Figure 1 . ( D ) Quantification of EGFP - LC3 puncta in MCF7 - EGFP - LC3 cells 56 h after transfection with the indicated siRNAs . When indicated , cells were exposed to 100 nM rapamycin for the last 3 h . RPTOR ( raptor ) and BECN1 ( beclin1 ) siRNAs served as positive and negative controls , respectively . ( E ) Autophagic flux was measured as the ratio between luciferase activities in MCF7 - RLuc - LC3WT and MCF7 - RLuc - LC3G120A cells transfected with the indicated siRNAs 56 h earlier and left untreated or treated with 100 nM rapamycin for the last 3 h . Error bars are SDs for a representative ( n = 5 ) triplicate experiment with a minimum of 4 × 10 randomly chosen areas / sample analyzed ( D ) or three independent experiments ( F ) . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 , as compared with similarly treated control siRNA - transfected samples .

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Figure 1.(D) Quantification of EGFP-LC3 puncta in MCF7-EGFP-LC3 cells 56 h after transfection with the indicated siRNAs. When indicated, cells were exposed to 100 nM rapamycin for the last 3 h. RPTOR (raptor) and BECN1 (beclin1) siRNAs served as positive and negative controls, respectively.(E) Autophagic flux was measured as the ratio between luciferase activities in MCF7-RLuc-LC3WT and MCF7-RLuc-LC3G120A cells transfected with the indicated siRNAs 56 h earlier and left untreated or treated with 100 nM rapamycin for the last 3 h. Error bars are SDs for a representative (n = 5) triplicate experiment with a minimum of 4 × 10 randomly chosen areas/sample analyzed (D) or three independent experiments (F). *, P < 0.05; **, P < 0.01; ***, P < 0.001, as compared with similarly treated control siRNA-transfected samples.


Figure 2 . ( A ) Representative immunoblots of indicated proteins from whole - cell lysates of MCF7 cells transfected with indicated siRNAs for 56 h and subjected to 0 - 40 min of amino acid starvation [ − AA ] ) . ( B ) Representative immunoblots of the indicated proteins from whole - cell lysates of MCF7 - EGFP - LC3 cells stably infected with control or CIP2Ar ( siRNA - resistant CIP2A ) - encoding lentivirus , transfected with the indicated siRNAs for 53 h , and treated with 100 nM rapamycin ( Rapa ) for 0 - 20 min . ( C ) MCF7 - EGFP - LC3 cells stably infected with control or CIP2Ar - encoding lentivirus were transfected with indicated siRNAs for 48 h and analyzed for EGFP - LC3 puncta formation . ( D ) MCF7 - EGFP - LC3 cells transfected with control ( − ) and CIP2A siRNAs together with an empty vector ( − ) or MYC - S62A cDNA as indicated were analyzed 56 h later for EGFP - LC3 puncta ( left ) and expression of indicated proteins ( right ) . ( E ) Representative immunoblots ( left ) of indicated proteins from whole - cell lysates of MCF7 cells treated with the control ( C ) , RPTOR ( RP ) , or CIP2A ( CIP ) siRNAs for 56 h and densitometric quantification of three independent experiments ( right ) . Error bars show SDs for three independent experiments . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 , compared with Student ' s t test as indicated ( C and D ) or with control siRNA - transfected samples ( E ) . Black lines indicate that intervening lanes have been spliced out .

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

Figure 2.(A) Representative immunoblots of indicated proteins from whole-cell lysates of MCF7 cells transfected with indicated siRNAs for 56 h and subjected to 0-40 min of amino acid starvation [−AA]).(B) Representative immunoblots of the indicated proteins from whole-cell lysates of MCF7-EGFP-LC3 cells stably infected with control or CIP2Ar (siRNA-resistant CIP2A)-encoding lentivirus, transfected with the indicated siRNAs for 53 h, and treated with 100 nM rapamycin (Rapa) for 0-20 min.(C) MCF7-EGFP-LC3 cells stably infected with control or CIP2Ar-encoding lentivirus were transfected with indicated siRNAs for 48 h and analyzed for EGFP-LC3 puncta formation.(D) MCF7-EGFP-LC3 cells transfected with control (−) and CIP2A siRNAs together with an empty vector (−) or MYC-S62A cDNA as indicated were analyzed 56 h later for EGFP-LC3 puncta (left) and expression of indicated proteins (right).(E) Representative immunoblots (left) of indicated proteins from whole-cell lysates of MCF7 cells treated with the control (C), RPTOR (RP), or CIP2A (CIP) siRNAs for 56 h and densitometric quantification of three independent experiments (right). Error bars show SDs for three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001, compared with Student's t test as indicated (C and D) or with control siRNA-transfected samples (E). Black lines indicate that intervening lanes have been spliced out.


Figure 3 . ( A , top left ) Quantification of EGFP - LC3 puncta in MCF7 - EGFP - LC3 cells infected with control or CIP2A lentiviral shRNAs . Representative immunofluorescence images ( right ) and immunoblots ( bottom ) are shown . ( B ) Mean cell diameters of MCF7 - EGFP - LC3 cells infected as in A were measured with a cytometer ( NucleoCounter NC - 3000 ; Chemometec AS ) equipped with Via1 - Cassettes ( Chemometec AS ) . ( C ) MCF7 cells infected with shRNAs as in A were plated in equal density and left untreated or treated with 100 nM rapamycin ( Rapa ) for 72 h . The viable cells were either visualized after fixation and crystal violet staining ( left ; representative figure , n = 4 ) or trypsinized and counted with NucleoCounter NC - 3000 equipped with Via1 - Cassettes ( right ) . ( D ) MCF7 cells infected with shRNAs as in A were left untreated or treated with 2 nM ConA , 2 µM MG132 , 5 µM siramesine ( Sira ) , or 100 µM etoposide ( Etop ) for 48 h , and the metabolic activity ( cell density ) was measured by the MTT assay ( left ) , and cell death was measured by the LDH assay ( right ) . ( E ) Light microscopy pictures of MCF10A cells at passage 8 after infection with control or CIP2A - encoding lentivirus . ( F ) Representative immunoblots of the indicated proteins from whole - cell lysates of MCF10A ( left ) and HEK - 293 ( right ) cells infected as in E and treated with 100 nM rapamycin for the indicated times . Black lines indicate that intervening lanes have been spliced out .

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

Figure 3.(A, top left) Quantification of EGFP-LC3 puncta in MCF7-EGFP-LC3 cells infected with control or CIP2A lentiviral shRNAs. Representative immunofluorescence images (right) and immunoblots (bottom) are shown. (B) Mean cell diameters of MCF7-EGFP-LC3 cells infected as in A were measured with a cytometer (NucleoCounter NC-3000; Chemometec AS) equipped with Via1-Cassettes (Chemometec AS).(C) MCF7 cells infected with shRNAs as in A were plated in equal density and left untreated or treated with 100 nM rapamycin (Rapa) for 72 h. The viable cells were either visualized after fixation and crystal violet staining (left; representative figure, n = 4) or trypsinized and counted with NucleoCounter NC-3000 equipped with Via1-Cassettes (right).(D) MCF7 cells infected with shRNAs as in A were left untreated or treated with 2 nM ConA, 2 µM MG132, 5 µM siramesine (Sira), or 100 µM etoposide (Etop) for 48 h, and the metabolic activity (cell density) was measured by the MTT assay (left), and cell death was measured by the LDH assay (right).(E) Light microscopy pictures of MCF10A cells at passage 8 after infection with control or CIP2A-encoding lentivirus.(F) Representative immunoblots of the indicated proteins from whole-cell lysates of MCF10A (left) and HEK-293 (right) cells infected as in E and treated with 100 nM rapamycin for the indicated times. Black lines indicate that intervening lanes have been spliced out.


Figure 4 . CIP2A expression correlates with mTORC1 activity in primary human breast tumors . TMAs including 33 normal breast tissues and 210 primary breast carcinoma samples were analyzed by semiquantitative immunohistochemistry for the expression of the indicated proteins and mean scores ( arbitrary units [ a . u . ] ) for duplicate samples were averaged and plotted according to mean CIP2A expression ( left ) . The red lines are the regression lines , and the stippled red lines define the 95 % confidence intervals . Statistical analysis of data was performed using NCSS 2007 and Prism version 5 . Examples of the staining of breast cancer ( a ) and normal ( b ) cores indicated in the P - T389 - S6K1 / CIP2A plot are shown on the right . rsp , Spearman ' s correlation coefficient .

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Figure 4.CIP2A expression correlates with mTORC1 activity in primary human breast tumors. TMAs including 33 normal breast tissues and 210 primary breast carcinoma samples were analyzed by semiquantitative immunohistochemistry for the expression of the indicated proteins and mean scores (arbitrary units [a.u.]) for duplicate samples were averaged and plotted according to mean CIP2A expression (left). The red lines are the regression lines, and the stippled red lines define the 95% confidence intervals. Statistical analysis of data was performed using NCSS 2007 and Prism version 5. Examples of the staining of breast cancer (a) and normal (b) cores indicated in the P-T389-S6K1/CIP2A plot are shown on the right. rsp, Spearman's correlation coefficient.


