A mRNA expression of FAM189A2 / ENTREP in human mammary epithelium HMEC human breast cancer lines . In qRT - PCR analyses , expression was normalized to immortalized normal human mammary epithelium HMEC4tertshp16 . Relative expression ratios shown as mean + SD from three biological replicates .

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

A The mRNA expression of FAM189A2/ENTREP in the primary human mammary epithelium HMEC and human breast cancer cell lines. In qRT-PCR analyses, the expression was normalized to the immortalized normal human mammary epithelium HMEC4tertshp16. Relative expression ratios shown as mean + SD from three biological replicates.


B Relative expression of FAM189A2 / ENTREP in normal ( ) cancer ( green ) tissues of breast ( Curtis Breast ) , lung ( Okayama Lung ) , colorectal ( TCGA Colorectal ) head neck ( Estilo Head - Neck ) . Data were downloaded from Oncomine database P - values obtained by Student ' s t - number of cases are listed on top bottom of figures , respectively . P < 0 . 05 was considered as statistically significant . Box - whisker plot represents interquartile range ( 25th 75th percentiles ) as a box median as a . maximn minimun values within 1 . 5 x interquartile range are shown as whiskers . Outlier data are plotted as dots . C Relative expression of FAM189A2 / ENTREP in ( green ) metastatic sites ( blue ) including lymph node , bone , liver , lung , soft tissues of prostatic cancer ( Grasso Prostate ) ( left ) in normal ( ) , ( green ) metastatic sites ( three cases of sentinel lymph node ) ( blue ) of breast cancer ( TCGA Breast ) ( right ) . Note that only three cases of metastatic breast cancer were available in dataset . Data were downloaded from Oncomine database P - values obtained by Student ' s t - number of cases are listed on top bottom of figures , respectively . P < 0 . 05 was considered as statistically significant . Box - whisker plot represents interquartile range ( 25th 75th percentiles ) as a box median as a . maximn minimun values within 1 . 5 x interquartile range are shown as whiskers . Outlier data are plotted as dots .

O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-TISSUE O O B-TISSUE O O B-TISSUE O O B-TISSUE O O B-TISSUE O O B-TISSUE O B-TISSUE I-TISSUE O O B-TISSUE O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O O O O B-DISEASE I-DISEASE O O B-TISSUE O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O B-DISEASE I-DISEASE O O B-TISSUE O O O O O O O O O O O O B-DISEASE I-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Relative expression of FAM189A2/ENTREP in the normal (white) and cancer (green) tissues of breast (Curtis Breast), lung (Okayama Lung), colorectal (TCGA Colorectal) and head and neck (Estilo Head-Neck). Data were downloaded from the Oncomine database P-values obtained by Student's t-tests and the number of cases are listed on the top and bottom of figures, respectively. P < 0.05 was considered as statistically significant. Box-whisker plot represents the interquartile range (25th and 75th percentiles) as a box and the median as a line. The maximn and minimun values within 1.5 x interquartile range are shown as whiskers. Outlier data are plotted as dots. C Relative expression of FAM189A2/ENTREP in the primary (green) and the metastatic sites (blue) including lymph node, bone, liver, lung, soft tissues of prostatic cancer (Grasso Prostate) (left) and in the normal (white), primary (green) and metastatic sites (three cases of sentinel lymph node) (blue) of breast cancer (TCGA Breast) (right). Note that only three cases of the metastatic breast cancer were available in the dataset. Data were downloaded from the Oncomine database P-values obtained by Student's t-tests and the number of cases are listed on the top and bottom of figures, respectively. P < 0.05 was considered as statistically significant. Box-whisker plot represents the interquartile range (25th and 75th percentiles) as a box and the median as a line. The maximn and minimun values within 1.5 x interquartile range are shown as whiskers. Outlier data are plotted as dots.


D - F Relapse - free survival ( RFS ) of breast cancer . Data were downloaded from Kaplan - Meier

D-F Relapse-free survival (RFS) of breast cancer patients. Data were downloaded from the Kaplan-Meier plotter


B , C immunoprecipitation . Co - precipitation was impaired by either WW domain deletion of ITCH or mutation in PPxY motifs of ENTREP . Data shown are representative of at least two independent experiments . IP , immunoprecipitation ; IB , immunoblot .

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

B, C The immunoprecipitation analysis. Co-precipitation was impaired by either the WW domain deletion of ITCH or the mutation in the PPxY motifs of ENTREP. Data shown are representative of at least two independent experiments. IP, immunoprecipitation; IB, immunoblot.


E Representative of proximity ligation ( PLA ) using Cos7 transfected with indicated vectors . PLA signals , granular dots of green color , were presented with DAPI . Arrowheads , plasma . Scale bar , 10mm . F box - - whisker plot presentation of PLA . A minimum of 10 per condition were counted from two independent experiments . P - values obtained by Student ' s t - P < 0 . 05 was considered as statistically significant . * P < 0 . 05 . Box - whisker plot represents interquartile range ( 25th 75th percentiles ) as a box median as a . maximn minimun values within 1 . 5 x interquartile range are shown as whiskers .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-CELL_LINE O O O O O B-EXP_ASSAY O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 images of the proximity ligation assay (PLA) using Cos7 cells transfected with indicated vectors. PLA signals, granular dots of green color, were presented with DAPI images. Arrowheads, the plasma membrane. Scale bar, 10mm. F The box-and-whisker plot presentation of the PLA. A minimum of 10 cells per condition were counted from two independent experiments. P-values obtained by Student's t-tests and P < 0.05 was considered as statistically significant. * P < 0.05. Box-whisker plot represents the interquartile range (25th and 75th percentiles) as a box and the median as a line. The maximn and minimun values within 1.5 x interquartile range are shown as whiskers.


A nickel - pull down using myc / HIS - tagged ENTREP along with HA - tagged ubiquitin . Arrows indicate HA - ubiquitin - incorporating ENTREP . Arrowhead indicates ENTREP without ubiquitination . Data shown are representative of three independent experiments .

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

A The nickel-pull down assay using myc/HIS-tagged ENTREP along with HA-tagged ubiquitin. Arrows indicate HA-ubiquitin-incorporating ENTREP. Arrowhead indicates ENTREP without ubiquitination. Data shown are representative of three independent experiments.


A immunoprecipitation . ENTREP ( wild or mut1 + 2 mutant ) co - precipitated with EPN1myc in presence of ITCH wild or C830A . blot bands were semi - quantified using ImageJ software . ENTREP was caluculated as a of ENTREP in myc - coprecipitaed sample to that of total lysate . ratios shown as mean ± SD from three biological replicates . P - values were obtained by Student ' s t - P < 0 . 005 was considered as statistically significant . No significant difference was observed .

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

A The immunoprecipitation analysis. The cytoplasmic ENTREP (wild or mut1+2 mutant) co-precipitated with EPN1myc in the presence of ITCH wild or C830A. The blot bands were semi-quantified using ImageJ software. ENTREP ratio was caluculated as a ratio of ENTREP in the myc-coprecipitaed sample to that of the total lysate. The ratios shown as mean ± SD from three biological replicates. P-values were obtained by Student's t-tests and P < 0.005 was considered as statistically significant. No significant difference was observed.


B Representative of proximity ligation ( PLA ) using Cos7 transfected with indicated vectors . PLA signals , granular dots of green color , were presented with DAPI . Scale bar , 10mm . C box - - whisker plot presentation of PLA . A minimum of 10 per condition were counted from two independent experiments . P - values obtained by Student ' s t - P < 0 . 05 was considered as statistically significant . * P < 0 . 05 . Box - whisker plot represents interquartile range ( 25th 75th percentiles ) as a box median as a . maximn minimun values within 1 . 5 x interquartile range are shown as whiskers .

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

B Representative images of the proximity ligation assay (PLA) using Cos7 cells transfected with indicated vectors. PLA signals, granular dots of green color, were presented with DAPI images. Scale bar, 10mm. C The box-and-whisker plot presentation of the PLA. A minimum of 10 cells per condition were counted from two independent experiments. P-values obtained by Student's t-tests and P < 0.05 was considered as statistically significant. * P < 0.05. Box-whisker plot represents the interquartile range (25th and 75th percentiles) as a box and the median as a line. The maximn and minimun values within 1.5 x interquartile range are shown as whiskers.


B immunoprecipitation . ENTREP co - precipitated with either ITCH C830A , delWW12 or delWW34 but not with delWW14 . Data shown are representative of at least two independent experiments . C immunoprecipitation . HA - CXCR4SS - DsRed hardly co - precipitated with either ITCHC830A or delWW mutants . HA - CXCR4DD - DsRed co - precipitated with ITCH C830A delWW12 but not with delWW14 delWW34 . Data shown are representative of at least two independent experiments .

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

B The immunoprecipitation analysis. Cytoplasmic ENTREP co-precipitated with either ITCH C830A, delWW12 or delWW34 but not with delWW14. Data shown are representative of at least two independent experiments. C The immunoprecipitation analysis. HA-CXCR4SS-DsRed hardly co-precipitated with either ITCHC830A or delWW mutants. HA-CXCR4DD-DsRed co-precipitated with ITCH C830A and delWW12 but not with delWW14 and delWW34. Data shown are representative of at least two independent experiments.


