Journal: BMC biology

Article Title: SETDB1 targeting SESN2 regulates mitochondrial damage and oxidative stress in renal ischemia-reperfusion injury.

doi: 10.1186/s12915-024-02048-z

Figure Lengend Snippet: Fig. 8 HP1β interacted with SETDB1 to jointly regulate SESN2. A CoIP results of SETDB1 and different HP1. B WB detection of HP1α and HP1β protein levels. C ChIP detection of the enrichment of HP1α and HP1β on SESN2 promoter in vivo. D Re-ChIP detection of the enrichment of SETDB1 combined with HP1β on SESN2 promoter in vivo. E WB detection of HP1β and Sestrin2 protein levels. F Detection of ROS levels in HK-2 cells by flow cytometry. G Representative images of Mito Tracker Red CMXRos in HK-2 cells (left) and related quantitative analysis (right). Bar = 30 μm. Values are expressed as the mean ± SEM. N = 3. The ** represents differences between groups, and p < 0.01

Article Snippet: To inhibit SETDB1 activity in vivo, (R,R)-59 (MedChemExpress, HY-141539, USA) was injected intraperitoneally at a concentration of 25 mg/kg or 50 mg/kg and a volume of 100 μl for 3 consecutive days before surgery in mice.

Techniques: In Vivo, Flow Cytometry

Journal: BMC biology

Article Title: SETDB1 targeting SESN2 regulates mitochondrial damage and oxidative stress in renal ischemia-reperfusion injury.

doi: 10.1186/s12915-024-02048-z

Figure Lengend Snippet: Fig. 9 SETDB1 collaborates with heterochromatin HP1β, facilitating the labeling of H3K9me3 on the SESN2 promoter and impeding SESN2 expression. Loss of Sestrin2 induces mitochondrial damage and subsequent oxidative stress

Article Snippet: To inhibit SETDB1 activity in vivo, (R,R)-59 (MedChemExpress, HY-141539, USA) was injected intraperitoneally at a concentration of 25 mg/kg or 50 mg/kg and a volume of 100 μl for 3 consecutive days before surgery in mice.

Techniques: Labeling, Expressing

Journal: Experimental and therapeutic medicine

Article Title: SETDB1 induces lenalidomide resistance in multiple myeloma cells via epithelial‑mesenchymal transition and PI3K/AKT pathway activation.

doi: 10.3892/etm.2023.11973

Figure Lengend Snippet: Figure 1. SETDB1 expression in MM is associated with poor survival and lenalidomide resistance. Kaplan‑Meier analysis of (A) progression‑free and (B) overall survival using GEO datasets (GSE136324). (C) Analysis of SETDB1 expression in lenalidomide‑resistant and ‑sensitive MM cell lines in a GEO cohort (GSE165557). SETDB1, SET domain bifurcated histone lysine methyltransferase 1; MM, multiple myeloma; GEO, Gene Expression Omnibus.

Article Snippet: Human SETDB1 (NM_001145415; Origene) was amplified and cloned into the lentiviral vector, pCDH‐CMV‐MCS‐EF1‐Pur (Asia‐Vector Biotechnology) and named SETDB1‐OE.

Techniques: Expressing, Gene Expression

Journal: Experimental and therapeutic medicine

Article Title: SETDB1 induces lenalidomide resistance in multiple myeloma cells via epithelial‑mesenchymal transition and PI3K/AKT pathway activation.

doi: 10.3892/etm.2023.11973

Figure Lengend Snippet: Figure 2. Construction of stable SETDB1‑OE and ‑knockdown multiple myeloma cell lines. (A) mRNA expression of SETDB1 in 293T cells was detected using reverse transcription‑quantitative PCR, SETDB1‑sh1 were selected for use in subsequent experiments. SETDB1 expression levels were normalized to GAPDH mRNA. ***P<0.001 vs. NC. #P<0.05, ###P<0.001 vs. shSETDB1‑NC. Western blot analysis following (B) OE and (C) knockdown of SETDB1. SETDB1, SET domain bifurcated histone lysine methyltrans‑ ferase 1; NC, negative control; OE, overexpression; sh, short hairpin RNA; Ctrl, control.

