F Immunoblot analysis of lysates from HEK293 cells cotransfected with Flag - AIM2 , - NLRP3 , - ASC and HA - CCDC50 or empty control vector . The expression levels of Flag - tagged proteins and Actin were quantitated using ImageJ software . Data information : actin was used as a loading control . All the experiments were repeated at least once .

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F Immunoblot analysis of lysates from HEK293 cells cotransfected with Flag- AIM2, -NLRP3, -ASC and HA-CCDC50 or empty control vector. The expression levels of Flag-tagged proteins and Actin were quantitated using ImageJ software. Data information: actin was used as a loading control. All the experiments were repeated at least once.


G Immunoblot analysis of HEK293 cells transfected with FLAG - NLRP3 and an increasing amount of HA - CCDC50 plasmids . Data information : actin was used as a loading control . All the experiments were repeated at least once .

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G Immunoblot analysis of HEK293 cells transfected with FLAG-NLRP3 and an increasing amount of HA-CCDC50 plasmids. Data information: actin was used as a loading control. All the experiments were repeated at least once.


H Immunoblot analysis of lysates from CCDC50 - KO and CCDC50 - WT THP - 1 cells induced with LPS plus ATP and then treated with cycloheximide ( CHX ) for indicated time points . The expression levels of NLRP3 and Actin were quantitated using ImageJ software . Data information : actin was used as a loading control . All the experiments were repeated at least once .

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H Immunoblot analysis of lysates from CCDC50-KO and CCDC50-WT THP-1 cells induced with LPS plus ATP and then treated with cycloheximide (CHX) for indicated time points. The expression levels of NLRP3 and Actin were quantitated using ImageJ software. Data information: actin was used as a loading control. All the experiments were repeated at least once.


I HEK293 cells were co - transfected with Flag - NLRP3 and HA - CCDC50 or empty control vector for 24 h and then treated with MG132 , NH4Cl , 3 - MA , CQ or DMSO for another 6 h . Data information : actin was used as a loading control . All the experiments were repeated at least once .

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I HEK293 cells were co-transfected with Flag-NLRP3 and HA-CCDC50 or empty control vector for 24 h and then treated with MG132, NH4Cl, 3-MA, CQ or DMSO for another 6 h. Data information: actin was used as a loading control. All the experiments were repeated at least once.


J Immunoblot analysis of NLRP3 in CCDC50 - WT and CCDC50 - KO THP - 1 cells stimulated with LPS in combination with ATP . Data information : actin was used as a loading control . All the experiments were repeated at least once .

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J Immunoblot analysis of NLRP3 in CCDC50-WT and CCDC50-KO THP-1 cells stimulated with LPS in combination with ATP. Data information: actin was used as a loading control. All the experiments were repeated at least once.


K Western blotting analysis of ATG5 wild - type or knockout HEK293T cells co - transfected with Flag - NLRP3 and HA - CCDC50 or empty control vector for 24 h . Data information : actin was used as a loading control . All the experiments were repeated at least once .

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K Western blotting analysis of ATG5 wild-type or knockout HEK293T cells co-transfected with Flag-NLRP3 and HA-CCDC50 or empty control vector for 24 h. Data information: actin was used as a loading control. All the experiments were repeated at least once.


L Immunoblot analyses of NLRP3 in HEK293 cells in the presence of CCDC50 or its mutant CCDC50 with the deletion of MIUs . Data information : actin was used as a loading control . All the experiments were repeated at least once .

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L Immunoblot analyses of NLRP3 in HEK293 cells in the presence of CCDC50 or its mutant CCDC50 with the deletion of MIUs. Data information: actin was used as a loading control. All the experiments were repeated at least once.


A Denature - IP ( with anti - Flag ) and immunoblot analysis of polyubiquitinated NLRP3 in HEK293 cells transfected with Flag - NLRP3 , HA - Ub ( ubiquitin ) as well as HA - CCDC50 or empty vector for 24 h .

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A Denature-IP (with anti-Flag) and immunoblot analysis of polyubiquitinated NLRP3 in HEK293 cells transfected with Flag-NLRP3, HA-Ub (ubiquitin) as well as HA- CCDC50 or empty vector for 24 h.


B Denature - IP analysis ( with anti - Flag ) of K63 - linked polyubiquitination of NLRP3 in cotransfected HEK293 cells left untreated or treated with CQ for 6 h .

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B Denature-IP analysis (with anti-Flag) of K63-linked polyubiquitination of NLRP3 in cotransfected HEK293 cells left untreated or treated with CQ for 6 h.


C The colocalization analysis of K63 - Ub ( Red ) , Flag - NLRP3 ( Purple ) and GFP - CCDC50 in HeLa cells ; scale bars , 5 μM .

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C The colocalization analysis of K63-Ub (Red), Flag-NLRP3 (Purple) and GFP- CCDC50 in HeLa cells; scale bars, 5 μM.


D , E Images of HeLa cells transfected with CCDC50 ( red ) ( D ) or K63 - Ub ( red ) ( E ) and Flag - NLRP3 ( Purple ) and GFP - LC3B for 24 h and then induced with LPS plus ATP ; scale bars , 5 μM .

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D,E Images of HeLa cells transfected with CCDC50 (red) (D) or K63-Ub (red) (E) and Flag-NLRP3 (Purple) and GFP-LC3B for 24 h and then induced with LPS plus ATP; scale bars, 5 μM.


F The colocalization analysis of NLRP3 and LC3B in CCDC50 - WT and CCDC50 - KO HeLa cells stimulated with LPS plus ATP ; scale bars , 5 μM . Data information : The nuclei were stained with DAPI ; data are representative of three individual experiments .

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F The colocalization analysis of NLRP3 and LC3B in CCDC50-WT and CCDC50- KO HeLa cells stimulated with LPS plus ATP; scale bars, 5 μM. Data information: The nuclei were stained with DAPI; data are representative of three individual experiments.


G The oligomerization analysis of NLRP3 in CCDC50 wild - type and knockout THP - 1 cells stimulated with LPS as well as ATP . LPS - primed ( 1 μg / ml , 3 h ) THP - 1 cells were stimulated with ATP ( 5 mM ) for another 30 min and then were collected and lysed . The cell lysates were centrifuged and separated into soluble and insoluble fractions . The soluble fraction was analyzed by SDS - PAGE . The insoluble fraction was analyzed by native - PAGE .

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G The oligomerization analysis of NLRP3 in CCDC50 wild-type and knockout THP-1 cells stimulated with LPS as well as ATP. LPS-primed (1 μg/ml, 3 h) THP-1 cells were stimulated with ATP (5 mM) for another 30 min and then were collected and lysed. The cell lysates were centrifuged and separated into soluble and insoluble fractions. The soluble fraction was analyzed by SDS-PAGE. The insoluble fraction was analyzed by native-PAGE.


H The oligomerization analysis of NLRP3 in HEK293 cells transfected with plasmids expressing Flag - NLRP3 , HA - CCDC50 or empty vector for 24 h , and then treated with DMSO or CQ for 6 h . After treatment , the cells were collected and lysed . The cell lysates were centrifuged and separated into soluble and insoluble fractions . The soluble fraction was analyzed by SDS - PAGE . The insoluble fraction was analyzed by native - PAGE .

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H The oligomerization analysis of NLRP3 in HEK293 cells transfected with plasmids expressing Flag-NLRP3, HA-CCDC50 or empty vector for 24 h, and then treated with DMSO or CQ for 6 h. After treatment, the cells were collected and lysed. The cell lysates were centrifuged and separated into soluble and insoluble fractions. The soluble fraction was analyzed by SDS-PAGE. The insoluble fraction was analyzed by native-PAGE.


I Co - IP ( with anti - HA ) and immunoblot analysis of the interaction between NLRP3 and ASC in HEK293 cells transfected with indicated plasmids for 24 h , and treated with or without CQ for 6 h .

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I Co-IP (with anti-HA) and immunoblot analysis of the interaction between NLRP3 and ASC in HEK293 cells transfected with indicated plasmids for 24 h, and treated with or without CQ for 6 h.


J Confocal microscopy analysis of colocalization between NLRP3 and ASC in CCDC50 - WT and CCDC50 - KO HeLa cells stimulated with LPS plus ATP ; scale bars , 5 μM . Data information : The nuclei were stained with DAPI ; data are representative of three individual experiments .

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J Confocal microscopy analysis of colocalization between NLRP3 and ASC in CCDC50-WT and CCDC50-KO HeLa cells stimulated with LPS plus ATP; scale bars, 5 μM. Data information: The nuclei were stained with DAPI; data are representative of three individual experiments.


K Confocal microscopy analysis of ASC speck formation in CCDC50 - WT and CCDC50 - KO HeLa cells stimulated with LPS plus ATP ; scale bars , 5 μM . Data information : The nuclei were stained with DAPI ; data are representative of three individual experiments .

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K Confocal microscopy analysis of ASC speck formation in CCDC50-WT and CCDC50-KO HeLa cells stimulated with LPS plus ATP; scale bars, 5 μM. Data information: The nuclei were stained with DAPI; data are representative of three individual experiments.


( A ) Expression patterns of Hoxa5 and Hoxc8 in MNs ( demarcated by white dashed lines ) along the rostrocaudal axis of the spinal cord ( cervical C4 to thoracic T1 ) from mouse embryos from E9 . 5 to E12 . 5 . Pink scale bars : 12 . 5 μm for E9 . 5 ; 12 . 5 μm for E10 . 5 ; 25 μm for E11 . 5 ; and 50 μm for E12 . 5 .

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(A) Expression patterns of Hoxa5 and Hoxc8 in MNs (demarcated by white dashed lines) along the rostrocaudal axis of the spinal cord (cervical C4 to thoracic T1) from mouse embryos from E9.5 to E12.5. Pink scale bars: 12.5 μm for E9.5; 12.5 μm for E10.5; 25 μm for E11.5; and 50 μm for E12.5.


Expression patterns of Hoxa5 and Hoxc8 mRNAs , as revealed by in situ hybridization , aligned with corresponding sections showing Hoxa5 and Hoxc8 immunostainings in MNs ( demarcated with pink / white dashed lines ) along the cervical spinal cord ( cervical C5 to C7 ) in mouse embryos at E12 . 5 . Pink scale bar , 50 μm . White and pink dash lines demarcate the postmitotic MN region .

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Expression patterns of Hoxa5 and Hoxc8 mRNAs, as revealed by in situ hybridization, aligned with corresponding sections showing Hoxa5 and Hoxc8 immunostainings in MNs (demarcated with pink/white dashed lines) along the cervical spinal cord (cervical C5 to C7) in mouse embryos at E12.5. Pink scale bar, 50 μm. White and pink dash lines demarcate the postmitotic MN region.