Figure 5 . ( A and B ) In vitro translated full - length CIP2A was incubated with purified human PP2A complex and HA - Raptor ( A ) or HA - S6K1 ( B ) purified from transiently transfected MCF7 cells . Protein complexes were immunoprecipitated with anti - HA antibody and analyzed by immunoblotting as indicated . Nonspecific IgG served as a control . Note that human reticulocyte ( RC ) lysate used for in vitro translation of CIP2A contained detectable amounts of PP2Ac . ( C ) Endogenous CIP2A protein complexes immunoprecipitated from MCF7 cells were analyzed by immunoblotting and PP2A activity assay . Immunoblots of corresponding cell lysates are shown on the right . ( D ) Endogenous raptor protein complexes immunoprecipitated from MCF7 cells stably infected with control ( Ctr ) or CIP2A shRNA lentiviruses ( Fig . 3 A ) and starved for amino acids for 20 min when indicated were analyzed by immunoblotting and PP2A activity assay . Immunoblots of corresponding cell lysates are shown on the right . ( E ) Raptor protein complexes immunoprecipitated from MCF7 cells treated with control ( C ) or PPP2R1A ( R1A ) siRNAs for 56 h were analyzed by immunoblotting with the indicated antibodies . Immunoblots of corresponding cell lysates are shown on the left . ( F ) HA - S6K1 protein complexes immunoprecipitated from MCF7 cells stably infected with control or CIP2A shRNA lentiviruses ( Fig . 3 A ) , transiently transfected with HA - RPS6K1 cDNA and starved for amino acids for 20 min when indicated , were analyzed by immunoblotting . Immunoblots of corresponding cell lysates are shown on the right . ( G ) Enzymatic activity of a purified PP2A holoenzyme incubated with human reticulocyte lysate with or without in vitro translated full - length CIP2A . Error bars are SDs for four ( D ) independent experiments or a representative triplicate experiment ( C and G ) . ( H ) Representative confocal microscopy images of untreated and amino acid - starved ( 4 h ) MCF7 cells stained for endogenous CIP2A and raptor . Black lines indicate that intervening lanes have been spliced out . − AA , amino acid starvation ; IP , immunoprecipitation ; a . u . , arbitrary unit . Bar , 10 µm .

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

Figure 5.(A and B) In vitro translated full-length CIP2A was incubated with purified human PP2A complex and HA-Raptor (A) or HA-S6K1 (B) purified from transiently transfected MCF7 cells. Protein complexes were immunoprecipitated with anti-HA antibody and analyzed by immunoblotting as indicated. Nonspecific IgG served as a control. Note that human reticulocyte (RC) lysate used for in vitro translation of CIP2A contained detectable amounts of PP2Ac.(C) Endogenous CIP2A protein complexes immunoprecipitated from MCF7 cells were analyzed by immunoblotting and PP2A activity assay. Immunoblots of corresponding cell lysates are shown on the right.(D) Endogenous raptor protein complexes immunoprecipitated from MCF7 cells stably infected with control (Ctr) or CIP2A shRNA lentiviruses (Fig. 3 A) and starved for amino acids for 20 min when indicated were analyzed by immunoblotting and PP2A activity assay. Immunoblots of corresponding cell lysates are shown on the right.(E) Raptor protein complexes immunoprecipitated from MCF7 cells treated with control (C) or PPP2R1A (R1A) siRNAs for 56 h were analyzed by immunoblotting with the indicated antibodies. Immunoblots of corresponding cell lysates are shown on the left.(F) HA-S6K1 protein complexes immunoprecipitated from MCF7 cells stably infected with control or CIP2A shRNA lentiviruses (Fig. 3 A), transiently transfected with HA-RPS6K1 cDNA and starved for amino acids for 20 min when indicated, were analyzed by immunoblotting. Immunoblots of corresponding cell lysates are shown on the right.(G) Enzymatic activity of a purified PP2A holoenzyme incubated with human reticulocyte lysate with or without in vitro translated full-length CIP2A. Error bars are SDs for four (D) independent experiments or a representative triplicate experiment (C and G).(H) Representative confocal microscopy images of untreated and amino acid-starved (4 h) MCF7 cells stained for endogenous CIP2A and raptor. Black lines indicate that intervening lanes have been spliced out. −AA, amino acid starvation; IP, immunoprecipitation; a.u., arbitrary unit. Bar, 10 µm.


Figure 6 . ( A ) Representative immunoblots of the indicated proteins from whole - cell lysates of MCF7 cells subjected to 100 nM rapamycin or amino acid starvation for 0 - 24 h . ( B ) qPCR analysis of CIP2A and MYC mRNA levels in MCF7 cells treated as in A . AA - starv , amino acid starvation ; a . u . , arbitrary unit . Error bars show SDs for three independent experiments . ( C ) Representative immunoblots of the indicated proteins from whole - cell lysates of MCF7 cells treated with ULK1 or SQSTM1 siRNAs for 54 h before the exposure to fresh medium ( 0 ) or amino acid starvation ( − AA ) for 12 h . Ctr , control . ( D ) Representative immunoblots of the indicated proteins from whole - cell lysates of MCF7 cells treated for 0 - 5 h with the indicated combinations of 100 µM cycloheximide ( CHX ) , 100 nM rapamycin , and 2 nM ConA . ( E ) Representative confocal images of MCF7 ( left and middle ) and MCF7 - LC3 - EGFP ( right ) cells left untreated or treated with 100 nM rapamycin for 4 h or 100 nM rapamycin and 2 nM ConA for 8 h , fixed , and stained with antibodies against CIP2A . Nuclei in MCF - LC3 - EGFP cells were visualized with Hoechst . Green arrowheads indicate cells with many LC3 - positive puncta and low CIP2A levels , and yellow arrowheads show cells in which LC3 and CIP2A colocalize . Bars , 10 µm . ( F ) Representative immunoblots of an endogenous CIP2A protein complex immunoprecipitated from lysates of MCF7 cells left untreated or treated for 4 h with 100 nM rapamycin ( Rapa ) and 2 nM ConA as indicated . Mouse IgG served as a negative control . ( G ) MCF7 cells were treated with 100 nM rapamycin , 2 nM ConA , and 2 µM MG132 for 24 h as indicated . Endogenous CIP2A was immunoprecipitated ( IP ) in stringent conditions ( no coimmunoprecipitation of p62 ) and analyzed by immunoblotting using antibodies against CIP2A , monoubiquitin , and polyubiquitin . Short ( < 100 kD ) , medium ( > 150 kD ) , and long ( 100 - 130 kD ) exposures are shown . ( H ) Proteins from MCF7 cell lysates were immunoprecipitated with the indicated antibodies in stringent conditions ( see G ) and analyzed by immunoblotting for CIP2A and monoubiquitin . ( I ) Lysates from MCF7 cells transfected with the indicated ubiquitin - HA constructs were subjected to anti - CIP2A immunopurification followed by immunoblotting with antibodies against CIP2A and HA . ( J ) Lysates from MCF7 cells transfected with the CIP2A - FLAG construct ( Junttila et al . , 2007 ) and the indicated ubiquitin - HA constructs were subjected to anti - FLAG immunopurification in denaturing conditions with 0 . 1 % SDS followed by immunoblotting with the indicated antibodies . Black lines indicate that intervening lanes have been spliced out . Ub , ubiquitin ; WT , wild type .

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Figure 6.(A) Representative immunoblots of the indicated proteins from whole-cell lysates of MCF7 cells subjected to 100 nM rapamycin or amino acid starvation for 0-24 h.(B) qPCR analysis of CIP2A and MYC mRNA levels in MCF7 cells treated as in A. AA-starv, amino acid starvation; a.u., arbitrary unit. Error bars show SDs for three independent experiments.(C) Representative immunoblots of the indicated proteins from whole-cell lysates of MCF7 cells treated with ULK1 or SQSTM1 siRNAs for 54 h before the exposure to fresh medium (0) or amino acid starvation (−AA) for 12 h. Ctr, control.(D) Representative immunoblots of the indicated proteins from whole-cell lysates of MCF7 cells treated for 0-5 h with the indicated combinations of 100 µM cycloheximide (CHX), 100 nM rapamycin, and 2 nM ConA.(E) Representative confocal images of MCF7 (left and middle) and MCF7-LC3-EGFP (right) cells left untreated or treated with 100 nM rapamycin for 4 h or 100 nM rapamycin and 2 nM ConA for 8 h, fixed, and stained with antibodies against CIP2A. Nuclei in MCF-LC3-EGFP cells were visualized with Hoechst. Green arrowheads indicate cells with many LC3-positive puncta and low CIP2A levels, and yellow arrowheads show cells in which LC3 and CIP2A colocalize. Bars, 10 µm.(F) Representative immunoblots of an endogenous CIP2A protein complex immunoprecipitated from lysates of MCF7 cells left untreated or treated for 4 h with 100 nM rapamycin (Rapa) and 2 nM ConA as indicated. Mouse IgG served as a negative control.(G) MCF7 cells were treated with 100 nM rapamycin, 2 nM ConA, and 2 µM MG132 for 24 h as indicated. Endogenous CIP2A was immunoprecipitated (IP) in stringent conditions (no coimmunoprecipitation of p62) and analyzed by immunoblotting using antibodies against CIP2A, monoubiquitin, and polyubiquitin. Short (<100 kD), medium (>150 kD), and long (100-130 kD) exposures are shown.(H) Proteins from MCF7 cell lysates were immunoprecipitated with the indicated antibodies in stringent conditions (see G) and analyzed by immunoblotting for CIP2A and monoubiquitin.(I) Lysates from MCF7 cells transfected with the indicated ubiquitin-HA constructs were subjected to anti-CIP2A immunopurification followed by immunoblotting with antibodies against CIP2A and HA.(J) Lysates from MCF7 cells transfected with the CIP2A-FLAG construct (Junttila et al., 2007) and the indicated ubiquitin-HA constructs were subjected to anti-FLAG immunopurification in denaturing conditions with 0.1% SDS followed by immunoblotting with the indicated antibodies. Black lines indicate that intervening lanes have been spliced out. Ub, ubiquitin; WT, wild type.


Figure 1C , Heatmap of DEGs between the Src - 1 / - 2 dKO and WT fetuses that are involved in the lung development pathway ( GO : 0030324 ) .

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Figure 1C, Heatmap of DEGs between the Src-1/-2 dKO and WT fetuses that are involved in the lung development pathway (GO: 0030324).


