( b ) Protein extracts from wild - type ( Ambra1 + / + ) and homozygous ( Ambra1gt / gt ) embryos for the Ambra1 gene - trap mutation at developmental day 14 . 5 were analysed by western blotting using anti - Ulk1 or anti - Hsp90 ( loading control ) antibodies . Quantification is shown on the right . n = 6 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 037 .

O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O B-ORGANISM O O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O 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) Protein extracts from wild-type (Ambra1+/+) and homozygous (Ambra1gt/gt) embryos for the Ambra1 gene-trap mutation at developmental day 14.5 were analysed by western blotting using anti-Ulk1 or anti-Hsp90 (loading control) antibodies. Quantification is shown on the right. n = 6 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.037.


( c ) AMBRA1 , ULK1 and actin ( as a protein loading control ) were analysed by western blotting in HeLa cells transiently transfected with scramble siRNA or AMBRA1 siRNA . Quantification is shown on the right . n = 5 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 007 .

O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O 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) AMBRA1, ULK1 and actin (as a protein loading control) were analysed by western blotting in HeLa cells transiently transfected with scramble siRNA or AMBRA1 siRNA. Quantification is shown on the right. n = 5 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.007.


( d ) HeLa cells stably interfered for AMBRA1 were transiently transfected with empty vector or AMBRA1 vector ; AMBRA1 , ULK1 , ATG13 , p - ATG13 and actin levels were analysed by western blotting . Quantification of ULK1 and pATG13 bands is shown on the right . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 006 for ULK1 and P = 0 . 044 and P = 0 . 011 for pATG13 .

O O O B-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O

(d) HeLa cells stably interfered for AMBRA1 were transiently transfected with empty vector or AMBRA1 vector; AMBRA1, ULK1, ATG13, p-ATG13 and actin levels were analysed by western blotting. Quantification of ULK1 and pATG13 bands is shown on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.006 for ULK1 and P = 0.044 and P = 0.011 for pATG13.


( e ) HeLa cells were transfected or not with vector encoding AMBRA1 and treated with 35 μM cycloheximide ( CHX ) for 6 and 8 h before extraction . ULK1 , actin and AMBRA1 were analysed by western blotting . Quantification is shown below . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 049 and P = 0 . 02 .

O O O B-CELL_LINE I-CELL_LINE O O O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O 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) HeLa cells were transfected or not with vector encoding AMBRA1 and treated with 35 μM cycloheximide (CHX) for 6 and 8 h before extraction. ULK1, actin and AMBRA1 were analysed by western blotting. Quantification is shown below. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.049 and P = 0.02.


( f ) HEK293 cells were transfected as in a ; ATG13 , pATG13 , ULK1 , AMBRA1 and actin were analysed by western blotting .

O O O B-CELL_LINE I-CELL_LINE O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O

(f) HEK293 cells were transfected as in a; ATG13, pATG13, ULK1, AMBRA1 and actin were analysed by western blotting.


( g ) Endogenous ULK1 is immunoprecipitated from HeLa cells transfected with β - galactosidase or AMBRA1 plasmid respectively using an anti - ULK1 antibody . As a control , rabbit IgG was used . The resulting precipitates were subjected to ULK1kinase assay using MBP as the substrate . Purified complexes were analysed by autoradiography and by western blotting using an anti - ULK1 antibody . Total extracts were analysed using an anti - AMBRA1 antibody .

O O O O B-GENEPROD O B-EXP_ASSAY O B-CELL_LINE I-CELL_LINE O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O

(g) Endogenous ULK1 is immunoprecipitated from HeLa cells transfected with β-galactosidase or AMBRA1 plasmid respectively using an anti-ULK1 antibody. As a control, rabbit IgG was used. The resulting precipitates were subjected to ULK1kinase assay using MBP as the substrate. Purified complexes were analysed by autoradiography and by western blotting using an anti-ULK1 antibody. Total extracts were analysed using an anti-AMBRA1 antibody.


( a ) Protein extracts from HeLa cells were immunoprecipitated using anti - ULK1 antibody or with IgG as a negative control and analysed by western blotting using anti - Lys63 specific ubiquitin and anti - ULK1 antibodies .

O O O O O O B-CELL_LINE O O B-EXP_ASSAY O O O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O B-GENEPROD O O

(a) Protein extracts from HeLa cells were immunoprecipitated using anti-ULK1 antibody or with IgG as a negative control and analysed by western blotting using anti-Lys63 specific ubiquitin and anti-ULK1 antibodies.


( b ) HeLa cells were co - transfected with ULK1 - Myc alone or in combination with wild - type ubiquitin or its mutant ( K48R or K63R ) HA - tagged constructs . Protein extracts were immunoprecipitated using an anti - Myc antibody . Purified complexes and total extracts were analysed by western blotting using anti - Myc and anti - HA antibodies .

O O O B-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O

(b) HeLa cells were co-transfected with ULK1-Myc alone or in combination with wild-type ubiquitin or its mutant (K48R or K63R) HA-tagged constructs. Protein extracts were immunoprecipitated using an anti-Myc antibody. Purified complexes and total extracts were analysed by western blotting using anti-Myc and anti-HA antibodies.


( c ) In HeLa cells , AMBRA1 was downregulated by RNAi . Protein extracts were immunoprecipitated using anti - ULK1 antibody or with IgG as a negative control . Purified complexes and total extracts were analysed by western blotting using anti - ULK1 , anti - Lys - 63 ubiquitin and anti - AMBRA1 antibodies . Of note , ULK1 levels in the co - immunoprecipitation experiment do not change in this case in the AMBRA1 siRNA lane , owing to saturation of the anti - ULK1 antibody ( see also Fig . 4h ) .

O O O O B-CELL_LINE O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O

(c) In HeLa cells, AMBRA1 was downregulated by RNAi. Protein extracts were immunoprecipitated using anti-ULK1 antibody or with IgG as a negative control. Purified complexes and total extracts were analysed by western blotting using anti-ULK1, anti-Lys-63 ubiquitin and anti-AMBRA1 antibodies. Of note, ULK1 levels in the co-immunoprecipitation experiment do not change in this case in the AMBRA1 siRNA lane, owing to saturation of the anti-ULK1 antibody (see also Fig. 4h).


( d ) HeLa cells were co - transfected with vectors encoding for ubiquitin Lys - 63 - HA alone or in combination with AMBRA1 . Protein extracts were immunoprecipitated using an anti - ULK1 antibody or with IgG as a negative control and Ub - Lys - 63 - HA , ULK1 and AMBRA1 were analysed by western blotting .

O O O B-CELL_LINE O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-SUBCELLULAR O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O

(d) HeLa cells were co-transfected with vectors encoding for ubiquitin Lys-63-HA alone or in combination with AMBRA1. Protein extracts were immunoprecipitated using an anti-ULK1 antibody or with IgG as a negative control and Ub-Lys-63-HA, ULK1 and AMBRA1 were analysed by western blotting.


( e , f ) HeLa cells were cultured in complete or starvation medium for 30 min or in the presence of 100 nM of rapamycin for 3 h and then protein extracts were immunoprecipitated using anti - ULK1 antibody or with IgG as a negative control . Purified complexes and total extracts were analysed by western blotting as in a . Quantifications are shown on the right . In both panels , n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 003 and P = 0 . 0015 , respectively .

O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,f) HeLa cells were cultured in complete or starvation medium for 30 min or in the presence of 100 nM of rapamycin for 3 h and then protein extracts were immunoprecipitated using anti-ULK1 antibody or with IgG as a negative control. Purified complexes and total extracts were analysed by western blotting as in a. Quantifications are shown on the right. In both panels, n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.003 and P = 0.0015, respectively.


( a ) HeLa cells were transfected with FLAG - TRAF6 or grown in starvation medium for 1 h . Some of them were treated with chloroquine . LC3 , p62 and actin were analysed by western blotting . Quantification of LC3II and p62 bands is shown on the right . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 017 for LC3 and P = 0 . 01 and P = 0 . 04 for p62 .

O O O B-CELL_LINE I-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O

(a) HeLa cells were transfected with FLAG-TRAF6 or grown in starvation medium for 1 h. Some of them were treated with chloroquine. LC3, p62 and actin were analysed by western blotting. Quantification of LC3II and p62 bands is shown on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.017 for LC3 and P = 0.01 and P = 0.04 for p62.


( b ) HeLa cells were transduced with AMBRA1 lentiviral shRNA and transfected or not with FLAG - TRAF6 . Quantification of LC3II and p62 bands is shown on the right . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 001 and P = 0 . 0018 for LC3 and P = 0 . 004 for p62 .

O O O B-CELL_LINE O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O B-GENEPROD O

(b) HeLa cells were transduced with AMBRA1 lentiviral shRNA and transfected or not with FLAG-TRAF6. Quantification of LC3II and p62 bands is shown on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.001 and P = 0.0018 for LC3 and P = 0.004 for p62.


( c ) Count of LC3 puncta in HeLa cells transduced as in b . Five different confocal microscopy images for each experiment were randomly chosen and GFP - positive dots were examined on the images with identical brightness and contrast settings . The results are represented as the average number of LC3 dots with 100 cells analysed per sample . Confocal images are shown in Supplementary Fig . S3b .

O O O O O B-GENEPROD O O B-CELL_LINE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD 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-GENEPROD O O O O O O O O O O O O O O O O O O

(c) Count of LC3 puncta in HeLa cells transduced as in b. Five different confocal microscopy images for each experiment were randomly chosen and GFP-positive dots were examined on the images with identical brightness and contrast settings. The results are represented as the average number of LC3 dots with 100 cells analysed per sample. Confocal images are shown in Supplementary Fig. S3b.


( d ) HEK293 cells were transfected with AMBRA1 and FLAG - TRAF6 as indicated . Protein extracts were immunoprecipitated using an anti - FLAG antibody .

O O O B-CELL_LINE I-CELL_LINE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O

(d) HEK293 cells were transfected with AMBRA1 and FLAG-TRAF6 as indicated. Protein extracts were immunoprecipitated using an anti-FLAG antibody.