Dot - plots showing expression of genes ( rows ) that distinguish MN subtypes ( columns ) . The height of each colored shape is proportional to the percentage of cells expressing the marker ( ≥ 1 UMI ) , and its width reflects the average transcript count within expressing cells .

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Dot-plots showing expression of genes (rows) that distinguish MN subtypes (columns). The height of each colored shape is proportional to the percentage of cells expressing the marker (≥1 UMI), and its width reflects the average transcript count within expressing cells.


Immunostaining ( C ) of EBs 96 h after ESC differentiation , and quantification ( D ) of Hoxa5on in MNs ( Hb9on ) .

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Immunostaining (C) of EBs 96 h after ESC differentiation, and quantification (D) of Hoxa5on in MNs (Hb9on).


Hoxa5 expression upon miR - 27 knockdown , with exposure to RAhi for 72 h , or with exposure to RAhi for 48 h , and then switching to RAlow plus RAR inhibitor ( 1 µM ) for a further 24 h , or with exposure to RAmed for 72 h .

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Hoxa5 expression upon miR-27 knockdown, with exposure to RAhi for 72 h, or with exposure to RAhi for 48 h, and then switching to RAlow plus RAR inhibitor (1 µM) for a further 24 h, or with exposure to RAmed for 72 h.


( C ) Immunostaining at cervical spinal cord sections reveals Hoxa5 expansion into the MNs of cervical C6 / 7 segments of miR - 23 ~ 27 ~ 24 double KO ( DKO ) mice . miR - 23a ~ 27a ~ 24 - 2 + / - ; miR - 23b ~ 27b ~ 24 - 1 - / - mice were used as a control ( ctrl ) . ( E ) Conversely , Hoxc8 is shifted into the MNs of cervical C5 segments of both miR - 196a2 / b DKO and miR - 196a1 / a2 / b triple knockout ( TKO ) embryos . Age - matched wild type embryos were used as controls . Scale bar represents 50 μm . White dash lines demarcate the postmitotoic MN domain ( D , F ) Quantifications of Hoxa5on , Hoxc8on , and Hoxa5on Hoxc8on cells in the cervical spinal cord of control and knockout embryos ( mean ± SD , N = 3 embryos , * p < 0 . 01 , Student ' s t - tests ) .

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(C) Immunostaining at cervical spinal cord sections reveals Hoxa5 expansion into the MNs of cervical C6/7 segments of miR-23~27~24 double KO (DKO) mice. miR-23a~27a~24-2+/-;miR-23b~27b~24-1-/- mice were used as a control (ctrl). (E) Conversely, Hoxc8 is shifted into the MNs of cervical C5 segments of both miR-196a2/b DKO and miR-196a1/a2/b triple knockout (TKO) embryos. Age-matched wild type embryos were used as controls. Scale bar represents 50 μm. White dash lines demarcate the postmitotoic MN domain (D, F) Quantifications of Hoxa5on, Hoxc8on, and Hoxa5on Hoxc8on cells in the cervical spinal cord of control and knockout embryos (mean ± SD, N=3 embryos, *p < 0.01, Student's t-tests ).


a , Ultrasonic vocalizations ( USV ) in isolated miR379 - 410 ko mouse pups emitted on postnatal day P3 , P6 , P9 and P12 . Left panel : total number of ultrasonic vocalizations ( USV ) in isolated miR379 - 410 wt and ko mouse pups emitted on average ( P 3 , 6 , 9 and 12 ) when isolated from the mother , wt n = 13 ( male n = 9 , female n = 4 ) , ko n = 20 ( male n = 11 , female n = 9 ) , t31 = 2 . 274 , * p = 0 . 0301 , unpaired Student ' s t - test . Right panel : developmental course for total number of USV , wt n = 13 ( male n = 9 , female n = 4 ) , ko n = 20 ( male n = 11 , female n = 9 ) , ( development : F3 , 87 = 25 . 463 , p < 0 . 001 ; genotype : F1 , 29 = 4 . 979 , p = 0 . 03 ; development x genotype : F3 , 87 = 1 . 403 , p = 0 . 22 , rm - ANOVA ) ; P 9 : t31 = 2 . 096 , * p = 0 . 044 ; unpaired Student ` s t - test .

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a, Ultrasonic vocalizations (USV) in isolated miR379-410 ko mouse pups emitted on postnatal day P3, P6, P9 and P12. Left panel: total number of ultrasonic vocalizations (USV) in isolated miR379-410 wt and ko mouse pups emitted on average (P 3, 6, 9 and 12) when isolated from the mother, wt n=13 (male n=9, female n=4), ko n=20 (male n=11, female n=9), t31=2.274, *p=0.0301, unpaired Student's t-test. Right panel: developmental course for total number of USV, wt n=13 (male n=9, female n=4), ko n=20 (male n=11, female n=9), (development: F3,87=25.463, p<0.001; genotype: F1,29=4.979, p=0.03; development x genotype: F3,87=1.403, p= 0.22, rm-ANOVA); P 9: t31=2.096, *p=0.044; unpaired Student`s t-test.


b , Reciprocal social interaction in juvenile miR379 - 410 wt - and ko mice pairs ( P23 ) . Aberrant time spending in social interaction activity in miR379 - 410 ko - pairs compared to their control wt littermate pairs . Left panel : wt - pairs male n = 13 , ko - pairs male n = 10 , t21 = 2 . 433 , * p = 0 . 0240 , unpaired Student ' s t - test . Middle panel : wt - pairs female n = 15 , ko - pairs female n = 13 , t26 = 1 . 873 , ns p = 0 . 0723 , unpaired Student ' s t - test . Right panel : pooled data of wt - pairs n = 28 ( male n = 13 , female n = 15 ) , ko - pairs n = 23 ( male n = 10 , female n = 13 ) , t49 = 2 . 969 , * * p = 0 . 005 ; unpaired Student ` s t - test .

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b, Reciprocal social interaction in juvenile miR379-410 wt- and ko mice pairs (P23). Aberrant time spending in social interaction activity in miR379-410 ko-pairs compared to their control wt littermate pairs. Left panel: wt-pairs male n=13, ko-pairs male n=10, t21=2.433, *p=0.0240, unpaired Student's t-test. Middle panel: wt-pairs female n=15, ko-pairs female n=13, t26=1.873, ns p=0.0723, unpaired Student's t-test. Right panel: pooled data of wt-pairs n=28 (male n=13, female n=15), ko-pairs n=23 (male n=10, female n=13), t49=2.969, **p=0.005; unpaired Student`s t-test.


c , Emitted USVs during social interaction activity in genotype - matched pairs in juvenile mice ( P23 ) . Left panel : wt - pairs male n = 13 , ko - pairs male n = 15 , t21 = 2 . 704 , * p = 0 . 0133 , unpaired Student ' s t - test . Middle panel : wt - pairs female n = 15 , ko - pairs female n = 13 , t26 = 2 . 869 , * * p = 0 . 0081 , unpaired Student ' s t - test . Right panel : pooled data of wt - pairs n = 28 ( male n = 13 , female n = 15 ) , ko - pairs n = 23 ( male n = 10 , female n = 13 ) , t49 = 3 . 992 , * * * p < 0 . 001 ; unpaired Student ` s t - test .

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c, Emitted USVs during social interaction activity in genotype-matched pairs in juvenile mice (P23). Left panel: wt-pairs male n=13, ko-pairs male n=15, t21=2.704, *p=0.0133, unpaired Student's t-test. Middle panel: wt-pairs female n=15, ko-pairs female n=13, t26=2.869, **p=0.0081, unpaired Student's t-test. Right panel: pooled data of wt-pairs n=28 (male n=13, female n=15), ko-pairs n=23 (male n=10, female n=13), t49=3.992, ***p<0.001; unpaired Student`s t-test.


d , Social preference index in adolescent mice - defined as the ratio of time sniffing a stranger mouse vs . an object - is shown . Left panel : wt male n = 26 , ko male n = 26 , t50 = 1 . 075 , ns p = 0 . 2876 , unpaired Student ' s t - test . Middle panel : wt female n = 27 , ko female n = 30 , t55 = 2 . 017 , * p = 0 . 0486 , unpaired Student ' s t - test . Right panel : pooled data of wt n = 53 ( male n = 26 , female n = 27 ) , ko n = 56 ( male n = 26 , female n = 30 ) , t107 = 2 . 283 , * p = 0 . 0244 ; unpaired Student ` s t - test .

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d, Social preference index in adolescent mice - defined as the ratio of time sniffing a stranger mouse vs. an object - is shown. Left panel: wt male n=26, ko male n=26, t50=1.075, ns p=0.2876, unpaired Student's t-test. Middle panel: wt female n=27, ko female n=30, t55=2.017, *p=0.0486, unpaired Student's t-test. Right panel: pooled data of wt n=53 (male n=26, female n=27), ko n=56 (male n=26, female n=30), t107=2.283, *p=0.0244; unpaired Student`s t-test.


A , B Single - molecule FISH ( smFISH ) on mouse hippocampal neurons . ( A ) Representative pictures of smFISH on BDNF - treated WT ( top ) and KO ( bottom ) mouse hippocampal neurons ( DIV6 ) using a probe directed against the Mirg transcript ( green ) . ( Middle ) Hoechst ( blue ) . ( Right ) MAP2 immunostaining ( red ) . Pictures show a zoom - in on the cell body , since the signal detected in WT neurons was restricted to the nuclei ( scale bar : 5μm ) . ( B ) Representative pictures of smFISH on mouse wild type hippocampal neuron cultures ( DIV6 ; basal ) using a probe specific for the Mirg transcript ( green ) . ( Middle ) Hoechst ( blue ) . ( Right ) MAP2 immunostaining ( red ) . Top : overview , scale bar : 10μm ; bottom : zoom - in , scale bar : 5μm .

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A, B Single-molecule FISH (smFISH) on mouse hippocampal neurons. (A) Representative pictures of smFISH on BDNF-treated WT (top) and KO (bottom) mouse hippocampal neurons (DIV6) using a probe directed against the Mirg transcript (green). (Middle) Hoechst (blue). (Right) MAP2 immunostaining (red). Pictures show a zoom-in on the cell body, since the signal detected in WT neurons was restricted to the nuclei (scale bar: 5μm). (B) Representative pictures of smFISH on mouse wild type hippocampal neuron cultures (DIV6; basal) using a probe specific for the Mirg transcript (green). (Middle) Hoechst (blue). (Right) MAP2 immunostaining (red). Top: overview, scale bar: 10μm; bottom: zoom-in, scale bar: 5μm.


