Figure 2 ( 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 .

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Figure 2(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.


Figure 3 ( 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 .

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Figure 3(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.


Figure 5 ( 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 . ( D ) PUB4 directly interacts with BIK1 in vitro . Immunoblots were analyzed using the indicated antibodies . Molecular weight ( kDa ) marker bands are indicated for reference .

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Figure 5(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.(D) PUB4 directly interacts with BIK1 in vitro. Immunoblots were analyzed using the indicated antibodies. Molecular weight (kDa) marker bands are indicated for reference.


Figure 6RipAC 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 .

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Figure 6RipAC 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.


Figure 7 ( 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 .

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Figure 7(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.


Figure 8 ( 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 . ( 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 .

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Figure 8(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.(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.


Figure 2D ) 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 .

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Figure 2D) 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.


Figure 3A ) 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 .

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Figure 3A) 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.


Figure 4A ) 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 . 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 .

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Figure 4A) 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.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.


Figure 5D ) 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 ] .

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Figure 5D) 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].


Figure 6A ) 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 .

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Figure 6A) 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.


Figure 7A ) 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 .

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Figure 7A) 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.


Figure 8B ) 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 .

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Figure 8B) 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.


Figure 1Validation 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 . 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 .

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Figure 1Validation 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.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.


Figure 2 ( 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 ) . ( 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 .

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Figure 2(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).(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.


Figure 3 ( 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 ) . ( 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 . 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 ) . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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 3(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).(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.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).(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).


Figure 4 ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4(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.(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.


Figure 5 ( 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 . ( 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 . ( 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 .

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Figure 5(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.(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.(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.


Figure 6 ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 6(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.(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.


Figure 7 ( 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 . ( 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 .

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Figure 7(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.(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.


Figure 8 ( 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 . ( 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 .

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Figure 8(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.(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.


Figure 1 ( 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 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 . ( 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 . ( 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 . 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 . 001BMDMs 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 . 001BMDMs 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 B-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O B-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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 1(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 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.(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.(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˂0.001(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˂0.001BMDMs 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˂0.001BMDMs 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.


Figure 2 ( 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 . ( 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 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 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 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 ) . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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 2(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.(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 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 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 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).(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.


Figure 3 ( 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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 3(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.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.


Figure 4 ( 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 . ( 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 ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4(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.(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 (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.(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).


Figure 5 ( 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 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 ; 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 . 001L929 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 . 001L929 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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(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 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;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˂0.001L929 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˂0.001L929 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.


Figure 6 ( 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 . ( 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 ( 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 . 0001F ) 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 . ( 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 .

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Figure 6(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.(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 (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˂0.0001F) 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.(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˂0.0001


Figure 1Western 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 . 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 .

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Figure 1Western 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.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.


Figure 2Genome 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 .

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Figure 2Genome 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.


Figure 3Heatmap 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 .

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Figure 3Heatmap 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.


Figure 4Immunofluorescence ( 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 .

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Figure 4Immunofluorescence (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.


Figure 5Genome 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 .

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Figure 5Genome 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.


Figure 1B . 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 .

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Figure 1B. 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.


Figure 2A . 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 . C . Phospho - Vimentin ( p - Vim ) staining in WT and RAB6A / B dKO E15 . 5 brains . Scale bar = 50µm . D . Percentage p - VIM + cells dividing ectopically , in the upper half of the cortex of WT and RAB6A / B dKO E15 . 5 brains . WT : 1713 cells from N = 6 brains . RAB6A / B dKO : 506 cells from N = 3 brains . Mann - Whitney U test . Data information : All error bars indicate SD . G . Electroporation of RAB6AloxP / loxP ; RAB6B - / - E14 . 5 embryos with mCherry ( control ) or mCherry + CRE ( RAB6A / B dKO ) and live imaging of delamination events at E17 . 5 . H . Apical endfoot detachment and retraction events during 20 hours movies in mCherry or mCherry + CRE electroporated cells at E17 . 5 . mCherry : N = 69 cells from 5 movies . mCherry + CRE : N = 52 cells from 4 movies . Fisher ' s exact test , * p ≤ 0 . 05 . Data information : All error bars indicate SD .

