EMBO/soda-nel huggingface dataset:
Single-cell transcriptomics reveals a senescence-associated IL-6/CCR6 axis driving radiodermatitis (2022)
Figure 5

Figure 5. CCR6 deficiency strongly reduces IRIAD in mice.

UMAP plots depicting relative expression and localization of Ccr6 and Ccl20 mRNAs in irradiated and naïve skin-derived cell clusters.

Quantification by real-time qPCR analysis of Ccr6 and Ccl20 mRNAs in whole skin of naïve and irradiated (15Gy) WT and IL-6-/- mice at indicated times post-IR, (n=5-7).

Photographic images showing the development of radiodermatitis and alopecia in irradiated (15 Gy) WT and CCR6-/- mice at indicated times post-IR. Escalating radiodermatitis, which manifests on day 7-10 as serous exudates on the chin and neck, is visible in both strains and progresses in WT mice to alopecia by day 14 post-IR but resolves spontaneously in the CCR6-/- mice. Quantification (lower right) of the area of hair loss at 14 days post-IR, (n = 6).

Histochemical staining (H&E) and immunostaining (red) show morphology and infiltration of neutrophils (Ly6b+) and T cells (CD3+) (red arrows) to the skin and to the hair follicle (HF) (dashed lines) in WT or CCR6-/- mice 14 days post-IR. Scale bars, 50 μm (black) and 25 μm (red).

Quantification of epidermal thickness, neutrophils (Ly6b+), total T cell (CD3+) infiltration to the skin, and (F) CD3+ cells infiltration to the hair follicle in (D), (n = 5-6).

MHC class I immunostaining (red) of skin thin sections prepared at 14 days post-IR from mice in (C), and quantification (right) of MHC I positive hair follicles, (n = 4-5). Scale bar, 50 μm.

Quantification by real-time qPCR analysis of Il6, Il17a, Ccl20, and Ccl3 mRNAs in the skin of naïve WT and irradiated (15Gy) WT and CCR6-/- mice at 14 days post-IR, (n = 3-5).

Data information: Data represent mean ± SD. *P < 0.05, **P < 0.01 by two-tailed Student's t test (E-Ly6b, H), or by Mann-Whitney test (all other panels). Two independent experiments. (See also Appendix Fig S13 - S15.)
Single-cell transcriptomics reveals a senescence-associated IL-6/CCR6 axis driving radiodermatitis (2022)
Figure 6

Figure 6. Cyclosporine A treatment prevents IR-induced alopecia and radiodermatitis.

Photographic images showing hair loss in control (PBS) and cyclosporine (CsA) treated WT mice at 14 days post-IR (15 Gy).

Quantification of the area of ventral hair loss in mice in (A), (n = 6).

Histochemical (H&E) and immunostaining (red) show T cell (CD3+) and neutrophil (Ly6b+) infiltration in the skin of mice in (A). Scale bar, 100 μm. (Images of CD3 and Ly6b immunostaining from CsA treated mice represent seperate analyses performed on serial thin sections.)

Quantification of epidermal thickness, and T cell (CD3+) and neutrophil (Ly6b+) infiltration (red staining) in skin thin sections from (C), (n = 6).

Quantification by real-time qPCR analysis of Il6, Il17a, II22, Ccl3, Ccr6, and Ccl20 mRNAs in whole skin of naïve and irradiated (15Gy) control (PBS) and CSA treated WT mice at 14 days post-IR, (n = 5).

Data information: Data represent mean ± SD. *P < 0.05, **P < 0.01 by two-tailed Mann-Whitney test; two independent experiments.

Expanded View Figure Legends

Table EV1 Cluster Analysis by IPA.
ZDHHC18 negatively regulates cGAS-mediated innate immunity through palmitoylation (2022)
Figure 1

Fig 1. Palmitoylation of cGAS suppresses its activation.

(A) Acyl-RAC assay of HEK293T cells transfected with the indicated plasmids for 24 h.

(B) Acyl-RAC assay of HEK293T cells transfected with the indicated plasmids for 24 h.

(C) Click chemistry was applied to detect endogenous cGAS palmitoylation in RAW264.7 cells.

(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.

(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.

(J) THP-1 cells were treated with palmitic acid (0, 50 μM, or 100 μM) for 12 h before a cGAMP bioassay.

(K-N) 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), Cxcl10 (M) and Rantes (N) expression.

(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.