Article Snippet: Human SETDB1 (NM_001145415; Origene) was amplified and cloned into the lentiviral vector, pCDH‐CMV‐MCS‐EF1‐Pur (Asia‐Vector Biotechnology) and named SETDB1‐OE.

Techniques: Expressing, Western Blot, Knockdown, Negative Control, Over Expression, shRNA, Control

Journal: Experimental and therapeutic medicine

Article Title: SETDB1 induces lenalidomide resistance in multiple myeloma cells via epithelial‑mesenchymal transition and PI3K/AKT pathway activation.

doi: 10.3892/etm.2023.11973

Figure Lengend Snippet: Figure 3. SETDB1 expression affects apoptosis of U266 and RPMI‑8226 cells and is associated with lenalidomide resistance. (A) Apoptosis was measured using flow cytometry. (B) Statistical analysis of the apoptosis. **P<0.01, ***P<0.001; ###P<0.001. (C) Cell viability was measured using Cell Counting Kit‑8 assay following lenalidomide treatment. (D) IC50 values. IC50, half maximal inhibitory concentration; SETDB1, SET domain bifurcated histone lysine methyltrans‑ ferase 1; NC, negative control; OE, overexpression; sh, short hairpin RNA.

Article Snippet: Human SETDB1 (NM_001145415; Origene) was amplified and cloned into the lentiviral vector, pCDH‐CMV‐MCS‐EF1‐Pur (Asia‐Vector Biotechnology) and named SETDB1‐OE.

Techniques: Expressing, Flow Cytometry, CCK-8 Assay, Concentration Assay, Negative Control, Over Expression, shRNA

Journal: Experimental and therapeutic medicine

Article Title: SETDB1 induces lenalidomide resistance in multiple myeloma cells via epithelial‑mesenchymal transition and PI3K/AKT pathway activation.

doi: 10.3892/etm.2023.11973

Figure Lengend Snippet: Figure 4. SETDB1 expression is associated with PI3K/AKT signaling and epithelial‑mesenchymal transition activation in multiple myeloma cells. (A) Gene set enrichment analysis illustrating that SETDB1 expression is positively associated with the PI3K/AKT/mTOR signaling pathway. Western blot analysis in (B) U266 and (C) RPMI‑8226 cells. ***P<0.001 vs. SETDB1‑NC. ##P<0.01, ###P<0.001 vs. shSETDB1‑NC. SETDB1, SET domain bifurcated histone lysine methyltransferase 1; NC, negative control; OE, overexpression; sh, short hairpin RNA; N‑cad, N‑cadherin; FDR, false discovery rate; NES, normalized enrichment score; p, phosphorylated.

Article Snippet: Human SETDB1 (NM_001145415; Origene) was amplified and cloned into the lentiviral vector, pCDH‐CMV‐MCS‐EF1‐Pur (Asia‐Vector Biotechnology) and named SETDB1‐OE.

Techniques: Expressing, Activation Assay, Western Blot, Negative Control, Over Expression, shRNA

Journal: Experimental and therapeutic medicine

Article Title: SETDB1 induces lenalidomide resistance in multiple myeloma cells via epithelial‑mesenchymal transition and PI3K/AKT pathway activation.

doi: 10.3892/etm.2023.11973

Figure Lengend Snippet: Figure 5. PI3K/AKT signaling and epithelial‑mesenchymal transition contribute to SETDB1‑induced lenalidomide resistance in multiple myeloma. (A) Flow cytometry analysis of apoptosis of U266 and RPMI‑8226 cells after treatment with LY294002 and SETDB1 overexpression. (B) Statistical analysis of apop‑ tosis in each group. **P<0.01, ***P<0.001 vs. SETDB1‑NC; ###P<0.001 vs. LY294002 + SETDB1‑NC. (C) IC50 values of lenalidomide were determined by Cell Counting Kit‑8 assay. LY294002 significantly reduced the IC50 value of lenalidomide, whereas SETDB1 overexpression attenuated this effect. (D) Western blot analysis revealed that the expression of N‑cadherin and vimentin, as well as the phosphorylation of PI3K and AKT, were inhibited by LY294002; these effects were counteracted by SETDB1 overexpression. ***P<0.001 vs. SETDB1‑NC; #P<0.05, ##P<0.01 and ###P<0.001 vs. LY294002 + SETDB1‑NC. SETDB1, SET domain bifurcated histone lysine methyltransferase 1; NC, negative control; OE, overexpression; IC50, half maximal inhibitory concentration; N‑cad, N‑cadherin; p, phosphorylation.