Figure 2A and C , mRNA levels of Arg1 and Ass1 in fetal lungs of mice with different genotypes at 18 . 5 dpc ; n = 8 biological replicates in each group for panel A and 7 biological replicates in each group for panel C . One - way ANOVA followed by Tukey ' s multiple comparison test was used to analyze the data . The data are shown as the mean ± s . e . m . , * P < 0 . 05 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , compared with the WT group . B and D , Representative immunoblots of ARG1 and ASS1 protein expression in fetal lungs from mice with different genotypes at 18 . 5 dpc . Densitometric quantitation of immunoblot scans ( relative to β - actin as a loading control ) is also shown as a bar graph ; n = 3 biological replicates in each group . One - way ANOVA followed by Tukey ' s multiple comparison test was used to analyze the data . The data are shown as the mean ± s . e . m . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , compared with the WT group . E and F , Immunohistochemical staining of ARG1 and ASS1 in fetal lungs of WT and Src - 1 / - 2 dKO mice at 18 . 5 dpc . PBS was used instead of the primary antibody as a negative control ( NC ) . Scale bars : 50 μm in E and F .

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 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 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

Figure 2A and C, mRNA levels of Arg1 and Ass1 in fetal lungs of mice with different genotypes at 18.5 dpc; n=8 biological replicates in each group for panel A and 7 biological replicates in each group for panel C. One-way ANOVA followed by Tukey's multiple comparison test was used to analyze the data. The data are shown as the mean ± s.e.m., *P<0.05, ***P<0.001, ****P<0.0001, compared with the WT group.B and D, Representative immunoblots of ARG1 and ASS1 protein expression in fetal lungs from mice with different genotypes at 18.5 dpc. Densitometric quantitation of immunoblot scans (relative to β-actin as a loading control) is also shown as a bar graph; n=3 biological replicates in each group. One-way ANOVA followed by Tukey's multiple comparison test was used to analyze the data. The data are shown as the mean ± s.e.m., *P<0.05, **P<0.01, ***P<0.001, compared with the WT group.E and F, Immunohistochemical staining of ARG1 and ASS1 in fetal lungs of WT and Src-1/-2 dKO mice at 18.5 dpc. PBS was used instead of the primary antibody as a negative control (NC). Scale bars: 50 μm in E and F.


Figure 3A and C , mRNA expression of Arg1 and Ass1 in the lungs of WT fetuses at gestational ages from 15 . 5 dpc to labor . n = 6 biological replicates for each gestational age in panel A ; n = 6 biological replicates for 15 . 5dpc , 17 . 5dpc , and labor group , n = 5 biological replicates for 16 . 5dpc and 18 . 5dpc group in panel C . Kruskal - Wallis test followed by Dunn ' s multiple comparison test was used to analyze the data . The data shown are the mean ± s . e . m . * * P < 0 . 01 , * * * * P < 0 . 0001 . B and D , Representative immunoblots of gestational changes in ARG1 and ASS1 protein expression in the lungs of WT fetuses at gestational ages from 15 . 5 dpc to labor . Densitometric quantitation of scans of immunoblots ( relative to tubulin as loading control ) are also shown as the mean ± s . e . m . in bar graph ; n = 4 biological replicates for each gestational age ; Kruskal - Wallis test followed by Dunn ' s multiple comparison test was used to analyze the data . * P < 0 . 05 compared with 15 . 5 dpc . E and F , Immunohistochemical staining of ARG1 + ( E ) or ASS1 + ( F ) cells in the fetal lungs of WT mice at 15 . 5 dpc , 17 . 5 dpc and labor . PBS was used instead of the primary antibody in the NC group . Scale bars : 100 μm in E and F .

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 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 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

Figure 3A and C, mRNA expression of Arg1 and Ass1 in the lungs of WT fetuses at gestational ages from 15.5 dpc to labor. n=6 biological replicates for each gestational age in panel A; n=6 biological replicates for 15.5dpc, 17.5dpc, and labor group, n=5 biological replicates for 16.5dpc and 18.5dpc group in panel C. Kruskal-Wallis test followed by Dunn's multiple comparison test was used to analyze the data. The data shown are the mean ± s.e.m. **P<0.01, ****P<0.0001.B and D, Representative immunoblots of gestational changes in ARG1 and ASS1 protein expression in the lungs of WT fetuses at gestational ages from 15.5 dpc to labor. Densitometric quantitation of scans of immunoblots (relative to tubulin as loading control) are also shown as the mean ± s.e.m. in bar graph; n=4 biological replicates for each gestational age; Kruskal-Wallis test followed by Dunn's multiple comparison test was used to analyze the data. *P<0.05 compared with 15.5 dpc.E and F, Immunohistochemical staining of ARG1+ (E) or ASS1+ (F) cells in the fetal lungs of WT mice at 15.5 dpc, 17.5 dpc and labor. PBS was used instead of the primary antibody in the NC group. Scale bars: 100 μm in E and F.


Figure 4D and E , Histograms showing the content of arginine , ornithine , ADMA and the ratio of arginine to ADMA in the fetal lungs of WT mice and Src - 1 / - 2 dKO mice at 18 . 5 dpc . Mann Whitney test was used to analyze the data in panel D , and unpaired t test was used to analyze the data in panel E . n = 5 biological replicates for each group , and the data are shown as the mean ± s . e . m . The central band indicates the median , the boxes indicate the upper quartile and lower quartile , and the whiskers indicate maximum value and minimum value of the data . * P < 0 . 05 , * * P < 0 . 01 , compared with the WT fetal lungs .

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

Figure 4D and E, Histograms showing the content of arginine, ornithine, ADMA and the ratio of arginine to ADMA in the fetal lungs of WT mice and Src-1/-2 dKO mice at 18.5 dpc. Mann Whitney test was used to analyze the data in panel D, and unpaired t test was used to analyze the data in panel E. n=5 biological replicates for each group, and the data are shown as the mean ± s.e.m. The central band indicates the median, the boxes indicate the upper quartile and lower quartile, and the whiskers indicate maximum value and minimum value of the data. *P<0.05, **P<0.01, compared with the WT fetal lungs.


Figure 5B , Representative immunoblots of ARG1 expression in the fetal lungs from the AAV - shArg1 - injected group and the AAV - 2 / 9 - injected group . Densitometric quantitation of scans of immunoblots ( normalized to tubulin ) is also shown as a bar graph . Unpaired t test was used to analyze the data and n = 3 biological replicates for each group . The data are shown as the mean ± s . e . m . * * P < 0 . 01 compared with the AAV - 2 / 9 - injected group . C , Representative immunoblots and densitometric quantitation of Ki67 , caspase3 precursor ( pro - caspase3 ) and cleaved caspase3 expression in the fetal lungs from the AAV - shArg1 - injected group and AAV - 2 / 9 - injected group . Unpaired t test was used to analyze the data and n = 3 biological replicates for each group . The data are shown as the mean ± s . e . m . * * P < 0 . 01 compared with the AAV - 2 / 9 - injected group ; n . s indicates non - significance . E , Multiple immunofluorescence staining and densitometric quantitation of TUNEL with PDPN antibody ( type I alveolar epithelial cells marker ) on sections of fetal lungs from AAV - shArg1 - injected mice vs . AAV - 2 / 9 - injected mice . Blue : DAPI ( nuclei ) ; Green : PDPN ( type I cells ) ; Red : TUNEL positive . Unpaired t test was used to analyze the quantitation data and n = 3 biological replicates generated from the mean intensity of 3 randomly selected fields of view for each group . The data are shown as the mean ± s . e . m . * * * P < 0 . 001 compared with the AAV - 2 / 9 - injected group . F , Multiple immunofluorescence staining and densitometric quantitation of TUNEL with LPCAT1 antibody ( type II alveolar epithelial cells marker ) on sections of fetal lungs from AAV - shArg1 - injected mice vs . AAV - 2 / 9 - injected mice . Blue : DAPI ( nuclei ) ; Green : LPCAT1 ( type II cells ) ; Red : TUNEL positive . Unpaired t test was used to analyze the quantitation data and n = 3 biological replicates generated from the mean intensity of 3 randomly selected fields of view for each group . The data are shown as the mean ± s . e . m . * * * P < 0 . 001 compared with the AAV - 2 / 9 - injected group . Data information : Scale bars in E and F : 50 μm .

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

Figure 5B, Representative immunoblots of ARG1 expression in the fetal lungs from the AAV-shArg1-injected group and the AAV-2/9-injected group. Densitometric quantitation of scans of immunoblots (normalized to tubulin) is also shown as a bar graph. Unpaired t test was used to analyze the data and n=3 biological replicates for each group. The data are shown as the mean ± s.e.m. **P<0.01 compared with the AAV-2/9-injected group.C, Representative immunoblots and densitometric quantitation of Ki67, caspase3 precursor (pro-caspase3) and cleaved caspase3 expression in the fetal lungs from the AAV-shArg1-injected group and AAV-2/9-injected group. Unpaired t test was used to analyze the data and n=3 biological replicates for each group. The data are shown as the mean ± s.e.m. **P<0.01 compared with the AAV-2/9-injected group; n.s indicates non-significance.E, Multiple immunofluorescence staining and densitometric quantitation of TUNEL with PDPN antibody (type I alveolar epithelial cells marker) on sections of fetal lungs from AAV-shArg1-injected mice vs. AAV-2/9-injected mice. Blue: DAPI (nuclei); Green: PDPN (type I cells); Red: TUNEL positive. Unpaired t test was used to analyze the quantitation data and n=3 biological replicates generated from the mean intensity of 3 randomly selected fields of view for each group. The data are shown as the mean ± s.e.m. ***P<0.001 compared with the AAV-2/9-injected group. F, Multiple immunofluorescence staining and densitometric quantitation of TUNEL with LPCAT1 antibody (type II alveolar epithelial cells marker) on sections of fetal lungs from AAV-shArg1-injected mice vs. AAV-2/9-injected mice. Blue: DAPI (nuclei); Green: LPCAT1 (type II cells); Red: TUNEL positive. Unpaired t test was used to analyze the quantitation data and n=3 biological replicates generated from the mean intensity of 3 randomly selected fields of view for each group. The data are shown as the mean ± s.e.m. ***P<0.001 compared with the AAV-2/9-injected group. Data information: Scale bars in E and F: 50 μm.