Data information: EtOH: ethanol; HAM: hydroxylamine; 2-BP: 2-bromopalmitate; FL: full length; N: N-terminal domain; C: C-terminal domain; PA: palmitic acid; 17-ODYA: 17-octadecanoic acid. 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 (H-N).
ZDHHC18 negatively regulates cGAS-mediated innate immunity through palmitoylation (2022)
Figure 2

Fig 2. ZDHHC18 is a major acyltransferase catalyzing cGAS palmitoylation.

(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).

(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.

(E and F) Immunoprecipitation (with an anti-Flag antibody) and immunoblot analysis of HEK293T cells transfected with the indicated plasmids for 24 h.

(G) Schematic diagrams of ZDHHC18 and its truncation mutants.

(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.

(I) Schematic diagrams of cGAS and its truncation mutants.

(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.

(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).

(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.

Data information: NC: negative control; WCL: whole-cell lysate; IP: immunoprecipitation; FL: full length; TM: transmembrane domain. 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 and L).
ZDHHC18 negatively regulates cGAS-mediated innate immunity through palmitoylation (2022)
Figure 3

Fig 3. Palmitoylation inhibits DNA binding of cGAS.

(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.

(B and C) Atomic MD simulations of the human cGAS-DNA 2:2 complex in the resting and palmitoylation states. Comparison of the relative positions of S329-S345, F433-P450 and E478-S493 in the WT and palmitoylated cGAS proteins.

(D and E) The binding modes of the human cGAS-DNA 2:2 complex in the resting and palmitoylation states. Monomer A, monomer B and DNA-1 in the human cGAS-DNA 2:2 complex in the resting state are shown with pale cyan, light pink and cyan cartoons, respectively, while these components in the human cGAS-DNA 2:2 complex in the palmitoylation state are exhibited with light blue, yellow and slate cartoons, respectively. The C474 and S-palmitoylated C474 residues are highlighted by ruby sticks, while the dominant residues in the human cGAS-DNA 2:2 complex in both the resting and palmitoylation states are shown in sticks.

(F and 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.

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

Data information: WCL: whole-cell lysate; IP: immunoprecipitation. Data are representative of at least two independent experiments.
ZDHHC18 negatively regulates cGAS-mediated innate immunity through palmitoylation (2022)
Figure 4

Fig 4. Palmitoylation dampens cGAS dimerization.

(A and B) View of the first normal modes of monomer A and monomer B in the human cGAS-DNA 2:2 complex in the resting and palmitoylation states during the simulation. The colors of monomer A and monomer B in (A) and (B) are consistent with those in fig. 3, (D) and (E). PC1: the first principal components in PCA.

(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.

(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).

(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).

(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.

(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: WCL: whole-cell lysate; IP: immunoprecipitation; 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 and J).
ZDHHC18 negatively regulates cGAS-mediated innate immunity through palmitoylation (2022)
Figure 5

Fig 5. ZDHHC18 negatively regulates cGAS activation.

(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.

(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.

(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.

(E to I) 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), IFNb1 (H), and Cxcl10 (I) expression. The knockdown efficiency of ZDHHC18 is shown by RT-qPCR (E) and western blotting (F) results.

(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: 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; 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 (A, E, G-I).
ZDHHC18 negatively regulates cGAS-mediated innate immunity through palmitoylation (2022)
Figure 6

Fig 6. ZDHHC18 deficiency promotes antiviral activity against HSV-1 in mice.

(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).

(C and 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.

(E and F) Microscopy of BMDMs (Zdhhc18+/+ or Zdhhc18-/-) infected with GFP-HSV-1 (MOI=1) for 10 h. Scale bar: 200 μm. Frequency of GFP+ cells among infected cells (E).

(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.

(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: PFU: plaque-forming units; H&E: hematoxylin-eosin. 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, H-J).
Oncogenic KRAS signaling activates mTORC1 through COUP-TFII-mediated lactate production (2019)
Figure 1

Figure 1. Effects of COUP-TFII on lactate production and mTORC1 activity in KRAS activated cells.

(A) Levels of KRAS, COUP-TFII, LDHA, and phosphorylated S6K (T389) in KRASWT, KRASG12D, and KRAS-silenced KRASG12D MEF cells.

(B) Extracellular acidification rate (ECAR) in KRASWT, KRAS G12D, and KRAS- or COUP-TFII-silenced KRAS G12D MEF cells.

