E Wild - type ( WT ) and USP19 KO A549 cells were cultured in EBSS for indicated time points , and the protein expression levels of Beclin - 1 and ULK1 were detected by immunoblot .

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E Wild-type (WT) and USP19 KO A549 cells were cultured in EBSS for indicated time points, and the protein expression levels of Beclin-1 and ULK1 were detected by immunoblot.


F USP13 KO HEK293T cells were transfected with plasmids encoding Myc - USP5 , Myc - USP19 and Myc - USP38 and the cellular extracts were analyzed by immunoblot .

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F USP13 KO HEK293T cells were transfected with plasmids encoding Myc-USP5, Myc-USP19 and Myc-USP38 and the cellular extracts were analyzed by immunoblot.


G 293T cells transfected with plasmid expressing Flag - Beclin - 1 and empty vector or Myc - tagged USP19 ( WT , CS , CS / HA or ΔTMD ) , the lysates were analyzed by immunoblot .

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G 293T cells transfected with plasmid expressing Flag-Beclin-1 and empty vector or Myc-tagged USP19 (WT, CS, CS/HA or ΔTMD), the lysates were analyzed by immunoblot.


H 293T cells were transfected with plasmid encoding Flag - Beclin - 1 and treated with CHX ( 100 μg / ml ) alone or with MG132 ( 10 μM ) , 3MA ( 10 mM ) , CQ ( 50 μM ) as combination for 6 h or transfected with the HA - USP19 plasmid . The cell lysates were analyzed by immunoblot .

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H 293T cells were transfected with plasmid encoding Flag-Beclin-1 and treated with CHX (100 μg/ml) alone or with MG132 (10 μM), 3MA (10 mM), CQ (50 μM) as combination for 6 h or transfected with the HA-USP19 plasmid. The cell lysates were analyzed by immunoblot.


A HeLa cells were transfected with USP19 siRNA or scrambled siRNA and protein extracts were harvested after MG132 ( 10 μM ) treatment for 3 h . Protein extracts were immunoprecipitated using anti - Beclin - 1 antibody or with IgG as a negative control and analyzed by immunoblot using anti - ubiquitin and anti - Beclin - 1 antibodies .

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A HeLa cells were transfected with USP19 siRNA or scrambled siRNA and protein extracts were harvested after MG132 (10 μM) treatment for 3 h. Protein extracts were immunoprecipitated using anti-Beclin-1 antibody or with IgG as a negative control and analyzed by immunoblot using anti-ubiquitin and anti-Beclin-1 antibodies.


B Lysates of 293T cells transfected with plasmid expressing Flag - Beclin - 1 and HA - tagged ubiquitin ( HA - Ub ( wild type ) , K6 - linked - Ub , K11 - linked - Ub , K27 - linked - Ub , K29 - linked - Ub , K33 - linked - Ub , K48 - linked - Ub or K63 - linked - Ub ) , together with the empty vector or expression vector of Myc - USP19 and treated with MG132 ( 10 μM ) for 3 h were immunoprecipitated with anti - Flag and immunoblotted with anti - HA .

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B Lysates of 293T cells transfected with plasmid expressing Flag-Beclin-1 and HA-tagged ubiquitin (HA-Ub (wild type), K6-linked-Ub, K11-linked-Ub, K27-linked-Ub, K29-linked-Ub, K33-linked-Ub, K48-linked-Ub or K63-linked-Ub), together with the empty vector or expression vector of Myc-USP19 and treated with MG132 (10 μM) for 3 h were immunoprecipitated with anti-Flag and immunoblotted with anti-HA.


C HeLa cells were transfected with USP19 - specific siRNA and plasmid encoding Flag - Beclin - 1 , plus K11 - linked - Ub . Protein was harvested after MG132 ( 10 μM ) treatment for 3 h and immunoprecipitated with anti - Flag and immunoblotted with anti - HA .

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C HeLa cells were transfected with USP19-specific siRNA and plasmid encoding Flag-Beclin-1, plus K11-linked-Ub. Protein was harvested after MG132 (10 μM) treatment for 3 h and immunoprecipitated with anti-Flag and immunoblotted with anti-HA.


D 293T cells were transfected with plasmid for Flag - Beclin - 1 and HA - K11 - Ub , together with the empty vector or expression vector for Myc - USP19 ( WT , CS or CS / HA ) and treated with MG132 ( 10 μM ) . Cell lysates were subjected to immunoprecipitation with anti - Flag and immunoblot analysis with anti - HA .

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D 293T cells were transfected with plasmid for Flag-Beclin-1 and HA-K11-Ub, together with the empty vector or expression vector for Myc-USP19 (WT, CS or CS/HA) and treated with MG132 (10 μM). Cell lysates were subjected to immunoprecipitation with anti-Flag and immunoblot analysis with anti-HA.


E Purified ubiquitinated Beclin - 1 was incubated with immunopurified Flag - USP19 in vitro in deubiquitinating buffer . The immunoblot was probed with anti - HA .

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E Purified ubiquitinated Beclin-1 was incubated with immunopurified Flag-USP19 in vitro in deubiquitinating buffer. The immunoblot was probed with anti-HA.


A Lysates of 293T cells transfected with plasmid for Flag - Beclin - 1 and HA - K11 - linked - Ub , together with empty vector or expression vector for Myc - USP5 or Myc - USP19 were immunoprecipitated with anti - Flag and immunoblotted with anti - HA .

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A Lysates of 293T cells transfected with plasmid for Flag-Beclin-1 and HA-K11-linked-Ub, together with empty vector or expression vector for Myc-USP5 or Myc-USP19 were immunoprecipitated with anti-Flag and immunoblotted with anti-HA.


B Immunoprecipitation and immunoblot analysis of 293T cells transfected with vectors expressing Flag - Beclin - 1 and HA - K11 - linked ubiquitin .

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B Immunoprecipitation and immunoblot analysis of 293T cells transfected with vectors expressing Flag-Beclin-1 and HA-K11-linked ubiquitin.


C 293T cells were transfected with Flag - Beclin - 1 ( WT or K437R ) and treated with CHX ( 100 μg / ml ) for indicated time points . The expression levels of WT and K437R Flag - Beclin - 1 were analyzed by immunoblot . D Quantification of the expression levels of WT and K437R Flag - Beclin - 1 .

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C 293T cells were transfected with Flag-Beclin-1 (WT or K437R) and treated with CHX (100 μg/ml) for indicated time points. The expression levels of WT and K437R Flag-Beclin-1 were analyzed by immunoblot.D Quantification of the expression levels of WT and K437R Flag-Beclin-1.


E Immunoblot analysis of protein extracts of 293T cells transfected with empty vector or vector for HA - USP19 , together with plasmid expressing WT or K427R , K430R or K437R mutant of Flag - Beclin - 1 .

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E Immunoblot analysis of protein extracts of 293T cells transfected with empty vector or vector for HA-USP19, together with plasmid expressing WT or K427R, K430R or K437R mutant of Flag-Beclin-1.


F HeLa cells transfected with BECN1 sgRNA and restored with WT and K437R Flag - Beclin - 1 were stained by anti - LC3 antibody . Scale bar , 200 μm . G Quantification of LC3 puncta shown in ( F ) . Bars represented mean ± SEM of triplicate samples . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 .

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F HeLa cells transfected with BECN1 sgRNA and restored with WT and K437R Flag-Beclin-1 were stained by anti-LC3 antibody. Scale bar, 200 μm.G Quantification of LC3 puncta shown in (F). Bars represented mean ± SEM of triplicate samples. *p < 0.05, **p < 0.01 and ***p < 0.001.


A Luciferase activity in 293T cells transfected with an ISRE ( left ) or IFN - β ( right ) promoter - driven luciferase reporter and together with plasmid encoding RIG - I ( N ) or cGAS and STING .

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A Luciferase activity in 293T cells transfected with an ISRE (left) or IFN-β (right) promoter-driven luciferase reporter and together with plasmid encoding RIG-I (N) or cGAS and STING.


B Protein lysates of WT and USP19 KO A549 cells infected with SeV at indicated time points were immunoblotted with indicated antibodies .

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B Protein lysates of WT and USP19 KO A549 cells infected with SeV at indicated time points were immunoblotted with indicated antibodies.


C RT - PCR analysis of gene transcription in WT and USP19 KO A549 cells after infection with SeV at indicated time points .

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C RT-PCR analysis of gene transcription in WT and USP19 KO A549 cells after infection with SeV at indicated time points.


D Phase - contrast microscopy ( PH ) and fluorescence microscopy images of WT and USP19 KO A549 cells infected with VSV - eGFP at an MOI of 0 . 01 for the indicated time length . Scale bar , 50 μm .

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D Phase-contrast microscopy (PH) and fluorescence microscopy images of WT and USP19 KO A549 cells infected with VSV-eGFP at an MOI of 0.01 for the indicated time length. Scale bar, 50 μm.


E Human PBMCs were transfected with control or USP19 - specific siRNA , followed by treatment with influenza A / Puerto Rico / 8 / 34 ( H1N1 ) ( PR8 ) ( MOI = 5 ) at different time points , the lysates were analyzed with each antibody .

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E Human PBMCs were transfected with control or USP19-specific siRNA, followed by treatment with influenza A/Puerto Rico/8/34 (H1N1) (PR8) (MOI = 5) at different time points, the lysates were analyzed with each antibody.


F Human PBMCs were transfected with control or USP19 - specific siRNA were infected with H1N1 virus ( MOI = 5 ) at different time points . Relative expression levels of IFNB , ISG15 , ISG54 , ISG56 mRNA and H1N1 nucleoprotein ( NP ) RNA were measured by real - time PCR .

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F Human PBMCs were transfected with control or USP19-specific siRNA were infected with H1N1 virus (MOI = 5) at different time points. Relative expression levels of IFNB, ISG15, ISG54, ISG56 mRNA and H1N1 nucleoprotein (NP) RNA were measured by real-time PCR.


G Relative expression levels of IFNB mRNA and interferon - stimulated genes , including ISG54 and ISG56 in WT , BECN1 , ATG5 and SQSTM1 KO cells with or without USP19 .

