2 cells were transduced with either an empty vector (WT) or the same vector containing an shRNA against KAP1 (KD) prior to puromycin selection and transduction with increasing doses of GFP reporter vectors. GFP expression was analysed 72 hours post reporter transduction. A representative experiment of 2 experiments is shown. Two-tailed unpaired t test p-values: PBS-LTR-GFP: 0.0148; PBSChr15-LTR-GFP: 0.0377


( C ) same as ( B ) except that here KAP1 wildtype ( WT ) knockout ( KO ) 293T were used . P - values : PBS - LTR - GFP : 0 . 0044 ; PBSChr15 - LTR - GFP : 0 . 0077 .

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

(C) The same as (B) except that here KAP1 wildtype (WT) and knockout (KO) 293T cells were used. P-values: PBS-LTR-GFP: 0.0044; PBSChr15-LTR-GFP: 0.0077.


( D ) PBMCs were transduced with increasing doses of GFP reporter vectors ( vectors were normalized to same number of infectious units after titering them on permissive ) , left plot . Vectors were integrated at similar as measured by GFP Taqman qPCR , right plot . A 293T with a single vector copy integrant ( PGK - GFP ) was used as a control to estimate absolute copy numbers . A representative experiment of 3 experiments is shown here . Two - unpaired t p - value 0 . 0288 ( dose 3 ) . Error bars show SD .

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

(D) PBMCs were transduced with increasing doses of GFP reporter vectors (vectors were normalized to the same number of infectious units after titering them on permissive cells), left plot. Vectors were integrated at similar levels as measured by GFP Taqman qPCR, right plot. A 293T cell line with a single vector copy integrant (PGK-GFP) was used as a control to estimate the absolute copy numbers. A representative experiment of 3 experiments is shown here. Two-tailed unpaired t test p-value 0.0288 (dose 3). Error bars show SD.


( A ) qRT - PCR expression of endogenous repeats ( left ) ISGs ( right ) following shRNA - mediated KAP1 depletion in HeLa ( day 6 post transduction ) , normalized to B2M GAPDH respectively . n = 2 two - unpaired t p - values are : HERVK14C : 0 . 025 , 0 . 003 ; SVA VNTR : 0 . 00027 , 0 . 01 ; ISG56 : 0 . 002 ; CCL5 : 0 . 007 , 0 . 00003 ; CXCL10 : 0 . 0008 ; Mxa : 0 . 011 ; IKba : 0 . 008 , 0 . 01 . See also Fig S2AB .

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

(A) qRT-PCR expression of endogenous repeats (left) and ISGs (right) following shRNA-mediated KAP1 depletion in HeLa cells (day 6 post transduction), normalized to B2M and GAPDH respectively. n=2 and two-tailed unpaired t test p-values are: HERVK14C: 0.025, 0.003; SVA VNTR: 0.00027, 0.01; ISG56: 0.002; CCL5: 0.007, 0.00003; CXCL10: 0.0008; Mxa: 0.011; IKba: 0.008, 0.01. See also Fig S2AB.


( B ) qRT - PCR expression of endogenous repeats ( left ) ISGs ( right ) following 5 - AZA treatment of HeLa PBMCs ( day 6 post transduction ) . A representative experiment of two is shown in each case . Two - unpaired t were performed . HeLa p - values : ISG56 : 0 . 005 ; CCL5 : 0 . 0006 ; CXCL10 : 0 . 0007 ; Mxa : 0 . 0001 ; IFNb : 0 . 02 . For repeat expression ( left panel ) , p values not available as N = 2 . PBMC p - values : HERVK14C : 0 . 1 ; SVA D VNTR : 0 . 003 ; ISG56 : 0 . 001 ; CCL5 : 0 . 012 ; CXCL10 : 0 . 012 ; Mxa : 0 . 02 ; IKba : 0 . 08 . Results were normalized to GAPDH B2M ( GAPDH results shown ) . See also Fig S2C for results in 293T . error bars show SD .

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

(B) qRT-PCR expression of endogenous repeats (left) and ISGs (right) following 5-AZA treatment of HeLa cells and PBMCs (day 6 post transduction). A representative experiment of two is shown in each case. Two-tailed unpaired t tests were performed. HeLa cell p-values: ISG56: 0.005; CCL5: 0.0006; CXCL10: 0.0007; Mxa: 0.0001; IFNb: 0.02. For repeat expression (left panel), p values not available as N=2. PBMC p-values: HERVK14C: 0.1; SVA D VNTR: 0.003; ISG56: 0.001; CCL5: 0.012; CXCL10: 0.012; Mxa: 0.02; IKba: 0.08. Results were normalized to GAPDH and B2M (GAPDH results shown). See also Fig S2C for results in 293T cells. All error bars show SD.