C , D , Patch - clamp electrophysiological recordings in cultured mouse hippocampal neurons ( DIV 8 - 10 ) isolated from wt or miR379 - 410 ko animals ( n = 3 per group ) . ( C ) Cumulative distribution ( left panel , D = 0 . 25 , p < 0 . 0001 , KS - test ) and mean ( right panel , t16 = 2 . 736 , * p = 0 . 0146 , unpaired Student ` s t - test ) of mEPSC inter - event intervals ; wt n = 9 , ko n = 9 cells analyzed . ( D ) Cumulative distribution ( left panel , D = 0 . 34741 , p < 0 . 0001 , KS - test ) and mean ( right panel , t18 = 2 . 357 , * p = 0 . 0299 , unpaired Student ` s t - test ) of mEPSC amplitudes ; wt n = 10 , ko n = 10 cells analyzed .

O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, D, Patch-clamp electrophysiological recordings in cultured mouse hippocampal neurons (DIV 8-10) isolated from wt or miR379-410 ko animals (n=3 per group). (C) Cumulative distribution (left panel, D=0.25, p<0.0001, KS-test) and mean (right panel, t16=2.736, *p=0.0146, unpaired Student`s t-test) of mEPSC inter-event intervals; wt n=9, ko n=9 cells analyzed. (D) Cumulative distribution (left panel, D=0.34741, p<0.0001, KS-test) and mean (right panel, t18=2.357, *p=0.0299, unpaired Student`s t-test) of mEPSC amplitudes; wt n=10, ko n=10 cells analyzed.


e , Representative confocal images of CA1 pyramidal neuron apical dendrites from adult Thy1 - GFP / miR379 - 410 wt and Thy1 - GFP / miR379 - 410 ko mouse tissue . Scale bar : 10 µm . f , Spine density in CA1 pyramidal neurons of adult male Thy1 - GFP / miR379 - 410 wt and ko mice ; wt n = 5 , ko n = 6 animals . Mean of basal and apical neurons per mouse are shown , t9 = 3 . 662 , * * p = 0 . 0052 , unpaired Student ` s t - test . g , Spine volume in CA1 pyramidal neurons of adult Thy1 - GFP / miR379 - 410 wt and ko mice ; wt n = 5 , ko n = 6 animals . Mean of basal and apical neurons per mouse are shown , t9 = 2 . 265 , * p = 0 . 0497 , unpaired Student ` s t - test .

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

e, Representative confocal images of CA1 pyramidal neuron apical dendrites from adult Thy1-GFP/miR379-410 wt and Thy1-GFP/miR379-410 ko mouse tissue. Scale bar: 10 µm. f, Spine density in CA1 pyramidal neurons of adult male Thy1-GFP/miR379-410 wt and ko mice; wt n=5, ko n=6 animals. Mean of basal and apical neurons per mouse are shown, t9=3.662, **p=0.0052, unpaired Student`s t-test. g, Spine volume in CA1 pyramidal neurons of adult Thy1-GFP/miR379-410 wt and ko mice; wt n=5, ko n=6 animals. Mean of basal and apical neurons per mouse are shown, t9=2.265, *p=0.0497, unpaired Student`s t-test.


b , qPCR validation of predicted direct miR379 - 410 targets using RNA from hippocampi of juvenile ( P 22 - 24 ) mice , except of Shank 3 where adult miR - 379 - 410 wt and ko animals were measured . If not indicated , both , male and female samples were used . Cnih2 ( wt n = 12 , ko n = 9 , t19 = 2 . 344 , * p = 0 . 0301 ) , Dlgap3 ( wt n = 12 , ko n = 9 , t19 = 2 . 128 , * p = 0 . 0467 ) , Prr7 ( male wt n = 7 , ko n = 4 , t9 = 2 . 719 , * p = 0 . 0236 ) , Src ( wt n = 12 , ko n = 9 , t19 = 2 . 410 , * p = 0 . 0263 ) , Lzts2 ( male wt n = 7 , ko n = 4 , t9 = 2 . 554 , * p = 0 . 0310 ) , Mpp2 ( male wt n = 7 , ko n = 4 , t9 = 2 . 369 , * p = 0 . 0420 ) , Prr12 ( wt n = 6 , ko n = 4 , t8 = 2 . 375 , * p = 0 . 0449 ) , Shank1 ( wt n = 11 , ko n = 9 , t18 = 2 . 310 , * p = 0 . 0329 ) , Shb ( male wt n = 6 , ko n = 4 , t8 = 3 . 451 , * * p = 0 . 0087 ) and Shank3 ( adult male wt n = 3 , ko n = 5 , t6 = 2 . 538 , * p = 0 . 0442 ) ; unpaired Student ` s t - test . Data are presented as mean ± s . e . m .

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

b, qPCR validation of predicted direct miR379-410 targets using RNA from hippocampi of juvenile (P 22-24) mice, except of Shank 3 where adult miR-379-410 wt and ko animals were measured. If not indicated, both, male and female samples were used. Cnih2 (wt n=12, ko n=9, t19=2.344, *p=0.0301), Dlgap3 (wt n=12, ko n=9, t19=2.128, *p=0.0467), Prr7 (male wt n=7, ko n=4, t9=2.719, *p=0.0236), Src (wt n=12, ko n=9, t19=2.410, *p=0.0263), Lzts2 (male wt n=7, ko n=4, t9=2.554, *p=0.0310), Mpp2 (male wt n=7, ko n=4, t9=2.369, *p=0.0420), Prr12 (wt n=6, ko n=4, t8=2.375, *p=0.0449), Shank1 (wt n=11, ko n=9, t18=2.310, *p=0.0329), Shb (male wt n=6, ko n=4, t8=3.451, **p=0.0087) and Shank3 (adult male wt n=3, ko n=5, t6=2.538, *p=0.0442); unpaired Student`s t-test. Data are presented as mean ± s.e.m.


c , d , Cnih2 , Prr7 and Src are direct miR - 379 - 410 target mRNAs . ( C ) 3 ' - UTR luciferase reporter gene assays in cultured rat cortical neurons using the indicated 3 ' UTR reporter genes ( wt or seed mutant ) together with miRNA mimics . Cnih2 ( n = 3 per group ; wt : * * p = 0 . 0020 , mut : p = 0 . 9267 ; mimic : F1 , 8 = 19 . 92 , p = 0 . 0021 ; utr - construct : F1 , 8 = 4 . 760 , p = 0 . 0607 ; mimic x utr - construct : F1 , 8 = 12 . 99 , p = 0 . 0069 ) , Prr7 ( n = 3 per group ; wt : * * * * p < 0 . 0001 , mut : * p = 0 . 0103 ; wt vs . mut mimic miR329 : * * p = 0 . 0014 ; mimic : F1 , 8 = 153 . 1 , p < 0 . 0001 ; utr - construct : F1 , 8 = 5 . 631 , p = 0 . 0450 ; mimic x utr - construct : F1 , 8 = 38 . 54 , p = 0 . 0003 ) ; Src ( n = 3 per group ; wt : * p = 0 . 0110 , mut : p = 0 . 4724 ; mimic : F1 , 8 = 16 . 99 , p = 0 . 0033 ; utr - construct : F1 , 8 = 0 . 6809 , p = 0 . 4332 ; mimic vs . utr - construct : F1 , 8 = 3 . 920 , p = 0 . 0831 ) ; two - way ANOVA . ( D ) Same as in ( C ) , but in the presence of antimiRs ( pLNAs ) in hippocampal neurons after 48h of PTX stimulation . Cnih2 ( n = 4 per group ; wt : p = 0 . 2688 ; mut : p = 0 . 8668 ; wt ctr vs . mut ctr : p = 0 . 6463 ; anti - miRNA : F1 , 12 = 3 . 629 , p = 0 . 0810 ; utr - construct : F1 , 12 = 0 . 7407 , p = 0 . 4063 ; anti - miRNA x utr - construct : F1 , 12 = 0 . 6679 , p = 0 . 4297 ) , Prr7 ( n = 4 per group ; wt : * p = 0 . 0123 ; mut : p = 0 . 9917 ; wt ctr vs . mut ctr : * p = 0 . 0152 ; anti - miRNA : F1 , 12 = 6 . 082 , p = 0 . 0297 ; utr - construct : F1 , 12 = 5 . 271 ; p = 0 . 0405 ; anti - miRNA x utr - construct : F1 , 12 = 8 . 212 , p = 0 . 0142 ) ; Src ( n = 3 per group ; wt : * p = 0 . 0370 ; mut : p = 0 . 3540 ; wt ctr vs . mut ctr : p = 0 . 9906 ; anti - miRNA : F1 , 12 = 13 . 43 , p = 0 . 0064 ; utr - construct : F1 , 12 = 0 . 5672 , p = 0 . 4730 ; anti - miRNA x utr - construct : F1 , 12 = 1 . 362 , p = 0 . 2768 ) ; two - way ANOVA . Data are presented as mean ± s . d .

O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, d, Cnih2, Prr7 and Src are direct miR-379-410 target mRNAs. (C) 3'-UTR luciferase reporter gene assays in cultured rat cortical neurons using the indicated 3'UTR reporter genes (wt or seed mutant) together with miRNA mimics. Cnih2 (n=3 per group; wt: **p=0.0020, mut: p=0.9267; mimic: F1,8=19.92, p=0.0021; utr-construct: F1,8=4.760, p=0.0607; mimic x utr-construct: F1,8=12.99, p=0.0069), Prr7 (n=3 per group; wt: ****p<0.0001, mut: *p=0.0103; wt vs. mut mimic miR329: **p=0.0014; mimic: F1,8=153.1, p<0.0001; utr-construct: F1,8=5.631, p=0.0450; mimic x utr-construct: F1,8=38.54, p=0.0003); Src (n=3 per group; wt: *p=0.0110, mut: p=0.4724; mimic: F1,8=16.99, p=0.0033; utr-construct: F1,8=0.6809, p=0.4332; mimic vs. utr-construct: F1,8=3.920, p=0.0831); two-way ANOVA. (D) Same as in (C), but in the presence of antimiRs (pLNAs) in hippocampal neurons after 48h of PTX stimulation. Cnih2 (n=4 per group; wt: p=0.2688; mut: p=0.8668; wt ctr vs. mut ctr: p=0.6463; anti-miRNA: F1,12=3.629, p=0.0810; utr-construct: F1,12=0.7407, p=0.4063; anti-miRNA x utr-construct: F1,12=0.6679, p=0.4297), Prr7 (n=4 per group; wt: *p=0.0123; mut: p=0.9917; wt ctr vs. mut ctr: *p=0.0152; anti-miRNA: F1,12=6.082, p=0.0297; utr-construct: F1,12=5.271; p=0.0405; anti-miRNA x utr-construct: F1,12=8.212, p=0.0142); Src (n=3 per group; wt: *p=0.0370; mut: p=0.3540; wt ctr vs. mut ctr: p=0.9906; anti-miRNA: F1,12=13.43, p=0.0064; utr-construct: F1,12=0.5672, p=0.4730; anti-miRNA x utr-construct: F1,12=1.362, p=0.2768); two-way ANOVA. Data are presented as mean ± s.d.