F nickel - pull down using indicated vectors along with HISx6 - tagged wild type ubiquitin vector . HA - tagged DsRed was used as a control for HA - CXCR4DD - DsRed . Arrowhead , FLAG - tagged ITCH ( wild or C830A ) ; arrow , FLAG - ENTREP wild cyt . Data shown are representative of at least two independent experiments . G Co - localization of ENTREP CXCR4 in endosome . Cos7 were transfected with ENTREP - FLAG , CXCR4 - DsRed Halo - ITCH wild vectors treated with CXCL12 . ENTREP - FLAG CXCR4 - DsRed co - localized in RAB7 - positive endosome . Inserts , high - magnification of marked indicating colocalizaion of ENTREP - FLAG , CXCR4 - DsRed RAB7 . Scale bar 10mm . image is representative of at least two independent experiments .

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

F The nickel-pull down assay using indicated vectors along with HISx6-tagged wild type ubiquitin vector. HA-tagged DsRed was used as a control for HA-CXCR4DD-DsRed. Arrowhead, FLAG-tagged ITCH (wild or C830A); arrow, FLAG-ENTREP wild cyt. Data shown are representative of at least two independent experiments. G Co-localization of ENTREP and CXCR4 in the endosome. Cos7 cells were transfected with ENTREP-FLAG, CXCR4-DsRed and Halo-ITCH wild vectors and treated with CXCL12. ENTREP-FLAG and CXCR4-DsRed co-localized in the RAB7-positive endosome. Inserts, the high-magnification images of the marked area indicating the colocalizaion of ENTREP-FLAG, CXCR4-DsRed and RAB7. Scale bar 10mm. The image is representative of at least two independent experiments.


C expression of endogenous CXCR4 in lentivirally - transduced MCF - 7 ENTREP - KO . CXCL12 ( + ) ( - ) indicate presence absence of CXCL12 treatment . In CXCL12 - treated ENTREP - EGFP expressing , a non - negligible amount of CXCR4 was observed in cytoplasm , in which CXCR4 was overlapped with ENTREP - EGFP . CXCR4 expression on surface still remained ( arrows ) . Scale bar 10 mm . are representative of at least three independet experiments .

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

C The expression of endogenous CXCR4 in lentivirally-transduced MCF-7 ENTREP-KO cells. CXCL12 (+) and (-) indicate the presence and absence of CXCL12 treatment. In CXCL12-treated ENTREP-EGFP expressing cells, a non-negligible amount of CXCR4 was observed in the cytoplasm, in which CXCR4 was overlapped with ENTREP-EGFP. CXCR4 expression on the cell surface still remained (arrows). Scale bar 10 mm. The images are representative of at least three independet experiments.


A Dot plot presentation of chemotaxis analyses using Boyden chamber . CXCL12 ( + ) ( - ) indicate presence absence of CXCL12 as an attractant in bottom chambers . ENTREP - knockout enhanced chemotaxis toward CXCL12 , which was blocked by pre - treatment with AMD3100 ( upper panel ) . doxycyclin ( DOX ) - induced ENTREP expression suppressed chemotaxis of mouse 4T1 - Luc toward CXCL12 ( lower panel ) . of migrated into bottom chamber against applied on top chambers was normalized by results of non - treated ( n = 7 , upper panel , n = 9 , lower panel , from at least two independent experiments , respectively ) . P - values were obtained by Student ' s t - P < 0 . 005 was considered as statistically significant . Insert , immunoblot of DOX - induced ENTREP expression in 4T1 - Luc . NS , not significant .

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

A Dot plot presentation of the chemotaxis analyses using the Boyden chamber. CXCL12 (+) and (-) indicate the presence and absence of CXCL12 as an attractant in the bottom chambers. ENTREP-knockout enhanced the chemotaxis toward CXCL12, which was blocked by pre-treatment with AMD3100 (upper panel). The doxycyclin (DOX)-induced ENTREP expression suppressed the chemotaxis of mouse 4T1-Luc cells toward CXCL12 (lower panel). Ratio of migrated cells into the bottom chamber against the applied cells on the top chambers was normalized by the results of non-treated cells (n=7, upper panel, and n=9, lower panel, from at least two independent experiments, respectively). P-values were obtained by Student's t-tests and P < 0.005 was considered as statistically significant. Insert, the immunoblot analysis of the DOX-induced ENTREP expression in 4T1-Luc cells. NS, not significant.


B Dot plot presentation of mammosphere ( left panel ) of mammosphere ( right panel ) . Each mark in dot plot presentation indicates number of mammosphere from 3 , 000 per well in an ultra - low attachment plate from two independent experiments . To estimate effect of CXCL12 / CXCR4 pathway , 100 ng / ml of CXCL12 12 μM of AMD3100 were applied as indicated . For statistical , variance was assessed using two - way ANOVA significance was calculated using Tukey post hoc correcting for multiple comparison . NS , not significant . Scale bar in of mammosphere 100 mm .

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

B Dot plot presentation of mammosphere assay (left panel) and the images of mammosphere (right panel). Each mark in dot plot presentation indicates the number of mammosphere from 3,000 cells per well in an ultra-low attachment plate from two independent experiments. To estimate the effect of CXCL12/CXCR4 pathway, 100 ng/ml of CXCL12 and 12 μM of AMD3100 were applied as indicated. For statistical analysis, variance was assessed using two-way ANOVA and significance was calculated using Tukey post hoc test correcting for multiple comparison. NS, not significant. Scale bar in the images of mammosphere 100 mm.


C Co - crystal structure of gephyrin E - domain ( blue ) bound GlyR ß - loop ( red - PDB code 2FTS ( Kim et al . , 2006 ) ) . mutated gephyrin Gly375 residue is shown in orange .

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

C Co-crystal structure of gephyrin E-domain (blue) and bound GlyR ß-loop (red - PDB code 2FTS (Kim et al., 2006)). The mutated gephyrin Gly375 residue is shown in orange.


A Representative of GFP - gephyrin GFP - G375D , expressed in hippocampal neurons at 9 + 4 days in vitro . Inset shows higher magnification . Scale bar = 20 µm . B Relative distribution of cluster density of gephyrin variants on dendrites from transfected neurons . Number of gephyrin clusters in each dendrite is normalized to 20 µm segments .

O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O B-GENEPROD O O O O O O O O O O O

A Representative images of GFP-gephyrin and GFP-G375D, expressed in hippocampal neurons at 9 + 4 days in vitro. Inset shows higher magnification. Scale bar = 20 µm.B Relative distribution of cluster density of gephyrin variants on dendrites from transfected neurons. Number of gephyrin clusters in each dendrite is normalized to 20 µm segments.


C Representative segments of dendrites expressing GFP - gephyrin or GFP - G375D immunostained with presynaptic marker VGAT . Arrowheads show co - localization of gephyrin VGAT clusters . Scale bar = 10 µm . D of co - localization between gephyrin VGAT clusters ( gephyrin - VGAT 77 . 4 ± 2 . 9 % , G375D - VGAT 30 ± 4 . 4 % ) . 31 GFP - gephyrin 30 GFP - G375D neurons from three independent cultures were used for quantifications . Results are expressed as mean ±

O O O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O O O

C Representative segments of dendrites expressing GFP-gephyrin or GFP-G375D and immunostained with the presynaptic marker VGAT. Arrowheads show co-localization of gephyrin and VGAT clusters. Scale bar = 10 µm.D Quantification of co-localization between gephyrin and VGAT clusters (gephyrin-VGAT 77.4±2.9 %, G375D-VGAT 30±4.4 %). 31 GFP-gephyrin and 30 GFP-G375D neurons from three independent cultures were used for quantifications. Results are expressed as mean ± SEM


A Representative dendritic segment of a neuron expressing GFP - G375D 3B11 - immunostained endogenous gephyrin ( red ) . B of density ( C ) of endogenous gephyrin clusters in neurons expressing GFP , GFP - G375D or non - transfected neighboring neurons . 20 GFP - G375 non - transfected neurons 15 GFP - expressing neurons from three independent cultures were used for quantifications . Results are expressed as mean ± data were analyzed by t - .

O O B-SUBCELLULAR O O O B-CELL_TYPE O B-GENEPROD O O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O O O O O O O B-CELL_TYPE O O B-GENEPROD O O O O O B-CELL_TYPE O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O

A Representative dendritic segment of a neuron expressing GFP-G375D and 3B11-immunostained endogenous gephyrin (red).B Quantification of density and (C) size of endogenous gephyrin clusters in neurons expressing GFP, GFP-G375D or non-transfected neighboring neurons. 20 GFP-G375 and non-transfected neurons and 15 GFP-expressing neurons from three independent cultures were used for quantifications. Results are expressed as mean ± SEM and data were analyzed by t-test.


A Representative neurons expressing myc - tagged gephyrin or G375D pHluorin - tagged GABAAR γ2 . Surface - expressed γ2 - subunits were immunolabelled with GFP - antibodies in red , pHluorin - ( total γ2 ) are shown in green . B C ( fl . ) of surface - expressed γ2 clusters in myc - gephyrin myc - G375D - expressing dendrites ( n = 20 myc - gephyrin 21 myc - G375D neurons in B 18 myc - gephyrin myc - G375D neurons in C ) D Mean γ2 - pHluorin in myc - gephyrin myc - G375D - expressing dendrites ( n = 20 18 myc - gephyrin myc - G375D expressing neurons ) . E Number of surface γ2 clusters on dendrites normalized to 20 µm segments ( n = 18 myc - gephyrin myc - G375D expressing neurons ) . At least three independent cultures were used for each experiment . Results are expressed as mean ± ; Data were analyzed by t - .