Article Snippet: Human SETDB1 (NM_001145415; Origene) was amplified and cloned into the lentiviral vector, pCDH‐CMV‐MCS‐EF1‐Pur (Asia‐Vector Biotechnology) and named SETDB1‐OE.

Techniques: Flow Cytometry, Over Expression, CCK-8 Assay, Western Blot, Expressing, Phospho-proteomics, Negative Control, Concentration Assay

Journal: Nucleic Acids Research

Article Title: SPOC1 modulates DNA repair by regulating key determinants of chromatin compaction and DNA damage response

doi: 10.1093/nar/gks868

Figure Lengend Snippet: SPOC1 protein interacts with KAP-1 and enzymatically active H3K9 KMTs. ( A ) In GST-pulldown experiments with GST-SPOC1 wt, GST-SPOC1 (amino acids 1–100) or GST alone and chromatin extracts from U2OS cells, only the GST-SPOC1-full-length protein precipitated the KAP-1 protein, as detected on the immunoblot (lower panel). Amounts of GST proteins used were visualized by Amido black staining of the blots (upper panel). Asterisks mark the positions of full-length GST-proteins. ( B ) LUMIER-assay for mapping the KAP-1/SPOC1 interaction. The indicated combinations of SPOC1 and KAP-1, ProteinA (ProtA) and luciferase (Luc) fusion proteins were coexpressed. Luc activity coprecipitated with IgG-beads was measured (Z-scores). Significant KAP-1-interaction is only evident between both full-length proteins, or the C-terminal half of SPOC1 and the N-terminal 400 amino acids of KAP1, as detected by significant Luc activity in the corresponding pellets. Z -score ≥ 0.5 indicates an interaction. Mean values ± SD of three independent experiments each performed in triplicate are given. ( C ) Immunoprecipitation of SPOC1 from HeLa nuclear extracts with anti-SPOC1 (lane 2) or with control antibodies (lane 1) and analysis of pelleted material by immunoblotting shows specific coprecipitation of G9a and SETDB1 with SPOC1. ( D ) Immunoprecipitation (IP) of endogenous GLP or SUV39H1 from C2C12 cell extracts and immunoblotting with the indicated Abs (IB Abs) against GLP (PP-B0422-00), SUV39H1 (07-550), SETDB1 (ab12317) and SPOC1 (6F6). Unlike the control Ab, GLP and SUV39H1 Abs precipitated all associated KMTs as well as SPOC1. ( E ) Nuclear extracts of control C2C12 cells (lane 1) or cells expressing HA-FLAG-tagged SETDB1 wt (lane 2) or SETDB1 H1224K mutant (lane 3) were analyzed on immunoblots (IB Ab.) with antibodies against FLAG (F1804), G9A (D141-3), GLP (PP-B0422-00), SUV39H1 (07-550) and SPOC1 (6F6). After anti-FLAG-SETDB1 immunoprecipitation (IP) the immunoblot detected specific coprecipitation of SPOC1 by wt SETDB1 (lane 5) but much less by enzymatically inactive SETDB1 H1224K (lane 6). (F) In vitro analysis of histone H3 methlylation by proteins immunoprecipitated by control, SPOC1, G9A, or SETDB1 antibodies. Left panel: autoradiography and SimplyBlue staining of the assay products separated by SDS–PAGE. Right panel: immunoblot which demonstrates that SPOC1, G9A and SETDB1 were immunoprecipitated specifically from the HeLa nuclear extracts. ( G ) In total lysates from HeLa-S3 cell lines constitutively expressing HA-FLAG-SETDB1 (HA-SETDB1), lentiviral-mediated SPOC1wt expression elevates H3K9me3 levels compared to control cells (REV), as detected by immunblotting with Abs against the indicated proteins (left). The SPOC1 PHD mutant (SPOC1mut) unable to bind to H3K4me3 only marginally increased H3K9me3 levels compared to SPOC1 wt.