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G Relative expression levels of IFNB mRNA and interferon-stimulated genes, including ISG54 and ISG56 in WT, BECN1, ATG5 and SQSTM1 KO cells with or without USP19.


H , I Luciferase activity in WT and BECN1 KO 293T cells transfected with an ISRE ( H ) or IFN - β ( I ) promoter - driven luciferase reporter after infection with SeV for indicated time points .

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H, I Luciferase activity in WT and BECN1 KO 293T cells transfected with an ISRE (H) or IFN-β (I) promoter-driven luciferase reporter after infection with SeV for indicated time points.


A Luciferase activity in WT and BECN1 KO 293T cells transfected with an ISRE ( left ) or IFN - β ( right ) promoter - driven luciferase reporter after infection with SeV for 12 h .

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A Luciferase activity in WT and BECN1 KO 293T cells transfected with an ISRE (left) or IFN-β (right) promoter-driven luciferase reporter after infection with SeV for 12 h.


B WT and BECN1 KO 293T cells were infected with SeV for indicated time points , then protein extracts were analyzed by immunoblot using the indicated antibodies .

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B WT and BECN1 KO 293T cells were infected with SeV for indicated time points, then protein extracts were analyzed by immunoblot using the indicated antibodies.


C Protein extracts of THP - 1 cells infected with SeV for various time points ( upper lanes ) were subjected to immunoprecipitation with anti - Beclin - 1 and immunoblot analysis with antibodies shown on the left .

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C Protein extracts of THP-1 cells infected with SeV for various time points (upper lanes) were subjected to immunoprecipitation with anti-Beclin-1 and immunoblot analysis with antibodies shown on the left.


D Co - immunoprecipitation and immunoblot analysis of protein lysates of A549 cells infected with SeV .

D Co-immunoprecipitation and immunoblot analysis of protein lysates of A549 cells infected with SeV.


E 293T cells were transfected with vectors encoding HA - Beclin - 1 and Flag - MAVS or Flag - MAVSΔCARD , followed by IP with anti - Flag beads and immunoblot analysis with anti - HA .

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E 293T cells were transfected with vectors encoding HA-Beclin-1 and Flag-MAVS or Flag-MAVSΔCARD, followed by IP with anti-Flag beads and immunoblot analysis with anti-HA.


F Co - immunoprecipitation and immunoblot analysis of WT and BECN1 KO 293T cells transfected with HA - MAVS plasmid along with vector encoding Flag - RIG - I ( N ) in the absence or presence of USP19 .

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F Co-immunoprecipitation and immunoblot analysis of WT and BECN1 KO 293T cells transfected with HA-MAVS plasmid along with vector encoding Flag-RIG-I (N) in the absence or presence of USP19.


G Protein extracts of scramble or USP19 - specific siRNA treated 293T cells transfected with HA - MAVS plasmid along with vector encoding for Flag - RIG - I ( N ) in the absence or presence of Beclin - 1 were subjected to immunoprecipitation and immunoblot analysis .

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G Protein extracts of scramble or USP19-specific siRNA treated 293T cells transfected with HA-MAVS plasmid along with vector encoding for Flag-RIG-I (N) in the absence or presence of Beclin-1 were subjected to immunoprecipitation and immunoblot analysis.


H Luciferase activity in 293T cells transfected with scramble or USP19 - specific siRNA and ISRE luciferase reporter after SeV infection for indicated time points with or without Beclin - 1 .

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H Luciferase activity in 293T cells transfected with scramble or USP19-specific siRNA and ISRE luciferase reporter after SeV infection for indicated time points with or without Beclin-1.


I Luciferase activity in 293T cells transfected with scramble or USP19 - specific siRNA and IFN - β luciferase reporter after SeV infection for indicated time points with or without Beclin - 1 .

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I Luciferase activity in 293T cells transfected with scramble or USP19-specific siRNA and IFN-β luciferase reporter after SeV infection for indicated time points with or without Beclin-1.


( B ) Violinplots showing the number of detected mRNAs and lincRNAs across distinct cell types by 10x Genomics . Total 7 , 935 cells were collected from 2 replicates of 6 mice .

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(B) Violinplots showing the number of detected mRNAs and lincRNAs across distinct cell types by 10x Genomics. Total 7,935 cells were collected from 2 replicates of 6 mice.


( C ) Boxplot showing the number of detected lincRNAs across distinct neuron types by two scRNA - seq methods . Total 1 , 818 neurons from 10x Genomics were collected from 2 replicates of 6 mice . And 197 neurons from Smart - seq2 ( analyzed in B , C and D ) were collected from lumbar 5 DRG from five adult male mice as described in previous study .

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(C) Boxplot showing the number of detected lincRNAs across distinct neuron types by two scRNA-seq methods. Total 1,818 neurons from 10x Genomics were collected from 2 replicates of 6 mice. And 197 neurons from Smart-seq2 (analyzed in B, C and D) were collected from lumbar 5 DRG from five adult male mice as described in previous study.


( E ) Representative images ( Left ) of smFISH in DRG section , tSNE plot of 10x Genomics ( Middle ) , and quantitative result ( Right ) showed that C530044C16Rik was co - localized with Gal - Cre ; Ai9 tdTomato neurons ( n = 7 ) , but not with Sst - Cre neurons ( n = 6 ) . ( F ) Representative smFISH images ( Left ) , tSNE plot of 10x Genomics ( Middle ) , and quantitative result ( Right ) showed that CLAP was co - localized with Sst - Cre ; Ai9 tdTomato neurons ( n = 6 ) , but not with Gal - Cre neurons ( n = 29 ) . ( G ) Representative smFISH images ( Left ) , tSNE plot of 10x Genomics ( Middle ) , and quantitative result ( Right ) showed that Gm11549 was co - localized with Slc17a8 - Cre ; Ai9 tdTomato neurons ( n = 5 ) , but not with Mrgprd - Cre neurons ( n = 6 ) . ( H ) Representative smFISH images ( Left ) , tSNE plot of 10x Genomics ( Middle ) , and quantitative result ( Right ) showed that 2310002F09Rik was co - localized with Slc17a8 - Cre ; Ai9 tdTomato neurons ( n = 6 ) , but not with Slc17a8 - Cre neurons ( n = 6 ) .

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(E) Representative images (Left) of smFISH in DRG section, tSNE plot of 10x Genomics (Middle), and quantitative result (Right) showed that C530044C16Rik was co-localized with Gal-Cre; Ai9 tdTomato neurons (n = 7), but not with Sst-Cre neurons (n = 6). (F) Representative smFISH images (Left), tSNE plot of 10x Genomics (Middle), and quantitative result (Right) showed that CLAP was co-localized with Sst-Cre; Ai9 tdTomato neurons (n = 6), but not with Gal-Cre neurons (n = 29). (G) Representative smFISH images (Left), tSNE plot of 10x Genomics (Middle), and quantitative result (Right) showed that Gm11549 was co-localized with Slc17a8-Cre; Ai9 tdTomato neurons (n = 5), but not with Mrgprd-Cre neurons (n = 6). (H) Representative smFISH images (Left), tSNE plot of 10x Genomics (Middle), and quantitative result (Right) showed that 2310002F09Rik was co-localized with Slc17a8-Cre; Ai9 tdTomato neurons (n = 6), but not with Slc17a8-Cre neurons (n = 6).


( I - K ) Representative images ( Right ) of double smFISH showed that 2310002F09Rik was rarely co - localized with C530044C16Rik ( I ) , CLAP ( J ) and Gm11549 ( K ) . Scale bar , 10 µm .

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(I-K) Representative images (Right) of double smFISH showed that 2310002F09Rik was rarely co-localized with C530044C16Rik (I), CLAP (J) and Gm11549 (K). Scale bar, 10 µm.


( D ) Representative smFISH images in DRG sections and quantitative results showed that 2310002F09Rik and Gm14273 were more enriched in the nucleus , whereas CLAP and Gm11549 were mainly distributed in the cytoplasm . DAPI served as a nuclear marker . Scale bar , 10 μm .

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(D) Representative smFISH images in DRG sections and quantitative results showed that 2310002F09Rik and Gm14273 were more enriched in the nucleus, whereas CLAP and Gm11549 were mainly distributed in the cytoplasm. DAPI served as a nuclear marker. Scale bar, 10 μm.


( B ) Sequence conservation analysis of lincRNA CLAP and upstream mRNA Pirt in 9 different species including mouse , human , chimpanzee , rhesus monkey , rat , cow , dog , chicken and zebrafish , which were visualized with the VISTA browser . CDS , coding sequence . UTR , untranslated region .

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(B) Sequence conservation analysis of lincRNA CLAP and upstream mRNA Pirt in 9 different species including mouse, human, chimpanzee, rhesus monkey, rat, cow, dog, chicken and zebrafish, which were visualized with the VISTA browser. CDS, coding sequence. UTR, untranslated region.


( D ) qPCR showed that CLAP was highly expressed in the DRG and other visceral organs such as small intestine , stomach and lung . The results are presented as mean ± SEM ( n = 3 ) . ( E ) qPCR showed that the expression of CLAP was gradually increased from E13 . 5 to adult DRGs . The results are presented as mean ± SEM ( n = 3 ) .

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(D) qPCR showed that CLAP was highly expressed in the DRG and other visceral organs such as small intestine, stomach and lung. The results are presented as mean ± SEM (n = 3). (E) qPCR showed that the expression of CLAP was gradually increased from E13.5 to adult DRGs. The results are presented as mean ± SEM (n = 3).


( F ) Representative bright - field and fluorescent images showing the Sst - cre ; Ai9 neurons for calcium imaging following siCLAP transfection . Scale bar , 10 μm .

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(F) Representative bright-field and fluorescent images showing the Sst-cre; Ai9 neurons for calcium imaging following siCLAP transfection. Scale bar, 10 μm.


( G ) qPCR showed that the expression of CLAP was decreased by siRNA treatment in cultured Sst - Cre ; Ai9 DRG neurons . The results are presented as mean ± SEM ( n = 3 ) . * * P < 0 . 01 versus siNC .