(C) Lactate levels in MEF cells shown in (B). Data are expressed as the mean ± SEM of three independent experiments and normalized against values measured in KRASWT MEF. ***p<0.001.

(D) Levels of COUP-TFII, LDHA, and phosphorylated S6K (T389) in COUP-TFII-silenced KRASG12D MEF cells.
Oncogenic KRAS signaling activates mTORC1 through COUP-TFII-mediated lactate production (2019)
Figure 2

Figure 2. Effects of COUP-TFII-induced lactate production on mTORC1 activation and cell proliferation in KRAS activated human cancer cells.

(A) Levels of KRAS, COUP-TFII, LDHA, and phosphorylated S6K (T389) in KRAS-silenced HCT116 and DLD-1 cells.

(B) Levels of COUP-TFII, LDHA, and phosphorylated S6K (T389) in COUP-TFII-silenced HCT116 and DLD-1 cells.

(C) Effect of lactate on phosphorylated S6K (T389), 4E-BP1 (T37/46), ULK1 (S757) in COUP-TFII-silenced HCT116 cells.

(D) Effects of COUP-TFII and the inhibition of LDHA on phosphorylated S6K (T389) and Raptor (S792) in KRAS-silenced HCT116 cells. Asterisk indicates exogenous HA-COUP-TFII. Arrow indicates phosphorylated Raptor (S792).

(E) Inhibition of COUP-TFII-silenced HCT116, DLD-1, and MIA PaCa-2 cell proliferation and its recovery by treatment with lactate. Data are expressed as the mean ± SEM of three independent experiments and normalized against values measured in control. *p<0.05, **p<0.01, and ***p<0.001.

(F) Clonogenic assay of COUP-TFII-silenced HCT116, DLD-1, and MIA PaCa-2 cells in the presence or absence of lactate.
Oncogenic KRAS signaling activates mTORC1 through COUP-TFII-mediated lactate production (2019)
Figure 3

Figure 3. Effects of lactate on TSC2 and Rheb interaction and TSC2 lysosomal localization.

(A) Effect of lactate on the ERK/TSC2 and AKT/TSC2 signaling pathway in serum-starved HCT116 cells.

(B) Effect of lactate on phosphorylated S6K (T389) inhibited by TSC2 overexpression in HCT116 cells.

(C) Molecular interaction between TSC2 and Rheb in the presence or absence of lactate in HA-Rheb-overexpressing HCT116 cells after serum deprivation overnight.

(D) Molecular interaction between TSC2 and recombinant GDP-loaded Rheb in the presence or absence of lactate in vitro.

(E) Effect of lactate on Rheb activation in serum-starved HCT116 cells.

(F) Interaction between endogenous Rheb and mTOR in the presence or absence of lactate in HCT116 cells after serum deprivation overnight.

(G) Immunofluorescence analysis of staining for TSC2 and LAMP2 in the presence or absence of lactate in HCT116 cells after serum deprivation overnight.
Oncogenic KRAS signaling activates mTORC1 through COUP-TFII-mediated lactate production (2019)
Figure 4

Figure 4. Effects of COUP-TFII on TSC2 and Rheb interaction, TSC2 lysosomal localization, and Rheb-GTP levels.

(A) Interaction between endogenous TSC2 and Rheb in the presence or absence of lactate in COUP-TFII-silenced HCT116 cells.

(B) Effect of lactate on Rheb activation in COUP-TFII-silenced HCT 116 cells.

(C) Immunofluorescence analysis of staining for TSC2 and LAMP2 in HCT116 cells shown in (B). The degree of colocalization of TSC2 and LAMP2 is graphed as the mean ± SEM. ***p<0.001.

(D) Molecular interaction between TSC2 and Rheb in the presence or absence of lactate in HA-Rheb- and/or COUP-TFII-overexpressing HCT116 cells treated with 25 nM trametinib for 24 h.

(E) Effects of lactate and COUP-TFII overexpression on Rheb activation in HCT116 cells treated with 25 nM trametinib for 24 h.

(F) Immunofluorescence analysis of staining for TSC2 and LAMP2 in HCT116 cells shown in (E). The degree of colocalization of TSC2 and LAMP2 is graphed as the mean ± SEM. ***p<0.001.
Oncogenic KRAS signaling activates mTORC1 through COUP-TFII-mediated lactate production (2019)
Figure 5

Figure 5. In vivo effects of COUP-TFII on tumor growth.