Figure 6B , Quantitative analysis of the gel area containing hTERT - HM cells treated with L - arginine or vehicle control . Mann Whitney test was used to analyze the data , and n = 9 biological replicates in control group , n = 12 biological replicates in L - Arginine - treated group . The data are shown as the mean ± s . e . m . * * P < 0 . 01 . Representative images of spontaneous contractions of the gel disks are shown . C , Representative western blots and quantitative analysis of CAP expression in the myometrium of pregnant mice injected intraplacentally with AAV - shArg1 or AAV control viruses . Mann Whitney test was used to analyze CX43 and PTGFR data and unpaired t test was used to analyze COX2 and OXTR data . n = 4 biological replicates for CX43 , COX2 and PTGFR groups , and n = 5 biological replicates for OXTR group . The data are shown as the mean ± s . e . m . * P < 0 . 05 . D , Representative western blots and quantitative analysis of NF - κB p - p65 ( phospho S536 ) and NF - κB p65 expression in the myometrium of pregnant mice injected intraplacentally with AAV - shArg1 or AAV control viruses . Unpaired t test was used and n = 4 biological replicates for each group . The data are shown as the mean ± s . e . m . * * P < 0 . 01 .

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 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 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

Figure 6B, Quantitative analysis of the gel area containing hTERT-HM cells treated with L-arginine or vehicle control. Mann Whitney test was used to analyze the data, and n=9 biological replicates in control group, n=12 biological replicates in L-Arginine-treated group. The data are shown as the mean ± s.e.m. **P < 0.01. Representative images of spontaneous contractions of the gel disks are shown.C, Representative western blots and quantitative analysis of CAP expression in the myometrium of pregnant mice injected intraplacentally with AAV-shArg1 or AAV control viruses. Mann Whitney test was used to analyze CX43 and PTGFR data and unpaired t test was used to analyze COX2 and OXTR data. n=4 biological replicates for CX43, COX2 and PTGFR groups, and n=5 biological replicates for OXTR group. The data are shown as the mean ± s.e.m. *P < 0.05.D, Representative western blots and quantitative analysis of NF-κB p-p65 (phospho S536) and NF-κB p65 expression in the myometrium of pregnant mice injected intraplacentally with AAV-shArg1 or AAV control viruses. Unpaired t test was used and n=4 biological replicates for each group. The data are shown as the mean ± s.e.m. **P < 0.01.


Figure 7Chromatin immunoprecipitation ( ChIP ) was performed with WT or Src - 1 / - 2 dKO fetal lung tissues using antibodies against GR ( B ) Enrichment of these transcription factors at the GRE or C / EBPβ binding sites within the Arg1 promoter were determined by quantitative PCR . Unpaired t test was used for panel B and n = 6 biological replicates for each group . The data are presented as the mean ± s . e . m . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Chromatin immunoprecipitation ( ChIP ) was performed with WT or Src - 1 / - 2 dKO fetal lung tissues using antibodies against SRC - 1 ( C ) , SRC - 2 ( D ) , or C / EBPβ ( E ) . Enrichment of these transcription factors at the GRE or C / EBPβ binding sites within the Arg1 promoter were determined by quantitative PCR . Mann Whitney test was used for Panel C - E . n = 6 biological replicates for each group . The data are presented as the mean ± s . e . m . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . F , Quantitative PCR results for GRE and C / EBP binding site using " input " ( positive control ) DNA without being immunoprecipitated with any specific antibody . n = 6 biological replicates for each group . The data are presented as the mean ± s . e . m .

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

Figure 7Chromatin immunoprecipitation (ChIP) was performed with WT or Src-1/-2 dKO fetal lung tissues using antibodies against GR (B) Enrichment of these transcription factors at the GRE or C/EBPβ binding sites within the Arg1 promoter were determined by quantitative PCR. Unpaired t test was used for panel B and n=6 biological replicates for each group. The data are presented as the mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001.Chromatin immunoprecipitation (ChIP) was performed with WT or Src-1/-2 dKO fetal lung tissues using antibodies against SRC-1 (C), SRC-2 (D), or C/EBPβ (E). Enrichment of these transcription factors at the GRE or C/EBPβ binding sites within the Arg1 promoter were determined by quantitative PCR. Mann Whitney test was used for Panel C-E. n=6 biological replicates for each group. The data are presented as the mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001. F, Quantitative PCR results for GRE and C/EBP binding site using "input" (positive control) DNA without being immunoprecipitated with any specific antibody. n=6 biological replicates for each group. The data are presented as the mean ± s.e.m.


Figure 2DNase - Seq tag density plots showing all peaks detected in replicate 1 of TB IL - 2 and TB IL - 2nil ( 32694 ) ordered by increasing fold change of tag count for TB IL - 2 compared to TB IL - 2nil . The sequence tag density is also shown in parallel at the same sites for the DNase - Seq analyses of CD4 naïve T cells ( TN ) , TB IL - 2 DMSO and TB IL - 2 Ruxolitinib and ATAC samples from in vivo derived Th2 ( Shih et al . , 2016 ) , Th1 and Th17 cells . The location of the 1000 IL - 2 pDHSs is shown to the right . Average DNase - Seq tag density profile for the 1000 IL - 2 pDHSs in both replicates of TB IL - 2 and TB IL - 2nil samples , and for naïve T cells ( TN ) . Number of IL - 2 pDHSs which are detected in in vivo purified CD4 T cell subsets ( Chen et al . , 2020 , Garg et al . , 2019 , Shih et al . , 2016 ) . UCSC genome browser tracks with examples of IL - 2 pDHSs within the Locus Control Region ( LCR ) within the Rad50 gene which controls the Th2 cytokines Il13 , Il4 and Il5 , plus IL - 2 pDHSs within the Tnfrsf9 and Cish loci . IL - 2 pDHSs are highlighted with blue boxes .

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

Figure 2DNase-Seq tag density plots showing all peaks detected in replicate 1 of TB IL-2 and TB IL-2nil (32694) ordered by increasing fold change of tag count for TB IL-2 compared to TB IL-2nil. The sequence tag density is also shown in parallel at the same sites for the DNase-Seq analyses of CD4 naïve T cells (TN), TB IL-2 DMSO and TB IL-2 Ruxolitinib and ATAC samples from in vivo derived Th2 (Shih et al., 2016), Th1 and Th17 cells. The location of the 1000 IL-2 pDHSs is shown to the right.Average DNase-Seq tag density profile for the 1000 IL-2 pDHSs in both replicates of TB IL-2 and TB IL-2nil samples, and for naïve T cells (TN).Number of IL-2 pDHSs which are detected in in vivo purified CD4 T cell subsets (Chen et al., 2020, Garg et al., 2019, Shih et al., 2016).UCSC genome browser tracks with examples of IL-2 pDHSs within the Locus Control Region (LCR) within the Rad50 gene which controls the Th2 cytokines Il13, Il4 and Il5, plus IL-2 pDHSs within the Tnfrsf9 and Cish loci. IL-2 pDHSs are highlighted with blue boxes.


Figure 3Jund mRNA levels in TB IL - 2 and TB IL - 2nil . Standard deviation is shown from the mean of 3 samples . P - values were calculated from the RNA - seq data using Limma with Benjamini - Hochberg correction for multiple testing . Western blot analyses showing JUND and pSTAT5 protein levels in TB IL - 2 and TB IL - 2nil ( left panel ) with the signal calculated from 2 replicates relative to TB IL - 2 in the right panel . DNA motif density plots ( right ) showing the location of binding motifs for RUNX , ETS , STAT and AP - 1 transcription factors at the 32694 DHSs detected in TB IL - 2 and TB IL - 2nil , as depicted in Figure 2A , ordered according to fold enrichment of DNase - Seq tag counts in TB IL - 2 compared to TB IL - 2nil ( left ) . DNA sequence tag density plots from ChIP - Seq assays of RUNX1 , ETS1 , STAT5 and JUND in TB IL - 2 , and STAT5 and JUND in TB IL - 2nil ordered as in ( D ) . UCSC genome browser tracks at the Il13 / Il4 / Il5 Th2 cytokine locus LCR ( indicated by the grey box within the Rad50 gene ) , Gzmb and Il24 showing DNase - Seq and ChIP - Seq for STAT5 and JUND in TB IL - 2 and TB IL - 2nil . Replicate JUND and STAT5 ChIP - Seq tracks are shown for TB IL - 2 .

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

Figure 3Jund mRNA levels in TB IL-2 and TB IL-2nil. Standard deviation is shown from the mean of 3 samples. P-values were calculated from the RNA-seq data using Limma with Benjamini-Hochberg correction for multiple testing.Western blot analyses showing JUND and pSTAT5 protein levels in TB IL-2 and TB IL-2nil (left panel) with the signal calculated from 2 replicates relative to TB IL-2 in the right panel.DNA motif density plots (right) showing the location of binding motifs for RUNX, ETS, STAT and AP-1 transcription factors at the 32694 DHSs detected in TB IL-2 and TB IL-2nil, as depicted in Figure 2A, ordered according to fold enrichment of DNase-Seq tag counts in TB IL-2 compared to TB IL-2nil (left).DNA sequence tag density plots from ChIP-Seq assays of RUNX1, ETS1, STAT5 and JUND in TB IL-2, and STAT5 and JUND in TB IL-2nil ordered as in (D).UCSC genome browser tracks at the Il13/Il4/Il5 Th2 cytokine locus LCR (indicated by the grey box within the Rad50 gene), Gzmb and Il24 showing DNase-Seq and ChIP-Seq for STAT5 and JUND in TB IL-2 and TB IL-2nil. Replicate JUND and STAT5 ChIP-Seq tracks are shown for TB IL-2.


Figure 4DNase - Seq tag density plots showing all peaks detected in replicate 1 of TB IL - 2 and TB + IL - 2 ( A , 38645 ) and in TB + IL - 2 and TB + IL - 2nil ( B , 36067 ) , ordered by increasing fold change in tag count as indicated . The position of the 1015 IL - 2 - dependent inducible DHSs is shown alongside . Average DHS signal at the 1015 IL - 2 - dependent inducible DHSs in TB IL - 2 , TB + IL - 2 , TB + IL - 2nil and naïve T cells stimulated with PMA / I ( TN + ) . Bar graph showing the number of IL - 2 iDHSs found within 25 kb of an IL2 pDHS . Examples of IL - 2 iDHSs which are proximal to IL - 2 pDHSs at the Csf2 / Il2 and Il10 loci . IL - 2 iDHSs are highlighted in red and IL - 2 pDHSs in blue .