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(G) qPCR showed that the expression of CLAP was decreased by siRNA treatment in cultured Sst-Cre; Ai9 DRG neurons. The results are presented as mean ± SEM (n = 3). ** P < 0.01 versus siNC.


The percentage of Sst - Cre ; Ai9 neurons evoked by histamine ( I ) was decreased by knockdown of CLAP , whereas the percentage of these neurons affected by 5 - HT ( J ) , chloroquine ( K ) , capsaicin ( L ) were not changed . The results are presented as mean ± SEM ( n = 4 ) . * * P < 0 . 01 versus siNC .

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The percentage of Sst-Cre; Ai9 neurons evoked by histamine (I) was decreased by knockdown of CLAP, whereas the percentage of these neurons affected by 5-HT (J), chloroquine (K), capsaicin (L) were not changed. The results are presented as mean ± SEM (n = 4). ** P < 0.01 versus siNC.


( B ) qPCR showed that the CLAP level was significantly decreased by siCLAP in the DRG following intrathecal injection . The results are presented as mean ± SEM ( siNC = 12 , siCLAP = 15 ) . * * * P < 0 . 001 versus siNC .

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(B) qPCR showed that the CLAP level was significantly decreased by siCLAP in the DRG following intrathecal injection. The results are presented as mean ± SEM (siNC = 12, siCLAP = 15). *** P < 0.001 versus siNC.


( C - E ) Knockdown of CLAP reduced itch scratching evoked by histamine ( C ) , but not 5 - HT ( D ) and chloroquine ( E ) . The results are presented as mean ± SEM ( For histamine and 5 - HT , siNC = 12 ， siCLAP = 15 ; for chloroquine , siNC = 5 ， siCLAP = 8 ) . * * P < 0 . 01 versus siNC .

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(C-E) Knockdown of CLAP reduced itch scratching evoked by histamine (C), but not 5-HT (D) and chloroquine (E). The results are presented as mean ± SEM (For histamine and 5-HT, siNC = 12，siCLAP = 15; for chloroquine, siNC = 5，siCLAP = 8 ). ** P < 0.01 versus siNC.


( D ) The expression changes of C2 - specific and itch - related genes upon CLAP knockdown by RNA - seq . Each dot represents an RNA - seq sample , and results are presented as mean ± SEM ( n = 3 ) . * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 versus siNC .

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(D) The expression changes of C2-specific and itch-related genes upon CLAP knockdown by RNA-seq. Each dot represents an RNA-seq sample, and results are presented as mean ± SEM (n = 3). * P < 0.05, ** P < 0.01 and *** P < 0.001 versus siNC.


( F ) qPCR showed that downregulated CLAP and C2 - specific genes including Sst , Plcb3 and Cysltr2 by siCLAP was fully rescued by overexpression of CLAPR in L4 / 5 DRGs of mice . The results are presented as mean ± SEM ( siNC , n = 10 ; siCLAP , n = 6 ; siCLAP + CLAPR , n = 12 ) . * P < 0 . 01 versus siNC and # P < 0 . 05 , # # P < 0 . 01 versus the indicated treatment .

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(F) qPCR showed that downregulated CLAP and C2-specific genes including Sst, Plcb3 and Cysltr2 by siCLAP was fully rescued by overexpression of CLAPR in L4/5 DRGs of mice. The results are presented as mean ± SEM (siNC, n = 10; siCLAP, n = 6; siCLAP + CLAPR, n = 12). * P < 0.01 versus siNC and # P < 0.05, ## P < 0.01 versus the indicated treatment.


( I ) qPCR showed that downregulated Plcb3 by siCLAP was fully rescued by knockdown of MSI2 in L4 / 5 DRGs of mice . The results are presented as mean ± SEM ( siNC , n = 8 ; siCLAP , n = 6 ; siCLAP + siMSI2 , n = 5 ) . * P < 0 . 05 , * * * P < 0 . 001 versus siNC and # # P < 0 . 01 versus the indicated treatment .

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(I) qPCR showed that downregulated Plcb3 by siCLAP was fully rescued by knockdown of MSI2 in L4/5 DRGs of mice. The results are presented as mean ± SEM (siNC, n = 8; siCLAP, n = 6; siCLAP + siMSI2, n = 5). * P < 0.05, *** P < 0.001 versus siNC and ## P < 0.01 versus the indicated treatment.


Human tissue sections were probed with Siglec ‐ 5 Fc and Siglec ‐ 5 R119A Fc to determine the presence of potential sialic acid ‐ independent ligands . Positive signals ( in blue ) for such ligands were identified in the lumen of the lung bronchioles and prostate ducts , as well as in pancreatic tissues .

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Human tissue sections were probed with Siglec‐5 Fc and Siglec‐5 R119A Fc to determine the presence of potential sialic acid‐independent ligands. Positive signals (in blue) for such ligands were identified in the lumen of the lung bronchioles and prostate ducts, as well as in pancreatic tissues.


Pancreatic tissue lysate or lung BAL were incubated with Siglec ‐ 5 R119A Fc ( Sig5 ‐ RA Fc ) or Siglec ‐ 7 Fc ( Sig7Fc ) immobilized onto Protein G beads . The proteins captured by these complexes were identified by mass spectrometric analysis . Hsp70 was the only protein that was captured by the Sig5 ‐ RA Fc bead complex from pancreas lysate and bronchoalveolar lavage , but not captured by Sig7Fc bead complex .

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Pancreatic tissue lysate or lung BAL were incubated with Siglec‐5 R119A Fc (Sig5‐RA Fc) or Siglec‐7 Fc (Sig7Fc) immobilized onto Protein G beads. The proteins captured by these complexes were identified by mass spectrometric analysis. Hsp70 was the only protein that was captured by the Sig5‐RA Fc bead complex from pancreas lysate and bronchoalveolar lavage, but not captured by Sig7Fc bead complex.


Co ‐ localization of Hsp70 with Siglec ‐ 5 and Siglec ‐ 14 ligands in lung bronchiolesImmunofluorescence of human lung and prostate sections probed with Siglec ‐ 5 R119A Fc or Siglec ‐ 14 R119A Fc ( red ) with an anti ‐ Hsp70 antibody ( green ) . The co ‐ localized signals for the Siglec ‐ Fc probes with an anti ‐ Hsp70 antibody suggest that Hsp70 is one of the possible sialic acid ‐ independent ligands of Siglec ‐ 5 and Siglec ‐ 14 in the lung bronchiole . White dashes mark the lung bronchiole epithelium . All images are representative of at least 2 - 3 replicates .

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Co‐localization of Hsp70 with Siglec‐5 and Siglec‐14 ligands in lung bronchiolesImmunofluorescence of human lung and prostate sections probed with Siglec‐5 R119A Fc or Siglec‐14 R119A Fc (red) with an anti‐Hsp70 antibody (green). The co‐localized signals for the Siglec‐Fc probes with an anti‐Hsp70 antibody suggest that Hsp70 is one of the possible sialic acid‐independent ligands of Siglec‐5 and Siglec‐14 in the lung bronchiole. White dashes mark the lung bronchiole epithelium. All images are representative of at least 2-3 replicates.


Immunoblot probed with Siglec ‐ 5 Fc and Siglec ‐ 14 Fc to detect direct binding to Hsp70 . B6N domain of GBS β ‐ protein was used as a positive control , and clusterin was used as the negative control .

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Immunoblot probed with Siglec‐5 Fc and Siglec‐14 Fc to detect direct binding to Hsp70. B6N domain of GBS β‐protein was used as a positive control, and clusterin was used as the negative control.


ELISA demonstrates direct binding of Siglec ‐ 5 Fc and Siglec ‐ 14 Fc , but not Siglec ‐ 7 Fc , to Hsp70 . Bovine serum albumin ( BSA ) was used as the negative control .

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ELISA demonstrates direct binding of Siglec‐5 Fc and Siglec‐14 Fc, but not Siglec‐7 Fc, to Hsp70. Bovine serum albumin (BSA) was used as the negative control.


Siglec ‐ 5 or Siglec ‐ 14 Fc immobilized on magnetic beads was incubated with heat ‐ shocked HEK293A cell lysates . A Western blot of the captured proteins using an anti ‐ Hsp70 antibody demonstrates Hsp70 endogenously produced by HEK293A cells interacts with Siglec ‐ 5 and Siglec ‐ 14 .

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Siglec‐5 or Siglec‐14 Fc immobilized on magnetic beads was incubated with heat‐shocked HEK293A cell lysates. A Western blot of the captured proteins using an anti‐Hsp70 antibody demonstrates Hsp70 endogenously produced by HEK293A cells interacts with Siglec‐5 and Siglec‐14.


HEK293A cells were transfected with expression plasmids for Siglec ‐ 7 , Siglec ‐ 5 or Siglec ‐ 14 , and then subsequently incubated with ( red ) or without ( gray ) recombinant Hsp70 ( bottom panels ) . Flow cytometry analysis reveals Hsp70 binding to cell surfaces of HEK293A cells expressing Siglec ‐ 5 and Siglec ‐ 14 , but not Siglec ‐ 7 . Histogram of Siglec expression after transfection is shown as blue lines ( top panels ) .

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HEK293A cells were transfected with expression plasmids for Siglec‐7, Siglec‐5 or Siglec‐14, and then subsequently incubated with (red) or without (gray) recombinant Hsp70 (bottom panels). Flow cytometry analysis reveals Hsp70 binding to cell surfaces of HEK293A cells expressing Siglec‐5 and Siglec‐14, but not Siglec‐7. Histogram of Siglec expression after transfection is shown as blue lines (top panels).


Using flow cytometry , undifferentiated or PMA ‐ differentiated THP1 cells with empty vector ( EV ) , Siglec ‐ 5 ( Sig5 ) , or Siglec ‐ 14 ( Sig14 ) overexpression were assessed for the presence of endogenously secreted Hsp70 bound on the cell surface . The mean fluorescence intensity ( MFI ) of signals from an anti ‐ Hsp70 antibody on unpermeabilized THP1 cells is shown .

