Figure 5 . TGFβ signaling activity in smooth muscle cells in human left main coronary arteries with various degrees of atherosclerosis . ( A & C & E ) Representative images of immunofluorescence staining for TGF ( red ) , p - Smad2 ( red ) , or p - Smad3 ( red ) from patients with No / mild , moderate , or severe disease . Nuclei were counter - stained with DAPI ( blue ) . Scale bar : 16 m . ( B & D & F ) Medial TGF area and percentage of medial p - Smad2 , and p - Smad3 positive SMCs ( * * * p < 0 . 001 compared to No / mild disease ; one - way ANOVA with Newman - Keuls post hoc test for multiple comparison correction ) . A full table of p - values for this figure is shown in Appendix Table S1 .

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

Figure 5. TGFβ signaling activity in smooth muscle cells in human left main coronary arteries with various degrees of atherosclerosis. (A&C&E) Representative images of immunofluorescence staining for TGF (red), p-Smad2 (red), or p-Smad3 (red) from patients with No/mild, moderate, or severe disease. Nuclei were counter-stained with DAPI (blue). Scale bar: 16 m. (B&D&F) Medial TGF area and percentage of medial p-Smad2, and p-Smad3 positive SMCs (***p<0.001 compared to No/mild disease; one-way ANOVA with Newman-Keuls post hoc test for multiple comparison correction). A full table of p-values for this figure is shown in Appendix Table S1.


( A ) Dissected mouse aorta demonstrating lipid - rich plaques in brachiocephalic artery after 4 months of high fat diet compared to the normal diet in Apoe - / - mice . ( b ) & ( d ) Cross - section of brachiocephalic artery from ( a ) & ( c ) stained with Oil Red O . L : lumen . Scale bar : 4 mm . 3 mice per group .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Dissected mouse aorta demonstrating lipid-rich plaques in brachiocephalic artery after 4 months of high fat diet compared to the normal diet in Apoe-/- mice. (b) & (d) Cross-section of brachiocephalic artery from (a) & (c) stained with Oil Red O. L: lumen. Scale bar: 4 mm. 3 mice per group.


( B ) Histological analysis of mouse normal artery or atherosclerotic plaque in brachiocephalic artery with anti - SM - actin , anti - Notch3 , and anti - SM - MHC antibodies . Nuclei were counterstained with DAPI ( blue ) . L : lumen . Scale bar : 62 m . 3 mice per group .

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

(B) Histological analysis of mouse normal artery or atherosclerotic plaque in brachiocephalic artery with anti-SM -actin, anti-Notch3, and anti-SM-MHC antibodies. Nuclei were counterstained with DAPI (blue). L: lumen. Scale bar: 62 m. 3 mice per group.


( C - F ) Analysis of brachiocephalic artery of Apoe - / - mice maintained for 4 months on either normal or high fat diet using anti - CD31 ( green ) , anti - p - FGFR1 ( red ) , anti - FGFR1 ( red ) , anti - p - Smad2 ( red ) , and anti - p - Smad3 ( red ) antibodies . Nuclei counterstained with DAPI ( blue ) . Scale bar : 62 m . L : lumen . M : Media . 6 mice per group .

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

(C-F) Analysis of brachiocephalic artery of Apoe-/- mice maintained for 4 months on either normal or high fat diet using anti-CD31 (green), anti-p-FGFR1 (red), anti-FGFR1 (red), anti-p-Smad2 (red), and anti-p-Smad3 (red) antibodies. Nuclei counterstained with DAPI (blue). Scale bar: 62 m. L: lumen. M: Media. 6 mice per group.


( G - J ) Quantification of the number of medial smooth muscle cells expressing p - FGFR1 , FGFR1 , p - Smad2 , and p - Smad3 ( * * * p < 0 . 001 compared to ND , NS : not significant compared to ND ; unpaired two - tailed Student ' s t test . ) . ND : Normal diet . HFD : High fat diet . A full table of p - values for this figure is shown in Appendix Table S1 .

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

(G-J) Quantification of the number of medial smooth muscle cells expressing p-FGFR1, FGFR1, p-Smad2, and p-Smad3 (***p<0.001 compared to ND, NS: not significant compared to ND; unpaired two-tailed Student's t test.). ND: Normal diet. HFD: High fat diet. A full table of p-values for this figure is shown in Appendix Table S1.


( A ) Representative photomicrographs of Oil Red O - stained atherosclerotic lesions in the aortic arch , of Apoe - / - or Frs2SMCKO / Apoe - / - mice after 16 weeks of high fat diet . 3 mice per group . Scale bar : 5 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Representative photomicrographs of Oil Red O-stained atherosclerotic lesions in the aortic arch, of Apoe-/- or Frs2SMCKO/Apoe-/- mice after 16 weeks of high fat diet. 3 mice per group. Scale bar: 5 mm.


( B ) ( Left ) Microphotographs of aortas ( en face ) from Apoe - / - and Frs2SMCKO / Apoe - / - mice after 16 weeks of high fat diet after staining with Oil Red O . ( Right ) Lesion area quantification . All data shown as mean ± SD . ( * * * p < 0 . 001 compared to Apoe - / - ; unpaired two - tailed Student ' s t test ) . 12 mice per group .

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

(B) (Left) Microphotographs of aortas (en face) from Apoe-/- and Frs2SMCKO/Apoe-/- mice after 16 weeks of high fat diet after staining with Oil Red O. (Right) Lesion area quantification. All data shown as mean ± SD. (***p<0.001 compared to Apoe-/-; unpaired two-tailed Student's t test). 12 mice per group.


( C - D ) Representative cross - sections of brachiocephalic arteries Apoe - / - and Frs2SMCKO / Apoe - / - mice stained with hematoxylin and eosin ( H & amp ; E ) ( C ) and Movat ( D ) . ( b ) & amp ; ( d ) are high - magnification view from ( a ) & amp ; ( c ) . NC : necrotic core . Apoe - / - mice N = 9 , Frs2SMCKO / Apoe - / - mice N = 12 .

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

(C-D) Representative cross-sections of brachiocephalic arteries Apoe-/- and Frs2SMCKO/Apoe-/- mice stained with hematoxylin and eosin (H&E) (C) and Movat (D). (b)&(d) are high-magnification view from (a)&(c). NC: necrotic core. Apoe-/- mice N=9, Frs2SMCKO/Apoe-/- mice N=12.


( E ) Histological analysis of atheosclerotic plaque with anti - Ki67 antibody . Nuclei were counterstained with DAPI ( blue ) . ( b ) & ( d ) are high - magnification images from ( a ) & ( c ) . Scale bar : 62 m ( low - magnification images ) ; 10 m ( high - magnification images ) . Apoe - / - mice N = 9 , Frs2SMCKO / Apoe - / - mice N = 12 .

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

(E) Histological analysis of atheosclerotic plaque with anti-Ki67 antibody. Nuclei were counterstained with DAPI (blue). (b)&(d) are high-magnification images from (a)&(c). Scale bar: 62 m (low-magnification images); 10 m (high-magnification images). Apoe-/- mice N=9, Frs2SMCKO/Apoe-/- mice N=12.


( F ) Quantification of plaque cellularity ; Apoe - / - mice N = 9 , Frs2SMCKO / Apoe - / - mice N = 12 ( * * * p < 0 . 001 compared to Apoe - / - ; unpaired two - tailed Student ' s t test ) .

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

(F) Quantification of plaque cellularity; Apoe-/- mice N=9, Frs2SMCKO/Apoe-/- mice N=12 (***p<0.001 compared to Apoe-/-; unpaired two-tailed Student's t test).


( G ) Quantifications of the extent of fibrous cap and necrotic areas in brachiocephalic artery of Apoe - / - and Frs2SMCKO / Apoe - / - mice . ( * p < 0 . 05 , * * p < 0 . 01 compared to Apoe - / - ; unpaired two - tailed Student ' s t test ) .