( e ) HeLa cells were transfected as in b and FLAG - TRAF6 was purified as in d . Quantification of immunoprecipitated TRAF6 is shown on the right . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 0003 .

O O O B-CELL_LINE O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-EXP_ASSAY B-GENEPROD O O O O O O O O O O O O O O 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) HeLa cells were transfected as in b and FLAG-TRAF6 was purified as in d. Quantification of immunoprecipitated TRAF6 is shown on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.0003.


( f ) In HeLa cells , TRAF6 was downregulated by RNAi . AMBRA1 , ULK1 , TRAF6 , ATG13 and actin were analysed by western blotting .

O O O O B-CELL_LINE O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O

(f) In HeLa cells, TRAF6 was downregulated by RNAi. AMBRA1, ULK1, TRAF6, ATG13 and actin were analysed by western blotting.


( g ) HeLa cells were co - transfected with vectors encoding ULK1 - Myc , FLAG - TRAF6 and Ub - Lys - 63 - HA , as indicated . Protein extracts were immunoprecipitated using an anti - Myc antibody .

O O O B-CELL_LINE O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O

(g) HeLa cells were co-transfected with vectors encoding ULK1-Myc, FLAG-TRAF6 and Ub-Lys-63-HA, as indicated. Protein extracts were immunoprecipitated using an anti-Myc antibody.


( h ) HeLa cells stably interfered for AMBRA1 were transfected with FLAG - TRAF6 and Ub - Lys - 63 - HA as indicated . Protein extracts were immunoprecipitated using anti - ULK1 antibody .

O O O B-CELL_LINE O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O

(h) HeLa cells stably interfered for AMBRA1 were transfected with FLAG-TRAF6 and Ub-Lys-63-HA as indicated. Protein extracts were immunoprecipitated using anti-ULK1 antibody.


( i ) Endogenous ULK1 is immunoprecipitated from HeLa cells transfected as in b . The resulting precipitates were subjected to a ULK1 kinase assay using MBP as the substrate . Purified complexes were analysed by autoradiography and by western blotting using an anti - ULK1 antibody .

O O O O B-GENEPROD O B-EXP_ASSAY O B-CELL_LINE O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O

(i) Endogenous ULK1 is immunoprecipitated from HeLa cells transfected as in b. The resulting precipitates were subjected to a ULK1 kinase assay using MBP as the substrate. Purified complexes were analysed by autoradiography and by western blotting using an anti-ULK1 antibody.


( a - c ) Two canonical TRAF6 - binding sites in AMBRA1 are essential for its interaction with TRAF6 . HEK293 cells were co - transfected with vectors encoding FLAG - TRAF6 together with AMBRA1WT and mutant constructs . Protein extracts were immunoprecipitated using an anti - FLAG antibody . AMBRA1 and FLAG - TRAF6 were analysed by western blotting .

O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-GENEPROD O B-CELL_LINE I-CELL_LINE O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O

(a-c) Two canonical TRAF6-binding sites in AMBRA1 are essential for its interaction with TRAF6. HEK293 cells were co-transfected with vectors encoding FLAG-TRAF6 together with AMBRA1WT and mutant constructs. Protein extracts were immunoprecipitated using an anti-FLAG antibody. AMBRA1 and FLAG-TRAF6 were analysed by western blotting.


( d ) HeLa cells were transfected with AMBRA1 RNAi oligonucleotide ( siRNA AMBRA1 ) . Then , cells were co - transfected with vectors encoding ubiquitin - HA and AMBRA1WT or AMBRA1AA , respectively . Protein extracts were immunoprecipitated using anti - ULK1 antibody ; ubiquitin - HA , ULK1 and AMBRA1 were analysed by western blotting .

O O O B-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O

(d) HeLa cells were transfected with AMBRA1 RNAi oligonucleotide (siRNA AMBRA1). Then, cells were co-transfected with vectors encoding ubiquitin-HA and AMBRA1WT or AMBRA1AA, respectively. Protein extracts were immunoprecipitated using anti-ULK1 antibody; ubiquitin-HA, ULK1 and AMBRA1 were analysed by western blotting.


( e ) HeLa cells stably interfered for AMBRA1 were transduced with lentiviral vectors encoding β - galactosidase ( negative control ) AMBRA1WT or AMBRA1AA respectively ; protein extracts were analysed by western blotting using anti - ULK1 , anti - AMBRA1 anti - ATG13 , anti - pATG13 and anti - actin antibodies .

O O O B-CELL_LINE O O O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O O O O B-GENEPROD O O

(e) HeLa cells stably interfered for AMBRA1 were transduced with lentiviral vectors encoding β-galactosidase (negative control) AMBRA1WT or AMBRA1AA respectively; protein extracts were analysed by western blotting using anti-ULK1, anti-AMBRA1 anti-ATG13, anti-pATG13 and anti-actin antibodies.


( f ) HeLa cells were transfected as in e and some of them were treated with chloroquine for 1 h . LC3 , p62 , AMBRA1 and actin were analysed by western blotting . Quantification of LC3II and p62 are shown on the right . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 03 for LC3 and P = 0 . 045 for p62 .

O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O B-GENEPROD O

(f) HeLa cells were transfected as in e and some of them were treated with chloroquine for 1 h. LC3, p62, AMBRA1 and actin were analysed by western blotting. Quantification of LC3II and p62 are shown on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.03 for LC3 and P = 0.045 for p62.


( g ) GFP - LC3 MEF cells from wild - type ( Ambra1 + / + ) and homozygous ( Ambra1gt / gt ) embryos were transduced with the indicated lentiviral vectors and quantification of LC3 puncta per cells is shown on the right . Bars represent mean ± s . e . m . of triplicate samples with 50 cells analysed per sample ; n = 3 independent experiments . Significance is P = 0 . 001 and P = 0 . 003 ( n = 3 ) . Scale bar , 10 μm .

O O O B-GENEPROD O B-GENEPROD B-CELL_LINE I-CELL_LINE O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(g) GFP-LC3 MEF cells from wild-type (Ambra1+/+) and homozygous (Ambra1gt/gt) embryos were transduced with the indicated lentiviral vectors and quantification of LC3 puncta per cells is shown on the right. Bars represent mean±s.e.m. of triplicate samples with 50 cells analysed per sample; n = 3 independent experiments. Significance is P = 0.001 and P = 0.003 (n = 3). Scale bar, 10 μm.


( a ) HEK293 cells were co - transfected with vectors encoding ULK1 - HA and ULK1 - Myc and then grown either in normal ( Ctrl ) or in starvation ( EBS ) medium for 1 h . Protein extracts were immunoprecipitated using an anti - Myc antibody , and analysed by western blotting using anti - HA , and anti - Myc antibodies . Quantification is shown on the right . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 001 .

O O O B-CELL_LINE I-CELL_LINE O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD 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-EXP_ASSAY O O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O 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) HEK293 cells were co-transfected with vectors encoding ULK1-HA and ULK1-Myc and then grown either in normal (Ctrl) or in starvation (EBS) medium for 1 h. Protein extracts were immunoprecipitated using an anti-Myc antibody, and analysed by western blotting using anti-HA, and anti-Myc antibodies. Quantification is shown on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.001.


( b ) HEK293 cells were transfected with TRAF6 RNAi oligonucleotide ( TRAF6 siRNA ) or unrelated oligonucleotides as a control ( Ctrl siRNA ) . Then , cells were co - transfected with vectors encoding ULK1 - HA and ULK1 - Myc and protein complexes were immunoprecipitated using anti - Myc antibody . Protein extracts were analysed by western blotting using anti - HA , anti - Myc and anti - TRAF6 antibodies . Quantification of ULK1 - HA relative to ULK1 - Myc is reported on the right . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 037 .

O O O B-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O 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) HEK293 cells were transfected with TRAF6 RNAi oligonucleotide (TRAF6 siRNA) or unrelated oligonucleotides as a control (Ctrl siRNA). Then, cells were co-transfected with vectors encoding ULK1-HA and ULK1-Myc and protein complexes were immunoprecipitated using anti-Myc antibody. Protein extracts were analysed by western blotting using anti-HA, anti-Myc and anti-TRAF6 antibodies. Quantification of ULK1-HA relative to ULK1-Myc is reported on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.037.


( c ) HEK293 cells were transfected with AMBRA1 RNAi oligonucleotide ( AMBRA1 siRNA ) or unrelated oligonucleotides as a control ( Ctrl siRNA ) . Then , cells were co - transfected and analysed as in a . Quantification is shown on the right . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 044 .

O O O B-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HEK293 cells were transfected with AMBRA1 RNAi oligonucleotide (AMBRA1 siRNA) or unrelated oligonucleotides as a control (Ctrl siRNA). Then, cells were co-transfected and analysed as in a. Quantification is shown on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.044.


( d ) HEK293 cells were co - transfected with vectors encoding for ULK1 - HA , ULK1 - Myc and AMBRA1WT or AMBRA1AA respectively . Protein complexes were immunoprecipitated and analysed as in a . Quantification is shown on the right . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 014 .

O O O B-CELL_LINE I-CELL_LINE O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O 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) HEK293 cells were co-transfected with vectors encoding for ULK1-HA, ULK1-Myc and AMBRA1WT or AMBRA1AA respectively. Protein complexes were immunoprecipitated and analysed as in a. Quantification is shown on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.014.


( a ) Highly purified mTOR was incubated with radiolabelled ATP without substrate , with purified FLAG - AMBRA1 and purified FLAG - AMBRA1S52A and analysed by autoradiography . The lower panels show a FLAG and mTOR western blot ( WB ) .

O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O

(a) Highly purified mTOR was incubated with radiolabelled ATP without substrate, with purified FLAG-AMBRA1 and purified FLAG-AMBRA1S52A and analysed by autoradiography. The lower panels show a FLAG and mTOR western blot (WB).


( b ) HEK293 cells expressing FLAG - RAPTOR were subjected to FLAG immunoprecipitation . Protein complexes were analysed by western blotting using anti - FLAG and anti - AMBRA1 antibodies .