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Using flow cytometry, undifferentiated or PMA‐differentiated THP1 cells with empty vector (EV), Siglec‐5 (Sig5), or Siglec‐14 (Sig14) overexpression were assessed for the presence of endogenously secreted Hsp70 bound on the cell surface. The mean fluorescence intensity (MFI) of signals from an anti‐Hsp70 antibody on unpermeabilized THP1 cells is shown.


A comparison of the relative binding affinities between Siglec ‐ 5 WT versus R119A Fc , and Siglec ‐ 14 WT versus R119A Fc to Hsp70 by ELISA . The arginine ‐ mutated Siglecs display lower affinity for Hsp70 in comparison with their wild ‐ type sequence .

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A comparison of the relative binding affinities between Siglec‐5 WT versus R119A Fc, and Siglec‐14 WT versus R119A Fc to Hsp70 by ELISA. The arginine‐mutated Siglecs display lower affinity for Hsp70 in comparison with their wild‐type sequence.


A lysine ‐ to ‐ alanine mutation in residue 134 in Siglec ‐ 14 Fc ( K134A ) also abrogates its affinity for Hsp70 , as determined by ELISA . Lysine 134 was identified as a possible residue that changes binding affinity by aligning the sequences of Siglec ‐ 5 and Siglec ‐ 14 with Siglec ‐ 7 . The Siglec ‐ 14 R119A and K134A double mutant ( DM ) Fc reduces the interaction with Hsp70 to background levels .

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A lysine‐to‐alanine mutation in residue 134 in Siglec‐14 Fc (K134A) also abrogates its affinity for Hsp70, as determined by ELISA. Lysine 134 was identified as a possible residue that changes binding affinity by aligning the sequences of Siglec‐5 and Siglec‐14 with Siglec‐7. The Siglec‐14 R119A and K134A double mutant (DM) Fc reduces the interaction with Hsp70 to background levels.


Antibodies against Siglec ‐ 5 and Siglec ‐ 14 neutralized the interaction between Siglec ‐ 14 Fc to Hsp70 , as determined by ELISA .

Antibodies against Siglec‐5 and Siglec‐14 neutralized the interaction between Siglec‐14 Fc to Hsp70, as determined by ELISA.


Co ‐ incubation of B6N peptide with Siglec ‐ 5 and Siglec ‐ 14 Fc decreased binding of each Siglec to Hsp70 in an ELISA .

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Co‐incubation of B6N peptide with Siglec‐5 and Siglec‐14 Fc decreased binding of each Siglec to Hsp70 in an ELISA.


Secretion of pro ‐ inflammatory cytokine TNFα by THP1 cells transfected with an empty vector ( EV ) or Siglec ‐ 5 ( Sig5 ) expression plasmid was evaluated by ELISA after concurrent exposure with LPS and 10 μg / ml Hsp70 or LPS alone . Secretion of TNFα was reduced only in THP1 Sig5 cells exposed to both LPS and Hsp70 but not LPS alone .

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Secretion of pro‐inflammatory cytokine TNFα by THP1 cells transfected with an empty vector (EV) or Siglec‐5 (Sig5) expression plasmid was evaluated by ELISA after concurrent exposure with LPS and 10 μg/ml Hsp70 or LPS alone. Secretion of TNFα was reduced only in THP1 Sig5 cells exposed to both LPS and Hsp70 but not LPS alone.


Similarly , IL ‐ 8 secretion from THP1 EV and Sig5 cells was evaluated by ELISA after concurrent exposure with LPS and 10 μg / ml Hsp70 . Secretion of IL ‐ 8 was also reduced in only THP1 Sig5cells exposed to both LPS and Hsp70 but not LPS alone .

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Similarly, IL‐8 secretion from THP1 EV and Sig5 cells was evaluated by ELISA after concurrent exposure with LPS and 10 μg/ml Hsp70. Secretion of IL‐8 was also reduced in only THP1 Sig5cells exposed to both LPS and Hsp70 but not LPS alone.


THP1 Sig5 cells were incubated with Hsp70 , and the levels of SHP ‐ 1 recruitment to Siglec ‐ 5 were evaluated by immunoprecipitation of Siglec ‐ 5 and Western blot for SHP ‐ 1 . Higher levels of SHP ‐ 1 were co ‐ immunoprecipitated with Siglec ‐ 5 when the cells were incubated with Hsp70 .

THP1 Sig5 cells were incubated with Hsp70, and the levels of SHP‐1 recruitment to Siglec‐5 were evaluated by immunoprecipitation of Siglec‐5 and Western blot for SHP‐1. Higher levels of SHP‐1 were co‐immunoprecipitated with Siglec‐5 when the cells were incubated with Hsp70.


Western blots demonstrate phosphorylated p38 ( p ‐ p38 ) was reduced in THP1Siglec ‐ 5cells when exposed to both LPS and Hsp70 in comparison with LPS alone . This reduction was also greater in THP1Siglec ‐ 5cells in comparison with the control THP1cells . Numbers below immunoblots indicate densitometric analysis of each band normalized to the respective cell line ' s unstimulated control group , divided by the respective loading control ( total p38 ) .

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Western blots demonstrate phosphorylated p38 (p‐p38) was reduced in THP1Siglec‐5cells when exposed to both LPS and Hsp70 in comparison with LPS alone. This reduction was also greater in THP1Siglec‐5cells in comparison with the control THP1cells. Numbers below immunoblots indicate densitometric analysis of each band normalized to the respective cell line's unstimulated control group, divided by the respective loading control (total p38).


Secretion of pro ‐ inflammatory cytokine IL ‐ 8 by PMA ‐ differentiated THP1 EV or THP Sig5 cells was evaluated by ELISA after concurrent exposure with 10 ng / ml LPS with an anti ‐ Hsp70 antibody or isotype ‐ matched antibody . Secretion of IL ‐ 8 was increased in only THP1 Sig5 cells exposed to LPS and an anti ‐ Hsp70 antibody in comparison with the isotype control antibody , but this increase was not replicated in THP1 EV cells .

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Secretion of pro‐inflammatory cytokine IL‐8 by PMA‐differentiated THP1 EV or THP Sig5 cells was evaluated by ELISA after concurrent exposure with 10 ng/ml LPS with an anti‐Hsp70 antibody or isotype‐matched antibody. Secretion of IL‐8 was increased in only THP1 Sig5 cells exposed to LPS and an anti‐Hsp70 antibody in comparison with the isotype control antibody, but this increase was not replicated in THP1 EV cells.


Secretion of TNFα by THP1 cells transfected with a Siglec ‐ 5 or Siglec ‐ 14 expression vector was evaluated by ELISA after exposure to Hsp70 at the various concentrations in the presence of endotoxin ‐ neutralizing agent polymyxin B . Only THP1 Siglec ‐ 14 cells exhibited significant secretion of TNFα in an Hsp70 dose ‐ dependent manner .

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Secretion of TNFα by THP1 cells transfected with a Siglec‐5 or Siglec‐14 expression vector was evaluated by ELISA after exposure to Hsp70 at the various concentrations in the presence of endotoxin‐neutralizing agent polymyxin B. Only THP1 Siglec‐14 cells exhibited significant secretion of TNFα in an Hsp70 dose‐dependent manner.


IL ‐ 8 secretion was also evaluated after THP1Siglec ‐ 5 and Siglec ‐ 14cells were exposed to Hsp70 . Similarly , only THP1Siglec ‐ 14cells exhibited significant secretion of IL ‐ 8 in an Hsp70 dose ‐ dependent manner .

IL‐8 secretion was also evaluated after THP1Siglec‐5 and Siglec‐14cells were exposed to Hsp70. Similarly, only THP1Siglec‐14cells exhibited significant secretion of IL‐8 in an Hsp70 dose‐dependent manner.


Western blot analysis demonstrates a greater increase in phosphorylated p38 ( p ‐ p38 ) from THP1 Siglec ‐ 14 cells after exposure to Hsp70 in comparison with control THP1 EV cells in a time ‐ dependent manner . Graph below immunoblots indicates densitometric analysis of each band normalized to the respective cell line ' s unstimulated control group , divided by the respective loading control .

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Western blot analysis demonstrates a greater increase in phosphorylated p38 (p‐p38) from THP1 Siglec‐14 cells after exposure to Hsp70 in comparison with control THP1 EV cells in a time‐dependent manner. Graph below immunoblots indicates densitometric analysis of each band normalized to the respective cell line's unstimulated control group, divided by the respective loading control.


Exposure to Hsp70 also yielded greater degradation of the NF ‐ κB inhibitor , IκBα , in THP1 Siglec ‐ 14 cells in comparison with control THP1 cells , augmentation of the pro ‐ inflammatory response . Graph below immunoblots indicates densitometric analysis of each band normalized to the respective cell line ' s unstimulated control group , divided by the respective loading control .

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Exposure to Hsp70 also yielded greater degradation of the NF‐κB inhibitor, IκBα, in THP1 Siglec‐14 cells in comparison with control THP1 cells, augmentation of the pro‐inflammatory response. Graph below immunoblots indicates densitometric analysis of each band normalized to the respective cell line's unstimulated control group, divided by the respective loading control.


Secretion of pro ‐ inflammatory cytokine IL ‐ 8 by PMA ‐ differentiated THP1 EV or THP Sig 14 cells was evaluated by ELISA after concurrent exposure with an anti ‐ Hsp70 antibody or isotype ‐ matched antibody . Secretion of IL ‐ 8 was decreased in only THP1 Sig 14 cells exposed to LPS and an anti ‐ Hsp70 antibody in comparison with the isotype control antibody , but this change was not observed in THP1 EV cells .

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Secretion of pro‐inflammatory cytokine IL‐8 by PMA‐differentiated THP1 EV or THP Sig 14 cells was evaluated by ELISA after concurrent exposure with an anti‐Hsp70 antibody or isotype‐matched antibody. Secretion of IL‐8 was decreased in only THP1 Sig 14 cells exposed to LPS and an anti‐Hsp70 antibody in comparison with the isotype control antibody, but this change was not observed in THP1 EV cells.