Article Snippet: Indirect immunofluroescence and immunoblotting were performed using antibodies to γH2AX (05-636; Millipore), 53BP1 (NB100-304; Novus), β-actin (A-5441; Sigma), ATM (1549-1, Epitomics), pATM-S1981 (2152-1; Epitomics), Chk2 (sc-56297; Santa Cruz), pChk2-T68 (2661; Cell signaling), CtIP (D4) (sc-271339; Sigma), FLAG (F1804; Sigma), G9A (D141-3; Clinisciences), G9A (A300-933A; Bethyl), GAPDH (sc-32233; Santa Cruz), GLP (PP-B0422-00; R&D Systems), GLP (Bethyl; A301-642A), H3 (1326-1; Epitomics), H3K9me3 (07-442; Upstate), HP1-α (05-689; Upstate), HP1-β (ab10811; Abcam), HP1-γ (ab10817; Abcam), Tif1-β/KAP-1 (MAB3662; Millipore), pKAP-1-S824 (A300-767A-1; Bethyl), SETDB1 (ab12317; Abcam), SETDB1 (A300-121A; Bethyl), SUV39H1 (05-615; Upstate), SUV39H1 (07-550; Upstate).

Techniques: Western Blot, Staining, Luciferase, Activity Assay, Immunoprecipitation, Expressing, Mutagenesis, In Vitro, Autoradiography, SDS Page

Journal: eLife

Article Title: IFI16, a nuclear innate immune DNA sensor, mediates epigenetic silencing of herpesvirus genomes by its association with H3K9 methyltransferases SUV39H1 and GLP

doi: 10.7554/eLife.49500

Figure Lengend Snippet: List of antibodies used and their dilutions for different applications.

Article Snippet: SETDB1 , Rabbit polyclonal , Novus Biologicals (#NBP2-20322) , WB: 1:500 IP: 1:30 ChIP: 1:30 IFA: 1:70 PLA: 1:50.

Techniques:

Journal: Nature Communications

Article Title: Identification of a lipid oxygen radical defense pathway and its epigenetic control

doi: 10.1038/s41467-025-67304-4

Figure Lengend Snippet: a Ontologies (GREAT analysis) derived from ZNF354A ChIP-seq peaks in HEK293T cells. Histogram displays enriched annotation terms ranked by binomial p value, indicating functional categories significantly associated with ZNF354A-bound genomic regions . b Volcano plot of RNA-seq analysis from U2OS cells (siControl-Ctrl vs. siZNF354A, 72 h), displaying expression changes of 22586 transcripts. Log2 fold change (FC) vs. −log10 P value plotted. Highlighted genes involved in GSH synthesis (MAT1A, SLC7A11, GSTO2, MGST, CTH), iron metabolism (HMOX1, FTH), and mevalonate pathway (HMGCR). Black dots: significantly upregulated genes (FC > 2, P < 0.05). P values were corrected for multiple testing using the Benjamini-Hochberg’s method c Gene Set Enrichment Analysis (GSEA) dot plot highlighting significantly enriched GO-terms in siZNF354A cells. Gene count, gene ratio, and specific p-values indicated. Enrichment scores and p values were computed by permutation testing (10,000 permutations) using ranked gene lists. d GSEA plots for Biological Process BP and Cellular Component CC GO categories. e Model overview of ZNF354A-regulated selenium-/thiol-dependent and independent pathways in phospholipid peroxide detoxification. ZNF354A targets highlighted in green. Main proteins/pathways upregulated by ZNF354A depletion are indicated (see Supplementary Table ). In thiol-dependent pathways GPX4 converts phospholipid hydroperoxides (PL-OOH) to alcohols (PL-OH), dependent on glutathione (GSH) synthesis via cystine uptake and reduction. The methionine cycle contributes with precursors. In parallel the NAD(P)H/FSP1/ubiquinone system reduces peroxyl radicals in phospholipids (PLOO•). SLC7A11 (xCT) cystine/glutamate antiporter; TXNRD1 thioredoxin reductase-1; CTH γ-cystathionase; MAT1A methionine adenosyltransferase; GSTs GSH-S-transferases; GSR GSH-disulfide reductase; GPX4 glutathione peroxidase-4; GSSG oxidized glutathione; FSP1 ferroptosis suppressor protein; CoQ10H2 ubiquinol; CoQ10(H), ubiquinone; SEPHS2 selenophosphate synthetase-2; FTH ferritin heavy chain-1; HMGC , 3-hydroxy-3-methylglutaryl-CoA reductase. Created in BioRender. Abrami, L. ( https://BioRender.com/mmywn2r ). Source data provided as a Source Data file. See Data Availability or ( https://tronoapps.epfl.ch/web/krabopedia/ ) for full datasets.