O O B-CELL_TYPE O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O B-SUBCELLULAR O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-CELL_TYPE O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O B-SUBCELLULAR O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O B-CELL_TYPE O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Representative neurons expressing myc-tagged gephyrin or G375D and pHluorin-tagged GABAAR γ2. Surface-expressed γ2-subunits were immunolabelled with GFP-antibodies in red, and the pHluorin-fluorescence (total γ2 levels) are shown in green.B Size and C fluorescence (fl.) intensity of surface-expressed γ2 clusters in myc-gephyrin and myc-G375D-expressing dendrites (n = 20 myc-gephyrin and 21 myc-G375D neurons in B and 18 myc-gephyrin and myc-G375D neurons in C)D Mean γ2-pHluorin fluorescence in myc-gephyrin and myc-G375D-expressing dendrites (n = 20 and 18 myc-gephyrin and myc-G375D expressing neurons).E Number of surface γ2 clusters on dendrites normalized to 20 µm segments (n = 18 myc-gephyrin and myc-G375D expressing neurons). At least three independent cultures were used for each experiment. Results are expressed as mean ± SEM; Data were analyzed by t-test.


F Representative traces of miniature recordings on cultured hippocampal neurons transfected either with GFP - gephyrin or GFP - G375D . G mIPSC amplitudes from neurons expressing GFP - gephyrin or GFP - G375D . Numbers show analyzed - numbers . Data were analyzed by one - way ANOVA post - hoc Tukey . H Frequency of mIPSCs from neurons expressing GFP - gephyrin or GFP - G375D . horizontal within boxes indicates median frequency for represented condition ( bottom / top boundary indicates 25th / 75th percentile ) . Top bottom error bars indicate 90th 10th percentiles , respectively . by Kruskal - Wallis followed by post - hoc Mann - Whitney .

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

F Representative traces of miniature recordings on cultured hippocampal neurons transfected either with GFP-gephyrin or GFP-G375D.G mIPSC amplitudes from neurons expressing GFP-gephyrin or GFP-G375D. Numbers show analyzed cell-numbers. Data were analyzed by one-way ANOVA and post-hoc Tukey test.H Frequency of the mIPSCs from neurons expressing GFP-gephyrin or GFP-G375D. The horizontal line within the boxes indicates the median frequency for the represented condition (bottom/top boundary indicates the 25th /75th percentile). Top and bottom error bars indicate the 90th and 10th percentiles, respectively. Analysis by Kruskal-Wallis test followed by post-hoc Mann-Whitney test.


A Western blot of HEK293 expressing GFP - tagged gephyrin or G375D using GFP - specific polyclonal gephyrin antibodies . Note that both gephyrin variants are stably expressed show no degradation in . Asterisk in poly - gephyrin blot shows endogenous gephyrin .

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A Western blot of HEK293 cells expressing GFP-tagged gephyrin or G375D using GFP-specific and polyclonal gephyrin antibodies. Note that both gephyrin variants are stably expressed and show no degradation in the cells. Asterisk in the poly-gephyrin blot shows endogenous gephyrin.


B SDS - PAGE of E . coli expressed purified 6His - gephyrins CD spectra . Mean residue ellipticity of gephyrin G375D show profiles with minima at 208 nm 222 nm .

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B SDS-PAGE of E. coli expressed and purified 6His-gephyrins and CD spectra. Mean residue ellipticity of gephyrin and G375D show profiles with minima at 208 nm and 222 nm.


C - exclusion chromatography of purified 6His - gephyrin isolated E - domain variants . Full - gephyrins isolated E - domains were loaded onto a Superose 6 gel filtration column elution of was monitored by measuring absorbance at 280 nm ( mAU , milliabsorbance units ) .

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C Size-exclusion chromatography of purified 6His-gephyrin and isolated E-domain variants. Full-length gephyrins and isolated E-domains were loaded onto a Superose 6 gel filtration column and elution of the protein was monitored by measuring absorbance at 280 nm (mAU, milliabsorbance units).


D Co - immunoprecipitation of HEK293 lysates expressing myc - tagged gephyrin together with GFP - tagged gephyrin or G375D using myc - specific antibodies . Control IgG were used to show specificity of .

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D Co-immunoprecipitation of HEK293 cell lysates expressing myc-tagged gephyrin together with GFP-tagged gephyrin or G375D using myc-specific antibodies. Control IgG were used to show specificity of the assay.


B Representative pulldown - experiment of 6His - tagged gephyrin G375D using immobilized α3 - peptide . 6His - tagged gephyrin variants were expressed in E . coli , purified to homogeneity incubated with α3 - peptides immobilized to NeutrAvidin beads . n = 3 , normalized to 6His - gephyrin . 38 . 9 ± 1 . 5 % G375D were pulled down .

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B Representative pulldown-experiment and quantification of 6His-tagged gephyrin and G375D using immobilized α3-peptide. 6His-tagged gephyrin variants were expressed in E. coli cells, purified to homogeneity and incubated with α3-peptides immobilized to NeutrAvidin beads. n = 3, normalized to 6His-gephyrin. 38.9±1.5% G375D were pulled down.


C Representative SPR sensograms of 6His - gephyrin 6His - G375D binding to immobilized GABAAR α3 - peptide . Response units are plotted against time . Injected gephyrin concentrations were 0 . 5 , 2 , 10 , 25 , 40 µM .

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C Representative SPR sensograms of 6His-gephyrin and 6His-G375D binding to immobilized GABAAR α3-peptide. Response units are plotted against time. Injected gephyrin concentrations were 0.5, 2, 10, 25, 40 µM.


D SPR binding isotherms of a representative experiment as in C . Calculated KD values of 3 . 2 10 µM for gephyrin G375D , respectively , are indicated by vertical lines . experiment was repeated three times with two independently purified batches two sensor chips .

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D SPR binding isotherms of a representative experiment as in C. Calculated KD values of 3.2 and 10 µM for gephyrin and G375D, respectively, are indicated by vertical lines. The experiment was repeated three times with two independently purified protein batches and two sensor chips.


E Representative ITC binding isotherms of loop of GlyR ß - subunit ( GlyR - ßL , residue 378 - 426 ) 6His - tagged gephyrin or G375D . GlyR - ßL - gephyrin binding data ( black ) were fitted in a two - side model , whereas G375D binding could only be fitted to a single binding site . Dissociation constants KD stoichiometry ( N = number of binding sites ) are given for each binding component . n = 5 from two independently purified batches . Results are expressed as mean ± .

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

E Representative ITC binding isotherms of the cytoplasmic loop of the GlyR ß-subunit (GlyR-ßL, residue 378-426) and 6His-tagged gephyrin or G375D. The GlyR-ßL-gephyrin binding data (black) were fitted in a two-side model, whereas G375D binding could only be fitted to a single binding site. Dissociation constants KD and stoichiometry (N = number of binding sites) are given for each binding component. n = 5 from two independently purified protein batches. Results are expressed as mean ± SEM.


A Biosynthesis pathway of Moco with stable intermediates . Gephyrin G - E - domain catalyze last two steps as depicted . B In vitro Moco - synthesis using 150 pmol purified 6His - tagged gephyrins . D580A is an activity - deficient gephyrin mutation previously identified in Moco - deficient ( Reiss et al . , 2011 ) . Assays without addition of gephyrin ( - gephyrin ) or molybdenum ( - Mo ) served as internal controls of ( see also ( Belaidi Schwarz , 2013 ) ) . C Experiment as in B but with pre - incubation of 6His - gephyrin / G375D in a 1 : 1 to simulate heterozygous mutation . n = at least 3 with two independently purified batches . Results are expressed as mean ± .

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

A Biosynthesis pathway of Moco with all stable intermediates. Gephyrin G- and E-domain catalyze the last two steps as depicted.B In vitro Moco-synthesis assay using 150 pmol purified 6His-tagged gephyrins. D580A is an activity-deficient gephyrin mutation previously identified in Moco-deficient patients (Reiss et al., 2011). Assays without the addition of gephyrin (-gephyrin) or molybdenum (-Mo) served as internal controls of the assay (see also (Belaidi Schwarz, 2013)).C Experiment as in B but with pre-incubation of 6His-gephyrin/G375D in a 1:1 ratio to simulate the heterozygous mutation. n = at least 3 with two independently purified protein batches. Results are expressed as mean ± SEM.


( b ) Parkin is recruited selectively to depolarized mitochondria directs mitophagy . HeLa transfected with HA - Parkin were treated with CCCP for indicated times . Mitochondria were stained by anti - TOM20 ( pseudo coloured ; blue ) a ΔΨm dependent MitoTracker ( red ) . Parkin was stained with anti - HA ( green ) . Without treatment , mitochondria are intact stained by both mitochondrial markers , whereas Parkin is equally distributed in cytoplasm . After 2 h of CCCP treatment , mitochondria are depolarized as shown by loss of MitoTracker . Parkin completely translocates to mitochondria clustering at perinuclear regions . After 24 h of CCCP treatment , massive loss of mitochondria is observed as shown by disappearance of mitochondrial marker . Only Parkin - positive show mitochondrial clustering clearance , in contrast to adjacent untransfected . Scale bars , 10 μm .

O O O B-GENEPROD O O O O O B-SUBCELLULAR O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O O O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O B-GENEPROD O O O O B-SUBCELLULAR O O O O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O B-SMALL_MOLECULE O O O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O O O O O O O O O

(b) Parkin is recruited selectively to depolarized mitochondria and directs mitophagy. HeLa cells transfected with HA-Parkin were treated with CCCP for the indicated times. Mitochondria were stained by anti-TOM20 (pseudo coloured; blue) and a ΔΨm dependent MitoTracker (red). Parkin was stained with anti-HA (green). Without treatment, mitochondria are intact and stained by both mitochondrial markers, whereas Parkin is equally distributed in the cytoplasm. After 2 h of CCCP treatment, mitochondria are depolarized as shown by the loss of MitoTracker staining. Parkin completely translocates to mitochondria clustering at perinuclear regions. After 24 h of CCCP treatment, massive loss of mitochondria is observed as shown by the disappearance of the mitochondrial marker. Only Parkin-positive cells show mitochondrial clustering and clearance, in contrast to adjacent untransfected cells. Scale bars, 10 μm.