O O O B-CELL_LINE I-CELL_LINE O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O

(b) HEK293 cells expressing FLAG-RAPTOR were subjected to FLAG immunoprecipitation. Protein complexes were analysed by western blotting using anti-FLAG and anti-AMBRA1 antibodies.


( c ) HEK293 cells were treated with Torin1 for 3 h or cultured in starvation medium for 2 h and protein extracts were analysed by western blotting using anti - pSer - 52 and anti - AMBRA1 antibodies .

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(c) HEK293 cells were treated with Torin1 for 3 h or cultured in starvation medium for 2 h and protein extracts were analysed by western blotting using anti-pSer-52 and anti-AMBRA1 antibodies.


( d ) HeLa cells stably interfered for AMBRA1 were transduced with the indicated lentiviral vectors encoding for β - galactosidase , AMBRA1WT or AMBRA1S52A respectively and some of them were treated with chloroquine for 1 h . Protein extracts were analysed by western blotting using anti - LC3 , anti - p62 and anti - actin antibodies . Quantification of LC3II and p62 bands is shown below . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 038 .

O O O B-CELL_LINE O O O O B-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O 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) HeLa cells stably interfered for AMBRA1 were transduced with the indicated lentiviral vectors encoding for β-galactosidase, AMBRA1WT or AMBRA1S52A respectively and some of them were treated with chloroquine for 1 h. Protein extracts were analysed by western blotting using anti-LC3, anti-p62 and anti-actin antibodies. Quantification of LC3II and p62 bands is shown below. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.038.


( e ) GFP - LC3 MEF cells from wild - type ( Ambra1 + / + ) and homozygous ( Ambra1gt / gt ) embryos were transduced with indicated lentiviral vectors and quantification of LC3 puncta per cell is shown on the right . Bars represent mean ± s . e . m . of triplicate samples and 50 cells analysed per sample . n = 3 independent experiments . Significance is P = 0 . 001 and P = 0 . 003 . Scale bar , 10

O O O B-GENEPROD O B-GENEPROD B-CELL_LINE I-CELL_LINE O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) GFP-LC3 MEF cells from wild-type (Ambra1+/+) and homozygous (Ambra1gt/gt) embryos were transduced with indicated lentiviral vectors and quantification of LC3 puncta per cell is shown on the right. Bars represent mean±s.e.m. of triplicate samples and 50 cells analysed per sample. n = 3 independent experiments. Significance is P = 0.001 and P = 0.003. Scale bar, 10 μm


( f ) HeLa cells stably interfered for AMBRA1 were co - transfected with vectors encoding Ub - Lys - 63 - HA and AMBRA1WT or AMBRA1S52A , respectively . Protein extracts were immunoprecipitated using an anti - ULK1 antibody and ubiquitin Lys - 63 - HA , ULK1 , AMBRA1 and actin were analysed by western blotting .

O O O B-CELL_LINE O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O

(f) HeLa cells stably interfered for AMBRA1 were co-transfected with vectors encoding Ub-Lys-63-HA and AMBRA1WT or AMBRA1S52A, respectively. Protein extracts were immunoprecipitated using an anti-ULK1 antibody and ubiquitin Lys-63-HA, ULK1, AMBRA1 and actin were analysed by western blotting.


( g ) HeLa cells stably interfered for AMBRA1 were transduced as in d ; protein extracts were analysed by western blotting using anti - AMBRA1 , anti - ATG13 , anti - pATG13 and anti - actin antibodies . Quantification of the p - ATG13 band is shown below .

O O O B-CELL_LINE O O O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O

(g) HeLa cells stably interfered for AMBRA1 were transduced as in d; protein extracts were analysed by western blotting using anti-AMBRA1, anti-ATG13, anti-pATG13 and anti-actin antibodies. Quantification of the p-ATG13 band is shown below.


( h ) HeLa cells transfected with ULK1 - HA , ULK1 - Myc and different AMBRA1 constructs , respectively . Protein complexes were immunoprecipitated using anti - HA antibody and analysed by western blotting using anti - HA , anti - Myc and anti - AMBRA1 antibodies .

O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O

(h) HeLa cells transfected with ULK1-HA, ULK1-Myc and different AMBRA1 constructs, respectively. Protein complexes were immunoprecipitated using anti-HA antibody and analysed by western blotting using anti-HA, anti-Myc and anti-AMBRA1 antibodies.


( B ) Phase - contrast images of wild - type or PABPC4KO HeLa cells depleted of PABPC1 by siRNA - mediated knockdown . Scale bar , 100 μm . ( C ) Total numbers of viable cells in ( B ) were quantified with acridine orange and propidium iodide staining and graphed as a percentage of wild - type HeLa cells . Error bars represent the SEM of three biological replicates .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD B-CELL_LINE O O O B-GENEPROD O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O

(B) Phase-contrast images of wild-type or PABPC4KO HeLa cells depleted of PABPC1 by siRNA-mediated knockdown. Scale bar, 100 μm. (C) Total numbers of viable cells in (B) were quantified with acridine orange and propidium iodide staining and graphed as a percentage of wild-type HeLa cells. Error bars represent the SEM of three biological replicates.


( C ) Phase - contrast images of PABPDHFR cells , or PABPDHFR cells transfected with a plasmid coding for wild - type PABPC1 , 48 hours post - TMP removal . Scale bar , 100 μm .

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(C) Phase-contrast images of PABPDHFR cells, or PABPDHFR cells transfected with a plasmid coding for wild-type PABPC1, 48 hours post-TMP removal. Scale bar, 100 μm.


( D ) Western blot analysis of lysates derived from cells imaged in ( C ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O

(D) Western blot analysis of lysates derived from cells imaged in (C).


( B ) SUnSET assay of PABPDHFR cells grown in the presence or absence of TMP for 12 hours to maintain or deplete PABP . Cells were subsequently pulsed with either puromycin or puromycin and cycloheximide ( control ) , lysed and equal protein amounts were resolved by SDS - PAGE . Western blot analysis was performed using a monoclonal antibody against puromycin or actin ( loading control ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O

(B) SUnSET assay of PABPDHFR cells grown in the presence or absence of TMP for 12 hours to maintain or deplete PABP. Cells were subsequently pulsed with either puromycin or puromycin and cycloheximide (control), lysed and equal protein amounts were resolved by SDS-PAGE. Western blot analysis was performed using a monoclonal antibody against puromycin or actin (loading control).


( D ) Scatter plot comparing log2 fold - changes [ ( - ) PABP vs ( + ) PABP ] in polysome - associated mRNA ( y - axis ) to corresponding changes in total cytoplasmic mRNA . Transcripts identified as regulated via altered translation efficiency ( orange and red ) or abundance ( light and dark green ) according to anota2seq are visualized together with non - regulated transcripts ( grey ) .

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(D) Scatter plot comparing log2 fold-changes [(-) PABP vs (+) PABP] in polysome-associated mRNA (y-axis) to corresponding changes in total cytoplasmic mRNA. Transcripts identified as regulated via altered translation efficiency (orange and red) or abundance (light and dark green) according to anota2seq are visualized together with non-regulated transcripts (grey).


Boxplots of 5 ´ UTR ( A ) , 3 ´ UTR ( B ) lengths for mRNAs whose abundance decreased ( ' DOWN ' , dark green ) or increased ( ' UP ' , light green ) in PABP - depleted cells as compared to cells expressing PABP . Solid horizonal middle lines in box plots denote the median ; lower and upper box limits correspond to first and third quartiles ; whiskers extend from box limits to the most extreme values . Wilcoxon - Mann - Whitney tests ( two - sided ) were used to determine differences between upregulated mRNAs and downregulated mRNAs relative to each other and to the background ( mRNAs whose abundance did not change in the absence of PABP relative to PABP - expressing cells ) .

O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O B-GENEPROD O O O O O

Boxplots of 5´UTR (A), 3´UTR (B) lengths for mRNAs whose abundance decreased ('DOWN', dark green) or increased ('UP', light green) in PABP-depleted cells as compared to cells expressing PABP. Solid horizonal middle lines in box plots denote the median; lower and upper box limits correspond to first and third quartiles; whiskers extend from box limits to the most extreme values. Wilcoxon-Mann-Whitney tests (two-sided) were used to determine differences between upregulated mRNAs and downregulated mRNAs relative to each other and to the background (mRNAs whose abundance did not change in the absence of PABP relative to PABP-expressing cells).


( A ) Boxplots of mRNA half - lives ( hrs ) assessed by metabolic labeling of normal human umbilical vein endothelial cells for mRNAs whose abundance decreased ( ' DOWN ' , dark green ) or increased ( ' UP ' , light green ) in PABP - depleted cells as compared to cells expressing PABP comparing ( Tiana et al . , 2020 ) . Solid horizonal middle lines in box plots denote the median ; lower and upper box limits correspond to first and third quartiles ; whiskers extend from box limits to the most extreme values but not further than 1 . 5 × the inter - quartile range ( IQR ) . ( B - H ) PABPDHFR cells were cultured in the presence or absence of TMP for 12 hours . The stabilities of select mRNAs was assessed by using actinomycin D ( 5 μg / ml ) for the indicated amounts of time . Total RNA was isolated , reverse transcribed with random hexamer oligonucleotide primers and quantified by qPCR . mRNA decay rates were normalized to an in vitro synthesized spike - in RNA with the zero time point set at 1 . Error bars represent the SEM of three independent experiments . First order exponential decay trend lines were generated by non - linear regression analysis .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-ORGANISM B-TISSUE I-TISSUE B-CELL_TYPE I-CELL_TYPE 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-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O 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 of mRNA half-lives (hrs) assessed by metabolic labeling of normal human umbilical vein endothelial cells for mRNAs whose abundance decreased ('DOWN', dark green) or increased ('UP', light green) in PABP-depleted cells as compared to cells expressing PABP comparing (Tiana et al., 2020). Solid horizonal middle lines in box plots denote the median; lower and upper box limits correspond to first and third quartiles; whiskers extend from box limits to the most extreme values but not further than 1.5× the inter-quartile range (IQR). (B- H) PABPDHFR cells were cultured in the presence or absence of TMP for 12 hours. The stabilities of select mRNAs was assessed by using actinomycin D (5 μg/ml) for the indicated amounts of time. Total RNA was isolated, reverse transcribed with random hexamer oligonucleotide primers and quantified by qPCR. mRNA decay rates were normalized to an in vitro synthesized spike-in RNA with the zero time point set at 1. Error bars represent the SEM of three independent experiments. First order exponential decay trend lines were generated by non-linear regression analysis.