Expression of USP28 ( left ) and TP63 ( right ) in human lung squamous cell carcinomas ( SCC , n = 498 ) , adenocarcinomas ( ADC , n = 513 ) and normal non - transformed tissue ( normal SCC = 338 , normal ADC = 348 ) . Xena UCSC software . In box plots , the centre line reflects the median , the cross represents the mean and the upper and lower box limits indicates the first and third quartile . Whiskers extend 1 . 5x the IQR and outliers are marked as dots .

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

Expression of USP28 (left) and TP63 (right) in human lung squamous cell carcinomas (SCC, n=498), adenocarcinomas (ADC, n=513) and normal non-transformed tissue (normal SCC=338, normal ADC=348). Xena UCSC software. In box plots, the centre line reflects the median, the cross represents the mean and the upper and lower box limits indicates the first and third quartile. Whiskers extend 1.5x the IQR and outliers are marked as dots.


Correlation of mRNA expression of USP28 and TP63 in lung SCC ( left , n = 498 ) , ADC ( right , n = 513 ) and normal , non - transformed tissue ( normal SCC = 338 , normal ADC = 348 ) . R : Spearmans correlation coefficient ; m = Slope . Xena UCSC software .

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

Correlation of mRNA expression of USP28 and TP63 in lung SCC (left, n=498), ADC (right, n=513) and normal, non-transformed tissue (normal SCC=338, normal ADC=348). R: Spearmans correlation coefficient; m=Slope. Xena UCSC software.


IHC analysis of USP28 and ∆ Np63 protein abundance in lung cancer and non - transformed human samples ( n = 300 ) . The staining intensity was quantified in arbitrary units from 0 up to 3 by three independent pathologists . In box plots , the centre line reflects the median , the cross represents the mean and the upper and lower box limits indicates the first and third quartile . Whiskers extend 1 . 5x the IQR and outliers are marked as dots . p - values were calculated using two - tailed t - test .

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

IHC analysis of USP28 and ∆Np63 protein abundance in lung cancer and non-transformed human samples (n=300). The staining intensity was quantified in arbitrary units from 0 up to 3 by three independent pathologists. In box plots, the centre line reflects the median, the cross represents the mean and the upper and lower box limits indicates the first and third quartile. Whiskers extend 1.5x the IQR and outliers are marked as dots. p-values were calculated using two-tailed t-test.


Kaplan - Meier estimator of NSCLC patients stratified by USP28 ( left , n = 1145 ) and TP63 ( right , n = 1926 ) expression . p - values were calculated using log - rank test . HR : hazard ratio . Kmplot software .

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

Kaplan-Meier estimator of NSCLC patients stratified by USP28 (left, n=1145) and TP63 (right, n=1926) expression. p-values were calculated using log-rank test. HR: hazard ratio. Kmplot software.


Kaplan - Meier estimator of lung SCC patients stratified by USP28 expression ( n = 271 ) . The p - value was calculated using a logrank test . HR : hazard ratio . Kmplot software .

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

Kaplan-Meier estimator of lung SCC patients stratified by USP28 expression (n=271). The p-value was calculated using a logrank test. HR: hazard ratio. Kmplot software.


Co - immunoprecipitation of exogenous HA - USP28 and FLAG - ΔNp63 in HEK293 cells . Either HA - USP28 or FLAG - ΔNp63 were precipitated and blotted against FLAG - ΔNp63 or HA - USP28 . The input corresponds to 10 % of the total protein amount used for the IP ( ACTIN as loading control ) .

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

Co-immunoprecipitation of exogenous HA-USP28 and FLAG-ΔNp63 in HEK293 cells. Either HA-USP28 or FLAG-ΔNp63 were precipitated and blotted against FLAG-ΔNp63 or HA-USP28. The input corresponds to 10% of the total protein amount used for the IP (ACTIN as loading control).


Ni - NTA His - ubiquitin pulldown in control transfected or HA - USP28 overexpressing HEK293 cells , followed by immunoblot against exogenous ΔNp63 . The input corresponds to 10 % of the total protein amount used for the pull down . Relative ubiquitination of the representative immunoblot was calculated using undefined for normalization .

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

Ni-NTA His-ubiquitin pulldown in control transfected or HA-USP28 overexpressing HEK293 cells, followed by immunoblot against exogenous ΔNp63. The input corresponds to 10% of the total protein amount used for the pull down. Relative ubiquitination of the representative immunoblot was calculated using undefined for normalization.


Ni - NTA His - ubiquitin pulldown K48 or K63 in control and HA - USP28 overexpressing HEK293 cells , followed by immunoblot against exogenous ΔNp63 . The input corresponds to 10 % of the total protein amount used for the pulldown . Relative ubiquitination of the representative immunoblot was calculated using VINCULIN for normalization .

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

Ni-NTA His-ubiquitin pulldown K48 or K63 in control and HA-USP28 overexpressing HEK293 cells, followed by immunoblot against exogenous ΔNp63. The input corresponds to 10% of the total protein amount used for the pulldown. Relative ubiquitination of the representative immunoblot was calculated using VINCULIN for normalization.


Co - immunoprecipitation of exogenous FLAG - USP28 C171A and FLAG - ΔNp63 in HEK293 cells . ΔNp63 was precipitated and blotted against FLAG - USP28 or ΔNp63 . The input corresponds to 10 % of the total protein amount used for the IP ( ACTIN as loading control ) .

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

Co-immunoprecipitation of exogenous FLAG-USP28 C171A and FLAG-ΔNp63 in HEK293 cells. ΔNp63 was precipitated and blotted against FLAG-USP28 or ΔNp63. The input corresponds to 10% of the total protein amount used for the IP (ACTIN as loading control).


Ni - NTA His - ubiquitin pulldown in control , FLAG - USP28 or FLAG - USP28 C171A transfected HEK293 cells , followed by immunoblot against exogenous ΔNp63 . The input corresponds to 10 % of the total protein amount used for the pulldown . Relative ubiquitination of the representative immunoblot was calculated using ACTIN for normalization .

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

Ni-NTA His-ubiquitin pulldown in control, FLAG-USP28 or FLAG-USP28 C171A transfected HEK293 cells, followed by immunoblot against exogenous ΔNp63. The input corresponds to 10% of the total protein amount used for the pulldown. Relative ubiquitination of the representative immunoblot was calculated using ACTIN for normalization.


CHX chase assay ( 100ug / ml ) of control , FLAG - USP28 or FLAG - USP28 C171A transfected HEK293 cells for indicated time points . Representative immunoblot analysis of FLAG ( USP28 ) and ∆ Np63 as well quantification of relative protein abundance ( ACTIN as loading control ) .

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

CHX chase assay (100ug/ml) of control, FLAG-USP28 or FLAG-USP28 C171A transfected HEK293 cells for indicated time points. Representative immunoblot analysis of FLAG (USP28) and ∆Np63 as well quantification of relative protein abundance (ACTIN as loading control).


Immunoblot of USP28 and ∆ Np63 in transfected HEK293 cells upon treatment with either DMSO or indicated concentrations of PR - 619 for 24 hours . Relative protein abundance was calculated ACTIN as loading control .

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

Immunoblot of USP28 and ∆Np63 in transfected HEK293 cells upon treatment with either DMSO or indicated concentrations of PR-619 for 24 hours. Relative protein abundance was calculated ACTIN as loading control.


Immunoblot of endogenous USP25 , USP28 and ∆ Np63 immunoprecipitated ( IP ) from A - 431 cells and co - precipitated ∆ Np63 or USP28 . Beads were coupled to specific USP25 , USP28 and ∆ Np63 antibodies or non - specific rabbit IgG as control . The input corresponds to 10 % of the total protein amount used for the IP ( TUBULIN as loading control ) .

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

Immunoblot of endogenous USP25, USP28 and ∆Np63 immunoprecipitated (IP) from A-431 cells and co-precipitated ∆Np63 or USP28. Beads were coupled to specific USP25, USP28 and ∆Np63 antibodies or non-specific rabbit IgG as control. The input corresponds to 10% of the total protein amount used for the IP (TUBULIN as loading control).


Inducible depletion of USP28 in A - 431 upon treatment with doxycycline ( 1µg / ml ) for 96h , western blot ( left , VINCULIN as loading control ) and qPCR analysis of USP28 and ∆ Np63 expression relative to ACTIN ( right ) was performed .

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

Inducible depletion of USP28 in A-431 upon treatment with doxycycline (1µg/ml) for 96h, western blot (left, VINCULIN as loading control) and qPCR analysis of USP28 and ∆Np63 expression relative to ACTIN (right) was performed.


Tandem ubiquitin binding entity ( TUBE ) pulldown of endogenous ubiquitylated ∆ Np63 in A - 431 cells upon DOX depletion of USP28 . Relative ubiquitination of the representative immunoblot was calculated using ACTIN for normalization .

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

Tandem ubiquitin binding entity (TUBE) pulldown of endogenous ubiquitylated ∆Np63 in A-431 cells upon DOX depletion of USP28. Relative ubiquitination of the representative immunoblot was calculated using ACTIN for normalization.


Cycloheximide ( CHX ) chase assay ( 100µg / ml ) of control or inducible sh - USP28 A - 431 cell line ( EtOH or 1µg / ml dox ) for indicated time points . Representative immunoblot ( left , VINCULIN as loading control ) of USP28 and ∆ Np63 and quantification of relative protein abundance ( right ) .

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

Cycloheximide (CHX) chase assay (100µg/ml) of control or inducible sh-USP28 A-431 cell line (EtOH or 1µg/ml dox) for indicated time points. Representative immunoblot (left, VINCULIN as loading control) of USP28 and ∆Np63 and quantification of relative protein abundance (right).


Doxycycline induced murine USP28 overexpression ( EtOH or 1ug / ml dox for 96 hours ) in A - 431 cells followed by immunoblot ( VINCULIN as loading control ) and qPCR analysis of USP28 and ∆ Np63 . For qPCR , human USP28 and murine USP28 ( mUSP28 ) primers were used . Relative mRNA was calculated using ∆ ∆ Ct analysis for human USP28 and ∆ Ct for mUSP28 ( ACTIN as housekeeping ) .

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

Doxycycline induced murine USP28 overexpression (EtOH or 1ug/ml dox for 96 hours) in A-431 cells followed by immunoblot (VINCULIN as loading control) and qPCR analysis of USP28 and ∆Np63. For qPCR, human USP28 and murine USP28 (mUSP28) primers were used. Relative mRNA was calculated using ∆∆Ct analysis for human USP28 and ∆Ct for mUSP28 (ACTIN as housekeeping).