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Figure 2A. 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.C. Phospho-Vimentin (p-Vim) staining in WT and RAB6A/B dKO E15.5 brains. Scale bar = 50µm. D. Percentage p-VIM+ cells dividing ectopically, in the upper half of the cortex of WT and RAB6A/B dKO E15.5 brains. WT: 1713 cells from N=6 brains. RAB6A/B dKO: 506 cells from N=3 brains. Mann-Whitney U test. Data information: All error bars indicate SD.G. Electroporation of RAB6AloxP/loxP; RAB6B-/- E14.5 embryos with mCherry (control) or mCherry + CRE (RAB6A/B dKO) and live imaging of delamination events at E17.5. H. Apical endfoot detachment and retraction events during 20 hours movies in mCherry or mCherry + CRE electroporated cells at E17.5. mCherry: N=69 cells from 5 movies. mCherry + CRE: N=52 cells from 4 movies. Fisher's exact test, *p ≤ 0.05. Data information: All error bars indicate SD.


Figure 3A . Localization of the Golgi apparatus ( ManII - GFP ) and the centrosome ( 𝛾 - tubulin ) in E15 . 5 mCherry - electroporated radial glial cell . The Golgi apparatus is localized basally , away from the centrosome . Scale bar = 5µm . B . Average distance between the apical - most part of the Golgi apparatus and the apical surface in aRG cells . N = 224 cells from 3 independent brains . E . Live imaging of GFP - RAB6A in aRG cells at E15 . 5 allows tracking of individual RAB6A + vesicles in situ , from the basal Golgi apparatus towards the apical surface . Scale bar = 5µm . Distance = 5µm , time = 30 seconds . F . RAB6A + vesicle directionality in apical processes of aRG cells over one - minute movies . G . Relative time spent by RAB6A + vesicles in apical , basal or static phases . H . Velocity of apically and basally moving RAB6A + vesicles . Data information : ( F , G , H ) N = 388 vesicles from 30 cells . Mann - Whitney U test * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 . All boxplots : whiskers indicate min and max , boxes indicate 25th and 75th percentile , and central band indicates the median . I Live imaging of GFP - RAB6A in control , dynarrestin - treated and CC1 - p150 - expressing aRG cells at E15 . 5 . Scale bars = 5µm . Distance = 5µm , time = 30 seconds . J . Number of RAB6A + vesicles in the apical process of DMSO and dynarrestin - treated mouse aRG cells . K . Relative time spent by RAB6A + vesicles in apical movement phase , in DMSO and dynarrestin - treated mouse aRG cells . L . Number of RAB6A + vesicles in the apical process of mCherry and CC1 - p150 - expressing aRG cells . M . Relative time spent by RAB6A + vesicles in apical movement phase , in mCherry and CC1 - p150 - expressing aRG cells . DMSO treatment slightly affected RAB6A dynamics , as compared to mCherry control . Data information : ( J , K , L , M ) 216 vesicles from N = 11 cells for DMSO , 145 vesicles from N = 25 cells for dynarrestin , 173 vesicles from N = 17 cells for mCherry control , 71 vesicles from N = 15 cells for CC1 - p150 . Mann - Whitney U test * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 . All boxplots : whiskers indicate min and max , boxes indicate 25th and 75th percentile , and central band indicates the median .