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

(G) Quantifications of the extent of fibrous cap and necrotic areas in brachiocephalic artery of Apoe-/- and Frs2SMCKO/Apoe-/- mice. (*p<0.05, **p<0.01 compared to Apoe-/-; unpaired two-tailed Student's t test).


( H ) Measurement of Ki67 + cells ( * p < 0 . 05 , * * * p < 0 . 001 compared to Apoe - / - ; unpaired two - tailed Student ' s t test ) .

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

(H) Measurement of Ki67+ cells (*p<0.05, ***p<0.001 compared to Apoe-/-; unpaired two-tailed Student's t test).


( D ) RipAC overexpression inhibits flg22 - triggered MAPK activation in Arabidopsis . 100 nM flg22Pto was used to treat Arabidopsis seedlings and the samples were collected at indicated time points . Immunoblots were analysed using anti - pMAPK and anti - RipAC antibodies . Coomassie brilliant blue ( CBB ) staining and a non - specific band were used as loading control . Molecular weight ( kDa ) marker bands are indicated for reference .

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

(D) RipAC overexpression inhibits flg22-triggered MAPK activation in Arabidopsis. 100 nM flg22Pto was used to treat Arabidopsis seedlings and the samples were collected at indicated time points. Immunoblots were analysed using anti-pMAPK and anti-RipAC antibodies. Coomassie brilliant blue (CBB) staining and a non-specific band were used as loading control. Molecular weight (kDa) marker bands are indicated for reference.


( A - B ) Interaction between RipAC and SlPUB4 / AtPUB4 was confirmed by Split - LUC assay qualitatively ( A ) and quantitatively ( B ) in Nicotiana benthamiana . Values are means ± SE ( n = 8 biological replicates ) . Asterisks indicate significant differences compared to RipAC - nLUC / cLUC - AtPIP2A negative control ( Student ' s t test , * * * * p < 0 . 0001 ) . RLU : relative luminescence units .

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

(A-B) Interaction between RipAC and SlPUB4/AtPUB4 was confirmed by Split-LUC assay qualitatively (A) and quantitatively (B) in Nicotiana benthamiana. Values are means ± SE (n=8 biological replicates). Asterisks indicate significant differences compared to RipAC-nLUC/cLUC-AtPIP2A negative control (Student's t test, **** p<0.0001). RLU: relative luminescence units.


( C ) Co - immunoprecipitation of SlPUB4 / AtPUB4 - GFP and RipAC - nLUC after co - expression in N . benthamiana . Two days after Agrobacterium infiltration , the plant tissues were collected and then subjected to anti - GFP immunoprecipitation . Immunoblots were analyzed using anti - LUC and anti - GFP antibodies . Molecular weight ( kDa ) marker bands are indicated for reference .

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

(C) Co-immunoprecipitation of SlPUB4/AtPUB4-GFP and RipAC-nLUC after co-expression in N. benthamiana. Two days after Agrobacterium infiltration, the plant tissues were collected and then subjected to anti-GFP immunoprecipitation. Immunoblots were analyzed using anti-LUC and anti-GFP antibodies. Molecular weight (kDa) marker bands are indicated for reference.


( E ) flg22 - triggered MAPK activation in pub4 T - DNA mutants . 100 nM flg22Pto was used to treat Arabidopsis seedlings and the samples were collected at indicated time points for western blots . Immunoblots were analyzed using anti - pMAPK and anti - FLS2 antibodies . Coomassie brilliant blue ( CBB ) staining was used as loading control . Molecular weight ( kDa ) marker bands are indicated for reference .

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

(E) flg22-triggered MAPK activation in pub4 T-DNA mutants. 100 nM flg22Pto was used to treat Arabidopsis seedlings and the samples were collected at indicated time points for western blots. Immunoblots were analyzed using anti-pMAPK and anti-FLS2 antibodies. Coomassie brilliant blue (CBB) staining was used as loading control. Molecular weight (kDa) marker bands are indicated for reference.


( B ) Co - immunoprecipitation of PUB4 and BIK1 in N . benthamiana . PUB4 - GFP or GFP - LTI6b were transiently co - expressed with BIK1 - HA in the leaves of N . benthamiana . After treatment with mock or 1 μM flg22Paer for 10 min , total proteins ( input ) were extracted and subjected for immunoprecipitation with anti - GFP beads .

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

(B) Co-immunoprecipitation of PUB4 and BIK1 in N. benthamiana. PUB4-GFP or GFP-LTI6b were transiently co-expressed with BIK1-HA in the leaves of N. benthamiana. After treatment with mock or 1 μM flg22Paer for 10 min, total proteins (input) were extracted and subjected for immunoprecipitation with anti-GFP beads.


( C ) Co - immunoprecipitation of PUB4 with PRR complex members in Arabidopsis . PUB4 co - immunoprecipitated with FLS2 and BAK1 specifically after 10 min of 1 μM flg22Paer treatment , and constantly with BIK1 . Total protein extracts ( input ) from Col - 0 and PUB4 - FLAG plants were subjected to immunoprecipitation with anti - FLAG beads .

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

(C) Co-immunoprecipitation of PUB4 with PRR complex members in Arabidopsis. PUB4 co-immunoprecipitated with FLS2 and BAK1 specifically after 10 min of 1 μM flg22Paer treatment, and constantly with BIK1. Total protein extracts (input) from Col-0 and PUB4-FLAG plants were subjected to immunoprecipitation with anti-FLAG beads.


( D ) PUB4 directly interacts with BIK1 in vitro . Immunoblots were analyzed using the indicated antibodies . Molecular weight ( kDa ) marker bands are indicated for reference .

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

(D) PUB4 directly interacts with BIK1 in vitro. Immunoblots were analyzed using the indicated antibodies. Molecular weight (kDa) marker bands are indicated for reference.


RipAC does not affect PUB4 association with PRR complex members . PUB4 - FLAG , PUB4 - FLAG RipAC - GFP and Col - 0 plants were treated for 10 min with water ( as mock ) or 1 μM flg22Paer and elf18Ecol treatment . Total protein extracts ( input ) were subjected for immunoprecipitation with anti - FLAG beads . Immunoblots were analyzed using the indicated antibodies . Molecular weight ( kDa ) marker bands are indicated for reference . Data information : The experiment was performed 3 times with similar results .

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

RipAC does not affect PUB4 association with PRR complex members. PUB4-FLAG, PUB4-FLAG RipAC-GFP and Col-0 plants were treated for 10 min with water (as mock) or 1 μM flg22Paer and elf18Ecol treatment. Total protein extracts (input) were subjected for immunoprecipitation with anti-FLAG beads. Immunoblots were analyzed using the indicated antibodies. Molecular weight (kDa) marker bands are indicated for reference. Data information: The experiment was performed 3 times with similar results.


( A ) PUB4 has auto - ubiquitination activity in vitro . PUB4 autoubiquitination assay using His - Ubiquitin , UBA1 ( E1 ) , HA - UBC8 ( E2 ) and MBP - PUB4 or MBP - PUB4 Cys239Ala inactive mutant reconstituted in bacteria . Ubiquitinated products were purified under denaturing conditions by immobilized metal ion chromatography ( IMAC ) . Input and precipitated proteins were resolved in 10 % and 8 % acrylamide gels , respectively , and analyzed by immunoblot with indicated antibodies . The line indicates autoubiquitinated PUB4 .