( B ) PABPDHFR cells were transfected with plasmids encoding RL - 6xB or RL - 6xBMUT . 24 hours after transfections , the cells were split and cultured for an additional 12 hours in the presence or absence of TMP . The stabilities of reporter mRNAs was assessed by using actinomycin D ( 5 μg / ml ) for the indicated amounts of time . Total RNA was isolated , reverse transcribed with random hexamer oligonucleotide primers and quantified by qPCR . mRNA decay rates were normalized to an in vitro synthesized spike - in RNA with the zero time point set at 1 . Error bars represent the SEM of three independent experiments . First order exponential decay trend lines were generated by non - linear regression analysis .

O O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O 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) PABPDHFR cells were transfected with plasmids encoding RL-6xB or RL-6xBMUT. 24 hours after transfections, the cells were split and cultured for an additional 12 hours in the presence or absence of TMP. The stabilities of reporter mRNAs was assessed by using actinomycin D (5 μg/ml) for the indicated amounts of time. Total RNA was isolated, reverse transcribed with random hexamer oligonucleotide primers and quantified by qPCR. mRNA decay rates were normalized to an in vitro synthesized spike-in RNA with the zero time point set at 1. Error bars represent the SEM of three independent experiments. First order exponential decay trend lines were generated by non-linear regression analysis.


( A ) Western blot analysis PABPDHFR cells depleted of LSM1 , DCP2 or XRN1 by siRNA - mediated knockdown . siGFP represents a negative control .

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(A) Western blot analysis PABPDHFR cells depleted of LSM1, DCP2 or XRN1 by siRNA-mediated knockdown. siGFP represents a negative control.


( B ) Phase - contrast images of PABPDHFR cells previously transfected with siRNAs targeting GFP ( control ) , LSM1 , DCP2 or XRN1 and cultured in the presence or absence of TMP for 48 hours . Total numbers of viable cells , denoted in the upper right hand corner of images , were quantified with acridine orange and propidium iodide and calculated as a percentage of PABPDHFR cells grown in the presence of TMP , along with the SEM . Scale bar , 100 μm .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O

(B) Phase-contrast images of PABPDHFR cells previously transfected with siRNAs targeting GFP (control), LSM1, DCP2 or XRN1 and cultured in the presence or absence of TMP for 48 hours. Total numbers of viable cells, denoted in the upper right hand corner of images, were quantified with acridine orange and propidium iodide and calculated as a percentage of PABPDHFR cells grown in the presence of TMP, along with the SEM. Scale bar, 100 μm.


( A ) Western blot analysis PABPDHFR cells depleted of LSM1 by siRNA - mediated knockdown . Transfected cells were subsequently cultured in the presence or absence of TMP to maintain or deplete endogenous PABP .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O

(A) Western blot analysis PABPDHFR cells depleted of LSM1 by siRNA-mediated knockdown. Transfected cells were subsequently cultured in the presence or absence of TMP to maintain or deplete endogenous PABP.


( C and D ) Poly ( A ) tail analysis of GAPDH and ACTB mRNAs from total RNA isolated from PABPDHFR cells described in ( B ) was determined by extension poly ( A ) tail ( ePAT ) . PCR products were run on high - resolution agarose gels . ' A12 ' represents the size of ePAT RT - PCR amplicons derived from a mRNA with a fixed ( A12 ) - tail .

O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O

(C and D) Poly(A) tail analysis of GAPDH and ACTB mRNAs from total RNA isolated from PABPDHFR cells described in (B) was determined by extension poly(A) tail (ePAT). PCR products were run on high-resolution agarose gels. 'A12' represents the size of ePAT RT-PCR amplicons derived from a mRNA with a fixed (A12)-tail.


( D - G ) Cell death mechanisms of C3H BMDMs transfected with miR - 342 - 3p mimic ( D ) , or B6 BMDMs transfected with miR - 342 - 3p inhibitor ( F ) , followed by Mtb infection for 36 hours . Cell viabilities of C3H BMDMs transfected with miR - 342 - 3p mimic ( E ) , or B6 BMDMs transfected with miR - 342 - 3p inhibitor ( G ) , followed by stimulation with Mtb or z - VAD ( 20 μM ) for 24 hours . Data are shown as the mean ± s . e . m . of n = 3 biological replicates .

O O O O O O O O O O B-CELL_TYPE O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-CELL_TYPE O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-ORGANISM O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-CELL_TYPE O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-ORGANISM O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O 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-G) Cell death mechanisms of C3H BMDMs transfected with miR-342-3p mimic (D), or B6 BMDMs transfected with miR-342-3p inhibitor (F), followed by Mtb infection for 36 hours. Cell viabilities of C3H BMDMs transfected with miR-342-3p mimic (E), or B6 BMDMs transfected with miR-342-3p inhibitor (G), followed by stimulation with Mtb or z-VAD (20 μM) for 24 hours. Data are shown as the mean ± s.e.m. of n = 3 biological replicates.


( A , B ) Survival of Mir342 + / + C3H mice and their wild - type littermate controls ( C3H ) ( n = 10 ) ( A ) , or Mir342 - / - B6 mice and their wild - type littermate controls ( B6 ) ( n = 10 ) ( B ) after aerosol infection with around 400 CFU Mtb . Data are shown as the Kaplan - Meier curves .

O O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-ORGANISM O 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-ORGANISM O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

(A, B) Survival of Mir342+/+C3H mice and their wild-type littermate controls (C3H) (n=10) (A), or Mir342-/-B6 mice and their wild-type littermate controls (B6) (n=10) (B) after aerosol infection with around 400 CFU Mtb. Data are shown as the Kaplan-Meier curves.


( G , H ) Secretion of cytokines TNF - α , IL - 1 , IL - 6 and CXCL15 in BMDMs obtained from Mir342 + / + C3H and their littermates ( C3H ) ( G ) , or from Mir342 - / - B6 and their littermates ( B6 ) ( H ) after Mtb stimulation , was detected by ELISA . Data are shown as the mean ± s . e . m . of n = 3 biological replicates .

O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-CELL_TYPE O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-ORGANISM O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O

(G, H) Secretion of cytokines TNF-α, IL-1, IL-6 and CXCL15 in BMDMs obtained from Mir342+/+C3H and their littermates (C3H) (G), or from Mir342-/-B6 and their littermates (B6) (H) after Mtb stimulation, was detected by ELISA. Data are shown as the mean ± s.e.m. of n = 3 biological replicates.


( C , D ) MiR - 342 - 3p mimic or inhibitor was transfected to NIH - 3T3 fibroblasts . After 48 hours , cells were collected for qRT - PCR ( C , data are shown as the mean ± s . e . m . of n = 3 biological replicates ) and western blotting ( D , representative blots from n = 3 biological replicates are shown ) to detect SOCS6 expression .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-CELL_TYPE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O

(C, D) MiR-342-3p mimic or inhibitor was transfected to NIH-3T3 fibroblasts. After 48 hours, cells were collected for qRT-PCR (C, data are shown as the mean ± s.e.m. of n = 3 biological replicates) and western blotting (D, representative blots from n = 3 biological replicates are shown) to detect SOCS6 expression.


( F - H ) C3H BMDMs were transfected with miR - 342 - 3p mimic or SOCS6 - overexpressing lentivirus , followed by stimulation with Mtb or z - VAD ( 20 μM ) for 24 hours . Cell death mechanisms ( F ) , cell viabilities ( G ) and Mtb growth rates ( H ) were analyzed respectively . Data are shown as the mean ± s . e . m . of n = 3 biological replicates .

O O O O O O B-CELL_TYPE O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O B-ORGANISM O O O O O B-ORGANISM O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM B-EXP_ASSAY O O O O 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-H) C3H BMDMs were transfected with miR-342-3p mimic or SOCS6-overexpressing lentivirus, followed by stimulation with Mtb or z-VAD (20 μM) for 24 hours. Cell death mechanisms (F), cell viabilities (G) and Mtb growth rates (H) were analyzed respectively. Data are shown as the mean ± s.e.m. of n = 3 biological replicates.


( A , B ) Survival of Mir342 - / - B6 and Mir342 - / - B6 mice supplemented with Socs6 siRNA ( n = 10 ) ( A ) , or Mir342 + / + C3H and Mir342 + / + C3H mice supplemented with SOCS6 - overexpressing vector ( n = 10 ) ( B ) , after aerosol infection with around 400 CFU Mtb . Data are shown as the Kaplan - Meier curves .

O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O O O O B-ORGANISM O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-ORGANISM O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

(A, B) Survival of Mir342-/-B6 and Mir342-/-B6 mice supplemented with Socs6 siRNA (n=10) (A), or Mir342+/+C3H and Mir342+/+C3H mice supplemented with SOCS6-overexpressing vector (n=10) (B), after aerosol infection with around 400 CFU Mtb. Data are shown as the Kaplan-Meier curves.


( C - F ) Relative expressions of chemokines CCL5 , CXCL10 , ICAM1 ( C ) , and Caspase 3 , Caspase 7 , Caspase 8 ( F ) in Mtb - stimulated Socs6 + / + or Socs6 - / - BMDMs were detected by qRT - PCR . Data are shown as the mean ± s . e . m . of n = 3 biological replicates . Cluster analysis of clinical samples .