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Figure 4DNase-Seq tag density plots showing all peaks detected in replicate 1 of TB IL-2 and TB+ IL-2 (A, 38645) and in TB+ IL-2 and TB+ IL-2nil (B, 36067), ordered by increasing fold change in tag count as indicated. The position of the 1015 IL-2-dependent inducible DHSs is shown alongside.Average DHS signal at the 1015 IL-2-dependent inducible DHSs in TB IL-2, TB+ IL-2, TB+ IL-2nil and naïve T cells stimulated with PMA/I (TN+).Bar graph showing the number of IL-2 iDHSs found within 25 kb of an IL2 pDHS.Examples of IL-2 iDHSs which are proximal to IL-2 pDHSs at the Csf2/Il2 and Il10 loci. IL-2 iDHSs are highlighted in red and IL-2 pDHSs in blue.


Figure 5Hierarchical clustering of RNA - Seq data from 3 replicates as described in ( A ) . MA plot showing the genes differentially regulated between TB IL - 2nil and TB IL - 2 . The number of genes with a fold change > 3 and adj p - value < 0 . 05 are highlighted . P - values were calculated from the RNA - seq data using Limma with Benjamini - Hochberg correction for multiple testing . log2 fold change in gene expression in TB IL - 2nil compared to TB IL - 2 for the nearest gene to the DHSs ranked according to the fold change in DNase - Seq tag count for TB IL - 2 / TB IL - 2nil . The DNase - Seq tag density plots are taken from Figure 2A for TB IL - 2 , TB IL - 2nil and TN . UCSC genome browser tracks showing DNase - seq and RNA - seq at the Socs1 ( E ) and Sell loci ( F ) .

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Figure 5Hierarchical clustering of RNA-Seq data from 3 replicates as described in (A).MA plot showing the genes differentially regulated between TB IL-2nil and TB IL-2. The number of genes with a fold change >3 and adj p-value <0.05 are highlighted. P-values were calculated from the RNA-seq data using Limma with Benjamini-Hochberg correction for multiple testing.log2 fold change in gene expression in TB IL-2nil compared to TB IL-2 for the nearest gene to the DHSs ranked according to the fold change in DNase-Seq tag count for TB IL-2/TB IL-2nil. The DNase-Seq tag density plots are taken from Figure 2A for TB IL-2, TB IL-2nil and TN.UCSC genome browser tracks showing DNase-seq and RNA-seq at the Socs1 (E) and Sell loci (F).


Figure 6Relative mRNA expression levels in TB + IL - 2 and TB + IL - 2nil ( A ) and TB + DMSO and TB + Ruxolitinib ( B ) . The expression level in the IL - 2 / DMSO TB + sample is set to 1 . ( n = 3 , error bars represent standard deviation , P - values were calculated using a paired t - test with * p < 0 . 05 , * * p < 0 . 005 , * * * p < 0 . 0005 . ) UCSC genome browser tracks showing DNase - Seq and RNA - Seq at the IL - 2 regulated genes Csf2 / Il3 and at the LCR within the Rad50 gene ( grey box ) which controls the Th2 cytokines Il13 , Il4 and Il5 . IL - 2 pDHSs are highlighted in blue and IL - 2 iDHSs in red .

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Figure 6Relative mRNA expression levels in TB+ IL-2 and TB+ IL-2nil (A) and TB+ DMSO and TB+ Ruxolitinib (B). The expression level in the IL-2/DMSO TB+ sample is set to 1. (n=3, error bars represent standard deviation, P-values were calculated using a paired t-test with *p<0.05, **p<0.005, ***p<0.0005.)UCSC genome browser tracks showing DNase-Seq and RNA-Seq at the IL-2 regulated genes Csf2/Il3 and at the LCR within the Rad50 gene (grey box) which controls the Th2 cytokines Il13, Il4 and Il5. IL-2 pDHSs are highlighted in blue and IL-2 iDHSs in red.


Figure 7DNase - Seq tag density plots showing all peaks detected in replicate 1 of TB IL - 2 and TB IL ‑ 2nil ( data taken from Figure 2A ) , duplicate DNase - seq samples for TB IL - 7 and TB IL - 7nil are shown in parallel for the same sites . Average DNase - Seq tag density profile for the 1000 IL - 2 pDHSs in both replicates of TB IL - 7 and TB IL - 7nil . UCSC genome browser tracks with DNase - Seq data for TB IL - 7 and TB IL - 7nil showing examples of IL - 2 pDHSs within the Locus Control Region ( LCR ) within the Rad50 gene which controls the Th2 cytokines Il13 , Il4 and Il5 , plus IL - 2 pDHSs within the Tnfrsf9 and Cish loci ( C ) IL - 2 pDHSs are highlighted with blue boxes . UCSC genome browser tracks with DNase - Seq data for TB IL - 7 and TB IL - 7nil showing examples of IL - 2 pDHSs within the Locus Control Region ( LCR ) within the non - primed control regions from the CD3 gene cluster and the Tbp locus ( D ) . IL - 2 pDHSs are highlighted with blue boxes . Average DHS signal at the 1015 IL - 2 regulated inducible DHSs in TB + IL - 7 and TB + IL - 7nil . Relative mRNA expression levels in TB + IL - 7 and TB + IL - 7nil . The expression level in the TB + IL - 7 is set to 1 . ( n = 3 , error bars represent standard deviation , P - values were calculated using a paired t - test with * p < 0 . 05 , * * p < 0 . 005 , * * * p < 0 . 0005 . )

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Figure 7DNase-Seq tag density plots showing all peaks detected in replicate 1 of TB IL-2 and TB IL‑2nil (data taken from Figure 2A), duplicate DNase-seq samples for TB IL-7 and TB IL-7nil are shown in parallel for the same sites.Average DNase-Seq tag density profile for the 1000 IL-2 pDHSs in both replicates of TB IL-7 and TB IL-7nil.UCSC genome browser tracks with DNase-Seq data for TB IL-7 and TB IL-7nil showing examples of IL-2 pDHSs within the Locus Control Region (LCR) within the Rad50 gene which controls the Th2 cytokines Il13, Il4 and Il5, plus IL-2 pDHSs within the Tnfrsf9 and Cish loci (C) IL-2 pDHSs are highlighted with blue boxes.UCSC genome browser tracks with DNase-Seq data for TB IL-7 and TB IL-7nil showing examples of IL-2 pDHSs within the Locus Control Region (LCR) within the non-primed control regions from the CD3 gene cluster and the Tbp locus (D). IL-2 pDHSs are highlighted with blue boxes.Average DHS signal at the 1015 IL-2 regulated inducible DHSs in TB+ IL-7 and TB+ IL-7nil.Relative mRNA expression levels in TB+ IL-7 and TB+ IL-7nil. The expression level in the TB+ IL-7 is set to 1. (n=3, error bars represent standard deviation, P-values were calculated using a paired t-test with *p<0.05, **p<0.005, ***p<0.0005.)


Figure 8Flow cytometric analysis of IL - 7Rα expression by 2W1S - specific CD44hi CD4 T cells in the presence or absence of cre recombinase . Average number of 2W1S - specific cells recovered / mouse after sorting 3 replicate samples of Il7rf / f compared to CD4creERT2 x Il7rf / f . Errors bars indicate the standard deviation . Average ATAC - Seq signal at the 411 IL - 2 pDHSs which contain a STAT binding motif . UCSC genome browser tracks at loci with IL - 2 pDHSs showing replicate ATAC - Seq samples for CD4creERT2 x Il7rf / f memory T cells , Il7rf / f control memory T cells , replicate DNase - Seq samples for TB IL - 2 and TB IL - 2nil and TB IL - 2 and TB IL ‑ 2nil STAT5 ChIP - Seq samples . The grey boxes signify IL - 2 pDHSs which diminish after disruption of the Il7r gene .

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Figure 8Flow cytometric analysis of IL-7Rα expression by 2W1S-specific CD44hi CD4 T cells in the presence or absence of cre recombinase.Average number of 2W1S-specific cells recovered/mouse after sorting 3 replicate samples of Il7rf/f compared to CD4creERT2 x Il7rf/f . Errors bars indicate the standard deviation.Average ATAC-Seq signal at the 411 IL-2 pDHSs which contain a STAT binding motif.UCSC genome browser tracks at loci with IL-2 pDHSs showing replicate ATAC-Seq samples for CD4creERT2 x Il7rf/f memory T cells, Il7rf/f control memory T cells, replicate DNase-Seq samples for TB IL-2 and TB IL-2nil and TB IL-2 and TB IL‑2nil STAT5 ChIP-Seq samples. The grey boxes signify IL-2 pDHSs which diminish after disruption of the Il7r gene.


Figure 9UCSC genome browser tracks showing ChIP - Seq and DNase - Seq at the LCR within the Rad50 gene which controls the Il4 / Il13 / Il5 Th2 cytokine locus , and also Il17a , Il12rb2 and Tbx21 . The grey boxes indicate IL - 2 pDHSs which bind STAT5 in Th0 cells and other STAT proteins in other T cell lineages .

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Figure 9UCSC genome browser tracks showing ChIP-Seq and DNase-Seq at the LCR within the Rad50 gene which controls the Il4/Il13/Il5 Th2 cytokine locus, and also Il17a, Il12rb2 and Tbx21. The grey boxes indicate IL-2 pDHSs which bind STAT5 in Th0 cells and other STAT proteins in other T cell lineages.