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

(A) PUB4 has auto-ubiquitination activity in vitro. PUB4 autoubiquitination assay using His-Ubiquitin, UBA1 (E1), HA-UBC8 (E2) and MBP-PUB4 or MBP-PUB4 Cys239Ala inactive mutant reconstituted in bacteria. Ubiquitinated products were purified under denaturing conditions by immobilized metal ion chromatography (IMAC). Input and precipitated proteins were resolved in 10% and 8% acrylamide gels, respectively, and analyzed by immunoblot with indicated antibodies. The line indicates autoubiquitinated PUB4.


( B ) PUB4 ubiquitinates non - active BIK1 , and does not ubiquitinate active BIK1 . Operon containing His - Ubiquitin , UBA1 ( E1 ) , HA - UBC8 ( E2 ) , and MBP - PUB4 were co - transformed with GST , GST - BIK1 , or GST - BIK1 kinase dead ( BIK1 - KD ) in the presence of PUB4 . Ubiquitinated products were purified under denaturing conditions by IMAC , and modified proteins further enriched by GSH chromatography . Input and precipitated proteins were resolved in 10 % and 8 % acrylamide gels , respectively , and analyzed by immunoblot with indicated antibodies . The line indicates the ubiquitination products .

O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) PUB4 ubiquitinates non-active BIK1, and does not ubiquitinate active BIK1. Operon containing His-Ubiquitin, UBA1 (E1), HA-UBC8 (E2), and MBP-PUB4 were co-transformed with GST, GST-BIK1, or GST-BIK1 kinase dead (BIK1-KD) in the presence of PUB4. Ubiquitinated products were purified under denaturing conditions by IMAC, and modified proteins further enriched by GSH chromatography. Input and precipitated proteins were resolved in 10% and 8% acrylamide gels, respectively, and analyzed by immunoblot with indicated antibodies. The line indicates the ubiquitination products.


( A ) Analysis of BIK1 - HA protein accumulation in BIK1 - HA + / - , pub4 - / - BIK1 - HA + / - , and PUB4 - FLAG + / - BIK1 - HA + / - in 4 - to 5 - week - old plants . Prior to protein extraction leave disks were treated for 6 h with 100 mM MG132 , 50 mM CHX , and for 10 min with water ( as mock ) or 1 mM flg22Paer . Proteins were extracted in SDS buffer and analysed by western blot . BAK1 was used as a loading control . Numbers correspond to the quantitation of the BIK1 - HA band , normalized to the quantitation of the BAK1 control band in the same sample .

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

(A) Analysis of BIK1-HA protein accumulation in BIK1-HA+/-, pub4-/- BIK1-HA+/-, and PUB4-FLAG+/- BIK1-HA+/- in 4- to 5-week-old plants. Prior to protein extraction leave disks were treated for 6 h with 100 mM MG132, 50 mM CHX, and for 10 min with water (as mock) or 1 mM flg22Paer. Proteins were extracted in SDS buffer and analysed by western blot. BAK1 was used as a loading control. Numbers correspond to the quantitation of the BIK1-HA band, normalized to the quantitation of the BAK1 control band in the same sample.


( B ) BIK1 - HA accumulation is reduced in PUB4 - FLAG + / - BIK1 - HA + / - line compared to BIK1 - HA + / - line in the absence of PAMP treatment . Total protein extracts were analysed by western blot . Tubulin was used as a loading control .

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

(B) BIK1-HA accumulation is reduced in PUB4-FLAG+/- BIK1-HA+/- line compared to BIK1-HA+/- line in the absence of PAMP treatment. Total protein extracts were analysed by western blot. Tubulin was used as a loading control.


( E ) BIK1 - HA accumulation is reduced in RipAC - GFP / BIK1 - HA line compared to Col - 0 / BIK1 - HA line . Total protein extracts were analysed by western blot . Actin was used as a loading control .

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

(E) BIK1-HA accumulation is reduced in RipAC-GFP/BIK1-HA line compared to Col-0/BIK1-HA line. Total protein extracts were analysed by western blot. Actin was used as a loading control.


D ) Cells expressing empty vector ( EV ) , or PRKAA1 - myc , PRKAB2 and PRKAG2 - FLAG ( AMPK ) alone or with myc - Ulk1 WT ( ULK ) were cultured in full medium ( F ) , EBSS ( St ) , or serum - free medium ( SFM ) alone or in the presence of 991 ( 1µM ) or MRT68921 ( 1μM ) for 90 minutes as indicated . 2 % Loading control and pull down samples were analysed by Western blot .

O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cells expressing empty vector (EV), or PRKAA1-myc, PRKAB2 and PRKAG2-FLAG (AMPK) alone or with myc-Ulk1 WT (ULK) were cultured in full medium (F), EBSS (St), or serum-free medium (SFM) alone or in the presence of 991 (1µM) or MRT68921 (1μM) for 90 minutes as indicated. 2% Loading control and pull down samples were analysed by Western blot.


A ) VPS15 - ZZ was co - expressed with Ulk1 and Ulk2 wild type ( WT ) or kinase inactive ( KI ) before analysis by Western blot . An electrophoretic mobility shift in VPS15 - ZZ was noted with WT only .

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

A) VPS15-ZZ was co-expressed with Ulk1 and Ulk2 wild type (WT) or kinase inactive (KI) before analysis by Western blot. An electrophoretic mobility shift in VPS15-ZZ was noted with WT only.


A ) Lysates from 3 control clones and 7 VPS15 CRISPR clones were analysed by Western blot . LC3 and p62 accumulation as well as depletion of VPS34 CI components was observed in all 6 sgA - derived clones .

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

A) Lysates from 3 control clones and 7 VPS15 CRISPR clones were analysed by Western blot. LC3 and p62 accumulation as well as depletion of VPS34 CI components was observed in all 6 sgA-derived clones.


B ) Controls and 6 VPS15 CRISPR KO clones were starved for 1 hr , fixed and imaged for WIPI2 ( green ) . Depletion of VPS15 was associated with a block in WIPI2 puncta formation and large vacuoles ( red asterisks ) . Quantification of WIPI2 spots , mean + / - SEM , n = 3 .

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

B) Controls and 6 VPS15 CRISPR KO clones were starved for 1 hr, fixed and imaged for WIPI2 (green). Depletion of VPS15 was associated with a block in WIPI2 puncta formation and large vacuoles (red asterisks). Quantification of WIPI2 spots, mean +/- SEM, n=3.


D ) Wild type ( WT ) , 6SA or 6SE VPS15 - HA , or empty vector ( EV ) , were expressed in VPS15 KO or HEK293A cells . Cells were starved for 1 hr and the distributions of WIPI2 ( green ) , HA ( red ) and LC3 ( blue ) were assessed , mean + / - SEM , n = 4 .

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

D) Wild type (WT), 6SA or 6SE VPS15-HA, or empty vector (EV), were expressed in VPS15 KO or HEK293A cells. Cells were starved for 1 hr and the distributions of WIPI2 (green), HA (red) and LC3 (blue) were assessed, mean +/- SEM, n=4.


D ) VPS34 CI components expressed with Ulk1 1 - 427 WT or KI as indicated . After starvation for 1 hr in the presence or absence of MRT68921 ( ULK inhibitor ) , cells were lysed and VPS34 CI coimmunoprecipitated via ATG14 - ZZ . VPS15 S861 phosphorylation was increased in the presence of active Ulk1 . VPS15 pS861 / total VPS15 was quantified , mean + / - SEM , n = 3 .

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

D) VPS34 CI components expressed with Ulk1 1-427 WT or KI as indicated. After starvation for 1 hr in the presence or absence of MRT68921 (ULK inhibitor), cells were lysed and VPS34 CI coimmunoprecipitated via ATG14-ZZ. VPS15 S861 phosphorylation was increased in the presence of active Ulk1. VPS15 pS861/total VPS15 was quantified, mean +/- SEM, n=3.