TUBE pulldown of endogenous ubiquitylated ∆ Np63 in A - 431 cells upon overexpression of mUSP28 for 96 hours ( EtOH or 1µg / ml dox ) . Relative ubiquitination of the representative immunoblot was calculated using ACTIN for normalization .

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

TUBE pulldown of endogenous ubiquitylated ∆Np63 in A-431 cells upon overexpression of mUSP28 for 96 hours (EtOH or 1µg/ml dox). Relative ubiquitination of the representative immunoblot was calculated using ACTIN for normalization.


CHX chase assay ( 100µg / ml ) of control or inducible mUSP28 overexpressing A - 431 cell line ( EtOH or 1µg / ml dox ) for indicated time points . Representative immunoblot analysis of USP28 and ∆ Np63 as well quantification of relative protein abundance ( VINCULIN as loading control ) .

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

CHX chase assay (100µg/ml) of control or inducible mUSP28 overexpressing A-431 cell line (EtOH or 1µg/ml dox) for indicated time points. Representative immunoblot analysis of USP28 and ∆Np63 as well quantification of relative protein abundance (VINCULIN as loading control).


Immunoblot of control ( sh - NTC ) and two independent shRNA targeting USP28 ( sh - USP28 # 1 and # 2 ) for ∆ Np63 and USP28 protein abundance in LUDLU - 1adh ( VINCULIN as loading control ) , followed by qPCR analysis of USP28 and ∆ Np63 expression relative to ACTIN .

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

Immunoblot of control (sh-NTC) and two independent shRNA targeting USP28 (sh-USP28#1 and #2) for ∆Np63 and USP28 protein abundance in LUDLU-1adh (VINCULIN as loading control), followed by qPCR analysis of USP28 and ∆Np63 expression relative to ACTIN.


Doxycycline induced mUSP28 overexpression ( EtOH or 1µg / ml dox for 96 hours ) in LUDLU - 1adh cells followed by immunoblot ( VINCULIN as loading control ) and qPCR analysis of USP28 , mUSP28 and ∆ Np63 . Relative mRNA was calculated using ∆ ∆ Ct analysis for human USP28 and ∆ Ct for mUSP28 ( ACTIN as housekeeping for the analysis ) .

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

Doxycycline induced mUSP28 overexpression (EtOH or 1µg/ml dox for 96 hours) in LUDLU-1adh cells followed by immunoblot (VINCULIN as loading control) and qPCR analysis of USP28, mUSP28 and ∆Np63. Relative mRNA was calculated using ∆∆Ct analysis for human USP28 and ∆Ct for mUSP28 (ACTIN as housekeeping for the analysis).


Correlation of gene expression changes upon constitutive transduction of A - 431 cells with either shRNA targeting USP28 ( sh - USP28 # 1 ) or ∆ Np63 ( sh - ∆ Np63 # 2 ) relative to non - targeting control ( sh - NTC ) . The diagonal line reflects a regression build on a linear model . R : Pearsons correlation coefficient , m : slope of the linear regression

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

Correlation of gene expression changes upon constitutive transduction of A-431 cells with either shRNA targeting USP28 (sh-USP28#1) or ∆Np63 (sh-∆Np63#2) relative to non-targeting control (sh-NTC). The diagonal line reflects a regression build on a linear model. R: Pearsons correlation coefficient, m: slope of the linear regression model


Gene set enrichment analysis ( GSEA ) of a gene set of significantly down - regulated genes in sh - ∆ Np63 # 2 transfected A - 431 cells ( " Down - regulated sh - ∆ NP63 " , Appendix Table S1 ) . The gene set was analysed in sh - ∆ Np63 # 2 ( left ) and sh - USP28 # 1 - depleted ( right ) A - 431 cells . ( N ) ES : ( normalised ) enrichment

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

Gene set enrichment analysis (GSEA) of a gene set of significantly down-regulated genes in sh-∆Np63#2 transfected A-431 cells ("Down-regulated sh-∆NP63", Appendix Table S1). The gene set was analysed in sh-∆Np63#2 (left) and sh-USP28#1-depleted (right) A-431 cells. (N)ES: (normalised) enrichment score


Gene set enrichment analysis ( GSEA ) of a gene set of significantly down - regulated genes in sh - USP28 # 1 transfected A - 431 cells ( " Down - regulated sh - USP28 " , Appendix Table S1 ) . The gene set was analysed in shUSP28 # 1 ( left ) and sh - ∆ Np63 # - depleted ( right ) A - 431 cells . ( N ) ES : ( normalised ) enrichment

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

Gene set enrichment analysis (GSEA) of a gene set of significantly down-regulated genes in sh-USP28#1 transfected A-431 cells ("Down-regulated sh-USP28", Appendix Table S1). The gene set was analysed in shUSP28#1 (left) and sh-∆Np63#-depleted (right) A-431 cells. (N)ES: (normalised) enrichment score


Immunoblot of endogenous ΔNp63 and USP28 in A - 431 cells stably transduced with constitutive shRNA - non - targeting control ( NTC ) or against USP28 and transiently transfected with exogenous ΔNp63 . ACTIN served as loading control . Representative western blot from three independent experiments .

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

Immunoblot of endogenous ΔNp63 and USP28 in A-431 cells stably transduced with constitutive shRNA-non-targeting control (NTC) or against USP28 and transiently transfected with exogenous ΔNp63. ACTIN served as loading control. Representative western blot from three independent experiments.


Cell growth of A - 431 cells stably transduced with constitutive shRNA - non - targeting control ( NTC ) or against USP28 and transiently transfected with exogenous ΔNp63 . Total cell number was measured and assessed at indicated time points . Quantitative graph is represented as mean ± SD of three experiments ( n = 3 ) . p - values were calculated using two - tailed t - test statistical analysis . * p - value < 0 . 05 , * * p - value < 0 .

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

Cell growth of A-431 cells stably transduced with constitutive shRNA-non-targeting control (NTC) or against USP28 and transiently transfected with exogenous ΔNp63. Total cell number was measured and assessed at indicated time points. Quantitative graph is represented as mean ± SD of three experiments (n=3). p-values were calculated using two-tailed t-test statistical analysis. *p-value < 0.05, **p-value < 0.01


Relative expression of SCC marker genes KRT5 , 14 and 19 in A - 431 cells stably transduced with constitutive shRNA - non - targeting control ( NTC ) , two independent shRNA - ΔNp63 , two independent shRNA - USP28 or ΔNp63 in shRNA - USP28 # 1 , normalised to ACTIN . Quantitative graph is represented as mean ± SD of three experiments ( n = 3 ) .

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

Relative expression of SCC marker genes KRT5, 14 and 19 in A-431 cells stably transduced with constitutive shRNA-non-targeting control (NTC), two independent shRNA-ΔNp63, two independent shRNA-USP28 or ΔNp63 in shRNA-USP28#1, normalised to ACTIN. Quantitative graph is represented as mean ± SD of three experiments (n=3).


Relative expression of epithelial marker genes KRT10 and GPCR5A in A - 431 cells stably transduced with constitutive shRNA - non - targeting control ( NTC ) , two independent shRNA - ΔNp63 and shRNA - USP28 # 1 , normalised to ACTIN . Quantitative graph is represented as mean ± SD of three experiments ( n = 3 ) .

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

Relative expression of epithelial marker genes KRT10 and GPCR5A in A-431 cells stably transduced with constitutive shRNA-non-targeting control (NTC), two independent shRNA-ΔNp63 and shRNA-USP28#1, normalised to ACTIN. Quantitative graph is represented as mean ± SD of three experiments (n=3).


Genomic signature of primary human lung SCC samples comprising USP28 , ∆ Np63 , KRT5 , KRT14 , KRT19 , DSG3 , MKI67 , PCNA and GPRC5A . Samples were sorted dependent on relative USP28 expression ( high to low ) . n = 553 . Xena UCSC software .

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

Genomic signature of primary human lung SCC samples comprising USP28, ∆Np63, KRT5, KRT14, KRT19, DSG3, MKI67, PCNA and GPRC5A. Samples were sorted dependent on relative USP28 expression (high to low). n=553. Xena UCSC software.


GSEA of consensus squamous cancer marker genes ( se Appendix Table S2 ) in A - 431 cells stably transduced with sh - ∆ Np63 # 2 or sh - NTC . ( N ) ES : ( normalised ) enrichment score GSEA of consensus squamous cancer marker genes in A - 431 cells stably transduced with sh - USP28 # 1 or sh - NTC . ( N ) ES : ( normalised ) enrichment

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

GSEA of consensus squamous cancer marker genes (se Appendix Table S2) in A-431 cells stably transduced with sh-∆Np63#2 or sh-NTC. (N)ES: (normalised) enrichment score GSEA of consensus squamous cancer marker genes in A-431 cells stably transduced with sh-USP28#1 or sh-NTC. (N)ES: (normalised) enrichment score


Correlation of mRNA expression of consensus squamous cancer marker genes and TP63 in lung SCC and non - transformed lung tissue ( Normal ) . R : Spearmans correlation coefficient . n = 836 . Gepia software . Correlation of mRNA expression of consensus squamous cancer marker genes and USP28 in lung SCC and non - transformed lung tissue ( Normal ) . R : Spearmans correlation coefficient . N = 836 . Gepia software .

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

Correlation of mRNA expression of consensus squamous cancer marker genes and TP63 in lung SCC and non-transformed lung tissue (Normal). R: Spearmans correlation coefficient. n=836. Gepia software. Correlation of mRNA expression of consensus squamous cancer marker genes and USP28 in lung SCC and non-transformed lung tissue (Normal). R: Spearmans correlation coefficient. N=836. Gepia software.


Representative haematoxylin and eosin ( H & E ) staining of tumour bearing animals 12 weeks post intratracheal infection . Boxes indicate highlighted tumour areas in ( C ) ( a , b , a ' and b ' ) . Scale bar = 2000µm ; nKPL = 6 and nKPLU = 5 . H = heart .

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Representative haematoxylin and eosin (H&E) staining of tumour bearing animals 12 weeks post intratracheal infection. Boxes indicate highlighted tumour areas in (C) (a, b, a' and b'). Scale bar = 2000µm; nKPL= 6 and nKPLU = 5. H=heart.


Representative IHC staining for ADC ( TTF - 1 ) and SCC ( KRT5 and ∆ Np63 ) marker expression as well as Usp28 abundance in KPL ( n = 6 ) and KPLU ( n = 5 ) lung tumours . Scale bar : 20µm .