Article Snippet: The antibodies and reagents were acquired from: ATF2 (Abcam: ab_131484; RRID: AB_11156678, rabbit: 1:2000x), ATF2 (Abcam: E243 ab32160, RRID: AB_2243137, rabbit: 1:2000x), phosphor-ATF2 (T69) (Abcam: 131106; RRID: AB_11157608;rabbit:1:1000x), GAPDH (Thermofisher: 398600; RRID: AB_2533438; mouse: 1:4000x), ZDHHC20 (Sigma: SAB4501054; RRID: AB_10744838; rabbit: 1:2000x), HA-HRP (Roche: 112013819001; RRID: AB_390917; rat: 1:5000x), KAP1 (Abcam: ab_109289; RRID: AB_10863057, rabbit: 1:1000x), phosphor-KAP1 S473 was generated in D. TRONO laboratory using the following peptide krsrSgegevsgl (rabbit: 1:500x), Nucleocapside N SARS-CoV-2 (Genetex: GTX135357; RRID: AB_2868464; rabbit 1:4000x), FLAG (Sigma: F3165; RRID: AB_259529; mouse: 1:2000x), phosphor-Threonine-Serine (BD: 612548; RRID: AB_399843; rabbit: 1:1000x), GPX4 for WB (Cell Signalling: 52455; RRID: AB_2924984, rabbit: 1:1000x), GPX4 for IP (MA5-49216, RRID: AB_3074874, mouse 1:2000x), SETDB1 (Novus Biologicals: NBP1_51677; RRID: AB_11002952, mouse: 1:2000x), NRF2 (Abnova: H00004780; RRID:566020, mouse: 1:2000x), V5-HRP (Sigma: V2260; RRID: AB_261857, mouse: 1:2000x), Actin (Millipore: MAB1501; RRID: AB_2223041, mouse: 1:4000x), MAT1A (Abcam: 129176; RRID: AB_11145300, rabbit: 1:2000x), ZNF354A (Santa Cruz: 81140; RRID: AB_1131557, mouse: 1:1000x), MAT1A (Abcam: ab1129176, RRID: AB_11145300, rabbit: 1:1000x), Myc (Millipore: 9E10, 05-419, RRID: AB_309725, mouse: 1:3000x), and HRP-conjugated secondary antibodies (GE-Healthcare).

Techniques: Derivative Assay, ChIP-sequencing, Functional Assay, RNA Sequencing, Expressing

Journal: Nature Communications

Article Title: Identification of a lipid oxygen radical defense pathway and its epigenetic control

doi: 10.1038/s41467-025-67304-4

Figure Lengend Snippet: a–c Lipid peroxides measured by flow cytometry (Bodipy-C11, Liperfluo) and membrane permeability (propidium iodide) in U2OS cells transfected with siControl or siZNF354A (72 h) ± RSL3 (3 μM, 24 h). Mean ± SEM; dots indicate biologically independent experiments.; p values compare to siControl, by two-way ANOVA, Tukey’s test. d Viability (ATP detection) of U2OS cells (siControl or siZNF354A, 72 h) ± RSL3 (3 μM) or H₂O₂ (50 µM, 16 h). Mean ± SD; representative assay (of three), n = 36.; p values compare to siControl, by two-way ANOVA, Sidak’s test. e , f Lipid peroxidation ( e ) and viability ( f ) of U2OS cells expressing wild-type (WT) or KRAB-binding mutant ZNF354A ± ferrostatin-1 (Fer-1, 15 µM, added at 0 and 48 h post-transfection). Mean ± SEM and dots represent biologically independent experiments ( e ). Mean ± SD normalized to untreated controls (empty plasmid) of representative assay (of three) n = 33 ( f ). p values compare untreated controls vs Fer-1, by two-way ANOVA, Holm-Sidak’s test. g Viability (as in f) of HeLa cells expressing ZNF354A, supplemented with FSP1, GPX4, GPX4 + N-Acetyl-Cysteine (NAC, 1 mM), or NAC alone. Mean ± SD; representative assay (of three), n = 33.; p values compare untreated controls at each time point, by two-way ANOVA, Sidak’s test. h Cellular level of GSH was measured in control cells and upon silencing of ZNF354A or GPX4 and upon over expression of ZNF354A. Mean ± SEM. and dots represent biologically independent experiments, p values obtained, by two-way ANOVA, Tukey’s test. i , Western blot of total cell extracts (TCE) and ZNF354A immunoprecipitates (IP) from indicated cancer cells, showing Short-S (42 kDa), 20 L isoforms ( ~ 49, 62, 69 kDa), GAPDH (loading control), total ZNF354A, and phospho-Ser/Thr proteins. j Viability (as in d) of cancer cells (from h) ± RSL3 (3 μM) or H₂O₂ (50 µM, 16 h). Viability normalized to untreated controls (100%). Mean ± SD; representative assay ( n = 35). p values compare each condition to HepG2 reference, by two-way ANOVA, Dunnett’s test.