E ) HEK293A were transfected with VPS34 CII components and Ulk1 1 - 427 WT or KI and treated as indicated before VPS34 CII co - immunopurification via UVRAG - ZZ . Samples were analysed by Western blot as in D , revealing that Ulk1 can similarly phosphorylate VPS15 at S861 when incorporated into CII . Quantifications show mean of 4 independent experiments + / - SEM , * * * p < 0 . 001 [ one - tailed ANOVA ] .

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

E) HEK293A were transfected with VPS34 CII components and Ulk1 1-427 WT or KI and treated as indicated before VPS34 CII co-immunopurification via UVRAG-ZZ. Samples were analysed by Western blot as in D, revealing that Ulk1 can similarly phosphorylate VPS15 at S861 when incorporated into CII. Quantifications show mean of 4 independent experiments +/-SEM, ***p<0.001 [one-tailed ANOVA].


A ) HEK293A , VPS15 KO control and stably rescued VPS15 KOs ( sgA - B3 ) were starved for 1 hr before WIPI2 ( green ) , LC3 ( red ) and DNA ( Hoechst , blue ) were visualised . Quantification of WIPI2 puncta number per cell , mean + / - SEM , n = 5 .

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

A) HEK293A, VPS15 KO control and stably rescued VPS15 KOs (sgA-B3) were starved for 1 hr before WIPI2 (green), LC3 (red) and DNA (Hoechst, blue) were visualised. Quantification of WIPI2 puncta number per cell, mean +/-SEM, n=5.


B ) Stably rescued VPS15 KO ( sgA - B3 ) , HEK293A and VPS15 KO control cells were starved for 1 hr with ( SB ) or without ( St ) 100nM Bafilomycin A1 , or cultured in full media ( F ) . Quantification shows LC3II / Actin , mean + / - SEM , n = 5 .

O O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O 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) Stably rescued VPS15 KO (sgA-B3), HEK293A and VPS15 KO control cells were starved for 1 hr with (SB) or without (St) 100nM Bafilomycin A1, or cultured in full media (F). Quantification shows LC3II/Actin, mean +/-SEM, n=5.


A ) HEK293A were treated with VPS34 - IN1 ( 1μM ) alone or in the presence of MRT68921 ( 1μM ) for the indicated time . Untreated VPS15 KOs were included as a positive control and lysates analysed by Western blot . Identical samples were loaded in parallel for VPS34 , ATG13 , PRKAB2 analysis , indicated by dashed line .

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

A) HEK293A were treated with VPS34-IN1 (1μM) alone or in the presence of MRT68921 (1μM) for the indicated time. Untreated VPS15 KOs were included as a positive control and lysates analysed by Western blot. Identical samples were loaded in parallel for VPS34, ATG13, PRKAB2 analysis, indicated by dashed line.


E ) HEK293A and VPS15 KO cells were fed , or starved for 2 hrs before fixation , and labelling with ULK1 ( green ) , ATG9A ( red ) and GM130 ( blue ) . Dashed boxes show magnified regions of interest .

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

E) HEK293A and VPS15 KO cells were fed, or starved for 2 hrs before fixation, and labelling with ULK1 (green), ATG9A (red) and GM130 (blue). Dashed boxes show magnified regions of interest.


B ) HEK293A were transfected with siRNAs targeting ATG14 , UVRAG or p62 , or with non - targeting siRNA ( RISC - free , RF ) and cells were treated with VPS34 - IN1 ( IN1 ; 1μM ) for 0 , 4 or 18 hrs as indicated . Quantification of ATG13 pS318 / Total ATG13 , mean + / - SEM , n = 3 . Identical samples were loaded onto 2 separate SDS PAGE gels for Western blot analysis , indicated by dashed line .

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

B) HEK293A were transfected with siRNAs targeting ATG14, UVRAG or p62, or with non-targeting siRNA (RISC-free, RF) and cells were treated with VPS34-IN1 (IN1; 1μM) for 0, 4 or 18 hrs as indicated. Quantification of ATG13 pS318/Total ATG13, mean +/- SEM, n=3. Identical samples were loaded onto 2 separate SDS PAGE gels for Western blot analysis, indicated by dashed line.


C ) Cells transfected as in B were treated with IN1 for 18 hours as indicated , fixed and labelled for ubiquitin ( green ) , FIP200 ( red ) and p62 ( blue ) . FIP200 - positive bodies were quantified from 4 independent experiments with mean + / - SEM plotted . Cells transfected with ATG14 siRNA generated significantly more FIP200 bodies per cell than all other conditions tested .

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

C) Cells transfected as in B were treated with IN1 for 18 hours as indicated, fixed and labelled for ubiquitin (green), FIP200 (red) and p62 (blue). FIP200-positive bodies were quantified from 4 independent experiments with mean +/- SEM plotted. Cells transfected with ATG14 siRNA generated significantly more FIP200 bodies per cell than all other conditions tested.


Validation of upregulated lncRNAs ( ΔCT ) in PBMC - derived M2 - like macrophages ( stimulated with IL - 4 stimulated for 24 hours , M2 ) . n = 6 biological replicates , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 compared with M1 - like macrophages ( LPS / IFNɣ - stimulated for 24 hours , M1 ) .

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

Validation of upregulated lncRNAs (ΔCT) in PBMC-derived M2-like macrophages (stimulated with IL-4 stimulated for 24 hours, M2). n = 6 biological replicates, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 compared with M1-like macrophages (LPS/IFNɣ-stimulated for 24 hours, M1).


Representative images of fluorescence in - situ hybridization of ADPGK - AS1 ( magenta ) and nuclei ( blue ) in IL - 4 stimulated M2 - like macrophages . β - actin served as the cytoplasmic control and MALAT1 as the nuclear control . Scale bar = 10 µm .

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

Representative images of fluorescence in-situ hybridization of ADPGK-AS1 (magenta) and nuclei (blue) in IL-4 stimulated M2-like macrophages. β-actin served as the cytoplasmic control and MALAT1 as the nuclear control. Scale bar = 10 µm.


RNA expression analysis of ADPGK - AS1 , 47S ribosomal RNA ( nuclear control ) , and β - Tubulin ( cytoplasmic control ) in IL - 4 stimulated M2 - like macrophages after subcellular fractionation . n = 3 independent experiments . Data represent the percentage of total detected RNA .

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

RNA expression analysis of ADPGK-AS1, 47S ribosomal RNA (nuclear control), and β-Tubulin (cytoplasmic control) in IL-4 stimulated M2-like macrophages after subcellular fractionation. n = 3 independent experiments. Data represent the percentage of total detected RNA.


Subcellular fractionation of IL - 4 - stimulated M2 - like macrophages and isolation and analysis of ADPGK - AS1 abundance in the cytoplasmic and mitochondrial fractions The mitochondrial encoded RNA mtATP6 served as the mitochondrial control and β - tubulin as the cytoplasmic control . n = 3 independent experiments , Data represent the percentage of total detected RNA .

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

Subcellular fractionation of IL-4-stimulated M2-like macrophages and isolation and analysis of ADPGK-AS1 abundance in the cytoplasmic and mitochondrial fractions The mitochondrial encoded RNA mtATP6 served as the mitochondrial control and β-tubulin as the cytoplasmic control. n = 3 independent experiments, Data represent the percentage of total detected RNA.


( A ) Validation of the ADPGK - AS1 - interacting protein MRPL35 by RNA immunoprecipitation ( RIP ) with followed by qPCR for ADPGK - AS1 . n = 4 independent experiments , * * * p ≤ 0 . 001 compared with IgG control .

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

(A) Validation of the ADPGK-AS1-interacting protein MRPL35 by RNA immunoprecipitation (RIP) with followed by qPCR for ADPGK-AS1. n = 4 independent experiments, ***p ≤ 0.001 compared with IgG control.