( c ) Pathogenic Parkin mutations abrogate mitochondrial translocation / or clearance . HeLa transfected with Flag - Parkin wild - type or pathogenic mutants were treated with CCCP stained with anti - Flag ( green ) , anti - TOM20 ( red ) Hoechst33342 ( blue ) . Representative merged are shown . Scale bars , 10 μm . Immunostaining of transfected with non - functional cysteine mutants functional Parkin variants is shown in Supplementary Information , Fig . S1a S1b , respectively .

O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O

(c) Pathogenic Parkin mutations abrogate mitochondrial translocation and/or clearance. HeLa cells transfected with Flag-Parkin wild-type or pathogenic mutants were treated with CCCP and stained with anti-Flag (green), anti-TOM20 (red) and Hoechst33342 (blue). Representative merged images are shown. Scale bars, 10 μm. Immunostaining of cells transfected with non-functional cysteine mutants and functional Parkin variants is shown in Supplementary Information, Fig. S1a and S1b, respectively.


( d , e ) on mitochondrial effects , Parkin mutants can be classified into functional non - functional classes . ( d ) of Parkin translocation to damaged mitochondria after 2 h of CCCP treatment , as defined by complete colocalization of Parkin with mitochondrial marker ( n = 3 ) . ( e ) of with uncleared mitochondria after 24 h of CCCP treatment , as shown by complete loss of mitochondrial ( n = 3 ) . Data represent mean ± s . d . , * P ≤ 0 . 05 , * * P ≤ 0 . 005 , * * * P ≤ 0 . 0005 compared with wild - type . WT , wild - type .

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(d, e) Based on the mitochondrial effects, Parkin mutants can be classified into functional and non-functional classes. (d) Quantification of Parkin translocation to damaged mitochondria after 2 h of CCCP treatment, as defined by complete colocalization of Parkin with the mitochondrial marker (n = 3). (e) Quantification of cells with uncleared mitochondria after 24 h of CCCP treatment, as shown by complete loss of mitochondrial staining (n = 3). Data represent the mean ± s.d., *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0005 compared with wild-type. WT, wild-type.


Stable Parkin wild - type or pathogenic mutants expressing SH - SY5Y lines were induced with doxycycline ( DOX ) for 72 h treated with CCCP for indicated times . were stained with anti - Parkin ( green ) , anti - TOM20 ( red ) Hoechst33342 ( blue ) . ( a - b ) CCCP treatment of stable SH - SY5Y for 0 h . Unstimulated control ( - DOX ) show relatively low of endogenous Parkin , compared to with induced expression of Parkin wild - type or K161N , R275W G430D mutants ( + DOX ) . Immunostaining ( a ) western blot ( b ) of induced demonstrate comparable of Parkin expression in selected clones . β - Actin served as a loading control .

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Stable Parkin wild-type or pathogenic mutants expressing SH-SY5Y cell lines were induced with doxycycline (DOX) for 72 h and treated with CCCP for the indicated times. Cells were stained with anti-Parkin (green), anti-TOM20 (red) and Hoechst33342 (blue). (a-b) CCCP treatment of stable SH-SY5Y cells for 0 h. Unstimulated control cells (- DOX) show relatively low levels of endogenous Parkin, compared to cells with induced expression of Parkin wild-type or K161N, R275W and G430D mutants (+ DOX). Immunostaining (a) and western blot analysis (b) of the induced cells demonstrate comparable levels of Parkin expression in the selected clones. β-Actin served as a loading control.


( c , d ) After 2 h of CCCP treatment , massive colocalization of wild - type Parkin mitochondrial clusters can be observed in induced when compared with unstimulated controls . Similarly , Parkin K161N R275W mutants translocate to mitochondria , whereas G430D does not .

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(c, d) After 2 h of CCCP treatment, massive colocalization of wild-type Parkin and mitochondrial clusters can be observed in the induced cells when compared with unstimulated controls. Similarly, Parkin K161N and R275W mutants translocate to mitochondria, whereas G430D does not.


( e , f ) After 24 h of CCCP treatment , induced wild - type Parkin - expressing show full clearance of damaged mitochondria . In contrast , pathogenic mutants analysed fail to execute mitophagy , therefore , damaged mitochondria remain uncleared . Representative of three independent experiments are shown . Scale bars , 10 μm ( a , c e ) . ( d , f ) Quantifications of observed mitochondrial effects in Parkin - expressing SH - SY5Y ( n = 3 ) . Data represent mean ± s . d . , * * * P ≤ 0 . 0005 compared with induced wild - type Parkin . WT , wild - type .

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(e, f) After 24 h of CCCP treatment, induced wild-type Parkin-expressing cells show full clearance of damaged mitochondria. In contrast, the pathogenic mutants analysed fail to execute mitophagy and, therefore, damaged mitochondria remain uncleared. Representative images of three independent experiments are shown. Scale bars, 10 μm (a, c and e). (d, f) Quantifications of the observed mitochondrial effects in Parkin-expressing SH-SY5Y cells (n = 3). Data represent the mean ± s.d., ***P ≤ 0.0005 compared with induced wild-type Parkin. WT, wild-type.


( b ) Co - immunoprecipitations of Parkin PINK1 after mitochondrial depolarization . Endogenous Parkin PINK1 were co - immunoprecipitated from neuronal SH - SY5Y after CCCP treatment with specific antibodies .

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(b) Co-immunoprecipitations of Parkin and PINK1 after mitochondrial depolarization. Endogenous Parkin and PINK1 were co-immunoprecipitated from neuronal SH-SY5Y cells after CCCP treatment with specific antibodies.


( c ) Western blot of PINK1 siRNA - transfected HeLa used in d - h demonstrates knockdown efficiency specificity of PINK1 silencing .

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(c) Western blot of PINK1 siRNA-transfected HeLa cells used in d-h demonstrates the knockdown efficiency and specificity of PINK1 silencing.


d - h ) PINK1 - specific siRNA abrogates Parkin translocation to mitochondria after 2 h , mitochondrial clearance after 24 h , of CCCP treatment . This can be rescued by re - transfection with wild - type PINK1 , but not with a kinase - dead mutant or a PINK1 variant lacking MTS . Control siRNA - or PINK1 siRNA - silenced HeLa were transfected with EGFP - Parkin wild - type PINK1 - V5 wild - type , 3 × KD or ΔMTS mutants . Use of appropriate empty vector controls is depicted . were treated with CCCP stained with anti - TOM20 ( pseudo coloured ; blue ) anti - V5 ( red ) to visualize mitochondrial re - transfected PINK1 , respectively . Wild - type Parkin was visualized by EGFP epifluorescence ( green ) . Parkin - positive are surrounded by a . Representative of four independent experiments are shown . Scale bars , 10 μm .

O O O O B-GENEPROD O O O O B-GENEPROD O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O

d-h) PINK1-specific siRNA abrogates Parkin translocation to mitochondria after 2 h, and mitochondrial clearance after 24 h, of CCCP treatment. This can be rescued by re-transfection with wild-type PINK1, but not with a kinase-dead mutant or a PINK1 variant lacking the MTS. Control siRNA- or PINK1 siRNA-silenced HeLa cells were transfected with EGFP-Parkin wild-type and PINK1-V5 wild-type, 3×KD or ΔMTS mutants. Use of appropriate empty vector controls is depicted. Cells were treated with CCCP and stained with anti-TOM20 (pseudo coloured; blue) and anti-V5 (red) to visualize mitochondrial morphology and re-transfected PINK1, respectively. Wild-type Parkin was visualized by EGFP epifluorescence (green). Parkin-positive cells are surrounded by a line. Representative images of four independent experiments are shown. Scale bars, 10 μm.


( e ) Wild - type Parkin colocalization with impaired mitochondria after 2 h of CCCP treatment was analysed ( n = 4 ) . ( g ) of results from in e .

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(e) Wild-type Parkin colocalization with impaired mitochondria after 2 h of CCCP treatment was analysed (n = 4). (g) Quantification of results from cells in e.


( f ) Uncleared mitochondria were analysed after 24 h of CCCP treatment ( n = 4 ) . ( h ) of results from in f . Data represent mean ± s . d . , * * P ≤ 0 . 005 , * * * P ≤ 0 . 0005 compared with control siRNA - transfected . Immunostaining of control siRNA - transfected at analysed time points is shown in Supplementary Information , Fig . S2 . WT , wild - type .

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(f) Uncleared mitochondria were analysed after 24 h of CCCP treatment (n = 4). (h) Quantification of results from cells in f. Data represent the mean ± s.d., **P ≤ 0.005, ***P ≤ 0.0005 compared with control siRNA-transfected cells. Immunostaining of control siRNA-transfected cells at all analysed time points is shown in Supplementary Information, Fig. S2. WT, wild-type.


a , b ) Stable Mito - DsRed ( pseudo coloured ; blue ) - expressing HeLa were transfected with Flag - Parkin wild - type or pathogenic mutants . were treated with CCCP before immunostaining of Parkin with anti - Flag ( green ) endogenous poly - ubiquitin with a specific antibody FK1 ( red ) . ( a ) In contrast to pathogenic mutations , wild - type Parkin shows colocalization with poly - ubiquitin after 6 h of CCCP treatment at site of condensed mitochondria . ( b ) After 24 h of CCCP treatment , K161N R275W Parkin mutants colocalize with both poly - ubiquitin mitochondria . In contrast , translocation - deficient Parkin mutants K211N , T240R G430D lack mitochondria - associated poly - ubiquitin signal . Representative are shown . Scale bars , 10 μm . Complementary Parkin of untreated of colocalization of Parkin , mitochondria poly - ubiquitin upon CCCP treatment are shown in Supplementary Information , Fig . S3a , b c , respectively .