O O O O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O O O B-ORGANISM O O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_TYPE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O 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-F) Relative expressions of chemokines CCL5, CXCL10, ICAM1 (C), and Caspase 3, Caspase 7, Caspase 8 (F) in Mtb-stimulated Socs6+/+ or Socs6-/- BMDMs were detected by qRT-PCR. Data are shown as the mean ± s.e.m. of n = 3 biological replicates. Cluster analysis of clinical samples.


( C ) Socs6 - / - BMDMs were transfected with plasmids expressing Myc - RIPK3 or Myc - RIPK3 K51A mutant for 24 hours . Afterwards , transfected cells were stimulated with Mtb for 12 hours , cell lysates were collected and immunoprecipitated using an anti - RIPK1 antibody . The recruitment of Caspase 8 , RIPK3 , FADD and MLKL was analyzed by immunoblot . The lower panel represents the immunoblot analysis of whole cell lysates . Representative blots from n = 3 biological replicates are shown .

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(C) Socs6-/- BMDMs were transfected with plasmids expressing Myc-RIPK3 or Myc-RIPK3 K51A mutant for 24 hours. Afterwards, transfected cells were stimulated with Mtb for 12 hours, cell lysates were collected and immunoprecipitated using an anti-RIPK1 antibody. The recruitment of Caspase 8, RIPK3, FADD and MLKL was analyzed by immunoblot. The lower panel represents the immunoblot analysis of whole cell lysates. Representative blots from n = 3 biological replicates are shown.


( D - F ) Cell viabilities ( D ) , cell death mechanisms ( E ) , or Mtb growth rates ( F ) of Mtb - infected Socs6 - / - BMDMs that were transfected with plasmids expressing Myc - RIPK3 or Myc - RIPK3 K51A mutant as indicated . Z - VAD treatment concentration was 20 μM and the treatment time was 24 hours . Data are shown as the mean ± s . e . m . of n = 3 biological replicates .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-ORGANISM B-EXP_ASSAY O O O O O B-ORGANISM O O B-GENEPROD O O O B-CELL_TYPE O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O 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-F) Cell viabilities (D), cell death mechanisms (E), or Mtb growth rates (F) of Mtb-infected Socs6-/- BMDMs that were transfected with plasmids expressing Myc-RIPK3 or Myc-RIPK3 K51A mutant as indicated. Z-VAD treatment concentration was 20 μM and the treatment time was 24 hours. Data are shown as the mean ± s.e.m. of n = 3 biological replicates.


( E - G ) RAW264 . 7 cells were transfected with plasmids expressing A20 and RIPK3 ( E ) , or A20 and RIPK3 mutants ( G ) as indicated , cell lysates were collected for immunoprecipitation and immunoblot . Myc - A20 or Flag - RIPK3 purified from transfected HEK - 293T cells was incubated with ATP , E1 , E2 and ubiquitin as indicated . The in vitro ubiquitination of RIPK3 was analyzed by immunoblot using an anti - Ub antibody ( F ) . Representative blots from n = 3 biological replicates are shown .

O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O

(E-G) RAW264.7 cells were transfected with plasmids expressing A20 and RIPK3 (E), or A20 and RIPK3 mutants (G) as indicated, cell lysates were collected for immunoprecipitation and immunoblot. Myc-A20 or Flag-RIPK3 purified from transfected HEK-293T cells was incubated with ATP, E1, E2 and ubiquitin as indicated. The in vitro ubiquitination of RIPK3 was analyzed by immunoblot using an anti-Ub antibody (F). Representative blots from n = 3 biological replicates are shown.


( A , B ) Production of cytokines TNF - α , IL - 1 , IL - 6 , CXCL15 ( A ) , or chemokines Ccl5 , Cxcl10 , Icam1 ( B ) in the A20 - / - Socs6 + / + , A20 + / + Socs6 + / + , A20 - / - Socs6 - / - and A20 + / + Socs6 - / - BMDMs after Mtb stimulation . Data are shown as the mean ± s . e . m . of n = 3 biological replicates .

O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O B-CELL_TYPE O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O

(A, B) Production of cytokines TNF-α, IL-1, IL-6, CXCL15 (A), or chemokines Ccl5, Cxcl10, Icam1 (B) in the A20-/-Socs6+/+, A20+/+Socs6+/+, A20-/-Socs6-/- and A20+/+Socs6-/- BMDMs after Mtb stimulation. Data are shown as the mean ± s.e.m. of n = 3 biological replicates.


( B ) Percentage infectivity of mouse tail fibroblasts ( MTFs ) infected with WT , RH , RHEV and EV CHIKV ( MOI 10 , n = 3 per group ) harvested at 12 hpi . Data are presented as the means ± SEM .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O O O O O O O B-ORGANISM 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) Percentage infectivity of mouse tail fibroblasts (MTFs) infected with WT, RH, RHEV and EV CHIKV (MOI 10, n=3 per group) harvested at 12 hpi. Data are presented as the means ± SEM.


( C ) Viremia progression in virus - infected mice was monitored over 2 weeks ( n = 11 per group ) . Viremia is detected with CHIKV nsP1 probe via qRT - PCR .

O O O B-EXP_ASSAY O O B-ORGANISM O O B-ORGANISM O O O O O O O O O O O O O B-EXP_ASSAY O O O B-ORGANISM B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

(C) Viremia progression in virus-infected mice was monitored over 2 weeks (n=11 per group). Viremia is detected with CHIKV nsP1 probe via qRT-PCR. Statistical


( D ) joint inflammation in virus - infected mice was monitored over 2 weeks ( n = 11 per group ) .

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(D) joint inflammation in virus-infected mice was monitored over 2 weeks (n=11 per group).


( E ) Compiled percentages of CHIKV - infected ( ZsGreen positive ) leukocytes at 2 days post infection ( dpi ) for the respective groups ( n = 5 per group ) . Background ZsGreen signals are indicated by the dotted line .

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(E) Compiled percentages of CHIKV-infected (ZsGreen positive) leukocytes at 2 days post infection (dpi) for the respective groups (n=5 per group). Background ZsGreen signals are indicated by the dotted line.


( F ) Dot - plots of the localized type - I interferon ( IFN - α and IFN - β ) concentrations at 15 hpi . Data are expressed as the means ± SD .

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(F) Dot-plots of the localized type-I interferon (IFN-α and IFN-β) concentrations at 15 hpi. Data are expressed as the means ± SD.


Cell lysates were collected from the joints of mice infected with WT CHIKV or CHIKV nsP - mutants at 15 hpi , 2 dpi or 6 dpi ( n = 5 per group ) . The concentrations of 36 immune mediators were quantified using a 36 - plex microbead - based immunoassay . Immune mediators are grouped based on function . Each color represents the relative concentration of a particular analyte . Blue and red indicates low and high concentration , respectively .

O O O O O O B-TISSUE O B-ORGANISM O O O B-ORGANISM O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY O O O

Cell lysates were collected from the joints of mice infected with WT CHIKV or CHIKV nsP-mutants at 15 hpi, 2 dpi or 6 dpi (n=5 per group). The concentrations of 36 immune mediators were quantified using a 36-plex microbead-based immunoassay. Immune mediators are grouped based on function. Each color represents the relative concentration of a particular analyte. Blue and red indicates low and high concentration, respectively.


Luminex analytes from WT CHIKV - infected joint cell lysates are categorized based on fold change states ( up , down at 2 dpi relative to mock , and 6 dpi relative to 2 dpi . The numbers in the heatmap represent Log2 fold changes of analytes or genes . Analytes in complete or partial agreement with corresponding gene expression values derived from RNA - sequencing ( RNA - seq ) data appeared predominantly in the ( A ) up then down state ( Mock to 2 dpi , then 2 dpi to 6 dpi , respectively ) ,

B-EXP_ASSAY O O O B-ORGANISM O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Luminex analytes from WT CHIKV-infected joint cell lysates are categorized based on fold change states (up, down at 2 dpi relative to mock, and 6 dpi relative to 2 dpi. The numbers in the heatmap represent Log2 fold changes of analytes or genes. Analytes in complete or partial agreement with corresponding gene expression values derived from RNA-sequencing (RNA-seq) data appeared predominantly in the (A) up then down state (Mock to 2 dpi, then 2 dpi to 6 dpi, respectively),


The study analytes and transcripts in the RNA - seq data from wild - type CHIKV - infected and mock - infected samples in the same states were used . ( C ,

O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O

The study analytes and transcripts in the RNA-seq data from wild-type CHIKV-infected and mock-infected samples in the same states were used. (C,


Luminex analytes from WT CHIKV - infected joint cell lysates are categorized based on fold change states no difference ( ND ) ) at 2 dpi relative to mock , and 6 dpi relative to 2 dpi . The numbers in the heatmap represent Log2 fold changes of analytes or genes . Analytes in complete or partial agreement with corresponding gene expression values derived from RNA - sequencing ( RNA - seq ) data appeared predominantly in the ( D ) up then ND state ( Mock to 2 dpi , then 2 dpi to 6 dpi , respectively ) . † indicates analyte involvement in the canonical pathways . Differences in analyte concentrations during attenuated CHIKV infection are indicated in the checkbox . Yellow represents lower while green represents higher concentrations relative to WT CHIKV .

B-EXP_ASSAY O O O B-ORGANISM O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O B-ORGANISM O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-ORGANISM O

Luminex analytes from WT CHIKV-infected joint cell lysates are categorized based on fold change states no difference (ND)) at 2 dpi relative to mock, and 6 dpi relative to 2 dpi. The numbers in the heatmap represent Log2 fold changes of analytes or genes. Analytes in complete or partial agreement with corresponding gene expression values derived from RNA-sequencing (RNA-seq) data appeared predominantly in the (D) up then ND state (Mock to 2 dpi, then 2 dpi to 6 dpi, respectively). † indicates analyte involvement in the canonical pathways. Differences in analyte concentrations during attenuated CHIKV infection are indicated in the checkbox. Yellow represents lower while green represents higher concentrations relative to WT CHIKV.