( B , C ) Co - staining for ADPGK - AS1 ( magenta ) and MRPL35 or MRPL15 ( yellow ) with respective fluorescence intensity across co - stained macrophages ( see white line ) for ADPGK - AS1 and MRPL35 or MRPL15 . Scale bar = 10 µm ( left ) , 5 µm ( right ) .

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

(B, C) Co-staining for ADPGK-AS1 (magenta) and MRPL35 or MRPL15 (yellow) with respective fluorescence intensity across co-stained macrophages (see white line) for ADPGK-AS1 and MRPL35 or MRPL15. Scale bar = 10 µm (left), 5 µm (right).


( E ) Number of M1 - like or M2 - like macrophages based on TMA data for lung cancer patients with either high ( above median ) or low ( below median ) expression of MRPL35 .

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

(E) Number of M1-like or M2-like macrophages based on TMA data for lung cancer patients with either high (above median) or low (below median) expression of MRPL35.


( F , G ) Kaplan - Meier curve estimate of disease - free survival ( DFS ) or overall survival ( OS ) of patients with high ( above median ) or low ( below median ) expression of MRPL35 in M2 - like macrophages . n = 200 biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, G) Kaplan-Meier curve estimate of disease-free survival (DFS) or overall survival (OS) of patients with high (above median) or low (below median) expression of MRPL35 in M2-like macrophages. n = 200 biological replicates.


( C , Representative images and quantification of mitochondria by MitoTracker ( red ) in THP1 control and ADPGK - AS1 OE cells . Nuclei were stained with DAPI ( blue ) . Scale bar = 200 µm ( left ) , 100 µm ( right , magnification ) .

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

(C, Representative images and quantification of mitochondria by MitoTracker (red) in THP1 control and ADPGK-AS1 OE cells. Nuclei were stained with DAPI (blue). Scale bar = 200 µm (left), 100 µm (right, magnification).


D ) Representative images and quantification of mitochondria by TOM20 ( green ) in THP1 control and ADPGK - AS1 OE cells . Nuclei were stained with DAPI ( blue ) . Scale bar = 200 µm ( left ) , 100 µm ( right , magnification ) .

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

D) Representative images and quantification of mitochondria by TOM20 (green) in THP1 control and ADPGK-AS1 OE cells. Nuclei were stained with DAPI (blue). Scale bar = 200 µm (left), 100 µm (right, magnification).


( E ) Representative immunoblot for mitochondrial protein expression ( TOM20 , αb - crystallin , PCG - 1 , MRPL35 , cytochrome C ) in THP1 control and ADPGK - AS1 OE cells . n = 4 independent experiments .

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

(E) Representative immunoblot for mitochondrial protein expression (TOM20, αb-crystallin, PCG-1, MRPL35, cytochrome C) in THP1 control and ADPGK-AS1 OE cells. n = 4 independent experiments.


( G ) Gene expression analysis ( fold change ) of mitochondrially encoded genes ( ND1 , ND2 , ND4 , ATP6 , ATP8 , CO1 , CO2 , CyB ) after isolation of mtDNA from THP1 control and ADPGK - AS1 OE cells . n = 3 independent experiments . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 compared with control .

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

(G) Gene expression analysis (fold change) of mitochondrially encoded genes (ND1, ND2, ND4, ATP6, ATP8, CO1, CO2, CyB) after isolation of mtDNA from THP1 control and ADPGK-AS1 OE cells. n = 3 independent experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 compared with control.


I ) full oxygen consumption plot ( n = 8 ) by the seahorse assay in THP1 control and ADPGK - AS1 OE cells . * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 compared with control .

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

I) full oxygen consumption plot (n = 8) by the seahorse assay in THP1 control and ADPGK-AS1 OE cells. **p ≤ 0.01, ***p ≤ 0.001 compared with control.


( K ) Representative confocal fluorescence images and quantification of mitochondrial shape with the mitochondrial marker TOM20 ( yellow ) in THP1 control and ADPGK - AS1 OE cells . Nuclei were stained with DAPI ( blue ) . n = 35 cells , Scale bar = 10µm ( left ) , 5µm ( magnification , right ) .

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

(K) Representative confocal fluorescence images and quantification of mitochondrial shape with the mitochondrial marker TOM20 (yellow) in THP1 control and ADPGK-AS1 OE cells. Nuclei were stained with DAPI (blue). n = 35 cells, Scale bar=10µm (left), 5µm (magnification, right).


( L ) Transmission electron microscopy images of mitochondrial shapes in THP1 control and ADPGK - AS1 OE cells . n = 4 cells , Scale bar = 5 µm ( left ) , 1 µm ( right , magnification ) .

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

(L) Transmission electron microscopy images of mitochondrial shapes in THP1 control and ADPGK-AS1 OE cells. n = 4 cells, Scale bar = 5 µm (left), 1 µm (right, magnification).


( B ) Expression analysis of ADPGK - AS1 and MRPL35 ( fold change ) in THP1 control and ADPGK - AS1 OE macrophages transfected with a siRNA against ADPGK - AS1 , MRPL35 or negative control . n = 4 independent experiments . * * * p ≤ 0 . 001 compared with ADPGK - AS1 OE siRNA control .

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

(B) Expression analysis of ADPGK-AS1 and MRPL35 (fold change) in THP1 control and ADPGK-AS1 OE macrophages transfected with a siRNA against ADPGK-AS1, MRPL35 or negative control. n = 4 independent experiments. ***p ≤ 0.001 compared with ADPGK-AS1 OE siRNA control.


( C ) Representative images of JC - 1 monomers ( green ) and aggregates ( red ) in THP1 control and ADPGK - AS1 overexpressing macrophages transfected with a siRNA against ADPGK - AS1 , MRPL35 , or negative control . Scale bar = 100 µm .

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

(C) Representative images of JC-1 monomers (green) and aggregates (red) in THP1 control and ADPGK-AS1 overexpressing macrophages transfected with a siRNA against ADPGK-AS1, MRPL35, or negative control. Scale bar = 100 µm.


( E ) Representative images ( left panel ) and quantification ( right panel ) of ROS accumulation in THP1 control and ADPGK - AS1 OE cells transfected with a siRNA against ADPGK - AS1 , MRPL35 , or negative control . n = 7 . * * * p ≤ 0 . 001 compared to control .

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

(E) Representative images (left panel) and quantification (right panel) of ROS accumulation in THP1 control and ADPGK-AS1 OE cells transfected with a siRNA against ADPGK-AS1, MRPL35, or negative control. n = 7. ***p ≤ 0.001 compared to control.


( F ) Spare capacity ( oxygen consumption rate ) measured with the seahorse assay in THP1 control and ADPGK - AS1 OE cells transfected with a siRNA against ADPGK - AS1 , MRPL35 , or negative control . n = 3 independent experiments . * * * p ≤ 0 . 001 compared with control , § p ≤ 0 . 05 , § § § p ≤ 0 . 001 compared with ADPGK - AS1 OE siRNA negative control .

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

(F) Spare capacity (oxygen consumption rate) measured with the seahorse assay in THP1 control and ADPGK-AS1 OE cells transfected with a siRNA against ADPGK-AS1, MRPL35, or negative control. n = 3 independent experiments. ***p ≤ 0.001 compared with control, §p ≤ 0.05, §§§p ≤ 0.001 compared with ADPGK-AS1 OE siRNA negative control.


( B ) mRNA expression analysis ( ΔCT ) of macrophage markers ( TNFα , IL - 8 , CD206 , CSF - 1R ) in co - cultured PBMC - derived macrophages with tumor cells or macrophage control ( M0 ) . n = 4 biological replicates . * p ≤ 0 . 05 , * * p ≤ 0 . 01 compared with M0 .