Figure 10mRNA expression levels of JUN family transcription factors . Normalised TPM is shown for TB IL - 2 , TB IL2nil , TB + IL - 2 and TB + IL - 2nil . Standard deviation is shown for 3 replicates . Average JUNB ChIP - Seq signal at the 1000 IL - 2 pDHSs in TB IL - 2 and TB + IL - 2 ( Bevington et al . , 2016 ) . Average JUND ChIP - Seq signal at the 1000 IL - 2 pDHSs in Junb + / + CD4Cre ( Junb WT ) and Junbf / fCD4Cre ( Junb KO ) Th17 cells . Genome browser tracks showing open chromatin ( DNase - Seq or ATAC - Seq ) and ChIP - Seq in Th0 , Th1 and Th17 cells at the Il17a locus ( Carr et al . , 2017 , Ciofani et al . , 2012 , Vahedi et al . , 2012 ) . The grey box indicates a DHS which binds JUND . Fold change in gene expression in TB + IL - 2 cells for Jund - / - compared to Jund + / + mice . Data from 2 replicates are shown . Genome browser tracks showing open chromatin ( DNase - Seq or ATAC - Seq ) and ChIP - Seq in Th0 , Th1 and Th17 cells at the Irf8 locus . The grey box indicates an IL - 2 pDHS which binds JUND .

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Figure 10mRNA expression levels of JUN family transcription factors. Normalised TPM is shown for TB IL-2, TB IL2nil, TB+ IL-2 and TB+ IL-2nil. Standard deviation is shown for 3 replicates.Average JUNB ChIP-Seq signal at the 1000 IL-2 pDHSs in TB IL-2 and TB+ IL-2 (Bevington et al., 2016).Average JUND ChIP-Seq signal at the 1000 IL-2 pDHSs in Junb+/+CD4Cre (Junb WT) and Junbf/fCD4Cre (Junb KO) Th17 cells.Genome browser tracks showing open chromatin (DNase-Seq or ATAC-Seq) and ChIP-Seq in Th0, Th1 and Th17 cells at the Il17a locus (Carr et al., 2017, Ciofani et al., 2012, Vahedi et al., 2012). The grey box indicates a DHS which binds JUND.Fold change in gene expression in TB+ IL-2 cells for Jund -/- compared to Jund+/+ mice. Data from 2 replicates are shown.Genome browser tracks showing open chromatin (DNase-Seq or ATAC-Seq) and ChIP-Seq in Th0, Th1 and Th17 cells at the Irf8 locus. The grey box indicates an IL-2 pDHS which binds JUND.


Figure 1B . In vitro modification reactions containing the six fragments and HeLa NE were performed . After nuclease P1 digestion , the products were analyzed by 1D - TLC with pU and pψ as controls . C . WT and mutant 7SK fragment 5 containing U250G were analyzed as in B . E . 7SK RNA , generated by either in vitro transcription ( IVT ) or purified from HeLa total RNA and then site - specifically labeled at the indicated positions by the procedure in D , was analyzed by 1D - TLC . F . Cyber Gold staining of 7SK obtained by either IVT or immunoprecipitation ( IP ) with the indicated antibodies . G . 2D - TLC analysis of 32p - labeled mononucleotides derived from IVT or CDK9 - bound 7SK RNA , with the first panel depicting a schematic migration pattern of the indicated 5 ' - phosphorylated mononucleotides . H . 7SK RNA , obtained by either IVT or anti - CDK9 IP and then site - specifically labeled at the indicated positions by the procedure in D , was analyzed by 1D - TLC .

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Figure 1B. In vitro modification reactions containing the six fragments and HeLa NE were performed. After nuclease P1 digestion, the products were analyzed by 1D-TLC with pU and pψ as controls.C. WT and mutant 7SK fragment 5 containing U250G were analyzed as in B.E. 7SK RNA, generated by either in vitro transcription (IVT) or purified from HeLa total RNA and then site-specifically labeled at the indicated positions by the procedure in D, was analyzed by 1D-TLC.F. Cyber Gold staining of 7SK obtained by either IVT or immunoprecipitation (IP) with the indicated antibodies.G. 2D-TLC analysis of 32p-labeled mononucleotides derived from IVT or CDK9-bound 7SK RNA, with the first panel depicting a schematic migration pattern of the indicated 5'-phosphorylated mononucleotides.H. 7SK RNA, obtained by either IVT or anti-CDK9 IP and then site-specifically labeled at the indicated positions by the procedure in D, was analyzed by 1D-TLC.


Figure 2C - F ; H - K ; and N - Q . The tagged WT or mutant 7SK RNA was co - transfected with the indicated Flag - tagged proteins into HeLa cells . Anti - Flag or immunoprecipitates ( IP ) were analyzed by immunoblotting for the indicated proteins and primer extension for the bound 7SK RNA . C - F ; H - K ; and N - Q . The tagged WT or mutant 7SK RNA was co - transfected with the indicated Flag - - tagged proteins into HeLa cells . Anti - Flag immunoprecipitates ( IP ) were analyzed by immunoblotting for the indicated proteins and primer extension for the bound 7SK RNA . C - F ; H - K ; and N - Q . The tagged WT or mutant 7SK RNA was co - transfected with the indicated Flag - tagged proteins into HeLa cells . Anti - Flag immunoprecipitates ( IP ) were analyzed by immunoblotting for the indicated proteins and primer extension for the bound 7SK RNA . C - F ; H - K ; and N - Q . The tagged WT or mutant 7SK RNA was co - transfected with the indicated HA - tagged proteins into HeLa cells . Anti - HA immunoprecipitates ( IP ) were analyzed by immunoblotting for the indicated proteins and primer extension for the bound 7SK RNA . R . The indicated 7SK were in vitro transcribed in the presence of 32p - UTP and subjected to in vitro modification reactions . The nuclease P1 - digested products were analyzed by 1D - TLC with pU and pΨ as controls .

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Figure 2C-F; H-K; and N-Q. The tagged WT or mutant 7SK RNA was co-transfected with the indicated Flag-tagged proteins into HeLa cells. Anti-Flag or immunoprecipitates (IP) were analyzed by immunoblotting for the indicated proteins and primer extension for the bound 7SK RNA.C-F; H-K; and N-Q. The tagged WT or mutant 7SK RNA was co-transfected with the indicated Flag--tagged proteins into HeLa cells. Anti-Flag immunoprecipitates (IP) were analyzed by immunoblotting for the indicated proteins and primer extension for the bound 7SK RNA.C-F; H-K; and N-Q. The tagged WT or mutant 7SK RNA was co-transfected with the indicated Flag-tagged proteins into HeLa cells. Anti-Flag immunoprecipitates (IP) were analyzed by immunoblotting for the indicated proteins and primer extension for the bound 7SK RNA.C-F; H-K; and N-Q. The tagged WT or mutant 7SK RNA was co-transfected with the indicated HA-tagged proteins into HeLa cells. Anti-HA immunoprecipitates (IP) were analyzed by immunoblotting for the indicated proteins and primer extension for the bound 7SK RNA.R. The indicated 7SK were in vitro transcribed in the presence of 32p-UTP and subjected to in vitro modification reactions. The nuclease P1-digested products were analyzed by 1D-TLC with pU and pΨ as controls.


Figure 3A . Anti - V5immunoprecipitates ( IP ) from HeLa cells expressing nothing ( - ) or V5 - DKC1 were analyzed by immunoblotting for V5 - DKC1 and qRT - PCR for the bound 7SK RNA . The error bars represent mean ± SD from three independent experiments . B . Anti - FlagIP from NE of HeLa or the HeLa - derived F1C2 cells expressing CDK9 - F were analyzed by Western blotting ( WB ) for the indicated proteins and primer extension ( PEx ) for 7SK RNA . C . WB analysis of DKC1 and CDK9 levels in two HeLa clones inducibly expressing shDKC1 upon doxycycline ( DOX ) treatment for 5 days . D . The 7SK RNA levels in these two clones were detected by qRT - PCR before and after the DOX treatment and normalized to the GAPDH mRNA levels . The error bars represent mean ± SD from three independent experiments . E . 1D - TLC analysis of Ψ250 in 7SK RNA from inducible DKC1 KD clone 1 before and after the DOX treatment . F . NE and anti - CDK9IP from NE of inducible DKC1 KD clone 1 , which was untreated or treated with DOX , were analyzed by WB for the indicated proteins and PEx for 7SK RNA . G . Clone 1 was treated with DOX for 3 days and then transfected with the Tat - Flag cDNA ( 20 ug / 150 - mm dish ) . Two days later , NE and anti - FlagIP from NE were analyzed as in F .

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Figure 3A. Anti-V5immunoprecipitates (IP) from HeLa cells expressing nothing (-) or V5-DKC1 were analyzed by immunoblotting for V5-DKC1 and qRT-PCR for the bound 7SK RNA. The error bars represent mean ± SD from three independent experiments.B. Anti-FlagIP from NE of HeLa or the HeLa-derived F1C2 cells expressing CDK9-F were analyzed by Western blotting (WB) for the indicated proteins and primer extension (PEx) for 7SK RNA.C. WB analysis of DKC1 and CDK9 levels in two HeLa clones inducibly expressing shDKC1 upon doxycycline (DOX) treatment for 5 days.D. The 7SK RNA levels in these two clones were detected by qRT-PCR before and after the DOX treatment and normalized to the GAPDH mRNA levels. The error bars represent mean ± SD from three independent experiments.E. 1D-TLC analysis of Ψ250 in 7SK RNA from inducible DKC1 KD clone 1 before and after the DOX treatment.F. NE and anti-CDK9IP from NE of inducible DKC1 KD clone 1, which was untreated or treated with DOX, were analyzed by WB for the indicated proteins and PEx for 7SK RNA.G. Clone 1 was treated with DOX for 3 days and then transfected with the Tat-Flag cDNA (20 ug/150-mm dish). Two days later, NE and anti-FlagIP from NE were analyzed as in F.