The study analytes and transcripts in the RNA - seq data from wild - type CHIKV - infected and mock - infected samples in the same states were used . F )

O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O

The study analytes and transcripts in the RNA-seq data from wild-type CHIKV-infected and mock-infected samples in the same states were used. F)


Joints from WT - CHIKV , RH - CHIKV , RHEV - CHIKV , EV - CHIKV and mock - infected WT C57BL / 6 mice were harvested at 6 dpi . ( A ) Absolute numbers of leukocytes present in the joints as determined by immune - phenotyping . Data are presented as the means ± SD ( n = 5 per group ) . No significance was obtained between the groups by Kruskal - Wallis test with Dunn ' s multiple comparisons .

B-TISSUE O O O B-ORGANISM O O O B-ORGANISM O O O B-ORGANISM O O O B-ORGANISM O O O O O O O O B-ORGANISM O O O O O O O O O O B-EXP_ASSAY O B-CELL_TYPE O O O B-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Joints from WT-CHIKV, RH-CHIKV, RHEV-CHIKV, EV-CHIKV and mock-infected WT C57BL/6 mice were harvested at 6 dpi. (A) Absolute numbers of leukocytes present in the joints as determined by immune-phenotyping. Data are presented as the means ± SD (n=5 per group). No significance was obtained between the groups by Kruskal-Wallis test with Dunn's multiple comparisons.


( B ) Jensen - Shannon divergence heatmap showing the level of divergence between infected groups .

(B) Jensen-Shannon divergence heatmap showing the level of divergence between infected groups.


( C ) Superimposed PhenoGraphs of UMAP transformed FACs data from immune - phenotyping of joint cells from WT - CHIKV , RH - CHIKV , RHEV - CHIKV , EV - CHIKV and mock - infected animals ( n = 5 per group ) .

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(C) Superimposed PhenoGraphs of UMAP transformed FACs data from immune-phenotyping of joint cells from WT-CHIKV, RH-CHIKV, RHEV-CHIKV, EV-CHIKV and mock-infected animals (n=5 per group).


( E ) Cluster ID annotation with heatmap . Cluster IDs are indicated in rows , while surface marker expression levels are indicated by the columns . The color represents the relative mean fluorescence intensity ( MFI ) of a surface marker . Blue and red represents low and high MFI , respectively . Red square and ovals highlight differences of several PhenoGraph clusters in infected joints at 6 dpi .

O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O 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-TISSUE O O O O

(E) Cluster ID annotation with heatmap. Cluster IDs are indicated in rows, while surface marker expression levels are indicated by the columns. The color represents the relative mean fluorescence intensity (MFI) of a surface marker. Blue and red represents low and high MFI, respectively. Red square and ovals highlight differences of several PhenoGraph clusters in infected joints at 6 dpi.


( A ) Anti - CHIKV IgG titers from the vaccination phase were determined with a CHIKV virion - based ELISA . Sera from four animals were pooled and diluted 1 : 500 for the assay . Statistical analyses were performed using two - tailed paired T - test ( * P = 0 . 029 RH - CHIKV 6 dpv , * P = 0 . 029 RH - CHIKV 90 dpv , * P = 0 . 029 RHEV - CHIKV 6 dpv , * P = 0 . 029 RHEV - CHIKV 90 dpv , * P = 0 . 029 EV - CHIKV 90 dpv ) .

O O O O O B-ORGANISM B-SUBCELLULAR B-EXP_ASSAY O O B-EXP_ASSAY O O O O O B-ORGANISM B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O O O O O O O O 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-ORGANISM O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O B-ORGANISM O O O O

(A) Anti-CHIKV IgG titers from the vaccination phase were determined with a CHIKV virion-based ELISA. Sera from four animals were pooled and diluted 1:500 for the assay. Statistical analyses were performed using two-tailed paired T-test (*P =0.029 RH-CHIKV 6 dpv, *P =0.029 RH-CHIKV 90 dpv, *P =0.029 RHEV-CHIKV 6 dpv, *P =0.029 RHEV-CHIKV 90 dpv, *P =0.029 EV-CHIKV 90 dpv).


( B ) Half maximum inhibitory concentration ( IC50 ) of pooled sera collected on 90 dpv were characterized in HEK293T cells infected with WT - CHIKV ( MOI 5 ) . Sera were serially diluted at 1 : 625000 , 1 : 125000 , 1 : 25000 , 1 : 16000 , 1 : 8000 , 1 : 5000 , 1 : 4000 , 1 : 2000 , 1 : 1000 , 1 : 500 , 1 : 250 . Sera dilution to obtain IC50 are shown in parenthesis .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-TISSUE O O O O O O O B-CELL_LINE O O O O O B-ORGANISM O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE O O O B-EXP_ASSAY O O O O O

(B) Half maximum inhibitory concentration (IC50) of pooled sera collected on 90 dpv were characterized in HEK293T cells infected with WT-CHIKV (MOI 5). Sera were serially diluted at 1:625000, 1:125000, 1:25000, 1:16000, 1:8000, 1:5000, 1:4000, 1:2000, 1:1000, 1:500, 1:250. Sera dilution to obtain IC50 are shown in parenthesis.


Viremia were monitored over 2 weeks . Viremia is detected with CHIKV nsP1 probe via qRT - PCR .

B-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O B-ORGANISM B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

Viremia were monitored over 2 weeks. Viremia is detected with CHIKV nsP1 probe via qRT-PCR.


( E ) severity of joint inflammation were monitored over 2 weeks .

O O O O O B-TISSUE B-EXP_ASSAY O O O O O O

(E) severity of joint inflammation were monitored over 2 weeks.


( A ) Representative hematoxylin and eosin ( H & E ) images of inflamed joint footpad at 6 dpc . Ed , region of edema ; red arrow , synovitis ; black arrow , normal synovial membrane ; blue arrow , mild synovial hyperplasia ; D , degeneration of muscle ; N , necrosis of muscle ; R , regeneration of muscle .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY B-TISSUE I-TISSUE O O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O B-TISSUE I-TISSUE O O O O O B-TISSUE B-EXP_ASSAY O O O B-EXP_ASSAY O B-TISSUE O O O B-EXP_ASSAY O B-TISSUE O O O B-EXP_ASSAY O B-TISSUE O

(A) Representative hematoxylin and eosin (H&E) images of inflamed joint footpad at 6 dpc. Ed, region of edema; red arrow, synovitis; black arrow, normal synovial membrane; blue arrow, mild synovial hyperplasia; D, degeneration of muscle; N, necrosis of muscle; R, regeneration of muscle.


( B ) Histopathological scoring of edema , inflammation in different regions of the joint footpad and muscle pathology of WT - CHIKV challenged animals ( n = 5 ) at 6 dpc , performed blind . Three sections from each joint footpad were scored . The data are expressed as the means ± SD . Data were analyzed by one - way ANOVA with Tukey post - test ; Edema ( * * * P = 0 . 000002 WT - CHIKV versus PBS , * * * P = 0 . 000004 RH - CHIKV versus PBS ) , Muscle necrosis ( * * P = 0 . 005521 WT - CHIKV versus PBS , * P = 0 . 02628 RH - CHIKV versus PBS ) , Muscle regeneration ( * * * P < 0 . 000001 WT - CHIKV versus PBS , * * * P < 0 . 000001 WT - CHIKV versus RH - CHIKV ) , Muscle inflammation ( * P = 0 . 040258 WT - CHIKV versus PBS , * P = 0 . 040258 RH - CHIKV versus PBS ) , Synovial membrane inflammation ( * P = 0 . 014457 WT - CHIKV versus PBS , * P = 0 . 014457 RH - CHIKV versus PBS ) , Subcutaneous area inflammation ( * * * P = 0 . 00004 WT - CHIKV versus PBS , * * * P = 0 . 000074 RH - CHIKV versus PBS ) .

O O O B-EXP_ASSAY O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O B-TISSUE I-TISSUE O B-TISSUE O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O 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-EXP_ASSAY O O O O O O O O O O O B-ORGANISM O B-SMALL_MOLECULE O O O O O O O O O O O B-ORGANISM O B-SMALL_MOLECULE O O B-TISSUE B-EXP_ASSAY O O O O O O O O O O B-ORGANISM O B-SMALL_MOLECULE O O O O O O O O O B-ORGANISM O B-SMALL_MOLECULE O O B-TISSUE B-EXP_ASSAY O O O O O O O O O O O B-ORGANISM O B-SMALL_MOLECULE O O O O O O O O O O O B-ORGANISM O O O B-ORGANISM O O B-TISSUE B-EXP_ASSAY O O O O O O O O O B-ORGANISM O B-SMALL_MOLECULE O O O O O O O O O B-ORGANISM O B-SMALL_MOLECULE O O B-TISSUE I-TISSUE B-EXP_ASSAY O O O O O O O O O B-ORGANISM O B-SMALL_MOLECULE O O O O O O O O O B-ORGANISM O B-SMALL_MOLECULE O O B-TISSUE O B-EXP_ASSAY O O O O O O O O O O O B-ORGANISM O B-SMALL_MOLECULE O O O O O O O O O O O B-ORGANISM O B-SMALL_MOLECULE O O

(B) Histopathological scoring of edema, inflammation in different regions of the joint footpad and muscle pathology of WT-CHIKV challenged animals (n=5) at 6 dpc, performed blind. Three sections from each joint footpad were scored. The data are expressed as the means ± SD. Data were analyzed by one-way ANOVA with Tukey post-test; Edema (***P =0.000002 WT-CHIKV versus PBS, ***P =0.000004 RH-CHIKV versus PBS), Muscle necrosis (**P =0.005521 WT-CHIKV versus PBS, *P =0.02628 RH-CHIKV versus PBS), Muscle regeneration (***P <0.000001 WT-CHIKV versus PBS, ***P <0.000001 WT-CHIKV versus RH-CHIKV), Muscle inflammation (*P =0.040258 WT-CHIKV versus PBS, *P =0.040258 RH-CHIKV versus PBS), Synovial membrane inflammation (*P =0.014457 WT-CHIKV versus PBS, *P =0.014457 RH-CHIKV versus PBS), Subcutaneous area inflammation (***P =0.00004 WT-CHIKV versus PBS, ***P =0.000074 RH-CHIKV versus PBS).