TNFα secretion from SIGLEC14 + / + monocytes was measured after exposure to LPS or Hsp70 in the presence or absence of endotoxin ‐ neutralizing compound polymyxin B . Hsp70 was able to induce secretion of TNFα even in the presence of polymyxin B , suggesting a mechanism independent of endotoxin contaminants . DnaK , the E . coli homolog of human Hsp70 , did not induce significant secretion of TNFα above background levels .

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TNFα secretion from SIGLEC14+/+ monocytes was measured after exposure to LPS or Hsp70 in the presence or absence of endotoxin‐neutralizing compound polymyxin B. Hsp70 was able to induce secretion of TNFα even in the presence of polymyxin B, suggesting a mechanism independent of endotoxin contaminants. DnaK, the E. coli homolog of human Hsp70, did not induce significant secretion of TNFα above background levels.


TNFα secretion from SIGLEC14 + / + monocytes by Hsp70 was reduced when the cells were exposed to an anti ‐ Siglec ‐ 5 / Siglec ‐ 14 blocking antibody .

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

TNFα secretion from SIGLEC14+/+ monocytes by Hsp70 was reduced when the cells were exposed to an anti‐Siglec‐5/Siglec‐14 blocking antibody.


Human monocytes isolated from SIGLEC14 + / + , SIGLEC14 + / − , and SIGLEC14 − / − individuals were exposed to varying concentrations of Hsp70 , and TNFα secretion was evaluated . The amount of TNFα secreted was normalized to the respective cells ' response to 1 ng / ml LPS to minimize the impact of other possible polymorphisms and factors that contribute to variation between individuals .

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

Human monocytes isolated from SIGLEC14+/+, SIGLEC14+/−, and SIGLEC14−/− individuals were exposed to varying concentrations of Hsp70, and TNFα secretion was evaluated. The amount of TNFα secreted was normalized to the respective cells' response to 1 ng/ml LPS to minimize the impact of other possible polymorphisms and factors that contribute to variation between individuals.


TNFα secretion from SIGLEC14 − / − monocytes stimulated with 15 ng / ml LPS and various concentrations of Hsp70 . TNFα secretion was reduced with increasing concentrations of Hsp70 .

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

TNFα secretion from SIGLEC14−/− monocytes stimulated with 15 ng/ml LPS and various concentrations of Hsp70. TNFα secretion was reduced with increasing concentrations of Hsp70.


( B ) B - RafE586K signals independently from Ras - GTP , but requires an intact AL . The MEK - ERK activation potential of the indicated HA - tagged B - Raf mutants was assessed by Western blotting using TCLs from transiently transfected Plat - E cells .

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

(B) B-RafE586K signals independently from Ras-GTP, but requires an intact AL. The MEK-ERK activation potential of the indicated HA-tagged B-Raf mutants was assessed by Western blotting using TCLs from transiently transfected Plat-E cells.


( C ) B - RafCAAX but not the B - RafV600E oncoprotein signals independently from Ras - GTP , but requires an intact AL . Same experimental set - up as in ( B ) . ( D ) Quantification of experiments shown in ( B ) and ( C ) . The signal elicited by the individual reference proteins ( B - Rafwt , B - RafE586K , B - RafV600E and B - RafCAAX ) was set in each analysis to 100 % . n = 3 , mean + SEM , t - test , * p < 0 . 05 , * * p < 0 .

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

(C) B-RafCAAX but not the B-RafV600E oncoprotein signals independently from Ras-GTP, but requires an intact AL. Same experimental set-up as in (B). (D) Quantification of experiments shown in (B) and (C). The signal elicited by the individual reference proteins (B-Rafwt, B-RafE586K, B-RafV600E and B-RafCAAX) was set in each analysis to 100%. n = 3, mean + SEM, t-test, * p < 0.05, ** p < 0.01


( A ) Braf - / - ; ERTmHRasG12V MEFs were reconstituted with HA - tagged hBRAF constructs or empty vector ( EV ) . Infected cells were either treated with 4 - HT ( 1µM ) to induce oncogenic Ras activation or with ethanol ( OH ) as a control . ( B ) Quantifications of pMEK ( left ) and pERK ( right ) normed to WT level . n = 3 , mean + SEM , t - test , * * p < 0 . 01 .

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(A) Braf-/-;ERTmHRasG12V MEFs were reconstituted with HA-tagged hBRAF constructs or empty vector (EV). Infected cells were either treated with 4-HT (1µM) to induce oncogenic Ras activation or with ethanol (OH) as a control. (B) Quantifications of pMEK (left) and pERK (right) normed to WT level. n = 3, mean + SEM, t-test, ** p < 0.01.


( C ) The AVKA mutation has no discernible influence on Ras - induced paradoxical ERK activation triggered by the D594A mutation . Experimental set - up as in ( A ) , shown are lysates from 4 - HT treated MEFs . ( D ) Quantification of pMEK and pERK levels normed to HA expression . n = 3 , mean + SEM , t - test , * * p < 0 . 01 .

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(C) The AVKA mutation has no discernible influence on Ras-induced paradoxical ERK activation triggered by the D594A mutation. Experimental set-up as in (A), shown are lysates from 4-HT treated MEFs. (D) Quantification of pMEK and pERK levels normed to HA expression. n = 3, mean + SEM, t-test, ** p < 0.01.


( E ) Top : Braf - / - ; ERTmHRasG12V MEFs were reconstituted with the indicated B - Raf constructs . Following induction of Ras signaling , HA - B - Raf proteins were purified using anti - HA antibodies and subject to Western blotting to analyze immunoprecipitation of Raf - 1 . Bottom : Quantification of co - immunoprecipitated Raf - 1 normed to HA - B - Raf levels . n = 3 , mean + SEM , t - test , * p < 0 . 05 .

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

(E) Top: Braf-/-;ERTmHRasG12V MEFs were reconstituted with the indicated B-Raf constructs. Following induction of Ras signaling, HA-B-Raf proteins were purified using anti-HA antibodies and subject to Western blotting to analyze immunoprecipitation of Raf-1. Bottom: Quantification of co-immunoprecipitated Raf-1 normed to HA-B-Raf levels. n = 3, mean + SEM, t-test, * p < 0.05.


( B ) Genomic PCR with the primers indicated as red arrows in ( A ) showing 3 out of 9 clones being positive for homologous recombination .

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(B) Genomic PCR with the primers indicated as red arrows in (A) showing 3 out of 9 clones being positive for homologous recombination.


( C ) Southern blot analysis of genomic DNA of parental W4 ES cells , wildtype MEFs and the ES cell clones # 286 and # 273 ( with recombined B - raffloxAVKA allele ) . Genomic DNA was digested by AseI and analyzed using the external probe indicated in ( A ) .

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(C) Southern blot analysis of genomic DNA of parental W4 ES cells, wildtype MEFs and the ES cell clones #286 and #273 (with recombined B-raffloxAVKA allele). Genomic DNA was digested by AseI and analyzed using the external probe indicated in (A).


( D ) Genomic PCR showing Cre - mediated recombination of the BraffloxAVKA allele in vivo using the primer pair E14fwd and E15rev ( indicated as blue arrows in A ) .

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(D) Genomic PCR showing Cre-mediated recombination of the BraffloxAVKA allele in vivo using the primer pair E14fwd and E15rev (indicated as blue arrows in A).


( A ) Weight at weaning of BrafAVKA mice from two different founders showed no significant differences . 215 : n = WT = 24 / Heterozygous = 32 / AVKA = 19 ; 273 : n = WT = 114 / Heterozygous = 117 / AVKA = 52 , mean + SEM , t -

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(A) Weight at weaning of BrafAVKA mice from two different founders showed no significant differences. 215: n = WT = 24/ Heterozygous = 32/ AVKA = 19; 273: n = WT = 114/ Heterozygous = 117/ AVKA = 52, mean + SEM, t-test


( B ) Homozygous BrafAVKA mice show reduced splenic cellularity . n = 7 , mean + SEM , t - test , * p < 0 . 05 .

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(B) Homozygous BrafAVKA mice show reduced splenic cellularity. n = 7, mean + SEM, t-test, * p < 0.05.


( C - E ) Relative number of CD8 + T cells . FACS analysis shows an increase in naïve cells ( C ) , whereas differentiated TEM ( D ) and Tregs ( E ) reveal a reduction in BrafAVKA mice compared to WT . n = 8 , mean + SEM , t - test , * p < 0 . 05 , * * p < 0 . 01 .

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(C-E) Relative number of CD8+ T cells. FACS analysis shows an increase in naïve cells (C), whereas differentiated TEM (D) and Tregs (E) reveal a reduction in BrafAVKA mice compared to WT. n = 8, mean + SEM, t-test, * p < 0.05, ** p < 0.01.


( F ) Primary B cells of BrafAVKA mice show reduced CD69 surface expression upon LPS and anti - IgM stimulation . n = 3 , mean + SEM , test , * * p < 0 . 01 .

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(F) Primary B cells of BrafAVKA mice show reduced CD69 surface expression upon LPS and anti-IgM stimulation. n = 3, mean + SEM, test, ** p < 0.01.


( G ) Primary B cells also show reduced proliferation upon stimulation compared with WT B cells . n = 3 , mean + SEM , test , * * p < 0 .

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(G) Primary B cells also show reduced proliferation upon stimulation compared with WT B cells. n = 3, mean + SEM, test, ** p < 0.01


( A ) Facial appearance of BrafAVKA mice .

(A) Facial appearance of BrafAVKA mice.


( B ) Top : In vitro kinase ( IVK ) assay of B - Raf complexes purified from either WT or BrafAVKA knock - in brains from both founder strains revealed a reduced MEK phosphorylation potential regardless of their purification from NLB or RIPA lysates . Botttom : Quantification of IVK differentials : # 215 samples indicated in black , # 273 samples in grey .

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

(B) Top: In vitro kinase (IVK) assay of B-Raf complexes purified from either WT or BrafAVKA knock-in brains from both founder strains revealed a reduced MEK phosphorylation potential regardless of their purification from NLB or RIPA lysates. Botttom: Quantification of IVK differentials: #215 samples indicated in black, #273 samples in grey.