Figure 4A . Luciferase activities were measured in HeLa cells transfected with the indicated reporter constructs , an empty vector ( - ) or the Tat cDNA , and either siDKC1 or a non - target siRNA ( siNT ) . For each reporter , the activity in cells expressing siNT was set to 1 . B , C , and E . The mRNA levels of luciferase ( B ) or Tat ( C E ) in cells expressing the indicated siRNAs were determined by qRT - PCR and normalized to the GAPDH mRNA and plotted . D . NH1 cells containing the integrated HIV - 1 LTR - luciferase reporter and expressing Tat were transfected with siDKC1 or siNT . ChIP - qPCR analysis was performed to determine the levels of the indicated factors bound to the HIV - 1 promoter . The signals were normalized to those of input and plotted . B , C , and E . The mRNA levels of Tat ( C E ) in cells expressing the indicated siRNAs were determined by qRT - PCR and normalized to the GAPDH mRNA and plotted . F . Western analysis of DKC1 and CDK9 levels in a Jurkat 2D10 - derived clone inducibly expressing shDKC1 upon exposure to DOX . G and H . The inducible DKC1 KD clone , treated or untreated with DOX for 4 days , was incubated with the indicated concentrations of JQ1 ( G ) or prostratin ( H ) for 24 h . The GFP expression was measured by flow cytometry and expressed as percentages of GFP ( + ) cells of the entire population . The error bars in all panels represent mean ± SD from three independent experiments , and the p values determined by the Student ' s t - test .

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Figure 4A. Luciferase activities were measured in HeLa cells transfected with the indicated reporter constructs, an empty vector (-) or the Tat cDNA, and either siDKC1 or a non-target siRNA (siNT). For each reporter, the activity in cells expressing siNT was set to 1.B, C, and E. The mRNA levels of luciferase (B) or Tat (C E) in cells expressing the indicated siRNAs were determined by qRT-PCR and normalized to the GAPDH mRNA and plotted.D. NH1 cells containing the integrated HIV-1 LTR-luciferase reporter and expressing Tat were transfected with siDKC1 or siNT. ChIP-qPCR analysis was performed to determine the levels of the indicated factors bound to the HIV-1 promoter. The signals were normalized to those of input and plotted.B, C, and E. The mRNA levels of Tat (C E) in cells expressing the indicated siRNAs were determined by qRT-PCR and normalized to the GAPDH mRNA and plotted.F. Western analysis of DKC1 and CDK9 levels in a Jurkat 2D10-derived clone inducibly expressing shDKC1 upon exposure to DOX.G and H. The inducible DKC1 KD clone, treated or untreated with DOX for 4 days, was incubated with the indicated concentrations of JQ1 (G) or prostratin (H) for 24 h. The GFP expression was measured by flow cytometry and expressed as percentages of GFP(+) cells of the entire population. The error bars in all panels represent mean ± SD from three independent experiments, and the p values determined by the Student's t-test.


Figure 4B , C Effects of H2A mutations ( B ) and H2A . B mutations ( C ) on nucleosome resistance to MNase digestion . MNase digested nucleosomal DNA are analyzed by 10 % Native - PAGE . All MNase digestion experiments are repeated twice . D , E Graph presentation of effects of H2A mutations ( D ) and H2A . B mutations ( E ) on nucleosome resistance to MNase digestion . MNase digestion efficiency for each sample is calculated from quantitation of the digested DNA against total DNA . All experiments are repeated twice . Data are mean ± SD , n = 2 .

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Figure 4B,C Effects of H2A mutations (B) and H2A.B mutations (C) on nucleosome resistance to MNase digestion. MNase digested nucleosomal DNA are analyzed by 10% Native-PAGE. All MNase digestion experiments are repeated twice.D,E Graph presentation of effects of H2A mutations (D) and H2A.B mutations (E) on nucleosome resistance to MNase digestion. MNase digestion efficiency for each sample is calculated from quantitation of the digested DNA against total DNA. All experiments are repeated twice. Data are mean ± SD, n = 2.


Figure 5B Effect of the canonical and noncanonical H2A ROF on histone octamer assembly . Left : Gel filtration chromatography profiles of octamers assembled by core histones including various H2A family members or mutants . Highlighted in blue shadow are chromatography fractions subject to SDS - PAGE analysis . Right : SDS - PAGE analysis of histone octamer integrity by visualizing histone stoichiometry . The stars indicate lnkH2B - H2A containing different H2A family members or mutants present in chromatography fractions . The triangles indicate the chromatography fractions containing the majority of the lnkH2B - H2A or their mutants . D , E Effects of the canonical and noncanonical H2A ROF on nucleosome resistance to MNase digestion . Displayed are Native - PAGE results ( D ) and graph of MNase digestion efficiency calculated from quantitation of the digested DNA against total DNA ( E ) . All experiments are repeated twice . Data are mean ± SD , n = 2 .

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Figure 5B Effect of the canonical and noncanonical H2A ROF on histone octamer assembly. Left: Gel filtration chromatography profiles of octamers assembled by core histones including various H2A family members or mutants. Highlighted in blue shadow are chromatography fractions subject to SDS-PAGE analysis. Right: SDS-PAGE analysis of histone octamer integrity by visualizing histone stoichiometry. The stars indicate lnkH2B-H2A containing different H2A family members or mutants present in chromatography fractions. The triangles indicate the chromatography fractions containing the majority of the lnkH2B-H2A or their mutants.D,E Effects of the canonical and noncanonical H2A ROF on nucleosome resistance to MNase digestion. Displayed are Native-PAGE results (D) and graph of MNase digestion efficiency calculated from quantitation of the digested DNA against total DNA (E). All experiments are repeated twice. Data are mean ± SD, n = 2.


Figure 6D SDS - PAGE analysis of SWR1 complex purified from S . cerevisiae . E Effect of H2A . Z . 2 . 2 ROF on facilitating histone replacement . Incorporations of yeast H2A . Z - H2B dimers containing either the wild type H2A . Z or Z . 2 . 2 - like H2A . Z were analyzed by SWR1 - catalyzed histone replacement assay . H2A . Z and Z . 2 . 2 - like H2A . Z incorporated into nucleosome were resolved by 6 % Native - PAGE and detected by SYBR Green staining and western blotting .

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Figure 6D SDS-PAGE analysis of SWR1 complex purified from S. cerevisiae.E Effect of H2A.Z.2.2 ROF on facilitating histone replacement. Incorporations of yeast H2A.Z-H2B dimers containing either the wild type H2A.Z or Z.2.2-like H2A.Z were analyzed by SWR1-catalyzed histone replacement assay. H2A.Z and Z.2.2-like H2A.Z incorporated into nucleosome were resolved by 6% Native-PAGE and detected by SYBR Green staining and western blotting.


Figure 1Plotting of the position of the 5 ' end of Alu RNA fragments across the Alu metagene to depict potential processing areas of Alu RNAs in all post - mortem hippocampal tissues ( PM hippo ) from patients from the CBB . Each row in the heatmap depicts the distribution of counts of the 5 ' ends of reads mapped across the Alu metagene for each patient . The x - axis represents a metagene combining all unique Alu RNA sequences ( ALUome ) aligned at the start site of their consensus sequence with numbers representing the distance from the Transcriptional Start Site ( TSS ) area . Heatmap density corresponds to normalized counts of the 5 ' end of the reads with red corresponding to higher density of these 5 ' ends at a specific position . XL1 , XR1 and XR2 denote the Alu processing areas defined by the high - density areas in the heatmap at specific positions of the Alu metagene , with the middle letter corresponding to the arm of the Alu RNA ( see Figure 1C , L = Left , R = Right ) , in which the area is located .

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Figure 1Plotting of the position of the 5'end of Alu RNA fragments across the Alu metagene to depict potential processing areas of Alu RNAs in all post-mortem hippocampal tissues (PM hippo) from patients from the CBB. Each row in the heatmap depicts the distribution of counts of the 5'ends of reads mapped across the Alu metagene for each patient. The x-axis represents a metagene combining all unique Alu RNA sequences (ALUome) aligned at the start site of their consensus sequence with numbers representing the distance from the Transcriptional Start Site (TSS) area. Heatmap density corresponds to normalized counts of the 5'end of the reads with red corresponding to higher density of these 5'ends at a specific position. XL1, XR1 and XR2 denote the Alu processing areas defined by the high-density areas in the heatmap at specific positions of the Alu metagene, with the middle letter corresponding to the arm of the Alu RNA (see Figure 1C, L=Left, R=Right), in which the area is located.


Figure 2Normalized counts of processed fragments mapping to the right arm of Alu RNAs that are differentially processed ( rows ) between AD and no AD for each CBB patient ( columns ) ( Dataset EV1 ) . Red corresponds to higher normalized counts of processed Alu RNA fragments . Boxplots depict differences in hippocampi of AD and no AD patients regarding SINE Alu RNA processing ratio ( left panel ) ( a p value of 0 . 05 was considered as threshold for statistical significance with p = 0 . 01 , n = 24 , unpaired non - directional t - test ) and in total Alu RNA levels ( right panel ) ( ns = not significant ) . In the boxplots , the central band ( the line that divides the box into 2 parts ) represents the median of the data , the ends of the box shows the upper ( Q3 ) and lower ( Q1 ) quartiles , the extreme line shows Q3 + 1 . 5xIQR to Q1 - 1 . 5xIQR ( the highest and lowest value excluding potential outliers ) , while dots beyond the extreme line show potential outliers .

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Figure 2Normalized counts of processed fragments mapping to the right arm of Alu RNAs that are differentially processed (rows) between AD and no AD for each CBB patient (columns) (Dataset EV1). Red corresponds to higher normalized counts of processed Alu RNA fragments.Boxplots depict differences in hippocampi of AD and no AD patients regarding SINE Alu RNA processing ratio (left panel) (a p value of 0.05 was considered as threshold for statistical significance with p=0.01 , n=24, unpaired non-directional t-test) and in total Alu RNA levels (right panel) (ns=not significant). In the boxplots, the central band (the line that divides the box into 2 parts) represents the median of the data, the ends of the box shows the upper (Q3) and lower (Q1) quartiles, the extreme line shows Q3+1.5xIQR to Q1-1.5xIQR (the highest and lowest value excluding potential outliers), while dots beyond the extreme line show potential outliers.