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

(B) mRNA expression analysis (ΔCT) of macrophage markers (TNFα, IL-8, CD206, CSF-1R) in co-cultured PBMC-derived macrophages with tumor cells or macrophage control (M0). n = 4 biological replicates. *p ≤ 0.05, **p ≤ 0.01 compared with M0.


( C ) Expression of ADPGK - AS1 lncRNA in macrophages co - cultured with tumor cells ( A549 ) or macrophage controls ( M0 ) . n = 4 biological replicates . * p ≤ 0 . 05 , compared with M0 .

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

(C) Expression of ADPGK-AS1 lncRNA in macrophages co-cultured with tumor cells (A549) or macrophage controls (M0). n = 4 biological replicates. *p ≤ 0.05, compared with M0.


( D ) Mass spectrometry analysis of TCA cycle metabolites ( fumarate , malate , citrate , succinate ) in macrophages co - cultured with tumor cells ( A549 ) and macrophage control ( M0 ) . n = 4 biological replicates . * p ≤ 0 . 05 , * * p ≤ 0 . 01 compared with M0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Mass spectrometry analysis of TCA cycle metabolites (fumarate, malate, citrate, succinate) in macrophages co-cultured with tumor cells (A549) and macrophage control (M0). n = 4 biological replicates. *p ≤ 0.05, **p ≤ 0.01 compared with M0.


( G ) Expression of IDH mRNA in A549 cells transfected with siRNA specific for IDH ( IDH KD ) or negative control . n = 5 independent experiments . * * * p ≤ 0 . 001 compared with A549 control .

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

(G) Expression of IDH mRNA in A549 cells transfected with siRNA specific for IDH (IDH KD) or negative control. n = 5 independent experiments. ***p ≤ 0.001 compared with A549 control.


( H ) RNA expression analysis of ADPGK - AS1 in THP1 macrophages stimulated to have an M1 - like or M2 - like phenotype , untreated ( M0 ) or treated with conditioned medium ( CM ) of A549 cells transfected with a siRNA specific for IDH ( IDH KD ) or negative control . n = 5 biological replicates . * * * p ≤ 0 . 001 , compared with tumor cell control CM .

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

(H) RNA expression analysis of ADPGK-AS1 in THP1 macrophages stimulated to have an M1-like or M2-like phenotype, untreated (M0) or treated with conditioned medium (CM) of A549 cells transfected with a siRNA specific for IDH (IDH KD) or negative control. n = 5 biological replicates. ***p ≤ 0.001, compared with tumor cell control CM.


( A , B ) mRNA expression analysis ( ΔCT ) of M1 ( TNFα , IL - 8 , CXCL10 ) and M2 ( CSF - 1R , CD206 , IL - 10 , ALOX15 , TGFβ , IL - 1Ra ) macrophage marker genes in THP1 control and ADPGK - AS1 OE cells . n = 4 independent experiments . * p ≤ 0 . 05 , * * * p ≤ 0 . 001 compared with control .

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

(A, B) mRNA expression analysis (ΔCT) of M1 (TNFα, IL-8, CXCL10) and M2 (CSF-1R, CD206, IL-10, ALOX15, TGFβ, IL-1Ra) macrophage marker genes in THP1 control and ADPGK-AS1 OE cells. n = 4 independent experiments. *p ≤ 0.05, ***p ≤ 0.001 compared with control.


( E ) Migration ( right panel ) of A549 cells treated with CM from THP1 control or ADPGK - AS1 OE macrophages . Representative membrane images are shown in the left panel . n = 5 independent experiments . * * * p ≤ 0 . 001 compared with control . Scale bar = 400 µm .

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

(E) Migration (right panel) of A549 cells treated with CM from THP1 control or ADPGK-AS1 OE macrophages. Representative membrane images are shown in the left panel. n = 5 independent experiments. ***p ≤ 0.001 compared with control. Scale bar = 400 µm.


( G - H ) mRNA expression analysis ( ΔCT ) of M1 ( TNFα , IL - 8 , CD80 , CCR7 ) and M2 ( CSF - 1R , CD206 , IL - 10 , TGFβ ) macrophage marker genes in primary M1 - like ( M1 ) and M2 - like ( M2 ) macrophages transfected with antisense LNA GapmeRs specific for ADPGK - AS1 or negative control . n = 5 biological replicates , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , compared to M2 control .

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

(G-H) mRNA expression analysis (ΔCT) of M1 (TNFα, IL-8, CD80, CCR7) and M2 (CSF-1R, CD206, IL-10, TGFβ) macrophage marker genes in primary M1-like (M1) and M2-like (M2) macrophages transfected with antisense LNA GapmeRs specific for ADPGK-AS1 or negative control. n = 5 biological replicates, *p ≤ 0.05, **p ≤ 0.01, compared to M2 control.


( K ) Migration ( right panel ) of tumor cells ( A549 ) treated with CM from PBMC - derived M1 - like ( M1 ) or M2 - like ( M2 ) macrophages transfected with antisense LNA GapmeRs specific for ADPGK - AS1 or negative control . Representative membrane images are shown in the left panel . n = 5 biological replicates . * p ≤ 0 . 05 , compared with M2 control . Scale bar = 400 µm .

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

(K) Migration (right panel) of tumor cells (A549) treated with CM from PBMC-derived M1-like (M1) or M2-like (M2) macrophages transfected with antisense LNA GapmeRs specific for ADPGK-AS1 or negative control. Representative membrane images are shown in the left panel. n = 5 biological replicates. *p ≤ 0.05, compared with M2 control. Scale bar = 400 µm.


( C ) Representative brightfield images of tumors ( scale bar = 2mm ) and fluorescence images ( Ki67 , cleaved caspase 3 , scale bar = 100 µm ; cleaved caspase 3 ( green ) and cytokeratin 18 ( magenta ) , scale bar = 50 µm ) and LDH enzyme activity ( scale bar = 25 µm ) after co - injection with M1 - like ( M1 ) or M2 - like ( M2 ) macrophages transfected with antisense LNA GapmeRs specific for ADPGK - AS1 or negative control ( ctrl ) together with A549 tumor cells or A549 cells alone as a control .

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

(C) Representative brightfield images of tumors (scale bar = 2mm) and fluorescence images (Ki67, cleaved caspase 3, scale bar = 100 µm; cleaved caspase 3 (green) and cytokeratin 18 (magenta), scale bar = 50 µm) and LDH enzyme activity (scale bar = 25 µm) after co-injection with M1-like (M1) or M2-like (M2) macrophages transfected with antisense LNA GapmeRs specific for ADPGK-AS1 or negative control (ctrl) together with A549 tumor cells or A549 cells alone as a control.


( D ) Quantification of Ki67 + cells and cleaved caspase 3 + of co - injected tumors . n = 5 biological replicates . * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 compared with M2 control .

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

(D) Quantification of Ki67+ cells and cleaved caspase 3+ of co-injected tumors. n = 5 biological replicates. **p ≤ 0.01, ***p ≤ 0.001 compared with M2 control.


( G ) Representative immunoblot of the mitochondrial - related proteins MRPL35 , Opa1 , Mfn1 , pDRP1 , and DRP1 about β - actin in isolated tumor tissue . n = 3 biological replicates .

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

(G) Representative immunoblot of the mitochondrial-related proteins MRPL35, Opa1, Mfn1, pDRP1, and DRP1 about β-actin in isolated tumor tissue. n = 3 biological replicates.


( C , D ) Representative fluorescence images of PCLS with proliferative ( EdU + ) and apoptotic ( TUNEL + ) cells ( red ) and nuclear dye ( DAPI + , blue ) in the tumor area ( cytokeratin + , green ) . Scale bar = 250 µm ( c , upper panel ) , 150 µm ( d , upper panel ) , and 50 µm ( c and d lower panel , magnification ) .