( C ) Western blot analysis showing reduced pMEK and pERK levels in BrafAVKA brains . n = 5 , mean + SEM , t - test , * * < 0 . 01 .

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(C) Western blot analysis showing reduced pMEK and pERK levels in BrafAVKA brains. n = 5, mean + SEM, t-test, ** < 0.01.


( D ) Immunohistochemistry showing reduced pERK levels in Purkinje cells of homozygous BrafAVKA mice . Scale bar indicates 10x magnification .

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(D) Immunohistochemistry showing reduced pERK levels in Purkinje cells of homozygous BrafAVKA mice. Scale bar indicates 10x magnification.


( A ) Total brain lysates of WT and BrafAVKA mice were subject to immunoprecipitation ( IP ) using either anti - B - Raf antibodies or non - specific IgG as negative control . The co - purified proteins were visualized using the indicated antibodies and quantified . Ksr1 is significantly decreased in B - RafAVKA complexes while the other proteins are equally bound to B - RafWT or B - RafAVKA . n = 3 , mean + SEM , t - test , * p < 0 . 05 .

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

(A) Total brain lysates of WT and BrafAVKA mice were subject to immunoprecipitation (IP) using either anti-B-Raf antibodies or non-specific IgG as negative control. The co-purified proteins were visualized using the indicated antibodies and quantified. Ksr1 is significantly decreased in B-RafAVKA complexes while the other proteins are equally bound to B-RafWT or B-RafAVKA. n = 3, mean + SEM, t-test, * p < 0.05.


( B ) Anti - Myc IP of Plat - E cells transfected with Myc - tagged Raf - 1 and different HA - B - Raf constructs . Western blot analysis showed that B - RafAVKA is not impaired in its ability to interact with Raf - 1 .

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

(B) Anti-Myc IP of Plat-E cells transfected with Myc-tagged Raf-1 and different HA-B-Raf constructs. Western blot analysis showed that B-RafAVKA is not impaired in its ability to interact with Raf-1.


( C ) Anti - Myc IP of Plat - E cells expressing different Myc - and HA - B - Raf proteins . Note that B - RafAVKA is not impaired in its ability to interact with other B - Raf proteins , while homo - dimerization of B - RafEVKD is enhanced .

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

(C) Anti-Myc IP of Plat-E cells expressing different Myc- and HA-B-Raf proteins. Note that B-RafAVKA is not impaired in its ability to interact with other B-Raf proteins, while homo-dimerization of B-RafEVKD is enhanced.


( D ) Top : Exemplary Western blot analysis of EGF stimulated primary astrocytes . Mutant astrocytes show drastic reduction in basic and stimulated levels of pMEK and pERK . Bottom : Quantification of all Western blot samples . WT : n = 7 , AVKA : n = 6 , mean + SEM , t - test , * p < 0 . 05 , * * p < 0 . 01 .

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(D) Top: Exemplary Western blot analysis of EGF stimulated primary astrocytes. Mutant astrocytes show drastic reduction in basic and stimulated levels of pMEK and pERK. Bottom: Quantification of all Western blot samples. WT: n = 7, AVKA: n= 6, mean + SEM, t-test, * p <0.05, ** p < 0.01.


( A ) MEFs expressing B - RafWT or B - RafAVKA were stimulated with EGF for the indicated time points ( min ) and analyzed by Western blotting .

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

(A) MEFs expressing B-RafWT or B-RafAVKA were stimulated with EGF for the indicated time points (min) and analyzed by Western blotting.


( B ) IP of EGF stimulated MEFs expressing B - RafWT or B - RafAVKA . Sorafenib was used to increase complex build up .

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

(B) IP of EGF stimulated MEFs expressing B-RafWT or B-RafAVKA. Sorafenib was used to increase complex build up.


( C ) Proliferation of two different MEF pools expressing either B - RafWT or B - RafAVKA . MEFs expressing B - RafAVKA show a strong reduction in growth rate . n = 3 , mean + SEM , t - test , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(C) Proliferation of two different MEF pools expressing either B-RafWT or B-RafAVKA. MEFs expressing B-RafAVKA show a strong reduction in growth rate. n = 3, mean + SEM, t-test, ** p < 0.01, *** p < 0.001.


( D ) Cell morphology of B - RafAVKA expressing MEFs are enlarged and present with abnormal

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(D) Cell morphology of B-RafAVKA expressing MEFs are enlarged and present with abnormal nuclei


( E ) MEFs expressing B - RafAVKA show a significant increase in caspase - 3 activity . n = 3 , mean + SEM , t - test , * * p < 0 . 01 .

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(E) MEFs expressing B-RafAVKA show a significant increase in caspase-3 activity. n = 3, mean + SEM, t-test, ** p < 0.01.


( F ) Decay of TAg expression in MEFs following dox withdrawal .

(F) Decay of TAg expression in MEFs following dox withdrawal.


( G ) Morphology of MEFs expressing B - Rafwt or B - RafAVKA in the presence or absence of TAg .

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(G) Morphology of MEFs expressing B-Rafwt or B-RafAVKA in the presence or absence of TAg.


( A ) MEFs were then infected with retroviral vectors encoding the indicated B - Raf proteins . Giemsa staining revealed transformed and hyper - proliferated cells by dark purple colour . ( B ) Quantification of Focus formation potential of the B - Raf mutants used in ( A ) . n = 3 , mean + SEM , t - test , * * * p < 0 . 001 .

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 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

(A) MEFs were then infected with retroviral vectors encoding the indicated B-Raf proteins. Giemsa staining revealed transformed and hyper-proliferated cells by dark purple colour. (B) Quantification of Focus formation potential of the B-Raf mutants used in (A). n = 3, mean + SEM, t-test, *** p < 0.001.


A . Example of littermate males of the indicated genotypes 26 days post partum . Additional examples are provided in Appendix Fig S2 .

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A. Example of littermate males of the indicated genotypes 26 days post partum. Additional examples are provided in Appendix Fig S2.


B . Weights of mice of the indicated genotypes from 18 to 120 days post partum . Boxplots indicate 5 - 95 percentile . A minimum of 8 animals is plotted at each age . Asterisks denote statistical significance ( NS , not significant , * P value < 0 . 05 , * * P value < 0 . 01 and * * * P value < 0 . 001 ) determined by the unpaired two - way Wilcoxon rank - sum test .

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B. Weights of mice of the indicated genotypes from 18 to 120 days post partum. Boxplots indicate 5-95 percentile. A minimum of 8 animals is plotted at each age. Asterisks denote statistical significance (NS, not significant, *P value <0.05, **P value <0.01 and ***P value <0.001) determined by the unpaired two-way Wilcoxon rank-sum test.


C . Example of dissected brains ( upper panel ) and coronal sections ( left and right panels ) stained with H & amp ; amp ; E of the indicated genotypes . Note the severe hydrocephaly and ventricular dilation of the lateral ventricle ( LV ) and third ventricle ( TV ) in a Gemc1 - / - mouse ( bottom right panel ) compared to a Gemc1 + / + littermate animal ( bottom left panel ) . Periventricular malacia ( asterisks ) and hippocampal atrophy ( arrow ) is also evident in the Gemc1 - / - sample . Scale bars = 500 µm .

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C. Example of dissected brains (upper panel) and coronal sections (left and right panels) stained with H&amp;E of the indicated genotypes. Note the severe hydrocephaly and ventricular dilation of the lateral ventricle (LV) and third ventricle (TV) in a Gemc1-/- mouse (bottom right panel) compared to a Gemc1+/+ littermate animal (bottom left panel). Periventricular malacia (asterisks) and hippocampal atrophy (arrow) is also evident in the Gemc1-/- sample. Scale bars = 500 µm.


D . Kaplan Meyer survival curve of a cohort of mice of the indicated genotypes and numbers ( n ) . The study was terminated at 16 months and mice were examined for histology . 97 % ( 31 / 32 ) of the Gemc1 - / - mice examined to date exhibited hydrocephaly ( additional histological examples in Appendix Fig S2 ) .

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D. Kaplan Meyer survival curve of a cohort of mice of the indicated genotypes and numbers (n). The study was terminated at 16 months and mice were examined for histology. 97% (31/32) of the Gemc1-/- mice examined to date exhibited hydrocephaly (additional histological examples in Appendix Fig S2).


E . RT - QPCR analysis of Gemc1 expression in murine tissues from Gemc1 + / + mice using a Taqman probe . Data is compiled from duplicate samples of 3 male mice per tissue and mean and standard deviation are plotted .

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E. RT-QPCR analysis of Gemc1 expression in murine tissues from Gemc1+/+ mice using a Taqman probe. Data is compiled from duplicate samples of 3 male mice per tissue and mean and standard deviation are plotted.


F . RT - QPCR analysis of Gemc1 in trachea , oviduct and ovary tissue from Gemc1 + / + and Gemc1 - / - mice shows that the knockout reduces Gemc1 mRNA to undetectable levels . Mean and standard deviation of duplicate samples from 2 female mice are plotted . Additional examples from other Gemc1 - / - tissues are shown in Appendix Fig S1 .

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F. RT-QPCR analysis of Gemc1 in trachea, oviduct and ovary tissue from Gemc1+/+ and Gemc1-/- mice shows that the knockout reduces Gemc1 mRNA to undetectable levels. Mean and standard deviation of duplicate samples from 2 female mice are plotted. Additional examples from other Gemc1-/- tissues are shown in Appendix Fig S1.


. Example of dissected testes from littermate mice of the indicated genotypes at 2 months of age .

. Example of dissected testes from littermate mice of the indicated genotypes at 2 months of age.


B . Testes from Gemc1 - / - mice have strongly reduced cellularity compared to Gemc1 + / + or Gemc1 + / - animals , as measured by disaggregation of the tissue and cell counting with a Neubauer Chamber ( n = 3 ) . Mean and standard deviation are indicated .

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B. Testes from Gemc1-/- mice have strongly reduced cellularity compared to Gemc1+/+ or Gemc1+/- animals, as measured by disaggregation of the tissue and cell counting with a Neubauer Chamber (n=3). Mean and standard deviation are indicated.