Figure 3Plotting of the position of the 5 ' end of Alu RNA fragments across the Alu metagene to depict potential processing areas of Alu RNAs in all post - mortem cortex tissues from patients from the MAP cohort . As in Figure 1B each row represents the distribution of normalized counts of the 5 ' ends of reads mapped across the Alu metagene for each patient . As in Figure 1B x - axis numbers represent the distance from the Transcriptional Start Site ( TSS ) area and heatmap density corresponds to normalized counts of the 5 ' end of the reads with red for higher density of these 5 ' ends at a specific position . XR1 as in Figure 1B .

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Figure 3Plotting of the position of the 5'end of Alu RNA fragments across the Alu metagene to depict potential processing areas of Alu RNAs in all post-mortem cortex tissues from patients from the MAP cohort. As in Figure 1B each row represents the distribution of normalized counts of the 5'ends of reads mapped across the Alu metagene for each patient. As in Figure 1B x-axis numbers represent the distance from the Transcriptional Start Site (TSS) area and heatmap density corresponds to normalized counts of the 5'end of the reads with red for higher density of these 5'ends at a specific position. XR1 as in Figure 1B.


Figure 4As in Figure 2B , normalized counts of processed fragments mapping to the right arm of Alu RNAs that are differentially processed ( rows ) between AD and no AD for each MAP patient ( columns ) ( Dataset EV2 ) . Red corresponds to higher normalized counts of processed Alu RNA fragments . Boxplots depict differences in cortex of AD and no AD patients regarding SINE Alu RNA processing ratio ( left panel ) ( a p value of 0 . 05 was considered as threshold for statistical significance with p < 0 . 001 , n = 118 , unpaired non - directional t - test ) and in full length Alu RNAs ( p = 0 . 005 ) . Boxplot representation as in subfigure 2D . Boxplots depict full length Alu RNA levels in cortex of AD and no AD patients separated into three age groups . No significant difference observed between the different age groups of either AD or no AD patients or for the comparisons between no AD and AD of each age group ( unpaired non - directional t - test , with p < 0 . 05 considered the threshold for statistical significance , n for no AD = 19 ( below 85 ) , 19 ( between 85 to 90 ) , 13 ( above 90 ) ; n for AD = 8 ( below 85 ) , 21 ( between 85 to 90 ) , 38 ( above 90 ) ) . Boxplot representation as in subfigure 2D . Boxplots depict differences in SINE Alu RNA processing ratio in cortex of AD and no AD patients separated into three age groups . No significant difference observed between the different age groups of either AD or no AD patients . A p value 0 . 05 was considered as threshold for statistical significance for the comparisons between no AD and AD of each age group ( unpaired non - directional t - test , with p = 0 . 003 for the comparison below 85 , and p < 0 . 001 for the other two comparisons and n numbers as in ( E ) ) . Boxplot representation as in subfigure 2D .

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Figure 4As in Figure 2B, normalized counts of processed fragments mapping to the right arm of Alu RNAs that are differentially processed (rows) between AD and no AD for each MAP patient (columns) (Dataset EV2). Red corresponds to higher normalized counts of processed Alu RNA fragments.Boxplots depict differences in cortex of AD and no AD patients regarding SINE Alu RNA processing ratio (left panel) (a p value of 0.05 was considered as threshold for statistical significance with p<0.001, n=118, unpaired non-directional t-test) and in full length Alu RNAs (p=0.005). Boxplot representation as in subfigure 2D.Boxplots depict full length Alu RNA levels in cortex of AD and no AD patients separated into three age groups. No significant difference observed between the different age groups of either AD or no AD patients or for the comparisons between no AD and AD of each age group (unpaired non-directional t-test, with p<0.05 considered the threshold for statistical significance, n for no AD=19 (below 85), 19 (between 85 to 90), 13 (above 90); n for AD=8 (below 85), 21 (between 85 to 90), 38 (above 90)). Boxplot representation as in subfigure 2D.Boxplots depict differences in SINE Alu RNA processing ratio in cortex of AD and no AD patients separated into three age groups. No significant difference observed between the different age groups of either AD or no AD patients. A p value 0.05 was considered as threshold for statistical significance for the comparisons between no AD and AD of each age group (unpaired non-directional t-test, with p=0.003 for the comparison below 85, and p < 0.001 for the other two comparisons and n numbers as in (E)). Boxplot representation as in subfigure 2D.


Figure 5Boxplots depict differences in SINE Alu RNA processing ratio in all MAP patients with regard to final clinical diagnosis ( p < 0 . 01 for the comparison between NCI and MCI , between NCI and AD , and between MCI and AD , unpaired , non directional t - test ) . NCI = no cognitive impairment ( n = 71 ) , MCI = mild cognitive impairment ( n = 67 ) , AD = Alzheimer ' s disease ( n = 87 ) . Boxplot representation as in subfigure 2D . Boxplots depict differences in SINE Alu RNA processing ratio in all MAP patients ( n = 241 ) with regard to Braak stage classified into three categories ( p = 0 . 004 for the comparison between 0 - 2 and 3 - 4 , and p < 0 . 001 for the comparisons between 3 - 4 and 5 - 6 and between 0 - 2 and 5 - 6 , unpaired , non directional t - test , n = 39 [ 0 - 2 ] , n = 148 [ 3 - 4 ] and n = 54 [ 5 - 6 ] ) . Boxplot representation as in subfigure 2D . Scatterplot depicting the positive correlation between TP53 expression values and Alu RNA processing ratio in AD patients ( r = 0 . 65 , p < 0 . 001 ) . Statistical test is based on Pearson ' s product moment correlation coefficient and follows a t distribution using the cor . test function in R package stats . Scatterplot depicting no correlation between TP53 expression values and full length Alu RNA levels in AD patients ( r = 0 . 06 , no correlation , ns = non - significant ) . Statistical test as in subfigure 5C .

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Figure 5Boxplots depict differences in SINE Alu RNA processing ratio in all MAP patients with regard to final clinical diagnosis (p < 0.01 for the comparison between NCI and MCI, between NCI and AD, and between MCI and AD, unpaired, non directional t-test). NCI=no cognitive impairment (n=71), MCI=mild cognitive impairment (n=67), AD=Alzheimer's disease (n=87). Boxplot representation as in subfigure 2D. Boxplots depict differences in SINE Alu RNA processing ratio in all MAP patients (n=241) with regard to Braak stage classified into three categories (p = 0.004 for the comparison between 0-2 and 3-4, and p < 0.001 for the comparisons between 3-4 and 5-6 and between 0-2 and 5-6, unpaired, non directional t-test, n=39[0-2],n=148[3-4] and n=54[5-6]). Boxplot representation as in subfigure 2D.Scatterplot depicting the positive correlation between TP53 expression values and Alu RNA processing ratio in AD patients (r=0.65, p < 0.001). Statistical test is based on Pearson's product moment correlation coefficient and follows a t distribution using the cor.test function in R package stats. Scatterplot depicting no correlation between TP53 expression values and full length Alu RNA levels in AD patients (r=0.06, no correlation, ns=non-significant). Statistical test as in subfigure 5C.


Figure 6Association between SINE Alu RNA ratio ( upper panel ) and gene expression levels ( lower panel ) of genes up - regulated in AD ( Dataset EV3 , | log2FoldChange | > 0 . 5 ) . Every column in both panels corresponds to the same MAP patient of either the no AD or AD group . Patients in each group are sorted from left to right in an ascending order with regard to Alu RNA processing ratio . Every row in the heatmap corresponds to one gene , with color density representing normalized FPKM values from RNA - seq for this gene for each patient ( columns ) ( red represents high and blue low expression levels ) . Association between full length Alu RNA levels ( upper panel ) and gene expression levels ( lower panel ) of genes down - regulated in AD ( Dataset EV4 , | log2FoldChange | > 0 . 5 ) . Every column in both panels corresponds to the same MAP patient of either the no AD or AD group . Patients in each group are sorted from left to right in an ascending order with regard to full length Alu RNA levels . Every row in the heatmap corresponds to one gene , with color density representing normalized FPKM values from RNA - seq for this gene for each patient ( columns ) ( red represents high and blue low expression levels ) . Scatterplot depicting the positive correlation between average gene expression values of upregulated genes in Figure 6B and Alu RNA processing ratio in AD patients ( r = 0 . 88 , p < 0 . 001 ) . Statistical test is based on Pearson ' s product moment correlation coefficient and follows a t distribution using the cor . test function in R package stats . Scatterplot depicting the negative correlation between average gene expression values of down - regulated genes in Figure 6C and full length Alu RNA levels in AD patients ( r = - 0 . 42 , p < 0 . 001 ) . Statistical test as in subfigure 6D .

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Figure 6Association between SINE Alu RNA ratio (upper panel) and gene expression levels (lower panel) of genes up-regulated in AD (Dataset EV3, |log2FoldChange|> 0.5). Every column in both panels corresponds to the same MAP patient of either the no AD or AD group. Patients in each group are sorted from left to right in an ascending order with regard to Alu RNA processing ratio. Every row in the heatmap corresponds to one gene, with color density representing normalized FPKM values from RNA-seq for this gene for each patient (columns) (red represents high and blue low expression levels). Association between full length Alu RNA levels (upper panel) and gene expression levels (lower panel) of genes down-regulated in AD (Dataset EV4, |log2FoldChange|> 0.5). Every column in both panels corresponds to the same MAP patient of either the no AD or AD group. Patients in each group are sorted from left to right in an ascending order with regard to full length Alu RNA levels. Every row in the heatmap corresponds to one gene, with color density representing normalized FPKM values from RNA-seq for this gene for each patient (columns) (red represents high and blue low expression levels).Scatterplot depicting the positive correlation between average gene expression values of upregulated genes in Figure 6B and Alu RNA processing ratio in AD patients (r = 0.88, p<0.001). Statistical test is based on Pearson's product moment correlation coefficient and follows a t distribution using the cor.test function in R package stats. Scatterplot depicting the negative correlation between average gene expression values of down-regulated genes in Figure 6C and full length Alu RNA levels in AD patients (r = -0.42, p<0.001). Statistical test as in subfigure 6D.