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

a, b) Stable Mito-DsRed (pseudo coloured; blue)-expressing HeLa cells were transfected with Flag-Parkin wild-type or pathogenic mutants. Cells were treated with CCCP before immunostaining of Parkin with anti-Flag (green) and endogenous poly-ubiquitin with a specific antibody FK1 (red). (a) In contrast to pathogenic mutations, wild-type Parkin shows colocalization with poly-ubiquitin after 6 h of CCCP treatment at the site of condensed mitochondria. (b) After 24 h of CCCP treatment, K161N and R275W Parkin mutants colocalize with both poly-ubiquitin and mitochondria. In contrast, translocation-deficient Parkin mutants K211N, T240R and G430D lack the mitochondria-associated poly-ubiquitin signal. Representative images are shown. Scale bars, 10 μm. Complementary Parkin of untreated cells and quantification of colocalization of Parkin, mitochondria and poly-ubiquitin upon CCCP treatment are shown in Supplementary Information, Fig. S3a, b and c, respectively.


( c , d ) Parkin - directed mitophagy specifically involves linkage of ubiquitin through lysines 27 63 . HeLa were transfected with EGFP - Parkin wild - type ubiquitin lysine variants treated with CCCP before immunostaining with anti - HtrA2 / Omi , as a mitochondrial marker ( pseudo coloured ; blue ) anti - His ( red ) to detect ectopic ubiquitin . Parkin was visualized by its fusion to EGFP ( green ) . ( c ) Note that only wild - type , K27 K63 6 × His - tagged ubiquitin variants are colocalized with condensed mitochondria EGFP - Parkin after 6 h of CCCP treatment . other ubiquitin mutants interfere with Parkin translocation to mitochondria . ( d ) Only co - expression of K27 or K63 ubiquitin recapitulates mitochondrial clearance similarly to 6 × His - tagged wild - type ubiquitin after 24 h of CCCP treatment . Representative are shown . Scale bars , 10 μm . Immunostaining of untreated other ubiquitin variant combinations tested are shown in Supplementary Information , Fig . S4a b , c , respectively . WT , wild - type ; PolyUb , poly - ubiquitin .

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(c, d) Parkin-directed mitophagy specifically involves linkage of ubiquitin through lysines 27 and 63. HeLa cells were transfected with EGFP-Parkin wild-type and ubiquitin lysine variants and treated with CCCP before immunostaining with anti-HtrA2/Omi, as a mitochondrial marker (pseudo coloured; blue) and anti-His (red) to detect ectopic ubiquitin. Parkin was visualized by its fusion to EGFP (green). (c) Note that only wild-type, K27 and K63 6×His-tagged ubiquitin variants are colocalized with condensed mitochondria and EGFP-Parkin after 6 h of CCCP treatment. All other ubiquitin mutants interfere with Parkin translocation to mitochondria. (d) Only co-expression of K27 or K63 ubiquitin recapitulates mitochondrial clearance similarly to 6×His-tagged wild-type ubiquitin after 24 h of CCCP treatment. Representative images are shown. Scale bars, 10 μm. Immunostaining of untreated cells and all other ubiquitin variant combinations tested are shown in Supplementary Information, Fig. S4a and b, c, respectively. WT, wild-type; PolyUb, poly-ubiquitin.


( a , b ) Stable Mito - DsRed ( pseudo coloured ; blue ) - expressing HeLa were transfected with Flag - Parkin wild - type or pathogenic mutants . were treated with CCCP for indicated times before immunostaining with anti - Flag ( green ) anti - p62 ( red ) . ( a ) In contrast to pathogenic mutants , wild - type Parkin colocalizes with p62 at site of clustered mitochondria , after 6 h of CCCP treatment . ( b ) After 24 h of CCCP treatment , wild - type Parkin - transfected show complete loss of mitochondria , whereas K161N R275W Parkin mutants colocalize with both p62 mitochondria . In contrast , translocation - compromised mutants K211N , T240R G430D , lacking mitochondria - associated poly - ubiquitin signal ( Fig . 4b ) , did not colocalize with p62 . Representative of four independent experiments are shown . Data from untreated control of colocalization of Parkin , mitochondria p62 upon CCCP treatment are shown in Supplementary Information , Fig . 5a b , c , respectively .

O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O B-SUBCELLULAR O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O B-GENEPROD O O O O B-GENEPROD B-SUBCELLULAR O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O

(a, b) Stable Mito-DsRed (pseudo coloured; blue)-expressing HeLa cells were transfected with Flag-Parkin wild-type or pathogenic mutants. Cells were treated with CCCP for the indicated times before immunostaining with anti-Flag (green) and anti-p62 (red). (a) In contrast to the pathogenic mutants, wild-type Parkin colocalizes with p62 at the site of clustered mitochondria, after 6 h of CCCP treatment. (b) After 24 h of CCCP treatment, wild-type Parkin-transfected cells show complete loss of mitochondria, whereas K161N and R275W Parkin mutants colocalize with both p62 and mitochondria. In contrast, translocation-compromised mutants K211N, T240R and G430D, lacking the mitochondria-associated poly-ubiquitin signal (Fig. 4b), did not colocalize with p62. Representative images of four independent experiments are shown. Data from untreated control cells and quantification of colocalization of Parkin, mitochondria and p62 upon CCCP treatment are shown in Supplementary Information, Fig. 5a and b, c, respectively.


( c , d ) Control siRNA - or p62 siRNA - silenced HeLa were transfected with EGFP - Parkin wild - type . were treated with CCCP for indicated times stained with anti - TOM20 ( pseudo coloured ; blue ) to visualize mitochondrial anti - p62 ( red ) to monitor silencing efficiency . Parkin wild - type was visualized by EGFP epifluorescence ( green ) . Parkin - positive with cleared mitochondria are surrounded by a . (

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(c, d) Control siRNA- or p62 siRNA-silenced HeLa cells were transfected with EGFP-Parkin wild-type. Cells were treated with CCCP for the indicated times and stained with anti-TOM20 (pseudo coloured; blue) to visualize mitochondrial morphology and anti-p62 (red) to monitor silencing efficiency. Parkin wild-type was visualized by EGFP epifluorescence (green). Parkin-positive cells with cleared mitochondria are surrounded by a line. (


( c - f ) Knockdown of p62 has no influence on Parkin translocation to mitochondria after 6 h of CCCP treatment ( c - e ) , but completely blocks final clearance after 24 h ( c , d , f ) . ( e , f ) Relative parkin translocation ( e ) mitochondrial clearance ( f ) after p62 knockdown CCCP treatment ( n = 4 ) . Data represent mean ± s . d . , * * * P ≤ 0 . 0005 compared with control siRNA - transfected . Representative of four independent experiments are shown . Scale bars , 10 μm . Immunostainings of control are shown in Supplementary Information , Fig . S5d , e .

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(c-f) Knockdown of p62 has no influence on Parkin translocation to mitochondria after 6 h of CCCP treatment (c-e), but completely blocks final clearance after 24 h (c, d, f). (e, f) Relative parkin translocation (e) and mitochondrial clearance (f) after p62 knockdown and CCCP treatment (n = 4). Data represent the mean ± s.d., ***P ≤ 0.0005 compared with control siRNA-transfected cells. Representative images of four independent experiments are shown. Scale bars, 10 μm. Immunostainings of control cells are shown in Supplementary Information, Fig. S5d, e.


( g ) Western blot of siRNA - transfected HeLa used in c - f demonstrates efficiency of p62 silencing . β - Actin probing served as a loading control . WT , wild - type .

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(g) Western blot of siRNA-transfected HeLa cells used in c-f demonstrates the efficiency of p62 silencing. β-Actin probing served as a loading control. WT, wild-type.


( a ) Time - course experiment of HeLa transfected with Flag - Parkin wild - type treated with CCCP for indicated periods . Total lysates were prepared , western blots were probed with specific anti - Parkin , anti - PINK1 , anti - TOM20 anti - VDAC1 antibodies . β - Actin probing served as a loading control . of detected are endogenously expressed , except Flag - Parkin .

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(a) Time-course experiment of HeLa cells transfected with Flag-Parkin wild-type and treated with CCCP for the indicated periods. Total lysates were prepared, and western blots were probed with specific anti-Parkin, anti-PINK1, anti-TOM20 and anti-VDAC1 antibodies. β-Actin probing served as a loading control. All of the proteins detected are endogenously expressed, except Flag-Parkin.