C . Histological analysis of tubules from 6 - week - old Gemc1 - / - mice revealed thinning of the spermatogenic cell layer and decreased numbers of elongated spermatids . Scale bars = 100 µm ( left panels ) and 50 µm ( right panels ) .

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C. Histological analysis of tubules from 6-week-old Gemc1-/- mice revealed thinning of the spermatogenic cell layer and decreased numbers of elongated spermatids. Scale bars = 100 µm (left panels) and 50 µm (right panels).


D . Sperm counts from the cauda epididymis of wild type and Gemc1 - / - mice revealed no sperm in mice lacking GEMC1 ( n = 3 ) . Mean and standard deviation are indicated . E . Examples of sperm morphology from spermatocyte spreads from 2 month old mice . Scale bars = 10 µm .

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D. Sperm counts from the cauda epididymis of wild type and Gemc1-/- mice revealed no sperm in mice lacking GEMC1 (n=3). Mean and standard deviation are indicated. E. Examples of sperm morphology from spermatocyte spreads from 2 month old mice. Scale bars = 10 µm.


F and G . Histological evaluation of the caput epididymis in sagittal ( F ) and coronal ( G ) orientation from 2 and 4 month old animals , respectively . Note the absence of luminal spermatozoa in the Gemc1 - / - mice compared to wild type . Scale bars = 100 µm ( F , left panels ) and 50 µm ( F , right panels and G ) .

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F and G. Histological evaluation of the caput epididymis in sagittal (F) and coronal (G) orientation from 2 and 4 month old animals, respectively. Note the absence of luminal spermatozoa in the Gemc1-/- mice compared to wild type. Scale bars = 100 µm (F, left panels) and 50 µm (F, right panels and G).


A . Examples of ovaries from Gemc1 + / + ( top panels ) and Gemc1 - / - mice ( bottom panels ) at 6 weeks of age . Ovaries were smaller in Gemc1 - / - mice and contained primarily degenerated antral follicles . A histologically normal antral follicle is indicated in the Gemc1 + / + ovary ( top right , black arrow ) and degenerated antral follicles with high levels of cell death are indicated in the Gemc1 - / - ovaries ( bottom right , red arrows ) . Scale bars = 200 µm ( left panels ) ) and 100 µm ( right top panels and right bottom panel respectively ) . B . Morphological abnormalities in the oviduct of 3 - week - old Gemc1 - / - mice ( right panels ) compared to Gemc1 + / + ( left panels ) . A clear lack of cilia ( bottom panels ) in Gemc1 - / - oviducts is observed by H & amp ; amp ; E staining . Scale bars = 50 µm ( top panels ) .

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A. Examples of ovaries from Gemc1+/+ (top panels) and Gemc1-/- mice (bottom panels) at 6 weeks of age. Ovaries were smaller in Gemc1-/- mice and contained primarily degenerated antral follicles. A histologically normal antral follicle is indicated in the Gemc1+/+ ovary (top right, black arrow) and degenerated antral follicles with high levels of cell death are indicated in the Gemc1-/- ovaries (bottom right, red arrows). Scale bars = 200 µm (left panels)) and 100 µm (right top panels and right bottom panel respectively). B. Morphological abnormalities in the oviduct of 3-week-old Gemc1-/- mice (right panels) compared to Gemc1+/+ (left panels). A clear lack of cilia (bottom panels) in Gemc1-/- oviducts is observed by H&amp;E staining. Scale bars = 50 µm (top panels).


C . Staining of frozen sections of oviduct with antibodies for Ac - tubulin ( cilia ) and γ - tubulin ( basal bodies ) reveals normal cilia organization in the wild type oviduct ( left ) while basal bodies and cilia are not detectable in Gemc1 - / - oviducts ( right ) . D . FOXJ1 and E . RFX3 staining that is characteristic of MCCs was clearly evident in Gemc1 + / + oviducts and absent in Gemc1 - / - mice . Scale bars = 50 µm ( C - E ) .

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C. Staining of frozen sections of oviduct with antibodies for Ac-tubulin (cilia) and γ-tubulin (basal bodies) reveals normal cilia organization in the wild type oviduct (left) while basal bodies and cilia are not detectable in Gemc1-/- oviducts (right). D. FOXJ1 and E. RFX3 staining that is characteristic of MCCs was clearly evident in Gemc1+/+ oviducts and absent in Gemc1-/- mice. Scale bars = 50 µm (C-E).


A . H & amp ; amp ; E staining of the nasal mucosa of E17 . 5 embryos of the indicated genotype . Normal respiratory pseudostratified columnar ciliated epithelium is shown in Gemc1 + / + embryos compared to the attenuated , flat columnar to squamous epithelium of Gemc1 - / - embryos . Scale bars = 20 µm .

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A. H&amp;E staining of the nasal mucosa of E17.5 embryos of the indicated genotype. Normal respiratory pseudostratified columnar ciliated epithelium is shown in Gemc1+/+ embryos compared to the attenuated, flat columnar to squamous epithelium of Gemc1-/- embryos. Scale bars = 20 µm.


B . PAS staining ( purple ) of the nasal mucosa of E18 . 5 embryos of the indicated genotype . Normal PAS positive goblet cells ( inset , red arrows ) are interspersed between MCCs ( inset , black arrows ) with their characteristic goblet or cup - like shape in Gemc1 + / + embryos . The Gemc1 - / - mucosa has abundant PAS positive materials that accumulate in the apical borders of almost every columnar epithelial cell ( inset , red arrows ) and lacks typical goblet cells . Scale bars = 20 µm .

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B. PAS staining (purple) of the nasal mucosa of E18.5 embryos of the indicated genotype. Normal PAS positive goblet cells (inset, red arrows) are interspersed between MCCs (inset, black arrows) with their characteristic goblet or cup-like shape in Gemc1+/+ embryos. The Gemc1-/- mucosa has abundant PAS positive materials that accumulate in the apical borders of almost every columnar epithelial cell (inset, red arrows) and lacks typical goblet cells. Scale bars = 20 µm.


C . Transmission electron micrographs of cross - sections of adult trachea from Gemc1 + / + animals revealed the presence of secretory cells ( SC ) and multiciliated cells ( MCC ) . The apical membrane of MCCs accumulated basal bodies ( BB ) nucleating multiple cilia ( C ) and microvilli ( MV ) . In the Gemc1 - / - airway epithelium , no MCCs are identifiable and only secretory - like cells with MV at the apical surface are detectable . Scale bars = 5µm . Higher magnifications of the boxed regions are shown below , scale bars = 0 . 5µm .

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C. Transmission electron micrographs of cross-sections of adult trachea from Gemc1+/+ animals revealed the presence of secretory cells (SC) and multiciliated cells (MCC). The apical membrane of MCCs accumulated basal bodies (BB) nucleating multiple cilia (C) and microvilli (MV). In the Gemc1-/- airway epithelium, no MCCs are identifiable and only secretory-like cells with MV at the apical surface are detectable. Scale bars = 5µm. Higher magnifications of the boxed regions are shown below, scale bars = 0.5µm.


D . Lack of Ac - tubulin ( top panel ) and RFX3 ( second panel from top ) staining in the trachea of 1 month old mice .

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D. Lack of Ac-tubulin (top panel) and RFX3 (second panel from top) staining in the trachea of 1 month old mice.


E . Cilia ( Ac - tubulin staining ) are not detectable in the ependymal cells of 2 month old Gemc1 - / - mice ( right panel ) . Choroid plexus ( CP ) and lateral ventricles ( LV ) are indicated . Scale bars = 20

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E. Cilia (Ac-tubulin staining) are not detectable in the ependymal cells of 2 month old Gemc1-/- mice (right panel). Choroid plexus (CP) and lateral ventricles (LV) are indicated. Scale bars = 20 µm


E and F . RT - QPCR analysis of individual genes related to MCC formation in the oviduct ( E ) and trachea ( F ) of Gemc1 - / - mice compared to wild type littermates , using 18S rRNA as a reference ( n = 2 ) . Genes examined generally fall into 3 classes : centriole duplication ( Cep152 , Plk4 , Cetn2 ) , Deuterosome mediated centriole expansion ( Deup1 , Ccdc78 , Ccno ) and transcription of genes required for MCC differentiation ( FoxJ1 , Myb , Mcidas ) .

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E and F. RT-QPCR analysis of individual genes related to MCC formation in the oviduct (E) and trachea (F) of Gemc1-/- mice compared to wild type littermates, using 18S rRNA as a reference (n=2). Genes examined generally fall into 3 classes: centriole duplication (Cep152, Plk4, Cetn2), Deuterosome mediated centriole expansion (Deup1, Ccdc78, Ccno) and transcription of genes required for MCC differentiation (FoxJ1, Myb, Mcidas).


A . GEMC1 interacts with E2F4 and DP1 . Western blot of HA or FLAG tagged protein expression in the input lysates are shown in the top panel . FLAGimmunoprecipiation ( IP ) of GEMC1co - IPs DP1 ( α - HA , lane 5 ) and E2F4 ( α - HA , lane 6 ) . The interaction is enhanced upon co - expression of HA tagged E2F4 and DP1 ( α - HA , lane 7 ) .

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A. GEMC1 interacts with E2F4 and DP1. Western blot of HA or FLAG tagged protein expression in the input lysates are shown in the top panel. FLAGimmunoprecipiation (IP) of GEMC1co-IPs DP1 (α-HA, lane 5) and E2F4 (α- HA, lane 6). The interaction is enhanced upon co-expression of HA tagged E2F4 and DP1 (α-HA, lane 7).


B . GEMC1 interacts with E2F5 but not E2F1 . Co - IP of E2F5 ( α - HA , lane 3 ) and E2F5 - DP1 ( α - HA , lane 6 ) but not E2F1 or E2F1 - DP1 ( α - HA , lanes 2 and 5 ) . In the FLAG - IP panels , a short ( top ) and long ( second from top ) exposure of the Western are shown .

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B. GEMC1 interacts with E2F5 but not E2F1. Co-IP of E2F5 (α-HA, lane 3) and E2F5-DP1 (α-HA, lane 6) but not E2F1 or E2F1-DP1 (α-HA, lanes 2 and 5). In the FLAG-IP panels, a short (top) and long (second from top) exposure of the Western are shown.