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

Representative IHC staining for ADC (TTF-1) and SCC (KRT5 and ∆Np63) marker expression as well as Usp28 abundance in KPL (n=6) and KPLU (n=5) lung tumours. Scale bar: 20µm.


Quantification of % tumour area ( normalised to total lung area ) in KPL ( n = 6 ) and KPLU ( n = 5 ) animals . Quantification of lung tumour numbers in KPL ( n = 6 ) and KPLU ( n = 5 ) animals .

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

Quantification of % tumour area (normalised to total lung area) in KPL (n=6) and KPLU (n=5) animals. Quantification of lung tumour numbers in KPL (n=6) and KPLU (n=5) animals.


Immunoblot of endogenous LKB1 , USP28 and ∆ Np63 in KPL and KPLU tumours . ( VINCULIN as loading control ) . Western blot shown are representative of three independent experiments ( n = 3 ) . Representative immunoblot of endogenous NOTCH1 , c - MYC and c - JUN in KPL and KPLU ( ACTIN as a control ) . n =

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

Immunoblot of endogenous LKB1, USP28 and ∆Np63 in KPL and KPLU tumours. (VINCULIN as loading control). Western blot shown are representative of three independent experiments (n=3). Representative immunoblot of endogenous NOTCH1, c-MYC and c-JUN in KPL and KPLU (ACTIN as a control). n=3


Kaplan - Meier survival curves comparing KPL ( n = 6 ) and KPLU ( n = 5 ) animals upon AAV intratracheal infection . The p - value was calculated using a log - rank test .

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Kaplan-Meier survival curves comparing KPL (n=6) and KPLU (n=5) animals upon AAV intratracheal infection. The p-value was calculated using a log-rank test.


Representative qPCR of SCC and ADC marker expression in two independent KP lung tumour clones , resulting in KPADC and KPSCC ; ( ACTIN served as loading control ) . Quantitative graph is represented as mean ± SD of three experiments ( n = 3 ) .

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

Representative qPCR of SCC and ADC marker expression in two independent KP lung tumour clones, resulting in KPADC and KPSCC; (ACTIN served as loading control). Quantitative graph is represented as mean ± SD of three experiments (n=3).


Immunoblot against endogenous USP28 in the murine KPSCC cell line upon targeting with two independent shRNA sequences . ACTIN served as loading control .

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

Immunoblot against endogenous USP28 in the murine KPSCC cell line upon targeting with two independent shRNA sequences. ACTIN served as loading control.


Representative haematoxylin and eosin ( H & E ) images of tumour bearing animals 8 weeks post intratracheal transplantation of 2 × 105 cells / animal . KPSCC sh - NTC ( n = 3 ) ; KPSCC sh - USP28 ( n = 3 ) , Scale bar =

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

Representative haematoxylin and eosin (H&E) images of tumour bearing animals 8 weeks post intratracheal transplantation of 2 × 105 cells/animal. KPSCC sh-NTC (n=3); KPSCC sh-USP28 (n=3), Scale bar = 5000µm


Quantification of % tumour area ( top , normalised to total lung area ) and lung tumour numbers ( bottom ) on KPSCC sh - NTC ( n = 6 ) and KPSCC sh - USP28 ( n = 6 ) animals .

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

Quantification of % tumour area (top, normalised to total lung area) and lung tumour numbers (bottom) on KPSCC sh-NTC (n=6) and KPSCC sh-USP28 (n=6) animals.


Representative IHC staining for ADC ( TTF - 1 ) and SCC ( KRT5 and ∆ Np63 ) marker expression as well as Usp28 and GFP abundance in KPSCC sh - NTC ( n = 3 ) and KPSCC sh - USP28 ( n = 3 ) lung tumours . Scale bar = 50

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

Representative IHC staining for ADC (TTF-1) and SCC (KRT5 and ∆Np63) marker expression as well as Usp28 and GFP abundance in KPSCC sh-NTC (n=3) and KPSCC sh-USP28 (n=3) lung tumours. Scale bar =50 µm


Analysis of occurring TP63 genetic alterations in lung squamous ( LUSC ) , cervical ( CESC ) , oesophagus ( ESCA ) , head and neck ( HNSC ) and pancreatic ( PAAD ) tumours . Cbioportal .

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Analysis of occurring TP63 genetic alterations in lung squamous (LUSC), cervical (CESC), oesophagus (ESCA), head and neck (HNSC) and pancreatic (PAAD) tumours. Cbioportal.


Expression of TP63 ( left ) and USP28 ( right ) in human Lung ( n = 498 ) , Cervix ( n = 254 ) , Oesophagus ( n = 96 ) and HNSC ( n = 522 ) SCC tumours and normal non - transformed tissue ( nLung = 338 nCervix = 3 , nOesophagus = 11 and nHNSC = 44 ) . In box plots , the centre line reflects the median , the cross represents the mean and the upper and lower box limits indicates the first and third quartile . Whiskers extend 1 . 5x the IQR . p - values were calculated using two - tailed t - test statistical analysis . Xena UCSC software .

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Expression of TP63 (left) and USP28 (right) in human Lung (n=498), Cervix (n=254), Oesophagus (n=96) and HNSC (n=522) SCC tumours and normal non-transformed tissue (nLung=338 nCervix=3, nOesophagus=11 and nHNSC=44). In box plots, the centre line reflects the median, the cross represents the mean and the upper and lower box limits indicates the first and third quartile. Whiskers extend 1.5x the IQR. p-values were calculated using two-tailed t-test statistical analysis. Xena UCSC software.


Correlation of mRNA expression of KRT14 / KRT7 and TP63 in ADC and SCC tumours for Lung ( nADC = 513 and nSCC = 498 ) , Cervix ( nADC = 47 and nSCC = 254 ) and Oesophagus ( nADC = 89 and nSCC = 96 ) . Blue dots : ADC ; Red dots : SCC ; R = Spearmans correlation coefficient ; m : Slope . Xena UCSC .

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

Correlation of mRNA expression of KRT14 / KRT7 and TP63 in ADC and SCC tumours for Lung (nADC=513 and nSCC=498), Cervix (nADC=47 and nSCC=254) and Oesophagus (nADC=89 and nSCC=96). Blue dots: ADC; Red dots: SCC; R= Spearmans correlation coefficient; m: Slope. Xena UCSC.


Immunoblot of control ( sh - NTC ) and two independent shRNA targeting USP28 ( sh - USP28 # 1 and # 2 ) for ∆ Np63 , KRT14 and USP28 protein abundance in H1299 , LUDLU - 1adh , HELA , SiHa , Ca Ski , PANC - 1 and BXPC - 3 ( ACTIN as loading control ) .

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

Immunoblot of control (sh-NTC) and two independent shRNA targeting USP28 (sh-USP28#1 and #2) for ∆Np63, KRT14 and USP28 protein abundance in H1299, LUDLU-1adh, HELA, SiHa, Ca Ski, PANC-1 and BXPC-3 (ACTIN as loading control).


Cells were seeded at equal cell density and counted after five days , Brigth field images of control or sh - USP28 # 2 infected H1299 , LUDLU - 1adh , Hela , CaSKI , PANC - 1 and BXPC - 3 cells before quantification . Scale bar = 30µm Relative number of H1299 , LUDLU - 1adh , Hela , CaSKI , SiHa , PANC - 1 and BXPC - 3 sh - USP28 # 2 cells compared with sh - NTC control cells . p - values were calculated using two - tailed t - test statistical analysis . SiHa * = Notably , the human SCC cell line SiHa was negative for ΔNp63 .

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

Cells were seeded at equal cell density and counted after five days, Brigth field images of control or sh-USP28#2 infected H1299, LUDLU-1adh, Hela, CaSKI, PANC-1 and BXPC-3 cells before quantification. Scale bar = 30µm Relative number of H1299, LUDLU-1adh, Hela, CaSKI, SiHa, PANC-1 and BXPC-3 sh-USP28#2 cells compared with sh-NTC control cells. p-values were calculated using two-tailed t-test statistical analysis. SiHa* = Notably, the human SCC cell line SiHa was negative for ΔNp63.


Immunoblot of endogenous USP28 in A - 431 cells upon treatment with warheads ( WH ) and either DMSO or indicated concentrations of AZ1 ( USP28 Inh . ) for 24 hours . USP28 Upper band : Active USP28 ; USP28 lower band : Inactive USP28 , VINCULIN as loading control .

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

Immunoblot of endogenous USP28 in A-431 cells upon treatment with warheads (WH) and either DMSO or indicated concentrations of AZ1 (USP28 Inh.) for 24 hours. USP28 Upper band: Active USP28; USP28 lower band: Inactive USP28, VINCULIN as loading control.


Immunoblot of endogenous USP28 , ∆ Np63 and KRT14 in A - 431 cells treated for 24 hours with either DMSO or indicated concentrations of AZ1 . VINCULIN served as loading control . TP63 and USP28 half maximal inhibitory protein abundance ( IC50 ) was calculated . Graphs are represented as mean ± SD ( n = 3 ) .

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

Immunoblot of endogenous USP28, ∆Np63 and KRT14 in A-431 cells treated for 24 hours with either DMSO or indicated concentrations of AZ1. VINCULIN served as loading control. TP63 and USP28 half maximal inhibitory protein abundance (IC50) was calculated. Graphs are represented as mean ± SD (n=3).


TUBE pulldown of endogenous ubiquitylated ∆ Np63 in A - 431 cells upon treatment with either DMSO or indicated concentrations of AZ1 for 24 hours . VINCULIN served as loading control for relative Ubiquitination .

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

TUBE pulldown of endogenous ubiquitylated ∆Np63 in A-431 cells upon treatment with either DMSO or indicated concentrations of AZ1 for 24 hours. VINCULIN served as loading control for relative Ubiquitination.


Immunoblot of endogenous USP28 , ∆ Np63 and KRT14 in A - 431 cells treated for 18 hours with either DMSO or 15µM AZ1 followed by 6 hours of treatment with 20µM proteasomal inhibitor MG - 132 ( 20µM ) or DMSO . VINCULIN served as loading control .

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

Immunoblot of endogenous USP28, ∆Np63 and KRT14 in A-431 cells treated for 18 hours with either DMSO or 15µM AZ1 followed by 6 hours of treatment with 20µM proteasomal inhibitor MG-132 (20µM) or DMSO. VINCULIN served as loading control.