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

(C, D) Representative fluorescence images of PCLS with proliferative (EdU+) and apoptotic (TUNEL+) cells (red) and nuclear dye (DAPI+, blue) in the tumor area (cytokeratin+, green). Scale bar = 250 µm (c, upper panel), 150 µm (d, upper panel), and 50 µm (c and d lower panel, magnification).


( F ) RNA expression analysis of ADPGK - AS1 and MRPL35 in tumor PCLS treated with antisense LNA GapmeRs specific for ADPGK - AS1 or a negative control or seeded with THP1 control or ADPGK - AS1 OE cells . n = 5 independent experiments . * p ≤ 0 . 05 , * * * p ≤ 0 . 001 compared with control GapmeR , § p ≤ 0 . 05 , § § § p ≤ 0 . 001 compared with EV control .

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

(F) RNA expression analysis of ADPGK-AS1 and MRPL35 in tumor PCLS treated with antisense LNA GapmeRs specific for ADPGK-AS1 or a negative control or seeded with THP1 control or ADPGK-AS1 OE cells. n = 5 independent experiments. *p ≤ 0.05, ***p ≤ 0.001 compared with control GapmeR, §p ≤ 0.05, §§§p ≤ 0.001 compared with EV control.


( G ) Multispectral flow cytometry analysis of M1 - like and M2 - like macrophage populations in tumor PCLS treated with antisense LNA GapmeRs specific for ADPGK - AS1 or a negative control or seeded with THP1 control or ADPGK - AS1 OE cells . n = 6 biological replicates . * p ≤ 0 . 05 compared with Gapmer negative control , § p ≤ 0 . 05 compared with EV control .

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

(G) Multispectral flow cytometry analysis of M1-like and M2-like macrophage populations in tumor PCLS treated with antisense LNA GapmeRs specific for ADPGK-AS1 or a negative control or seeded with THP1 control or ADPGK-AS1 OE cells. n = 6 biological replicates. *p ≤ 0.05 compared with Gapmer negative control, §p ≤ 0.05 compared with EV control.


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

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

O O O O O O O O O O O O O O O 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) 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 cells . Data information : PA : palmitic acid ; 17 - ODYA : 17 - octadecanoic acid .

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

(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 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 .

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

(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 analysis of palmitoylated peptides of cGAS .

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

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


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

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

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


( 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

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

O O O O O O O O O O O O O O O O O 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 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 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 :

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

(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 assay of HEK293T cells transfected with the indicated plasmids for 24 h . HAM : hydroxylamine .

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

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


( 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 (

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

O O O O O O O O O O O O O O O O O O O O O O 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 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 ) 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

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

(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 ) 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

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

(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 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 ) .

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

(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 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 .

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

(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 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 .

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 .

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

G) 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 O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O 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 the Cα atoms of the WT and palmitoylated cGAS proteins .

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

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


( 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 .

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

(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 ) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 (

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) 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 .

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

(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 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 ) .

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

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

(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 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

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

(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 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 ;

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

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

O O O O O O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR 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 efficiency of ZDHHC18 is shown by western blotting (F) results. Data information: NC: negative control.


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 .

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

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 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 .

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


( J ) L929 cells stably transfected with control shRNA or ZDHHC18 shRNA were transfected with HT - DNA ( 2 μg / mL ) for the indicated times before immunoblotting analysis with the indicated antibodies . Data information : HT - DNA : herring testis DNA ; NC : negative control .

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

(J) L929 cells stably transfected with control shRNA or ZDHHC18 shRNA were transfected with HT-DNA (2 μg/mL) for the indicated times before immunoblotting analysis with the indicated antibodies. Data information: HT-DNA: herring testis DNA; NC: negative control.


( A ) Survival analysis of Zdhhc18 + / + and Zdhhc18 - / - mice ( n = 8 each , 6 - 8 weeks old ) intranasally injected with VACV ( 5 × 107 PFU per mouse ) and monitored daily for survival for 13 days using Kaplan - Meier curves and a log - rank ( Mantel - Cox ) test . * * * , P ˂ 0 . 001 .

O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Survival analysis of Zdhhc18+/+ and Zdhhc18-/- mice (n = 8 each, 6-8 weeks old) intranasally injected with VACV (5 × 107 PFU per mouse) and monitored daily for survival for 13 days using Kaplan-Meier curves and a log-rank (Mantel-Cox) test. ***, P˂0.001.


( B ) Representative photomicrographs of H & E - stained liver and kidney tissue sections from mice ( Zdhhc18 + / + or Zdhhc18 - / - ) intravenously injected with HSV - 1 - FS ( 5 × 107 PFU per mouse ) or not after 2 days . Scale bars : 100 μm ( liver ) , 200 μm ( kidney ) . Data information : PFU : plaque - forming units ; H & E : hematoxylin - eosin . Data are representative of at least two independent experiments .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 photomicrographs of H&E-stained liver and kidney tissue sections from mice (Zdhhc18+/+ or Zdhhc18-/-) intravenously injected with HSV-1-FS (5 × 107 PFU per mouse) or not after 2 days. Scale bars: 100 μm (liver), 200 μm (kidney). Data information: PFU: plaque-forming units; H&E: hematoxylin-eosin. Data are representative of at least two independent experiments.


( C BMDMs ( Zdhhc18 + / + or Zdhhc18 - / - ) were infected with HSV - 1 ( MOI = 2 ) for 8 h , and viral titers in the supernatants were quantified by plaque assay . Data information : Data are representative of at least two independent experiments .

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

(C BMDMs (Zdhhc18+/+ or Zdhhc18-/-) were infected with HSV-1 (MOI=2) for 8 h, and viral titers in the supernatants were quantified by plaque assay. Data information: Data are representative of at least two independent experiments.


D ) BMDMs ( Zdhhc18 + / + or Zdhhc18 - / - ) were infected with HSV - 1 ( MOI = 2 ) for 8 h , and viral titers in the supernatants were quantified by plaque assay . Data information : Data are representative of at least two independent experiments . Mean ± SEM from triplicates of technical replicates , unpaired t test ; Ns : no significance ; * , P ˂ 0 . 05 ; * * , P ˂ 0 . 01 ; * * * * , P ˂ 0 . 0001 (

O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) BMDMs (Zdhhc18+/+ or Zdhhc18-/-) were infected with HSV-1 (MOI=2) for 8 h, and viral titers in the supernatants were quantified by plaque assay. Data information: Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; Ns: no significance; *, P˂0.05; **, P˂0.01; ****, P˂0.0001 (D


( E Frequency of GFP + cells among infected cells ( E ) . Data information : Data are representative of at least two independent experiments . Mean ± SEM from triplicates of technical replicates , unpaired t test ; Ns : no significance ; * , P ˂ 0 . 05 ; * * , P ˂ 0 . 01 ; * * * * , P ˂ 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Frequency of GFP+ cells among infected cells (E). Data information: Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; Ns: no significance; *, P˂0.05; **, P˂0.01; ****, P˂0.0001


F ) Microscopy of BMDMs ( Zdhhc18 + / + or Zdhhc18 - / - ) infected with GFP - HSV - 1 ( MOI = 1 ) for 10 h . Scale bar : 200 μm . Data information : Data are representative of at least two independent experiments .

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

F) Microscopy of BMDMs (Zdhhc18+/+ or Zdhhc18-/-) infected with GFP-HSV-1 (MOI=1) for 10 h. Scale bar: 200 μm. Data information: Data are representative of at least two independent experiments.


( G ) MEFs ( Zdhhc18 + / + or Zdhhc18 - / - ) were infected with VACV ( 1 : 200 ) for the indicated times before immunoblot analysis .

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

(G) MEFs (Zdhhc18+/+ or Zdhhc18-/-) were infected with VACV (1:200) for the indicated times before immunoblot analysis.