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Figure 3A. Localization of the Golgi apparatus (ManII-GFP) and the centrosome (𝛾-tubulin) in E15.5 mCherry-electroporated radial glial cell. The Golgi apparatus is localized basally, away from the centrosome. Scale bar = 5µm. B. Average distance between the apical-most part of the Golgi apparatus and the apical surface in aRG cells. N=224 cells from 3 independent brains.E. Live imaging of GFP-RAB6A in aRG cells at E15.5 allows tracking of individual RAB6A+ vesicles in situ, from the basal Golgi apparatus towards the apical surface. Scale bar = 5µm. Distance = 5µm, time = 30 seconds. F. RAB6A+ vesicle directionality in apical processes of aRG cells over one-minute movies. G. Relative time spent by RAB6A+ vesicles in apical, basal or static phases. H. Velocity of apically and basally moving RAB6A+ vesicles. Data information: (F, G, H) N= 388 vesicles from 30 cells. Mann-Whitney U test *p ≤ 0.05, **p ≤ 0.01, *** p ≤ 0.001. All boxplots: whiskers indicate min and max, boxes indicate 25th and 75th percentile, and central band indicates the median.I Live imaging of GFP-RAB6A in control, dynarrestin-treated and CC1-p150-expressing aRG cells at E15.5. Scale bars = 5µm. Distance = 5µm, time = 30 seconds. J. Number of RAB6A+ vesicles in the apical process of DMSO and dynarrestin-treated mouse aRG cells. K. Relative time spent by RAB6A+ vesicles in apical movement phase, in DMSO and dynarrestin-treated mouse aRG cells. L. Number of RAB6A+ vesicles in the apical process of mCherry and CC1-p150-expressing aRG cells. M. Relative time spent by RAB6A+ vesicles in apical movement phase, in mCherry and CC1-p150-expressing aRG cells. DMSO treatment slightly affected RAB6A dynamics, as compared to mCherry control. Data information: (J, K, L, M) 216 vesicles from N=11 cells for DMSO, 145 vesicles from N=25 cells for dynarrestin, 173 vesicles from N=17 cells for mCherry control, 71 vesicles from N=15 cells for CC1-p150. Mann-Whitney U test *p ≤ 0.05, **p ≤ 0.01, *** p ≤ 0.001. All boxplots: whiskers indicate min and max, boxes indicate 25th and 75th percentile, and central band indicates the median.


Figure 4C . RUSH assay for CRB3 - GFP in control ( mcherry ) and dynactin - inhibited radial glial cells ( CC1 - p150 - dsRed ) , electroporated at E . 15 . 5 and imaged at E16 . 5 . Scale bar = 5µm . D . CRB3 localization at the apical surface upon release . E . Percentage of cells with 100 % of CRB3 signal at the apical surface upon release . Data information : ( D , E ) mCherry ( control ) : N = 3 brains per timepoint ( 361 cells total ) . CC1 - p150 : N = 3 brains per timepoint ( 2 brains for T0 ) ( 268 cells total ) . Fisher ' s exact test and Benjamini - Hochberg procedure , * * * p ≤ 0 . 001 . All error bars indicate SD .

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Figure 4C. RUSH assay for CRB3-GFP in control (mcherry) and dynactin-inhibited radial glial cells (CC1-p150-dsRed), electroporated at E.15.5 and imaged at E16.5. Scale bar = 5µm. D. CRB3 localization at the apical surface upon release. E. Percentage of cells with 100% of CRB3 signal at the apical surface upon release. Data information: (D, E) mCherry (control): N= 3 brains per timepoint (361 cells total). CC1-p150: N=3 brains per timepoint (2 brains for T0) (268 cells total). Fisher's exact test and Benjamini-Hochberg procedure, *** p ≤ 0.001. All error bars indicate SD.


Figure 5A . PAX6 and phospho - Histone 3 ( p - H3 ) staining in WT and LIS1 KO E12 . 5 brains . Cortices were subdivided into 5 bins of equal size along the radial axis . Scale bar = 25µm . B . Percentage of PH3 + / PAX6 + cells located above the ventricular surface of WT and LIS1 KO E12 . 5 brains . WT : 1192 cells from N = 6 brains . LIS1 KO : 589 cells from N = 3 brains . Data information : ( B , Mann - Whitney U test , * p ≤ 0 . 05 . All error bars indicate SD . C . Phospho - Vimentin ( p - VIM ) staining in WT and LIS1 KO E12 . 5 brains . Scale bar = 25µm . D . Percentage p - VIM + cells dividing ectopically , away from the ventricular surface of WT and LIS1 KO E12 . 5 brains . WT : 1056 cells from N = 6 brains . LIS1 KO : 879 cells from N = 3 brains . Data information : D ) Mann - Whitney U test , * p ≤ 0 . 05 . All error bars indicate SD .