A - 431 cells were seeded at equal cell density and cultured in the presence of either DMSO , 1µM , 10µM or 30µM AZ1 for 48h . Cells were quantified with the Operetta imaging system using Hoechst staining . 50 % growth inhibition ( GI50 ) was calculated . Scale bar = 250µm . n = 30 fields

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-431 cells were seeded at equal cell density and cultured in the presence of either DMSO, 1µM, 10µM or 30µM AZ1 for 48h. Cells were quantified with the Operetta imaging system using Hoechst staining. 50% growth inhibition (GI50) was calculated. Scale bar = 250µm. n= 30 fields analysed


Immunoblot of endogenous USP28 , ∆ Np63 and KRT14 in LUDLU - 1adh , H1299 , Ca Ski , Hela and SiHa cells treated for 24 hours with either DMSO or indicated concentrations of AZ1 . VINCULIN served as loading

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

Immunoblot of endogenous USP28, ∆Np63 and KRT14 in LUDLU-1adh, H1299, Ca Ski, Hela and SiHa cells treated for 24 hours with either DMSO or indicated concentrations of AZ1. VINCULIN served as loading control


LUDLU - 1ADH , H1299 , Ca Ski , Hela and SiHa cells were seeded at equal cell density and cultured in the presence of either DMSO , 1µM , 10µM or 30µM AZ1 for 48h . Scale bar for LUDLU - 1adh and H1299 = 500µm . Scale bar for Ca Ski , Hela and SiHa = 250µm . SiHa * = Notably , the human SCC cell line SiHa was negative for ΔNp63 . Number of cells were quantified with the Operetta imaging system using Hoechst staining . 50 % growth inhibition ( GI50 ) was calculated . n = 30 fields

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

LUDLU-1ADH, H1299, Ca Ski, Hela and SiHa cells were seeded at equal cell density and cultured in the presence of either DMSO, 1µM, 10µM or 30µM AZ1 for 48h. Scale bar for LUDLU-1adh and H1299 = 500µm. Scale bar for Ca Ski, Hela and SiHa = 250µm. SiHa* = Notably, the human SCC cell line SiHa was negative for ΔNp63. Number of cells were quantified with the Operetta imaging system using Hoechst staining. 50% growth inhibition (GI50) was calculated. n=30 fields analysed


Immunofluorescence ( IF ) staining against endogenous ∆ Np63 in KP and KPL cell lines ( blue : DAPI [ staining of nuclei ] and red / magenta : ∆ Np63 ) , Scale bar = 32µm .

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Immunofluorescence (IF) staining against endogenous ∆Np63 in KP and KPL cell lines (blue: DAPI [staining of nuclei] and red/magenta: ∆Np63), Scale bar = 32µm.


GFP Fluorescent images of lungs treated with either PBS / DMSO / 0 , 1 % Tween80 or indicated concentrations of AZ1 . Tumour cells are positive for GFP ( green arrows ) . n = 3 Quantification of % tumour area ( normalised to total lung area ) in control ( n = 5 ) , 125 mg / kg AZ1 ( n = 4 ) and 375 mg / kg AZ1 ( n = 4 ) treated animals .

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GFP Fluorescent images of lungs treated with either PBS/DMSO/0,1% Tween80 or indicated concentrations of AZ1. Tumour cells are positive for GFP (green arrows). n=3 Quantification of % tumour area (normalised to total lung area) in control (n=5), 125 mg/kg AZ1 (n=4) and 375 mg/kg AZ1 (n=4) treated animals.


Representative staining of H & E and IHC for USP28 and ∆ Np63 of SCC tumours from mice treated with PBS / DMSO / 0 , 1 % Tween80 or indicated concentrations of AZ1 . Boxes indicate highlighted tumour areas . First line Scale bars = 500µm ; Lower scale bars = 20µm . n = 3 ∆ Np63 staining intensity of lung tumours treated with PBS / DMSO / 0 , 1 % Tween80 or indicated concentrations of AZ1 . nControl = 33712 cells ; nAZ1 125mg / kg = 7382 cells ; nAZ1 375mg / kg = 4967 cells . Cells were analysed from three independent animals .

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

Representative staining of H&E and IHC for USP28 and ∆Np63 of SCC tumours from mice treated with PBS/DMSO/0,1% Tween80 or indicated concentrations of AZ1. Boxes indicate highlighted tumour areas. First line Scale bars = 500µm; Lower scale bars = 20µm. n=3   ∆Np63 staining intensity of lung tumours treated with PBS/DMSO/0,1%Tween80 or indicated concentrations of AZ1. nControl = 33712 cells ; nAZ1 125mg/kg = 7382 cells; nAZ1 375mg/kg = 4967 cells. Cells were analysed from three independent animals.


A Western blot analysis STAT expression and phosphorylation . Bone marrow derived macrophages ( BMDMs ) of wild type ( WT ) , Stat1Y701F / + ( WT / YF ) or Stat1 - / - ( S1 ) mice were treated with 250 IU / mL of IFNβ or 5 ng / mL of IFNγ for 0 . 5 , 6 , 12 and 24 h . Whole cell extracts were collected and tested in western blot for levels of phosphorylation of STAT1 ( Y701 ) and STAT2 ( Y689 ) and total level of STAT1 and STAT2 . The blots are representative of more than 3 independent experiments .

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

A Western blot analysis STAT expression and phosphorylation. Bone marrow derived macrophages (BMDMs) of wild type (WT), Stat1Y701F/+ (WT/YF) or Stat1-/- (S1) mice were treated with 250 IU/mL of IFNβ or 5 ng/mL of IFNγ for 0.5, 6, 12 and 24 h. Whole cell extracts were collected and tested in western blot for levels of phosphorylation of STAT1 (Y701) and STAT2 (Y689) and total level of STAT1 and STAT2. The blots are representative of more than 3 independent experiments.


B Effect of STAT1Y701Fheterozygosity on the expression of type I IFN - induced genes ( ISG ) . BMDMs of wild type ( WT ) , Stat1Y701F / + ( WT / YF ) or Stat1 - / - ( S1 ) mice were treated with 250 IU / mL of IFNβ for 4 and 48 h . Gene expression was measured by Q - PCR and normalized to Gapdh and to the expression levels in untreated wild type cells . The bars represent mean values with the standard deviations ( SD ) of three independent experiments . C Effect of STAT1Y701Fheterozygosity on the expression of IFNγ - induced genes . BMDMs of wild type ( WT ) , Stat1Y701F / + ( WT / YF ) or Stat1 - / - ( S1 ) mice were treated with 5 ng / mL of IFNγ for 4 and 48 h . Gene expression was measured by Q - PCR and normalized to Gapdh and to the expression levels in untreated wild type cells . The bars represent mean values with the standard deviations ( SD ) of three independent experiments .

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

B Effect of STAT1Y701Fheterozygosity on the expression of type I IFN-induced genes (ISG). BMDMs of wild type (WT), Stat1Y701F/+ (WT/YF) or Stat1-/- (S1) mice were treated with 250 IU/mL of IFNβ for 4 and 48 h. Gene expression was measured by Q-PCR and normalized to Gapdh and to the expression levels in untreated wild type cells. The bars represent mean values with the standard deviations (SD) of three independent experiments.C Effect of STAT1Y701Fheterozygosity on the expression of IFNγ-induced genes. BMDMs of wild type (WT), Stat1Y701F/+ (WT/YF) or Stat1-/- (S1) mice were treated with 5 ng/mL of IFNγ for 4 and 48 h. Gene expression was measured by Q-PCR and normalized to Gapdh and to the expression levels in untreated wild type cells. The bars represent mean values with the standard deviations (SD) of three independent experiments.


A Effect of STAT1Y701F homozygosity on STAT1 expression in mouse cells and organs . Spleens , livers or bone marrow derived macrophages ( BMDMs ) were isolated from wild type ( WT ) , Stat1Y701F ( YF ) and Stat1 - / - ( S1 ) mice . Whole cell extracts were collected and tested for levels of total Stat1 in western blot . The blots are representative of more than 3 independent experiments .

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

A Effect of STAT1Y701F homozygosity on STAT1 expression in mouse cells and organs. Spleens, livers or bone marrow derived macrophages (BMDMs) were isolated from wild type (WT), Stat1Y701F (YF) and Stat1-/- (S1) mice. Whole cell extracts were collected and tested for levels of total Stat1 in western blot. The blots are representative of more than 3 independent experiments.


B Effect of STAT1Y701F homozygosity on STAT1 phosphorylation at Y701 . BMDMs were isolated from wild type ( WT ) , Stat1Y701F ( YF ) and Stat1 - / - ( S1 ) mice and stimulated for 30 min with 250 IU / mL of IFNβ or 5 ng / mL of IFNγ . Whole cell extracts were collected and tested for levels of STAT1 phosphorylation on Y701 in western blot . The blots are representative of more than 3 independent experiments .

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

B Effect of STAT1Y701F homozygosity on STAT1 phosphorylation at Y701. BMDMs were isolated from wild type (WT), Stat1Y701F (YF) and Stat1-/- (S1) mice and stimulated for 30 min with 250 IU/mL of IFNβ or 5 ng/mL of IFNγ. Whole cell extracts were collected and tested for levels of STAT1 phosphorylation on Y701 in western blot. The blots are representative of more than 3 independent experiments.


C Effect of STAT1Y701Fhomozygosity on the expression of IFNβ - induced genes . BMDMs of wild type ( WT ) , Stat1Y701F ( YF ) and Stat1 - / - ( S1 ) mice were treated with 5 ng / mL of IFNγ for 4 or 48 h . Gene expression was measured by Q - PCR and normalized to Gapdh and to the expression levels in untreated wild type cells . Bars represent a mean value of 3 independent experiments . Error bars represent standard error of the mean ( SEM ) ; asterisks denote the level of statistical significance ( ns , p > 0 . 05 ) ; the p - values were calculated using paired ratio t - test .

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

C Effect of STAT1Y701Fhomozygosity on the expression of IFNβ-induced genes. BMDMs of wild type (WT), Stat1Y701F (YF) and Stat1-/- (S1) mice were treated with 5 ng/mL of IFNγ for 4 or 48 h. Gene expression was measured by Q-PCR and normalized to Gapdh and to the expression levels in untreated wild type cells. Bars represent a mean value of 3 independent experiments. Error bars represent standard error of the mean (SEM); asterisks denote the level of statistical significance (ns, p>0.05); the p-values were calculated using paired ratio t-test.


D Effect of STAT1Y701Fhomozygosity on the expression of type I IFN - induced genes ( ISG ) . BMDMs were isolated from wild type ( WT ) , Stat1Y701F , Stat1 - / - , Stat2 - / - and IRF9 - / - mice treated with 250 IU / mL of IFNβ for 4 , 8 , 12 , 24 or 48 h . Gene expression was measured by Q - PCR and normalized to Gapdh and to the expression levels in untreated wild type cells . Bars represent a mean value of 3 independent experiments . Error bars represent standard error of the mean ( SEM ) ; asterisks denote the level of statistical significance ( ns , p > 0 . 05 ; * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 ) ; the p - values were calculated using paired ratio t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Effect of STAT1Y701Fhomozygosity on the expression of type I IFN-induced genes (ISG). BMDMs were isolated from wild type (WT), Stat1Y701F, Stat1-/-, Stat2-/- and IRF9-/- mice treated with 250 IU/mL of IFNβ for 4, 8, 12, 24 or 48 h. Gene expression was measured by Q-PCR and normalized to Gapdh and to the expression levels in untreated wild type cells. Bars represent a mean value of 3 independent experiments. Error bars represent standard error of the mean (SEM); asterisks denote the level of statistical significance (ns, p>0.05; *, p≤ 0.05; **, p≤ 0.01); the p-values were calculated using paired ratio t-test.