( C ) methylation at endogenous SVAs was measured over 18 CpGs in stated treatment groups ( day 5 post transductions , or day 2 post 5 - AZA treatment ) . Each point represents average methylation state of one with at least 10 sequences analyzed per group . Mann - Whitney were performed to compare shControl to shKAP1 ( p = 0 . 6593 ) DMSO to 5 - AZA ( 0 . 0027 ) . 5 - AZA was added to experiments at 7uM . numbers above bars depict fold changes compared to control ( to one decimal place ) .

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(C) DNA methylation at endogenous SVAs was measured over 18 CpGs in the stated treatment groups (day 5 post transductions, or day 2 post 5-AZA treatment). Each point represents the average methylation state of one sequence with at least 10 sequences analyzed per group. Mann-Whitney tests were performed to compare shControl to shKAP1 (p=0.6593) and DMSO to 5-AZA (0.0027). 5-AZA was added to all experiments at 7uM. All numbers above bars depict fold changes compared to control cells (to one decimal place).


( A ) H3K9me3 ChIP - PCR where error bars show SD of 3 independent IPs . Results show IP enrichment relative to total input normalized to GAPDH as a negative control region . IgG control IPs gave only background enrichments , shown .

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(A) H3K9me3 ChIP-PCR where error bars show SD of 3 independent IPs. Results show the IP enrichment relative to the total input normalized to GAPDH as a negative control region. IgG control IPs gave only background enrichments, shown.


( B ) 293T were transduced with stated shRNAs harvested 4 days after puro selection for Western Blot to verify KAP1 SETDB1 - depletion ( left ) ChIP ( right ) . numbers were normalized per treatment group before sonication IPs were performed in triplicate . Results show IP relative to total input GAPDH normalized . IgG control IPs gave only background enrichments shown . One representative experiment of two is shown . Unpaired t were performed . P values : ZNF239 : KAP1 , 0 . 0292 ; SETDB1 , 0 . 0137 ; HERVK14C _ 1 : KAP1 , 0 . 0227 ; SETDB1 , 0 . 0082 ; HERVK14C _ 2 : SETDB1 , 0 . 0462 ; SVA D VNTR , SETDB1 , 0 . 0064 . inlay shows qRTPCR expression of HERVK14C in same experiment , normalized to B2M or GAPDH as stated . error bars show SD .

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(B) 293T cells were transduced with the stated shRNAs and cells harvested 4 days after puro selection for Western Blot to verify KAP1 and SETDB1-depletion (left) and ChIP (right). Cell numbers were normalized per treatment group before sonication and IPs were performed in triplicate. Results show IP relative to total input and GAPDH normalized. IgG control IPs gave only background enrichments shown. One representative experiment of two is shown. Unpaired t tests were performed. P values: ZNF239: KAP1, 0.0292; SETDB1, 0.0137; HERVK14C_1: KAP1, 0.0227; SETDB1, 0.0082; HERVK14C_2: SETDB1, 0.0462; SVA D VNTR, SETDB1, 0.0064. The inlay shows qRTPCR expression analysis of HERVK14C in the same experiment, normalized to B2M or GAPDH as stated. All error bars show SD.


( C ) Venn diagram showing H3K9me3 enriched sites ( 18271 ) as those identified in both ChIP replicates in human K562 ( left ) . Right : % of ISGs or random genes intersecting a H3K9me3 or KAP1 ChIP - seq peak . 5kb upstream downstream of each gene was also included . N = 437 genes per group .

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(C) Venn diagram showing H3K9me3 enriched sites (18271) as those identified in both ChIP replicates in the human cell line K562 (left). Right: % of ISGs or random genes intersecting a H3K9me3 or KAP1 ChIP-seq peak. 5kb upstream and downstream of each gene was also included. N=437 genes per group.


( D ) An IFIT1 promoter - Luciferase reporter THP - 1 was used or 293T or HeLa transduced with an IFN - β promoter - Luciferase reporter . These lines were transduced with an shKAP1 or shControl vector or a vFLIP - expressing vector luciferase measured 6 days later . Error bars show SD . Unpaired t were used to compare shControl to shKAP1 samples : p values = 0 . 122 ( THP - 1 ) , 0 . 0001 ( HeLa ) , 0 . 0001 ( 293T ) .

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(D) An IFIT1 promoter-based Luciferase reporter THP-1 cell line was used or 293T cells or HeLa cells transduced with an IFN-β promoter-based Luciferase reporter. These cell lines were transduced with an shKAP1 or shControl vector or a vFLIP-expressing vector and luciferase measured 6 days later. Error bars show SD. Unpaired t tests were used to compare shControl to shKAP1 samples: p values = 0.122 (THP-1), 0.0001 (HeLa), 0.0001 (293T cells).


( E ) WT , STING KO MAVS KO THP - 1 were verified by Licor blot .

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(E) WT, STING KO and MAVS KO THP-1 cells were verified by Licor blot.