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Figure 5A. PAX6 and phospho-Histone 3 (p-H3) staining in WT and LIS1 KO E12.5 brains. Cortices were subdivided into 5 bins of equal size along the radial axis. Scale bar = 25µm. B. Percentage of PH3+/PAX6+ cells located above the ventricular surface of WT and LIS1 KO E12.5 brains. WT: 1192 cells from N=6 brains. LIS1 KO: 589 cells from N=3 brains. Data information: (B, Mann-Whitney U test, *p ≤ 0.05. All error bars indicate SD.C. Phospho-Vimentin (p-VIM) staining in WT and LIS1 KO E12.5 brains. Scale bar = 25µm. D. Percentage p-VIM+ cells dividing ectopically, away from the ventricular surface of WT and LIS1 KO E12.5 brains. WT: 1056 cells from N=6 brains. LIS1 KO: 879 cells from N=3 brains. Data information: D) Mann-Whitney U test, *p ≤ 0.05. All error bars indicate SD.


Figure 6A . CRB3 and PALS1 staining in WT , RAB6A / B dKO ( E15 . 5 ) and LIS1 KO ( E12 . 5 ) brains . Scale bar = 25µm . B . Quantification of CRB3 staining interruptions along the ventricular boundary of WT ( E12 . 5 and E15 . 5 ; N = 13 brains ) , LIS1 KO ( E12 . 5 , N = 4 brains ) ) and RAB6A / B dKO ( E15 . 5 , N = 4 brains ) . Data information : ( B , Kruskal - Wallis test with a Dunn post - hoc test and Benjamini - Hochberg procedure , * p ≤ 0 . 05 , * * * p ≤ 0 . 001 . All error bars indicate SD . C . N - Cadherin staining in WT , RAB6A / B dKO ( E15 . 5 ) and LIS1 KO ( E12 . 5 ) brains . Scale bar = 25µm . D . Quantification of N - Cadh staining interruptions along the ventricular boundary of WT ( E12 . 5 and E15 . 5 ; N = 6 brains ) , LIS1 KO ( E12 . 5 , N = 3 brains ) and RAB6A / B dKO ( E15 . 5 , N = 3 brains ) . Data information : D ) Kruskal - Wallis test with a Dunn post - hoc test and Benjamini - Hochberg procedure , * p ≤ 0 . 05 , * * * p ≤ 0 . 001 . All error bars indicate SD .

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Figure 6A. CRB3 and PALS1 staining in WT, RAB6A/B dKO (E15.5) and LIS1 KO (E12.5) brains. Scale bar = 25µm.B. Quantification of CRB3 staining interruptions along the ventricular boundary of WT (E12.5 and E15.5; N=13 brains), LIS1 KO (E12.5, N=4 brains)) and RAB6A/B dKO (E15.5, N=4 brains). Data information: (B, Kruskal-Wallis test with a Dunn post-hoc test and Benjamini-Hochberg procedure, *p ≤ 0.05, *** p ≤ 0.001. All error bars indicate SD.C. N-Cadherin staining in WT, RAB6A/B dKO (E15.5) and LIS1 KO (E12.5) brains. Scale bar = 25µm.D. Quantification of N-Cadh staining interruptions along the ventricular boundary of WT (E12.5 and E15.5; N=6 brains), LIS1 KO (E12.5, N=3 brains) and RAB6A/B dKO (E15.5, N=3 brains). Data information: D) Kruskal-Wallis test with a Dunn post-hoc test and Benjamini-Hochberg procedure, *p ≤ 0.05, *** p ≤ 0.001. All error bars indicate SD.