( b ) Pathogenic Parkin mutants fail to poly - ubiquitylate endogenous VDAC1 in response to CCCP treatment . HeLa were transfected with empty vector ( - ) , Flag - Parkin wild - type or pathogenic mutants along with 6 × His - tagged ubiquitin wild - type for 24 h . were treated with CCCP for 6 h subsequently lysates were prepared in urea buffer . Denaturing conditions of Ni - NTA affinity purifications were used to specifically pulldown only covalently modified with 6xHis - tagged ubiquitin . Western blots of lysates purified Ni - NTA agarose elutions were probed with anti - VDAC1 , anti - TOM20 anti - Parkin antibodies . Modification of VDAC1 was observed in expressing Parkin wild - type functional mutant G328E , whereas non - functional mutants did not have this effect . (

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(b) Pathogenic Parkin mutants fail to poly-ubiquitylate endogenous VDAC1 in response to CCCP treatment. HeLa cells were transfected with empty vector (-), Flag-Parkin wild-type or pathogenic mutants along with 6×His-tagged ubiquitin wild-type for 24 h. Cells were treated with CCCP for 6 h and subsequently protein lysates were prepared in urea buffer. Denaturing conditions of Ni-NTA affinity purifications were used to specifically pulldown only proteins covalently modified with 6xHis-tagged ubiquitin. Western blots of protein lysates and purified Ni-NTA agarose elutions were probed with anti-VDAC1, anti-TOM20 and anti-Parkin antibodies. Modification of VDAC1 was observed in cells expressing Parkin wild-type and the functional mutant G328E, whereas non-functional mutants did not have this effect. (


( c ) In response to CCCP treatment , Parkin catalyses predominantly K27 - linked poly - ubiquitin chains on endogenous VDAC1 is itself strongly poly - ubiquitylated . HeLa were transfected with an empty vector ( - ) or Flag - Parkin wild - type along with 6 × His - tagged ubiquitin variants for 24 h . Treatment of , preparation of lysates Ni - NTA purification were performed as described in c probed for Parkin , VDAC1 β - actin . WT , wild - type ; Ub , ubiquitin .

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

(c) In response to CCCP treatment, Parkin catalyses predominantly K27-linked poly-ubiquitin chains on endogenous VDAC1 and is itself strongly poly-ubiquitylated. HeLa cells were transfected with an empty vector (-) or Flag-Parkin wild-type along with 6×His-tagged ubiquitin variants for 24 h. Treatment of cells, preparation of lysates and Ni-NTA purification were performed as described in c and probed for Parkin, VDAC1 and β-actin. WT, wild-type; Ub, ubiquitin.


( a ) Western blot of parental SH - SY5Y treated with CCCP for indicated times . Time - course experiments of CCCP - treated neuronal reveal increasing modifications of VDAC1 over time . Total lysates were prepared , western blots were probed with specific anti - Parkin , anti - PINK1 , anti - TOM20 anti - VDAC1 antibodies . represent endogenous .

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(a) Western blot analysis of parental SH-SY5Y cells treated with CCCP for the indicated times. Time-course experiments of CCCP-treated neuronal cells reveal increasing modifications of VDAC1 over time. Total lysates were prepared, and western blots were probed with specific anti-Parkin, anti-PINK1, anti-TOM20 and anti-VDAC1 antibodies. All proteins represent endogenous levels.


( b ) Immunoprecipitations of Parkin VDAC1 from parental SH - SY5Y confirm ubiquitylation of both . CCCP treatment of neuronal increases ubiquitylated species of endogenous Parkin VDAC1 over time . Using specific antibodies , Parkin VDAC1 were immunoprecipitated from lysates of parental SH - SY5Y at different time points , western blotted probed with anti - ubiquitin , anti - Parkin anti - VDAC1 antibodies .

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(b) Immunoprecipitations of Parkin and VDAC1 from parental SH-SY5Y cells confirm ubiquitylation of both proteins. CCCP treatment of neuronal cells increases ubiquitylated species of endogenous Parkin and VDAC1 over time. Using specific antibodies, Parkin and VDAC1 were immunoprecipitated from lysates of parental SH-SY5Y cells at different time points, western blotted and probed with anti-ubiquitin, anti-Parkin and anti-VDAC1 antibodies.


( c ) Pathogenic Parkin mutations fail to poly - ubiquitylate endogenous VDAC1 in response to CCCP treatment . Western blot of a time - course experiment with inducible Parkin SH - SY5Y after CCCP treatment . Total lysates were prepared , western blots were probed with specific anti - Parkin anti - VDAC1 antibodies . β - Actin probing served as a loading control in a - c . WT , wild - type ; Ub , ubiquitin .

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(c) Pathogenic Parkin mutations fail to poly-ubiquitylate endogenous VDAC1 in response to CCCP treatment. Western blot analysis of a time-course experiment with inducible Parkin SH-SY5Y cells after CCCP treatment. Total lysates were prepared, and western blots were probed with specific anti-Parkin and anti-VDAC1 antibodies. β-Actin probing served as a loading control in a-c. WT, wild-type; Ub, ubiquitin.


VDAC1 - specific siRNA abrogates Parkin translocation to mitochondria after 2 h , mitochondrial clearance after 24 h , of CCCP treatment . Both effects can be rescued by re - transfection with wild - type VDAC1 . ( a ) Immunofluorescence ( b ) western blot of siRNA - transfected HeLa used in c - g demonstrate partial silencing of endogenous VDAC1 . ( a ) Silenced were stained with anti - HtrA2 / Omi , a mitochondrial marker ( pseudo coloured ; blue ) , anti - VDAC1 ( red ) antibodies . ( b ) Lysates from silenced were subjected to western blot probed with an anti - VDAC1 antibody , while TOM20 β - actin probing served as loading controls .

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VDAC1-specific siRNA abrogates Parkin translocation to mitochondria after 2 h, and mitochondrial clearance after 24 h, of CCCP treatment. Both effects can be rescued by re-transfection with wild-type VDAC1. (a) Immunofluorescence and (b) western blot of siRNA-transfected HeLa cells used in c-g demonstrate partial silencing of endogenous VDAC1. (a) Silenced cells were stained with anti-HtrA2/Omi, a mitochondrial marker (pseudo coloured; blue), and anti-VDAC1 (red) antibodies. (b) Lysates from silenced cells were subjected to western blot analysis and probed with an anti-VDAC1 antibody, while TOM20 and β-actin probing served as loading controls.


( c - e ) Control siRNA - or VDAC1 siRNA - silenced HeLa were transfected with indicated combinations of EGFP - Parkin wild - type , VDAC1 - Flag wild - type or appropriate empty vector controls . were treated with CCCP for 2 h 24 h stained with anti - HtrA2 / Omi ( pseudo coloured ; blue ) anti - Flag ( red ) to visualize mitochondrial re - transfected VDAC1 , respectively . Wild - type Parkin was visualized by EGFP epifluorescence ( green ) . Parkin - positive are surrounded by a . Representative pictures of three independent experiments are shown . Scale bars , 10 μm .

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

(c-e) Control siRNA- or VDAC1 siRNA-silenced HeLa cells were transfected with the indicated combinations of EGFP-Parkin wild-type, VDAC1-Flag wild-type or appropriate empty vector controls. Cells were treated with CCCP for 2 h and 24 h and stained with anti-HtrA2/Omi (pseudo coloured; blue) and anti-Flag (red) to visualize mitochondrial morphology and re-transfected VDAC1, respectively. Wild-type Parkin was visualized by EGFP epifluorescence (green). Parkin-positive cells are surrounded by a line. Representative pictures of three independent experiments are shown. Scale bars, 10 μm.


( f , g ) of d ( f ) e ( g ) . Parkin wild - type colocalization with impaired mitochondria after 2 h ( f ) uncleared mitochondria after 24 h ( g ) of CCCP treatment ( n = 3 ) . Data represent mean ± s . d . , * P ≤ 0 . 05 compared with control siRNA - transfected or with VDAC1 - Flag wild - type - transfected .

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(f, g) Quantification of d (f) and e (g). Parkin wild-type colocalization with impaired mitochondria after 2 h (f) and uncleared mitochondria after 24 h (g) of CCCP treatment (n = 3). Data represent the mean ± s.d., *P ≤ 0.05 compared with control siRNA-transfected or with VDAC1-Flag wild-type-transfected cells.


( A ) Acyl - RAC of HEK293T transfected with indicated plasmids for 24 h . Data information : EtOH : ethanol ; HAM : hydroxylamine ; 2 - BP : 2 -

(A) Acyl-RAC assay of HEK293T cells transfected with the indicated plasmids for 24 h. Data information: EtOH: ethanol; HAM: hydroxylamine; 2-BP: 2-bromopalmitate


( B ) Acyl - RAC of HEK293T transfected with indicated plasmids for 24 h . Data information : FL : full ; N : N - terminal domain ; C : C - terminal

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(B) Acyl-RAC assay of HEK293T cells transfected with the indicated plasmids for 24 h. Data information: FL: full length; N: N-terminal domain; C: C-terminal domain


( C ) Click chemistry was applied to detect endogenous cGAS palmitoylation in RAW264 . 7 . Data information : PA : palmitic acid ; 17 - ODYA : 17 - octadecanoic acid .

(C) Click chemistry was applied to detect endogenous cGAS palmitoylation in RAW264.7 cells. Data information: PA: palmitic acid; 17-ODYA: 17-octadecanoic acid.


( D E ) In vitro palmitoylation for indicated . NBD - Palm - CoA : ( N - [ ( 7 - nitro - 2 - 1 , 3 - benzoxadiazol - 4 - yl ) - methyl ] ) palmitoyl - CoA . recombinant cGAS used in was indicated by Coomassie blue .

(D and E) In vitro palmitoylation assay for the indicated proteins. NBD-Palm-CoA: (N-[(7-nitro-2-1,3-benzoxadiazol-4-yl)-methyl] amino) palmitoyl-CoA. The recombinant cGAS protein used in the assay was indicated by Coomassie blue staining.


( F ) LC - MS / MS of palmitoylated peptides of cGAS .

(F) LC-MS/MS analysis of palmitoylated peptides of cGAS.