E STAT1 and STAT2 phosphorylation in Stat1 - / - , Stat1Y701F , Stat2 - / - and Irf9 - / - macrophages . BMDMs were isolated from wild type ( WT ) , Stat1Y701F ( YF ) , Stat1 - / - ( S1 ) , Stat2 - / - ( S2 ) and IRF9 - / - ( IRF9 ) mice and treated with 250 IU / mL of IFNβ for 30 min or 6 , 12 or 24 h . The whole cell extracts were collected and tested in western blot for levels of phosphorylation of STAT1 on Y701 and of STAT2 on Y689 . The same cell extracts were tested for total levels of STAT1 and STAT2 . The blots are representative of more than 3 independent experiments .

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

E STAT1 and STAT2 phosphorylation in Stat1-/-, Stat1Y701F, Stat2-/- and Irf9-/- macrophages. BMDMs were isolated from wild type (WT), Stat1Y701F (YF), Stat1-/- (S1), Stat2-/- (S2) and IRF9-/- (IRF9) mice and treated with 250 IU/mL of IFNβ for 30 min or 6, 12 or 24 h. The whole cell extracts were collected and tested in western blot for levels of phosphorylation of STAT1 on Y701 and of STAT2 on Y689. The same cell extracts were tested for total levels of STAT1 and STAT2. The blots are representative of more than 3 independent experiments.


A IFNβ - stimulated binding of STAT2 to ISRE sequences of Mx2 and IRF7 promoters . Bone marrow derived macrophages ( BMDMs ) of wild type ( WT ) , Stat1Y701F ( YF ) , Stat1 - / - ( S1 ) , Stat2 - / - ( S2 ) and IRF9 - / - ( IRF9 ) mice were treated with 250 IU / mL of IFNβ for 2 or 24 h . Cells were crosslinked , sonicated and immunoprecipitated with STAT2 - specific antibody . The amount of precipitated DNA was measured by Q - PCR . Bars represent a mean value of 3 independent experiments . Error bars represent standard error of the mean ( SEM ) ; asterisks denote the level of statistical significance ( * * , p ≤ 0 . 01 ) ; the p - values were calculated using paired ratio t - test .

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

A IFNβ-stimulated binding of STAT2 to ISRE sequences of Mx2 and IRF7 promoters. Bone marrow derived macrophages (BMDMs) of wild type (WT), Stat1Y701F (YF), Stat1-/- (S1), Stat2-/- (S2) and IRF9-/- (IRF9) mice were treated with 250 IU/mL of IFNβ for 2 or 24 h. Cells were crosslinked, sonicated and immunoprecipitated with STAT2-specific antibody. The amount of precipitated DNA was measured by Q-PCR. Bars represent a mean value of 3 independent experiments. Error bars represent standard error of the mean (SEM); asterisks denote the level of statistical significance (**, p≤ 0.01); the p-values were calculated using paired ratio t-test.


B Impact of STAT2 deficiency on IFNβ - stimulated STAT1 association with the Mx2 ISRE . BMDMs of wild type ( WT ) , Stat1 - / - ( S1 ) and Stat2 - / - ( S2 ) mice were treated with 250 IU / mL of IFNβ for 2 or 24 h . Cells were crosslinked , sonicated and immunoprecipitated with STAT1 - specific antibody . The amount of precipitated DNA was measured by Q - PCR . Bars represent mean values of three independent experiments ; error bars represent standard error of mean ( SEM ) .

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

B Impact of STAT2 deficiency on IFNβ -stimulated STAT1 association with the Mx2 ISRE. BMDMs of wild type (WT), Stat1-/- (S1) and Stat2-/- (S2) mice were treated with 250 IU/mL of IFNβ for 2 or 24 h. Cells were crosslinked, sonicated and immunoprecipitated with STAT1-specific antibody. The amount of precipitated DNA was measured by Q-PCR. Bars represent mean values of three independent experiments; error bars represent standard error of mean (SEM).


C Simultaneous association of STAT1 and STAT2 with the Mx2 ISRE analyzed by ChIP - reChIP . BMDMs of wild type ( WT ) mice were treated with 250 IU / mL of IFNβ for 2 or 24 h . Cells were crosslinked , sonicated and immunoprecipitated with either STAT1 - specific antibody and re - immunoprecipitated with STAT2 - specific antibody or vice versa . The amount of precipitated DNA was measured by Q - PCR . Bars represent mean values of three independent experiments ; error bars represent standard deviation ( SD ) .

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

C Simultaneous association of STAT1 and STAT2 with the Mx2 ISRE analyzed by ChIP-reChIP. BMDMs of wild type (WT) mice were treated with 250 IU/mL of IFNβ for 2 or 24 h. Cells were crosslinked, sonicated and immunoprecipitated with either STAT1-specific antibody and re-immunoprecipitated with STAT2-specific antibody or vice versa. The amount of precipitated DNA was measured by Q-PCR. Bars represent mean values of three independent experiments; error bars represent standard deviation (SD).


D Impact of STAT1Y701F on delayed , STAT2 - mediated expression of IFN - induced genes . Stat1 - / - fibroblasts were transfected with plasmids driving expression of the indicated proteins . 24 h after transfection , 250 IU / ml of IFNβ was added to the transfected cells and ISG expression was determined by Q - PCR after 48h of cytokine treatment . Gene expression was measured by Q - PCR and normalized to Gapdh . Bars represent a mean value of 3 independent experiments . Error bars represent standard error of the mean ( SEM ) and asterisks denote the level of statistical significance ( * , p ≤ 0 . 05 ) ; the p - values were calculated using paired ratio t - test .

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

D Impact of STAT1Y701F on delayed, STAT2-mediated expression of IFN-induced genes. Stat1-/- fibroblasts were transfected with plasmids driving expression of the indicated proteins. 24 h after transfection, 250 IU/ml of IFNβ was added to the transfected cells and ISG expression was determined by Q-PCR after 48h of cytokine treatment. Gene expression was measured by Q-PCR and normalized to Gapdh. Bars represent a mean value of 3 independent experiments. Error bars represent standard error of the mean (SEM) and asterisks denote the level of statistical significance (*, p≤ 0.05); the p-values were calculated using paired ratio t-test.


A Analysis of STAT2 nuclear translocation by immunofluorescence . Bone marrow derived macrophages ( BMDMs ) of wild type ( WT ) , Stat1Y701F ( YF ) , Stat1 - / - ( S1 ) , Stat2 - / - ( S2 ) and IRF9 - / - ( IRF9 ) mice were seeded on cover slips and stimulated with 250 IU / mL of IFNβ for 30 min or 24 h . The cells were fixed and stained for STAT2 specific antibody followed by Alexafluor ® 488 conjugated secondary antibody ( green ) . Nuclei were stained with DAPI ( blue ) . Studies are representative of more than three independent experiments . The scale bars represent 10 µm . B Quantitative evaluation of STAT2nuclear translocation . The intensity of STAT2 - dependent immunofluorescence over DNA staining ( DAPI ) was quantified using ImageJ software in 20 cells from two independent experiments . Bars represent a mean with standard deviation ( SD ) and asterisks denote the level of statistical significance ( * * * , p ≤ 0 . 001 ) ; p - value was calculated using unpaired t - test .

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

A Analysis of STAT2 nuclear translocation by immunofluorescence. Bone marrow derived macrophages (BMDMs) of wild type (WT), Stat1Y701F (YF), Stat1-/- (S1), Stat2-/- (S2) and IRF9-/- (IRF9) mice were seeded on cover slips and stimulated with 250 IU/mL of IFNβ for 30 min or 24 h. The cells were fixed and stained for STAT2 specific antibody followed by Alexafluor® 488 conjugated secondary antibody (green). Nuclei were stained with DAPI (blue). Studies are representative of more than three independent experiments. The scale bars represent 10 µm.B Quantitative evaluation of STAT2nuclear translocation. The intensity of STAT2-dependent immunofluorescence over DNA staining (DAPI) was quantified using ImageJ software in 20 cells from two independent experiments. Bars represent a mean with standard deviation (SD) and asterisks denote the level of statistical significance (***, p≤ 0.001); p-value was calculated using unpaired t-test.


A Legionella pneumophila growth in unstimulated macrophages . Bone marrow derived macrophages ( BMDMs ) of wild type ( WT ) , Stat1Y701F , Stat1 - / - mice were seeded on in the 24 - well plates and infected with L . pneumophila ( JR32 Fla - , MOI 0 . 25 ) . The numbers of colony forming units ( CFUs ) were determined 24 h , 48 h and 72 h after infection on charcoal yeast extract plates ( CYE ) . The 0 time point was collected 1 . 5 h after the infection . B Legionella pneumophila growth in IFNβ - treated macrophages . Bone marrow derived macrophages ( BMDMs ) of wild type ( WT ) , Stat1Y701F , Stat1 - / - mice were seeded in 24 - well plates , treated with 500 U / mL of IFNβ and then infected with L . pneumophila ( JR32 Fla - , MOI 0 . 25 ) . The numbers of colony forming units ( CFUs ) were determined 24 h , 48 h and 72 h after infection on charcoal yeast extract plates ( CYE ) . The 0 time point was collected 1 . 5 h after the infection .

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

A Legionella pneumophila growth in unstimulated macrophages. Bone marrow derived macrophages (BMDMs) of wild type (WT), Stat1Y701F, Stat1-/- mice were seeded on in the 24-well plates and infected with L. pneumophila (JR32 Fla-, MOI 0.25). The numbers of colony forming units (CFUs) were determined 24 h, 48 h and 72 h after infection on charcoal yeast extract plates (CYE). The 0 time point was collected 1.5 h after the infection.B Legionella pneumophila growth in IFNβ-treated macrophages. Bone marrow derived macrophages (BMDMs) of wild type (WT), Stat1Y701F, Stat1-/- mice were seeded in 24-well plates, treated with 500 U/mL of IFNβ and then infected with L. pneumophila (JR32 Fla-, MOI 0.25). The numbers of colony forming units (CFUs) were determined 24 h, 48 h and 72 h after infection on charcoal yeast extract plates (CYE). The 0 time point was collected 1.5 h after the infection.