(A-C) Effects of COUP-TFII inhibition and lactate treatment on tumor growth in vivo. (A) Growth curve, (B) tumor volume at the experimental endpoint, and (C) Western blot analysis of the indicated proteins in tumors derived from xenografted shScramble or shCOUP-TFII-HCT116 cells and treated with sodium chloride or sodium L-lactate (60 l x g-1 body weight; 150 mM).

(D) Effects of trametinib treatment and overexpression of COUP-TFII on tumor growth in vivo. Tumors were derived from xenografted HCT116 expressing HA-COUP-TFII or carrying a control vector and treated with trametinib (3mg/kg).

(E) Western blot analysis of indicated proteins in tumors derived from xenografted HCT116 treated with trametinib with or without COUP-TFII overexpression. Asterisk indicates exogenous HA-COUP-TFII.

(F) Schematic showing COUP-TFII-mediated growth pathway in KRAS activated cancer cells.

Expanded View Figure legends
Effector loading onto the VgrG carrier activates type VI secretion system assembly (2019)
Figure 1

Figure 1. Presence of Tde effectors in the cell is critical for secretion of the cognate VgrG.

A. The structure of the imp and hcp operons and vgrG2 operon in A. tumefaciens C58 [10]. The arrows represent coding sequencing, with arrowheads depicting the direction of expression. The wider arrows represent genes in the T6SS associated operons. The vgrG2 operon is located distal to imp operon and hcp operon. The vgrG and toxin-immunity gene pairs are highlighted in colors.

B. T6SS secretion assay of A. tumefaciens strains: wild type C58, various mutants lacking one, two, or three toxin-immunity gene pairs, and a mutant lacking tssL.

C. T6SS secretion assay of various A. tumefaciens strains: wild type C58, ΔtssL, the tde double deletion mutant (Δtdei) containing pRL662 and pTrc200 empty vector (V) only or expression of pTdei1 (tde1-tdi1 expressed from pTrc200), pTde2* (catalytic site mutated tde2 expressed on pRL662), or pTdei1+ pTde2*.

D. A. tumefaciens antibacterial activity assay against E. coli. The strains of A. tumefaciens were co-cultured at a ratio of 30:1 with E. coli DH10B (+ pRL662) on LB agar. The survival of target E. coli cells was quantified by counting CFUs on gentamicin-containing LB agar plates. Data represent mean ± SEM of 6 biological replicates from three independent experiments. One-way ANOVA followed by Turkey HSD test was used for statistical analysis. Two groups with significant differences (P=0.0010053) are indicated with different letters (a and b).

Data information: In (B, C), cellular and extracellular fractions were collected from A. tumefaciens strains grown in liquid 523 medium. Western blots were probed with indicated antibodies; the α-VgrG antibody detects VgrG1 (upper band) and VgrG2 (lower band) while α-VgrG1 detects only VgrG1 [16]. RpoA is RNA polymerase subunit alpha, which is localized to the cytosol of A. tumefaciens. Molecular weight markers (in kDa) are indicated on the left.

Data are from one independent experiment and reproduced in at least two independent experiments.
Effector loading onto the VgrG carrier activates type VI secretion system assembly (2019)
Figure 2

Figure 2. Tde effector loading onto VgrG is required for secretion of the cognate VgrG.

A. T6SS secretion assay of A. tumefaciens strains: wild type C58, ΔtssL, Δtap-1, and Δatu3641 harboring a pTrc200 vector (V) or derivatives pTap-1 (tap-1 expressed from pTrc200), or p3641 (atu3641 expressed from pTrc200).

B. T6SS secretion assay of A. tumefaciens C58 Δtap-1Δatu3641 containing an empty pTrc200 vector (V) or its derivatives expressing tap-1, atu3641, tde1-tdi1, or tde2-tdi2.

C. T6SS secretion assay of A. tumefaciens C58ΔG2op mutant encoding full-length or truncated VgrG1 proteins.

Data information: In (A-C), cellular and extracellular fractions were collected from A. tumefaciens strains grown in liquid 523 medium. Western blots were probed with indicated antibodies; the α-VgrG antibody detects VgrG1 (upper band) and VgrG2 (lower band) while α-VgrG1 detects only VgrG1 [16]. RpoA is RNA polymerase subunit alpha, which is localized to the cytosol of A. tumefaciens. Molecular weight markers (in kDa) are indicated on the left.

Data are from one independent experiment and reproduced in at least two independent experiments.