Three - week - old WT C57BL / 6 mice were infected with 1E6 PFU of WT - ONNV at the metatarsal region of the footpad . ( A ) Viremia progression in ONNV - infected mice were monitored over 2 weeks ( n = 5 ) . Viremia is detected with ONNV nsP1 probe via qRT - PCR .

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Three-week-old WT C57BL/6 mice were infected with 1E6 PFU of WT-ONNV at the metatarsal region of the footpad. (A) Viremia progression in ONNV-infected mice were monitored over 2 weeks (n=5). Viremia is detected with ONNV nsP1 probe via qRT-PCR.


Three - week - old WT C57BL / 6 mice were infected with 1E6 PFU of WT - ONNV at the metatarsal region of the footpad . ( B ) joint inflammation in ONNV - infected mice were monitored over 2 weeks ( n = 5 ) .

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Three-week-old WT C57BL/6 mice were infected with 1E6 PFU of WT-ONNV at the metatarsal region of the footpad. (B) joint inflammation in ONNV-infected mice were monitored over 2 weeks (n=5).


Three - week - old WT C57BL / 6 mice were vaccinated subcutaneously with 1E6 PFU of WT - CHIKV or attenuated CHIKV ( RH ) at the metatarsal region of the footpad . Challenge with 1E6 PFU of WT - ONNV was performed via the same route at 3 months post - vaccination . Viremia progression of the mice were monitored over 2 weeks ( n = 5 per group ) . Viremia is detected with ONNV nsP1 probe via qRT - PCR .

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Three-week-old WT C57BL/6 mice were vaccinated subcutaneously with 1E6 PFU of WT-CHIKV or attenuated CHIKV (RH) at the metatarsal region of the footpad. Challenge with 1E6 PFU of WT-ONNV was performed via the same route at 3 months post-vaccination. Viremia progression of the mice were monitored over 2 weeks (n=5 per group). Viremia is detected with ONNV nsP1 probe via qRT-PCR.


D ) Three - week - old WT C57BL / 6 mice were vaccinated subcutaneously with 1E6 PFU of WT - CHIKV or attenuated CHIKV ( RH ) at the metatarsal region of the footpad . Challenge with 1E6 PFU of WT - ONNV was performed via the same route at 3 months post - vaccination . ( D ) joint inflammation of the mice were monitored over 2 weeks ( n = 5 per group ) .

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D) Three-week-old WT C57BL/6 mice were vaccinated subcutaneously with 1E6 PFU of WT-CHIKV or attenuated CHIKV (RH) at the metatarsal region of the footpad. Challenge with 1E6 PFU of WT-ONNV was performed via the same route at 3 months post-vaccination. (D) joint inflammation of the mice were monitored over 2 weeks (n=5 per group).


( A ) C - circle assay for tumor samples . The presence of C - circles was tested in 22 osteosarcomas by Rolling Circle Amplification ( RCA ) assay using Φ29 DNA polymerase . Φ29 DNA polymerase negative controls and Φ29 DNA polymerase - based reactions starting respectively with 75 , 37 . 5 , and 18 . 75 ng of DNA were applied to dot blots and C - circles were detected by hybridization with a 32P - ( CCCTAA ) 4 telomeric probe . U2OS and HeLa cells correspond to positive and negative control samples , respectively .

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(A) C-circle assay for tumor samples. The presence of C-circles was tested in 22 osteosarcomas by Rolling Circle Amplification (RCA) assay using Φ29 DNA polymerase. Φ29 DNA polymerase negative controls and Φ29 DNA polymerase-based reactions starting respectively with 75, 37.5, and 18.75 ng of DNA were applied to dot blots and C-circles were detected by hybridization with a 32P-(CCCTAA)4 telomeric probe. U2OS and HeLa cells correspond to positive and negative control samples, respectively.


( B ) Representative images of telomere FISH ( red ) and PML IF ( green ) colocalizations ( APBs ) in osteosarcoma tumor samples . DNA is counterstained blue with DAPI . APBs are indicated by white arrows . Scale bars are 5 μm .

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(B) Representative images of telomere FISH (red) and PML IF (green) colocalizations (APBs) in osteosarcoma tumor samples. DNA is counterstained blue with DAPI. APBs are indicated by white arrows. Scale bars are 5 μm.


( C ) Representative image of TeSLA Southern Blot of two representative samples of ALT - positive ( left ) and telomerase - positive ( right ) osteosarcomas . Nine TeSLA PCRs ( 30 pg each reaction ) were done for each DNA sample . DIG - labelled MW ladder has been added a posteriori . ( D ) Plot of cumulative TeSLA fragment sizes ( in kb ) in ALT - positive ( n = 16 ) and ALT - negative ( n = 6 ) osteosarcomas . Points and error bars ( ± SEM ) represent cumulative percentage of TeSLA fragments from all the TeSLA PCRs ( n = 9 per tumor sample ) from all the patients in both groups ( n = 16 in ALT - positive group , and n = 6 in ALT - negative group ) . The Kolmogorov - Smirnov test was applied to identify statistical differences in TeSLA fragments distributions ( p = 0 . 0016 ) . ( E ) Individual TeSLa fragment lengths in ALT - positive ( n = 16 ) and ALT - negative ( n = 6 ) osteosarcomas . Each dot represents a TeSLA fragment , bars represent the median value for individual patient ' s samples .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-SUBCELLULAR O O O O O B-DISEASE O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-EXP_ASSAY 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-DISEASE O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-DISEASE O O O O O B-EXP_ASSAY O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O

(C) Representative image of TeSLA Southern Blot of two representative samples of ALT-positive (left) and telomerase-positive (right) osteosarcomas. Nine TeSLA PCRs (30 pg each reaction) were done for each DNA sample. DIG-labelled MW ladder has been added a posteriori. (D) Plot of cumulative TeSLA fragment sizes (in kb) in ALT-positive (n=16) and ALT-negative (n=6) osteosarcomas. Points and error bars (± SEM) represent cumulative percentage of TeSLA fragments from all the TeSLA PCRs (n=9 per tumor sample) from all the patients in both groups (n=16 in ALT-positive group, and n=6 in ALT-negative group). The Kolmogorov-Smirnov test was applied to identify statistical differences in TeSLA fragments distributions (p=0.0016). (E) Individual TeSLa fragment lengths in ALT-positive (n=16) and ALT-negative (n=6) osteosarcomas. Each dot represents a TeSLA fragment, bars represent the median value for individual patient's samples.


( A ) Oncoprint graph showing the distribution of mutations ( green rectangles ) and copy number variations ( red rectangle , amplification ; blue rectangle , copy loss ) in all samples . Tumors are distributed according to ALT - positivity ( ALT + ) or - negativity ( ALT - ) and ATRX status ( ATRX - mutated or ATRX - wt ) .

O O O O O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O 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-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O O

(A) Oncoprint graph showing the distribution of mutations (green rectangles) and copy number variations (red rectangle, amplification; blue rectangle, copy loss) in all samples. Tumors are distributed according to ALT-positivity (ALT+) or -negativity (ALT-) and ATRX status (ATRX-mutated or ATRX-wt).


( B ) Tissue sections of two representative high - grade osteosarcomas with HES ( hematoxylin eosin saffron ) staining ( left panel ) , anti - ATRX ( HPA064684 ) immunochemistry ( middle panel ) , and anti - DAXX ( HPA008736 ) immunochemistry ( right panel ) , showing ATRX protein expression in two ALT - positive samples . In the top panel , intratumoral ATRX expression is negative ( positive control osteoclasts are shown ) . In the bottom panel , intratumoral ATRX expression is high . Scale bars are 250 μm .

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(B) Tissue sections of two representative high-grade osteosarcomas with HES (hematoxylin eosin saffron) staining (left panel), anti-ATRX (HPA064684) immunochemistry (middle panel), and anti-DAXX (HPA008736) immunochemistry (right panel), showing ATRX protein expression in two ALT-positive samples. In the top panel, intratumoral ATRX expression is negative (positive control osteoclasts are shown). In the bottom panel, intratumoral ATRX expression is high. Scale bars are 250 μm.


( A ) Copy - number - alteration ( CNA ) profiles of osteosarcomas . Representation of the GISTIC analysis with false discovery rate ( FDR ( q - value ) ) for all cases ( n = 22 ; left panel ) , ALT - positive / ATRX - wt cases ( n = 11 ; middle panel ) , and ALT - positive / ATRX - mutated cases ( n = 5 ; right panel ) .

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(A) Copy-number-alteration (CNA) profiles of osteosarcomas. Representation of the GISTIC analysis with false discovery rate (FDR (q-value)) for all cases (n=22; left panel), ALT-positive/ATRX-wt cases (n=11; middle panel), and ALT-positive/ATRX-mutated cases (n=5; right panel).


( B ) Gain / amplification of TOP3A gene region ( 17p11 ) is a genomic signature of ATRX - wt osteosarcomas . For each ATRX - mutated ( top part ) and ATRX - wt ( bottom part ) case , from the left to the right , the chromosome 17 and regional genomic profiles were established , with CGH Analytics software . A focus was made within the genomic interval ( 17 . 25 - 19 . 11 Mb ) of the short arm of chromosome 17 ( hg38 human genome mapping ; Build 38 from NCBI , December 2013 version ) including TOP3 . Color profiles corresponding to the different tumors are defined at the top of each group . Most ATRX - wt cases show gain or amplification of this region ( bottom part ) , whereas no ATRX - mutated cases display this alteration ( top part ) .

O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O B-DISEASE O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O B-EXP_ASSAY O O 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-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O

(B) Gain/amplification of TOP3A gene region (17p11) is a genomic signature of ATRX-wt osteosarcomas. For each ATRX-mutated (top part) and ATRX-wt (bottom part) case, from the left to the right, the chromosome 17 and regional genomic profiles were established, with CGH Analytics software. A focus was made within the genomic interval (17.25-19.11 Mb) of the short arm of chromosome 17 (hg38 human genome mapping; Build 38 from NCBI, December 2013 version) including TOP3. Color profiles corresponding to the different tumors are defined at the top of each group. Most ATRX-wt cases show gain or amplification of this region (bottom part), whereas no ATRX-mutated cases display this alteration (top part).