( H and I ) MEFs ( Zdhhc18 + / + or Zdhhc18 - / - ) were infected with VACV ( 1 : 200 ) for the indicated times before RT - qPCR analysis of Cxcl10 ( H ) and Ifnb1 ( I ) expression . Data information : Data are representative of at least two independent experiments . Mean ± SEM from triplicates of technical replicates , unpaired t test ; Ns : no significance ; * , P ˂ 0 . 05 ; * * , P ˂ 0 . 01 ; * * * * , P ˂ 0 .

O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and I) MEFs (Zdhhc18+/+ or Zdhhc18-/-) were infected with VACV (1:200) for the indicated times before RT-qPCR analysis of Cxcl10 (H) and Ifnb1 (I) expression. Data information: Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; Ns: no significance; *, P˂0.05; **, P˂0.01; ****, P˂0.0001


( J ) PBMCs from healthy people were transfected with scrambled siRNA or ZDHHC18 siRNA ( cocktail ) for 48 h and then infected with VACV ( 1 : 200 ) for the indicated times before RT - qPCR analysis of IFNB1 expression . Data information : Data are representative of at least two independent experiments . Mean ± SEM from triplicates of technical replicates , unpaired t test ; Ns : no significance ; * , P ˂ 0 . 05 ; * * , P ˂ 0 . 01 ; * * * * , P ˂ 0 .

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

(J) PBMCs from healthy people were transfected with scrambled siRNA or ZDHHC18 siRNA (cocktail) for 48 h and then infected with VACV (1:200) for the indicated times before RT-qPCR analysis of IFNB1 expression. Data information: Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; Ns: no significance; *, P˂0.05; **, P˂0.01; ****, P˂0.0001


Western blots showing changing protein levels on the path to senescence in IMR90 and HUVECs . Passage 6 cells represent the proliferating state , and passages 21 and 34 the senescent state for HUVECs and IMR90 , respectively .

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

Western blots showing changing protein levels on the path to senescence in IMR90 and HUVECs. Passage 6 cells represent the proliferating state, and passages 21 and 34 the senescent state for HUVECs and IMR90, respectively.


Super - resolution ( gSTED ) imaging of HMGB1 distribution in proliferating HUVEC nuclei counterstained with DAPI . Bar : 2μm .

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

Super-resolution (gSTED) imaging of HMGB1 distribution in proliferating HUVEC nuclei counterstained with DAPI. Bar: 2μm.


Representative immunofluorescence images of IMR90 and HUVECs ( left ) showing reduced HMGB1 levels in senescent nuclei ; bean plots quantify this reduction ( right ; N is the number of cells analyzed per each condition / cell type ) . Bars : 5 μm . * : P < 0 . 01 ; Wilcoxon - Mann - Whitney test .

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

Representative immunofluorescence images of IMR90 and HUVECs (left) showing reduced HMGB1 levels in senescent nuclei; bean plots quantify this reduction (right; N is the number of cells analyzed per each condition/cell type). Bars: 5 μm. *: P<0.01; Wilcoxon-Mann-Whitney test.


Genome browser view of HMGB1 ChIP - seq ( normalized to input ; mean from two replicates ) from proliferating HUVEC ( red ) or proliferating / senescent IMR90 ( grey / green ) in the TMEM92 locus .

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

Genome browser view of HMGB1 ChIP-seq (normalized to input; mean from two replicates) from proliferating HUVEC (red) or proliferating/senescent IMR90 (grey/green) in the TMEM92 locus.


Heatmap showing protein ( SASP , from http : / / www . saspatlas . com / ; log fold ratio ) and gene expression levels ( log2FC ) of IMR90 SASP - related genes embedded in high - HMGB1 ( red ) TADs like those in panel C . * * : genes bound by HMGB1 in ChIP - seq data are more than expected by chance ; P > 0 . 001 , hypergeometric test .

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

Heatmap showing protein (SASP, from http://www.saspatlas.com/; log fold ratio)and gene expression levels (log2FC) of IMR90 SASP-related genes embedded in high-HMGB1 (red) TADs like those in panel C. **: genes bound by HMGB1 in ChIP-seq data are more than expected by chance; P>0.001, hypergeometric test.


Immunofluorescence ( top ) , western blot ( middle ) and RT - qPCR analyses ( bottom ; mean fold - change ± S . D . from two biological replicates ) confirm HMGB1 knockdown in IMR90 .

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

Immunofluorescence (top), western blot (middle) and RT-qPCR analyses (bottom; mean fold-change ±S.D. from two biological replicates) confirm HMGB1 knockdown in IMR90.


Representative images of IMR90 overexpressing HMGB1 - GFP , immunostained for p21 and CTCF , and counterstained with DAPI . IMR90 transfected with empty vectors provide a control . Bar : 5 μm .

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

Representative images of IMR90 overexpressing HMGB1-GFP, immunostained for p21 and CTCF, and counterstained with DAPI. IMR90 transfected with empty vectors provide a control. Bar: 5 μm.


Genome browser views showing HMGB1 sCLIP data ( black ) along the ASH1L , CCNL2 and TET2 loci ; input tracks ( grey ) provide background levels . * : significantly - enriched peaks . The consensus motif for HMGB1 binding on RNA is also shown ( bottom right ) .

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

Genome browser views showing HMGB1 sCLIP data (black) along the ASH1L, CCNL2 and TET2 loci; input tracks (grey) provide background levels. *: significantly-enriched peaks. The consensus motif for HMGB1 binding on RNA is also shown (bottom right).


Bar graphs showing mean fold - change of selected mRNAs ( over ± S . D . from two biological replicates ) from ILF3 - ( purple ) , RBMX - ( white ) or PNN - knockdown experiments ( blue ) in proliferating IMR90 . * : significantly different to siRNA controls ; P < 0 . 01 , unpaired two - tailed Student ' s t - test . Dashed line indicates no change in expression .

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

Bar graphs showing mean fold-change of selected mRNAs (over ±S.D. from two biological replicates) from ILF3- (purple), RBMX- (white) or PNN-knockdown experiments (blue) in proliferating IMR90. *: significantly different to siRNA controls; P<0.01, unpaired two-tailed Student's t-test. Dashed line indicates no change in expression.


B . Western blot analysis of RAB6A / A ' and RAB6B protein levels in WT , Emx1 - Cre ; RAB6loxP / loxP ( RAB6A KO ) , RAB6B - / - ( RAB6B KO ) and Emx1 - Cre ; RAB6AloxP / loxP ; RAB6B - / - ( RAB6A / B dKO ) E15 . 5 cortical extracts .

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

B. Western blot analysis of RAB6A/A' and RAB6B protein levels in WT, Emx1-Cre; RAB6loxP/loxP (RAB6A KO), RAB6B-/- (RAB6B KO) and Emx1-Cre; RAB6AloxP/loxP; RAB6B-/- (RAB6A/B dKO) E15.5 cortical extracts.


A . PAX6 staining in WT and RAB6A / B dKO E15 . 5 brains . Scale bar = 50µm . B . Percentage of PAX6 + cells located in the upper half of the cortex of WT and RAB6A / B dKO E15 . 5 brains . WT : 4282 cells from N = 6 brains . RAB6A / B dKO : 1241 cells from N = 3 brains . Mann - Whitney U test . Data information : All error bars indicate SD .

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

A. PAX6 staining in WT and RAB6A/B dKO E15.5 brains. Scale bar = 50µm. B. Percentage of PAX6+ cells located in the upper half of the cortex of WT and RAB6A/B dKO E15.5 brains. WT: 4282 cells from N=6 brains. RAB6A/B dKO: 1241 cells from N=3 brains. Mann-Whitney U test. Data information: All error bars indicate SD.