( G ) Acyl - RAC of HEK293T transfected with indicated plasmids for 24 h . Data information : HAM :

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(G) Acyl-RAC assay of HEK293T cells transfected with the indicated plasmids for 24 h. Data information: HAM: hydroxylamine


( H - I ) THP - 1 were treated with DMSO or 2 - BP ( 50 μM ) ( H ) or palmitic acid ( 100 μM ) ( I ) for 12 h . Six hours after transfection with HT - ( 2 μg / mL ) , cGAMP was extracted quantified by cGAMP ELISA . Data information : 2 - BP : 2 - bromopalmitate Data are representative of at least two independent experiments . Mean ± from triplicates of technical replicates , unpaired t ; ns , not significant ; * * , P ˂ 0 . 005 ; * * * , P ˂ 0 .

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

(H-I) THP-1 cells were treated with DMSO or 2-BP (50 μM) (H) or palmitic acid (100 μM) (I) for 12 h. Six hours after transfection with HT-DNA (2 μg/mL), cGAMP was extracted and quantified by cGAMP ELISA. Data information: 2-BP: 2-bromopalmitate Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; ns, not significant; **, P˂0.005; ***, P˂0.001


( J ) THP - 1 were treated with palmitic acid ( 0 , 50 μM , or 100 μM ) for 12 h before a cGAMP bioassay . Data information : Data are representative of at least two independent experiments . Mean ± from triplicates of technical replicates , unpaired t ; ns , not significant ; * * , P ˂ 0 . 005 ; * * * , P ˂ 0 .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(J) THP-1 cells were treated with palmitic acid (0, 50 μM, or 100 μM) for 12 h before a cGAMP bioassay. Data information: Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; ns, not significant; **, P˂0.005; ***, P˂0.001


BMDMs were treated with palmitic acid ( 0 , 100 μM , or 200 μM ) for 12 h transfected with HT - ( 2 μg / mL ) for 6 h before RT - qPCR of Ifna4 ( K ) , IFNb1 ( L ) expression . Data information : Data are representative of at least two independent experiments . Mean ± from triplicates of technical replicates , unpaired t ; ns , not significant ; * * , P ˂ 0 . 005 ; * * * , P ˂ 0 .

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

BMDMs were treated with palmitic acid (0, 100 μM, or 200 μM) for 12 h and transfected with HT-DNA (2 μg/mL) for 6 h before RT-qPCR analysis of Ifna4 (K), IFNb1 (L) expression. Data information: Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; ns, not significant; **, P˂0.005; ***, P˂0.001


BMDMs were treated with palmitic acid ( 0 , 100 μM , or 200 μM ) for 12 h transfected with HT - ( 2 μg / mL ) for 6 h before RT - qPCR of , Cxcl10 ( M ) Rantes ( N ) expression . Data information : Data are representative of at least two independent experiments . Mean ± from triplicates of technical replicates , unpaired t ; ns , not significant ; * * , P ˂ 0 . 005 ; * * * , P ˂ 0 .

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

BMDMs were treated with palmitic acid (0, 100 μM, or 200 μM) for 12 h and transfected with HT-DNA (2 μg/mL) for 6 h before RT-qPCR analysis of , Cxcl10 (M) and Rantes (N) expression. Data information: Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; ns, not significant; **, P˂0.005; ***, P˂0.001


( O ) L929 were treated with BSA palmitic acid ( 100 μM ) transfected with HT - ( 2 μg / mL ) for indicated times before immunoblotting with indicated antibodies .

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

(O) L929 cells were treated with BSA and palmitic acid (100 μM) and transfected with HT-DNA (2 μg/mL) for the indicated times before immunoblotting analysis with the indicated antibodies.


( A ) Acyl - RAC of HEK293T transfected with Flag - cGAS 24 h after transfection with indicated shRNA . ( B ) of palmitoylation of Flag - cGAS in ( A ) . Data information :

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

(A) Acyl-RAC assay of HEK293T cells transfected with Flag-cGAS 24 h after transfection with the indicated shRNA. (B) Quantification of palmitoylation levels of Flag-cGAS in (A). Data information:


( C ) Acyl - RAC of HEK293T transfected with indicated plasmids for 24 h . HAM : hydroxylamine .

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

(C) Acyl-RAC assay of HEK293T cells transfected with the indicated plasmids for 24 h. HAM: hydroxylamine.


( D ) HEK293T were transfected with HA - cGAS ( WT or C474S mutant ) ( 200 ng ) , Flag - STING ( 200 ng ) Myc - ZDHHC18 ( 200 ng ) expression plasmids for 24 h before luciferase reporter assays . Data information : Data are representative of at least two independent experiments . Mean ± from triplicates of technical replicates , unpaired t ; * * , P ˂ 0 . 005 ; * * * , P ˂ 0 . 001 (

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

(D) HEK293T cells were transfected with HA-cGAS (WT or C474S mutant) (200 ng), Flag-STING (200 ng) and Myc-ZDHHC18 (200 ng) expression plasmids for 24 h before luciferase reporter assays. Data information: Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; **, P˂0.005; ***, P˂0.001 (D


( E F ) Immunoprecipitation ( with an anti - Flag antibody ) immunoblot of HEK293T transfected with indicated plasmids for 24 h . Data information : WCL : whole - lysate ; IP : immunoprecipitation ; FL : full

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

(E and F) Immunoprecipitation (with an anti-Flag antibody) and immunoblot analysis of HEK293T cells transfected with the indicated plasmids for 24 h. Data information: WCL: whole-cell lysate; IP: immunoprecipitation; FL: full length


( H ) Immunoprecipitation ( with an anti - Flag antibody ) immunoblot of HEK293T transfected with plasmids encoding HA - cGAS Flag - ZDHHC18 truncations or GFP for 24 h . Data information : WCL : whole - lysate ; IP : immunoprecipitation ; FL : full

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

(H) Immunoprecipitation (with an anti-Flag antibody) and immunoblot analysis of HEK293T cells transfected with plasmids encoding HA-cGAS and Flag-ZDHHC18 truncations or GFP for 24 h. Data information: WCL: whole-cell lysate; IP: immunoprecipitation; FL: full length


( J ) Immunoprecipitation ( with an anti - Flag antibody ) immunoblot of HEK293T transfected with plasmids encoding HA - ZDHHC18 Flag - cGAS truncates or GFP for 24 h . Data information : WCL : whole -

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

(J) Immunoprecipitation (with an anti-Flag antibody) and immunoblot analysis of HEK293T cells transfected with plasmids encoding HA-ZDHHC18 and Flag-cGAS truncates or GFP for 24 h. Data information: WCL: whole-cell lysate


( K ) Immunofluorescence of HA - cGAS ( green ) Flag - ZDHHC18 ( red ) in HT - - stimulated ( or not ) HeLa . Scale bars : 7 μm . ( L ) Colocalization ( merged volume of total cGAS signal ) of cGAS ZDHHC18 in ( K ) . Data information : Data are representative of at least two independent experiments . Mean ± from triplicates of technical replicates , unpaired t ; * * , P ˂ 0 . 005 ; * * * , P ˂ 0 . 001 L ) .

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

(K) Immunofluorescence analysis of HA-cGAS (green) and Flag-ZDHHC18 (red) in HT-DNA-stimulated (or not) HeLa cells. Scale bars: 7 μm. (L) Colocalization (merged volume of total cGAS signal) of cGAS and ZDHHC18 in (K). Data information: Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; **, P˂0.005; ***, P˂0.001 L).


( M - N ) Comparison of binding free energy ( M ) binding energy decomposition of a per - residue to binding affinity between ZDHHC18 cGAS ( N ) in different complexes .

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

(M-N) Comparison of the binding free energy (M) and binding energy decomposition of a per-residue to the binding affinity between ZDHHC18 and cGAS (N) in different complexes.


( A ) Immunofluorescence of GFP - cGAS , nucleus ( DAPI ) , ER ( calnexin ) , Golgi apparatus ( GM130 ) , early endosomes ( EEA1 ) in HeLa . 2 - BP : 2 - bromopalmitate ; ISD : IFN - stimulatory . Arrows indicate cGAS . Scale bars : 10 μm .

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

(A) Immunofluorescence analysis of GFP-cGAS, the nucleus (DAPI), the ER (calnexin), the Golgi apparatus (GM130), and early endosomes (EEA1) in HeLa cells. 2-BP: 2-bromopalmitate; ISD: IFN-stimulatory DNA. Arrows indicate cGAS puncta. Scale bars: 10 μm.


( F HEK293T were transfected with indicated plasmids . Biotin - conjugated ISD was transfected into 6 h before harvesting . Lysates were coprecipitated with streptavidin beads then assessed by immunoblot as shown . Data information : WCL : whole - lysate ; IP : immunoprecipitation . Data are representative of at least two independent experiments .

O O B-CELL_LINE O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O

(F HEK293T cells were transfected with the indicated plasmids. Biotin-conjugated ISD was transfected into cells 6 h before harvesting. Lysates were coprecipitated with streptavidin beads and then assessed by immunoblot analysis as shown. Data information: WCL: whole-cell lysate; IP: immunoprecipitation. Data are representative of at least two independent experiments.


G ) HEK293T were transfected with indicated plasmids . Biotin - conjugated ISD was transfected into 6 h before harvesting . Lysates were coprecipitated with streptavidin beads then assessed by immunoblot as shown . Data information : WCL : whole - lysate ; Data are representative of at least two independent experiments .

O O B-CELL_LINE O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O

G) HEK293T cells were transfected with the indicated plasmids. Biotin-conjugated ISD was transfected into cells 6 h before harvesting. Lysates were coprecipitated with streptavidin beads and then assessed by immunoblot analysis as shown. Data information: WCL: whole-cell lysate; Data are representative of at least two independent experiments.