( C ) Chromosome 17 copy number variation analysis showing amplification / gain of the TOP3A region that do not extend into the TP53 gene in ALT ATRX - wt tumors ( left panel ) , and amplification of the TOP3A region for which TP53 region is at the edge of the amplification / gain in ALT - positive ATRX - wt tumors ( right panel ) .

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(C) Chromosome 17 copy number variation analysis showing amplification/gain of the TOP3A region that do not extend into the TP53 gene in ALT ATRX-wt tumors (left panel), and amplification of the TOP3A region for which TP53 region is at the edge of the amplification/gain in ALT-positive ATRX-wt tumors (right panel).


( A ) Volcano plot of the supervised analysis of mRNA expression profiles of osteosarcomas according to ALT - positive vs . ALT - negative samples , showing upregulated ( in red ) and downregulated ( in green ) genes . Twelve tumors had sufficient RNA quality to be analyzed : seven ALT - positive ATRX - wt , two ALT - positive / ATRX - mutated and three ALT - negative .

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(A) Volcano plot of the supervised analysis of mRNA expression profiles of osteosarcomas according to ALT-positive vs. ALT-negative samples, showing upregulated (in red) and downregulated (in green) genes. Twelve tumors had sufficient RNA quality to be analyzed: seven ALT-positive ATRX-wt, two ALT-positive/ATRX-mutated and three ALT-negative.


( B ) TOP3A gene expression according to ALT and ATRX status : ALT + / ATRX - wt ( n = 7 ) , ALT + / ATRX - mut ( n = 2 ) and ALT - ( n = 3 ) . Box - and - whisker plot were defined with default parameters by median value ( central band at the 50th percentile ) , interquartile ranges ( IQR , box limited by 25th and 75th percentile ) and whisker boundaries defined by minimum and maximum value . An ANOVA statistical test , and Tukey ' s range test were used to compare modalities .

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(B) TOP3A gene expression according to ALT and ATRX status: ALT+/ATRX-wt (n=7), ALT+/ATRX-mut (n=2) and ALT- (n=3). Box-and-whisker plot were defined with default parameters by median value (central band at the 50th percentile), interquartile ranges (IQR, box limited by 25th and 75th percentile) and whisker boundaries defined by minimum and maximum value. An ANOVA statistical test, and Tukey's range test were used to compare modalities.


( A ) Western blot showing ATRX protein expression in nine osteosarcoma cell lines and HTC116 as positive control .

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(A) Western blot showing ATRX protein expression in nine osteosarcoma cell lines and HTC116 as positive control.


( B ) Western blot showing TOP3A protein expression in nine osteosarcoma cell lines and NY siTOP3A as control .

(B) Western blot showing TOP3A protein expression in nine osteosarcoma cell lines and NY siTOP3A as control.


( D ) C - circle assay showing inverse effect of ATRX ectopic expression on C - circle levels in ATRX - mutated and ATRX - wt cell lines . Error bars represent the mean ± SEM from n = 2 experiments , n . s . = non - significant , * p < 0 . 05 , Mann - Whitney test .

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(D) C-circle assay showing inverse effect of ATRX ectopic expression on C-circle levels in ATRX-mutated and ATRX-wt cell lines. Error bars represent the mean ± SEM from n = 2 experiments, n.s. = non-significant, *p < 0.05, Mann-Whitney test.


( E ) C - circle assay showing rescue of ATRX ectopic expression by TOP3A overexpression in U2OS osteosarcoma cells Error bars represent the mean ± SEM from n = 2 experiments , n . s . = non - significant , * p < 0 . 05 , Mann - Whitney test . Western blot and quantification of TOP3A expression is shown for U2OS cells overexpressing TOP3A .

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(E) C-circle assay showing rescue of ATRX ectopic expression by TOP3A overexpression in U2OS osteosarcoma cells Error bars represent the mean ± SEM from n = 2 experiments, n.s. = non-significant, *p < 0.05, Mann-Whitney test. Western blot and quantification of TOP3A expression is shown for U2OS cells overexpressing TOP3A.


( B ) Representative images of telomeric DNA ( green ) and PML protein ( red ) colocalizations ( APBs ) in osteosarcoma and in vitro - immortalized ATRX - mutated or ATRX - wt cell lines according to TOP3A KD . Scale bars are 5 μm . ( C ) Quantification of APB frequency in osteosarcoma and in vitro - immortalized ATRX - mutated or ATRX - wt cell lines according to TOP3A KD ; Error bars represent the mean ± SEM from n = 3 experiments , n = 150 cells scored per treatment , n . s . , non - significant ; Mann - Whitney test . ( D ) Effect of TOP3A KD on Telomere Dysfunction - Induced Foci ( TIFs ) in osteosarcoma and in vitro - immortalized cell lines ; Error bars represent the mean ± SEM from n = 3 experiments , n = 150 cells scored per treatment , * p < 0 . 05 , * * p < 0 . 005 ; Mann - Whitney test .

O O O O O O B-SUBCELLULAR O O O O O B-GENEPROD O O O O B-EXP_ASSAY O B-SUBCELLULAR O O B-DISEASE O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-SUBCELLULAR O O B-DISEASE O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O B-SUBCELLULAR O O O O O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative images of telomeric DNA (green) and PML protein (red) colocalizations (APBs) in osteosarcoma and in vitro-immortalized ATRX-mutated or ATRX-wt cell lines according to TOP3A KD. Scale bars are 5 μm. (C) Quantification of APB frequency in osteosarcoma and in vitro-immortalized ATRX-mutated or ATRX-wt cell lines according to TOP3A KD; Error bars represent the mean ± SEM from n = 3 experiments, n = 150 cells scored per treatment, n.s., non-significant; Mann-Whitney test. (D) Effect of TOP3A KD on Telomere Dysfunction-Induced Foci (TIFs) in osteosarcoma and in vitro-immortalized cell lines; Error bars represent the mean ± SEM from n = 3 experiments, n = 150 cells scored per treatment, *p < 0.05, **p < 0.005; Mann-Whitney test.


( E ) Distributions of TeSLA fragments in osteosarcoma and in vitro - immortalized ATRX - mutated or ATRX - wt cell lines according to TOP3A KD ( left panel ; each dot represents a TeSLA fragment ) , and representative TeSLA Southern Blot image for NY ( right panel ) .

O O O O O B-EXP_ASSAY O O B-DISEASE O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE O O O O O

(E) Distributions of TeSLA fragments in osteosarcoma and in vitro-immortalized ATRX-mutated or ATRX-wt cell lines according to TOP3A KD (left panel; each dot represents a TeSLA fragment), and representative TeSLA Southern Blot image for NY (right panel).


( A ) FISH - IF of telomeres ( in red ) and BLM ( in green ) in TOP3A - depleted cells BLM foci at telomeres are indicated by white arrows . Error bars represent the mean ± SEM from n = 3 experiments , n = 150 cells scored per treatment , n . s . = non - significant , * * p < 0 . 01 , Mann - Whitney test . Scale bars are 5 μm .

O O O B-EXP_ASSAY O B-EXP_ASSAY O B-SUBCELLULAR O O O O O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) FISH-IF of telomeres (in red) and BLM (in green) in TOP3A-depleted cells BLM foci at telomeres are indicated by white arrows. Error bars represent the mean ± SEM from n = 3 experiments, n = 150 cells scored per treatment, n.s. = non-significant, **p < 0.01, Mann-Whitney test. Scale bars are 5 μm.


( B ) ATSA assay assessing ALT - mediated telomeric DNA synthesis ( telomere in red , EdU in purple ) in PML ( in green ) according to TOP3A KD . EdU signal at APB ( telomere / PML ) are indicated by white arrows . Approximately 150 cells were divided into five groups ( 0 , 1 - 2 , 3 - 4 , 5 - 6n and ≥ 7 ) based on the number of EdU + APBs . Scale bars are 5 μm .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O B-SUBCELLULAR O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O O 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-SMALL_MOLECULE O B-SUBCELLULAR O O O O O O O O

(B) ATSA assay assessing ALT-mediated telomeric DNA synthesis (telomere in red, EdU in purple) in PML (in green) according to TOP3A KD. EdU signal at APB (telomere/PML) are indicated by white arrows. Approximately 150 cells were divided into five groups (0,1-2, 3-4, 5-6n and ≥7) based on the number of EdU+APBs. Scale bars are 5 μm.


Representative low ( left ) and high ( right ) power images of H & E - stained lung sections from KrasG12D , KrasG12D : Adam17ex / + and KrasG12D : Adam17ex / ex mice at 6 weeks post Ad - Cre inhalation . Scale bars , 3mm ( left ) and 300μm ( right ) . Quantification of lung parenchyma area occupied by tumor lesions ( B ) , and tumor incidence ( C ) , per whole mouse lung in the indicated genotypes ( n = 6 per genotype ) .

O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-TISSUE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O O O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O B-ORGANISM B-TISSUE O O O O O O O O O O O O

Representative low (left) and high (right) power images of H&E-stained lung sections from KrasG12D, KrasG12D:Adam17ex/+ and KrasG12D:Adam17ex/ex mice at 6 weeks post Ad-Cre inhalation. Scale bars, 3mm (left) and 300μm (right). Quantification of lung parenchyma area occupied by tumor lesions (B), and tumor incidence (C), per whole mouse lung in the indicated genotypes (n = 6 per genotype).


Representative images of TTF - 1 - stained lung sections from the indicated genotypes . Scale bar , 100μm . Quantification of TTF - 1 - positive cells / high power field ( HPF ) in lungs of the indicated genotypes ( n = 6 per genotype ) .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O

Representative images of TTF-1-stained lung sections from the indicated genotypes. Scale bar, 100μm. Quantification of TTF-1-positive cells/high power field (HPF) in lungs of the indicated genotypes (n = 6 per genotype).