C . Identification of the domain required for E2F4 interaction . GFP tagged GEMC1 and mutant forms were expressed and IPs were performed against GFP . Wild type GEMC1 ( lane 5 ) or GEMC1 lacking the CC domain ( lane 6 ) pulled down E2F4 - DP1 . A GEMC1 protein lacking the TIRT domain or GFP alone did not IP E2F4 - DP1 ( lanes 7 , 8 ) .

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C. Identification of the domain required for E2F4 interaction. GFP tagged GEMC1 and mutant forms were expressed and IPs were performed against GFP. Wild type GEMC1 (lane 5) or GEMC1 lacking the CC domain (lane 6) pulled down E2F4-DP1. A GEMC1 protein lacking the TIRT domain or GFP alone did not IP E2F4-DP1 (lanes 7, 8).


D . Mutation of the single amino acid analogous that identified in MCIDAS in human RGMC patients ( G313D ) impairs the interaction with E2F4 - DP1 ( compare land 5 and 6 ) .

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D. Mutation of the single amino acid analogous that identified in MCIDAS in human RGMC patients (G313D) impairs the interaction with E2F4-DP1 (compare land 5 and 6).


E . GEMC1 interacts with Multicilin through its CC domain . Wild type GEMC1 or a mutant lacking the TIRT domain pull down Myc - tagged Multicilin ( anti - Myc blot , lanes 4 and 6 ) while a CC domain mutant does not ( anti - Myc blot , lane 5 ) .

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E. GEMC1 interacts with Multicilin through its CC domain. Wild type GEMC1 or a mutant lacking the TIRT domain pull down Myc-tagged Multicilin (anti- Myc blot, lanes 4 and 6) while a CC domain mutant does not (anti-Myc blot, lane 5).


A . Transient overexpression of GEMC1 leads to increased levels of FOXJ1 and MCIDAS expression ( RT - QPCR ) in HEK293T cells . Results for MCIDAS are graphed on a smaller scale in the right panel : V = vector and G = GEMC1 . The mean and standard deviation of 3 individual experiments in HEK293T cells are graphed . Similar induction of FOXJ1 was observed in U2OS and T98G ( Fig EV3 ) .

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A. Transient overexpression of GEMC1 leads to increased levels of FOXJ1 and MCIDAS expression (RT-QPCR) in HEK293T cells. Results for MCIDAS are graphed on a smaller scale in the right panel: V = vector and G = GEMC1. The mean and standard deviation of 3 individual experiments in HEK293T cells are graphed. Similar induction of FOXJ1 was observed in U2OS and T98G (Fig EV3).


B . Both the CC and the TIRT domain of GEMC1 are required to activate FOXJ1 ( RT - QPCR ) . A corresponding Western blot showing relative expression levels of the mutant GFP tagged GEMC1 variants is shown from HEK293T cells . The mean and standard deviation of 3 individual experiments in HEK293T cells are graphed . Similar results were also observed in U2OS ( Fig EV3 ) .

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B. Both the CC and the TIRT domain of GEMC1 are required to activate FOXJ1 (RT-QPCR). A corresponding Western blot showing relative expression levels of the mutant GFP tagged GEMC1 variants is shown from HEK293T cells. The mean and standard deviation of 3 individual experiments in HEK293T cells are graphed. Similar results were also observed in U2OS (Fig EV3).


C . Activation of FOXJ1 by GEMC1 is inhibited by the co - expression of Geminin ( compare 3rd and 4th lanes ) . Example Western blot is shown below . Results graphed are the mean and standard deviation of 3 individual experiments in HEK293T cells .

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C. Activation of FOXJ1 by GEMC1 is inhibited by the co-expression of Geminin (compare 3rd and 4th lanes). Example Western blot is shown below. Results graphed are the mean and standard deviation of 3 individual experiments in HEK293T cells.


D . Co - expression of GEMC1 and Multicilin results in similar levels of FOXJ1 expression ( measured by RT - QPCR ) as Multicilin alone in HEK293T cells . The mean and standard deviation of 4 individual experiments in HEK293T cells are graphed .

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D. Co-expression of GEMC1 and Multicilin results in similar levels of FOXJ1 expression (measured by RT-QPCR) as Multicilin alone in HEK293T cells. The mean and standard deviation of 4 individual experiments in HEK293T cells are graphed.


E and F . The activation of FOXJ1 by GEMC1 is partially dependent on Multicilin . HEK293T cells were transfected with a control siRNA ( siC ) or 2 different siRNAs against MCIDAS ( si1 and si2 ) prior to being transfected with GFP or GFP - GEMC1 expression vectors . Relative MCIDAS ( E ) and FOXJ1 ( F ) expression levels were assessed by RT - QPCR . The mean and standard deviation of a representative experiment performed in triplicate is graphed . An additional experiment is presented in Fig EV3 .

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E and F. The activation of FOXJ1 by GEMC1 is partially dependent on Multicilin. HEK293T cells were transfected with a control siRNA (siC) or 2 different siRNAs against MCIDAS (si1 and si2) prior to being transfected with GFP or GFP-GEMC1 expression vectors. Relative MCIDAS (E) and FOXJ1 (F) expression levels were assessed by RT-QPCR. The mean and standard deviation of a representative experiment performed in triplicate is graphed. An additional experiment is presented in Fig EV3.


IL - 1β ELISA of supernatants from LPS - primed ( 1 μg / mL , 4hrs . ) MDMs pre - incubated with 0 . 1 % DMSO or P22077 ( 2 . 5 µM ) 15 mins . before treatment with either nigericin ( 10 μM , 45 mins . ) or CPPD crystals ( 250 μg / mL , 2 hrs . ) . Bars represent the mean , ± S . D . n = 13 and 11 independent blood donors for nigericin and CPPD , respectively . IL - 18 ELISA of supernatants from MDMs treated as in A . Bars represent the mean , ± S . D . n = 11 independent blood donors .

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IL-1β ELISA of supernatants from LPS-primed (1 μg/mL, 4hrs.) MDMs pre-incubated with 0.1% DMSO or P22077 (2.5 µM) 15 mins. before treatment with either nigericin (10 μM, 45 mins.) or CPPD crystals (250 μg/mL, 2 hrs.). Bars represent the mean, ± S.D. n = 13 and 11 independent blood donors for nigericin and CPPD, respectively. IL-18 ELISA of supernatants from MDMs treated as in A. Bars represent the mean, ± S.D. n = 11 independent blood donors.


LDH - release from MDMs treated as in A . Bars represent the mean percentage of LDH release relative to the total cells lysed , ± S . D . n = 10 and 11 independent blood donors for nigericin and CPPD , respectively .

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LDH-release from MDMs treated as in A. Bars represent the mean percentage of LDH release relative to the total cells lysed, ± S.D. n = 10 and 11 independent blood donors for nigericin and CPPD, respectively.


Western blots of supernatants and cell lysates from MDMs treated as in A . Bands in the figure represent : Pro - IL - 1β ( 31 kDa ) ; mature IL - 1β ( mIL - 1β , 17 kDa ) ; pro - IL - 18 ( 24 kDa ) , mature IL - 18 ( mIL - 18 , 18 kDa ) , pro - caspase - 1 ( pro - Casp - 1 ; 45 kDa ) and mature caspase - 1 ( mCasp - 1 ; 20 kDa ) . β - actin is shown as a loading control . Blots are representative of at least 3 independent blood donors .

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Western blots of supernatants and cell lysates from MDMs treated as in A. Bands in the figure represent: Pro-IL-1β (31 kDa); mature IL-1β (mIL-1β, 17 kDa); pro-IL-18 (24 kDa), mature IL-18 (mIL-18, 18 kDa), pro-caspase-1 (pro-Casp-1; 45 kDa) and mature caspase-1 (mCasp-1; 20 kDa). β-actin is shown as a loading control. Blots are representative of at least 3 independent blood donors.


Quantitative RT - PCR showing the mRNA expression levels of IL - 1β , IL - 18 , ASC , CASP1 and NLRP3 in MDMs treated with P22077 ( 2 . 5 µM ) and / or LPS ( 1 μg / mL ; 4 hrs . ) , as indicated . Bars represent the mean fold increase from an untreated ( UT )

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

Quantitative RT-PCR showing the mRNA expression levels of IL-1β, IL-18, ASC, CASP1 and NLRP3 in MDMs treated with P22077 (2.5 µM) and/or LPS (1 μg/mL; 4 hrs.), as indicated. Bars represent the mean fold increase from an untreated (UT) control


Western blots showing the relative abundance of NLRP3 , ASC , pro - caspase - 1 , pro - IL - 1β , pro - IL - 18 , USP7 and USP47 in cell lysates of MDMs treated with P22077 ( 2 . 5 µM ) and / or LPS ( 1 μg / mL ) for 4 hrs . , as indicated . β - actin is shown as a loading control . Blots are representative of at least 3 independent blood donors .

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

Western blots showing the relative abundance of NLRP3, ASC, pro-caspase-1, pro- IL-1β, pro-IL-18, USP7 and USP47 in cell lysates of MDMs treated with P22077 (2.5 µM) and/or LPS (1 μg/mL) for 4 hrs., as indicated. β-actin is shown as a loading control. Blots are representative of at least 3 independent blood donors.


IL - 18 ELISA of supernatants from LPS - primed ( 1 μg / mL , 10 mins . ) MDMs pre - incubated with either vehicle ( 0 . 1 % DMSO ) or P22077 ( 2 . 5 µM ) for 15 mins . before treatment with nigericin ( 10 μM , 45 mins . ) . Bars represent the mean , ± S . D . n = 9 independent blood donors .

B-MEASURED_VAR I-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

IL-18 ELISA of supernatants from LPS-primed (1 μg/mL, 10 mins.) MDMs pre-incubated with either vehicle (0.1% DMSO) or P22077 (2.5 µM) for 15 mins. before treatment with nigericin (10 μM, 45 mins.). Bars represent the mean, ± S.D. n = 9 independent blood donors.