( H ) Confocal microscopy of BMDMs ( Zdhhc18 + / + or Zdhhc18 − / − ) transfected with FITC - conjugated ISD for 6 h ( left ) . Scale bar : 20 μm .

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

(H) Confocal microscopy of BMDMs (Zdhhc18+/+ or Zdhhc18−/−) transfected with FITC-conjugated ISD for 6 h (left). Scale bar: 20 μm.


( C ) Cross - correlated motions of Cα atoms of WT palmitoylated cGAS .

(C) Cross-correlated motions of the Cα atoms of the WT and palmitoylated cGAS proteins.


( D ) Immunoprecipitation ( with an anti - Flag antibody ) immunoblot of HEK293T transfected with plasmids encoding Flag - cGAS , HA - cGAS , Myc - ZDHHC18 ( WT or CS mutant ) for 24 h . Data information : WCL : whole - lysate ; IP : immunoprecipitation ; Data are representative of at least two independent experiments .

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

(D) Immunoprecipitation (with an anti-Flag antibody) and immunoblot analysis of HEK293T cells transfected with plasmids encoding Flag-cGAS, HA-cGAS, and Myc-ZDHHC18 (WT or CS mutant) for 24 h. Data information: WCL: whole-cell lysate; IP: immunoprecipitation; Data are representative of at least two independent experiments.


( E ) Immunoprecipitation ( with an anti - Flag antibody ) immunoblot of HEK293T transfected with indicated plasmids followed by addition of DMSO , 2 - BP ( 50 μM ) or palmitic acid ( 100 μM ) . Data information : WCL : whole - lysate ; IP : immunoprecipitation ; 2 - BP : 2 - bromopalmitate . Data are representative of at least two independent experiments .

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

(E) Immunoprecipitation (with an anti-Flag antibody) and immunoblot analysis of HEK293T cells transfected with the indicated plasmids followed by the addition of DMSO, 2-BP (50 μM) or palmitic acid (100 μM). Data information: WCL: whole-cell lysate; IP: immunoprecipitation; 2-BP: 2-bromopalmitate. Data are representative of at least two independent experiments.


( F ) FRET of HeLa transfected with indicated plasmids with addition of DMSO , 2 - BP ( 50 μM ) or palmitic acid ( 100 μM ) . Scale bar : 8 μm . ( G ) FRET efficiency of cGAS - GFP cGAS - mCherry in ( F ) . Data information : 2 - BP : 2 - bromopalmitate . Data are representative of at least two independent experiments . Mean ± from triplicates of technical replicates , unpaired t ; * , P ˂ 0 . 01 . * * * , P ˂ 0 . 001 (

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

(F) FRET assay of HeLa cells transfected with the indicated plasmids with the addition of DMSO, 2-BP (50 μM) or palmitic acid (100 μM). Scale bar: 8 μm. (G) FRET efficiency of cGAS-GFP and cGAS-mCherry in (F). Data information: 2-BP: 2-bromopalmitate. Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; *, P˂0.01. ***, P˂0.001 (G


( H ) Immunoprecipitation ( with an anti - Flag antibody ) immunoblot of HEK293T transfected with Flag - cGAS HA - cGAS ( WT , C405S , C409S C474S ) expression plasmids . Data information : WCL : whole - lysate ; IP : immunoprecipitation Data are representative of at least two independent experiments .

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

(H) Immunoprecipitation (with an anti-Flag antibody) and immunoblot analysis of HEK293T cells transfected with Flag-cGAS and HA-cGAS (WT, C405S, C409S and C474S) expression plasmids. Data information: WCL: whole-cell lysate; IP: immunoprecipitation Data are representative of at least two independent experiments.


( I ) FRET of HeLa transfected with indicated plasmids . Scale bar : 8 μm . ( J ) FRET efficiency of cGAS - GFP cGAS - mCherry in ( I ) . Data information : Data are representative of at least two independent experiments . Mean ± from triplicates of technical replicates , unpaired t ; * , P ˂ 0 . 01 . * * * , P ˂ 0 . 001 J ) .

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

(I) FRET assay of HeLa cells transfected with the indicated plasmids. Scale bar: 8 μm. (J) FRET efficiency of cGAS-GFP and cGAS-mCherry in (I). Data information: Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; *, P˂0.01. ***, P˂0.001 J).


( A ) HEK293T ( 5 × 105 ) were transfected with HA - cGAS ( 200 ng ) , Flag - STING ( 200 ng ) Myc - ZDHHC18 ( WT or CS mutant ) expression plasmids ( 0 , 50 , 100 , or 200 ng ) for 24 h before luciferase reporter assays were conducted . Data information : ZDHHC18 ( CS ) : a catalytic mutant with a cysteine - to - serine substitution in DHHC motif of ZDHHC18 data are representative of at least two independent experiments . Mean ± from triplicates of technical replicates , unpaired t ; ns , not significant ; * * , P ˂ 0 . 01 ; * * * , P ˂ 0 . 001 (

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

(A) HEK293T cells (5×105) were transfected with HA-cGAS (200 ng), Flag-STING (200 ng) and Myc-ZDHHC18 (WT or CS mutant) expression plasmids (0, 50, 100, or 200 ng) for 24 h before luciferase reporter assays were conducted. Data information: ZDHHC18(CS): a catalytic mutant with a cysteine-to-serine substitution in the DHHC motif of ZDHHC18 All data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; ns, not significant; **, P˂0.01; ***, P˂0.001 (A


( B ) HEK293T ( 1 × 106 ) were transfected with HA - cGAS ( 500 ng ) , Flag - STING ( 500 ng ) Myc - ZDHHC18 ( 500 ng ) expression plasmids for 24 h . TBK1 phosphorylation was detected by immunoblotting . Data information : ZDHHC18 ( CS ) : a catalytic mutant with a cysteine - to - serine substitution in DHHC motif of

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

(B) HEK293T cells (1×106) were transfected with HA-cGAS (500 ng), Flag-STING (500 ng) and Myc-ZDHHC18 (500 ng) expression plasmids for 24 h. TBK1 phosphorylation was detected by immunoblotting. Data information: ZDHHC18(CS): a catalytic mutant with a cysteine-to-serine substitution in the DHHC motif of ZDHHC18


( C D ) HEK293T were transfected with indicated plasmids , amount of cGAMP in lysates was quantified by LC - MS / MS . Data information : ZDHHC18 ( CS ) : a catalytic mutant with a cysteine - to - serine substitution in DHHC motif of ZDHHC18 ; LC - MS / MS : liquid chromatography - tandem mass spectrometry ;

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

(C and D) HEK293T cells were transfected with the indicated plasmids, and the amount of cGAMP in the lysates was quantified by LC-MS/MS. Data information: ZDHHC18(CS): a catalytic mutant with a cysteine-to-serine substitution in the DHHC motif of ZDHHC18; LC-MS/MS: liquid chromatography-tandem mass spectrometry;


L929 stably transfected with control shRNA or ZDHHC18 shRNA knockdown efficiency of ZDHHC18 is shown by RT - qPCR ( E ) results . Data information : NC : negative control . data are representative of at least two independent experiments . Mean ± from triplicates of technical replicates , unpaired t ; ns , not significant ; * * , P ˂ 0 . 01 ; * * * , P ˂ 0 .

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

L929 cells stably transfected with control shRNA or ZDHHC18 shRNA The knockdown efficiency of ZDHHC18 is shown by RT-qPCR (E) results. Data information: NC: negative control. All data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; ns, not significant; **, P˂0.01; ***, P˂0.001


L929 stably transfected with control shRNA or ZDHHC18 shRNA efficiency of ZDHHC18 is shown by western blotting ( F ) results . Data information : NC : negative control .

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

L929 cells stably transfected with control shRNA or ZDHHC18 shRNA efficiency of ZDHHC18 is shown by western blotting (F) results. Data information: NC: negative control.


L929 stably transfected with control shRNA or ZDHHC18 shRNA were transfected with HT - ( 2 μg / mL ) for indicated times before RT - qPCR of Ifna4 ( G ) , expression . Data information : HT - : herring testis ; NC : negative control . data are representative of at least two independent experiments . Mean ± from triplicates of technical replicates , unpaired t ; ns , not significant ; * * , P ˂ 0 . 01 ; * * * , P ˂ 0 .

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

L929 cells stably transfected with control shRNA or ZDHHC18 shRNA were transfected with HT-DNA (2 μg/mL) for the indicated times before RT-qPCR analysis of Ifna4 (G), expression. Data information: HT-DNA: herring testis DNA; NC: negative control. All data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; ns, not significant; **, P˂0.01; ***, P˂0.001


L929 stably transfected with control shRNA or ZDHHC18 shRNA were transfected with HT - ( 2 μg / mL ) for indicated times before RT - qPCR of IFNb1 ( H ) , Cxcl10 ( I ) expression . Data information : HT - : herring testis ; NC : negative control . data are representative of at least two independent experiments . Mean ± from triplicates of technical replicates , unpaired t ; ns , not significant ; * * , P ˂ 0 . 01 ; * * * , P ˂ 0 .

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

L929 cells stably transfected with control shRNA or ZDHHC18 shRNA were transfected with HT-DNA (2 μg/mL) for the indicated times before RT-qPCR analysis of IFNb1 (H), and Cxcl10 (I) expression. Data information: HT-DNA: herring testis DNA; NC: negative control. All data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; ns, not significant; **, P˂0.01; ***, P˂0.001