Article Snippet: The antibodies and reagents were acquired from: ATF2 (Abcam: ab_131484; RRID: AB_11156678, rabbit: 1:2000x), ATF2 (Abcam: E243 ab32160, RRID: AB_2243137, rabbit: 1:2000x), phosphor-ATF2 (T69) (Abcam: 131106; RRID: AB_11157608;rabbit:1:1000x), GAPDH (Thermofisher: 398600; RRID: AB_2533438; mouse: 1:4000x), ZDHHC20 (Sigma: SAB4501054; RRID: AB_10744838; rabbit: 1:2000x), HA-HRP (Roche: 112013819001; RRID: AB_390917; rat: 1:5000x), KAP1 (Abcam: ab_109289; RRID: AB_10863057, rabbit: 1:1000x), phosphor-KAP1 S473 was generated in D. TRONO laboratory using the following peptide krsrSgegevsgl (rabbit: 1:500x), Nucleocapside N SARS-CoV-2 (Genetex: GTX135357; RRID: AB_2868464; rabbit 1:4000x), FLAG (Sigma: F3165; RRID: AB_259529; mouse: 1:2000x), phosphor-Threonine-Serine (BD: 612548; RRID: AB_399843; rabbit: 1:1000x), GPX4 for WB (Cell Signalling: 52455; RRID: AB_2924984, rabbit: 1:1000x), GPX4 for IP (MA5-49216, RRID: AB_3074874, mouse 1:2000x), SETDB1 (Novus Biologicals: NBP1_51677; RRID: AB_11002952, mouse: 1:2000x), NRF2 (Abnova: H00004780; RRID:566020, mouse: 1:2000x), V5-HRP (Sigma: V2260; RRID: AB_261857, mouse: 1:2000x), Actin (Millipore: MAB1501; RRID: AB_2223041, mouse: 1:4000x), MAT1A (Abcam: 129176; RRID: AB_11145300, rabbit: 1:2000x), ZNF354A (Santa Cruz: 81140; RRID: AB_1131557, mouse: 1:1000x), MAT1A (Abcam: ab1129176, RRID: AB_11145300, rabbit: 1:1000x), Myc (Millipore: 9E10, 05-419, RRID: AB_309725, mouse: 1:3000x), and HRP-conjugated secondary antibodies (GE-Healthcare).

Techniques: Flow Cytometry, Membrane, Permeability, Transfection, Expressing, Binding Assay, Mutagenesis, Plasmid Preparation, Control, Over Expression, Western Blot

Journal: Nature Communications

Article Title: Identification of a lipid oxygen radical defense pathway and its epigenetic control

doi: 10.1038/s41467-025-67304-4

Figure Lengend Snippet: a–d Incorporation of 3 H-palmitic acid ( 3 H-palm) in GPX4 from U2OS cells silenced for 72 h with pooled ( a ) or individual ( c ) ZDHHC siRNAs. Cells were metabolically labeled for 3 h at 37 °C with 3 H-palm; GPX4 was immunoprecipitated (IP-GPX4), resolved by SDS–PAGE, and analyzed by Western blot (WB) and autoradiography ( 3 H-palm). The levels of 3 H-palm incorporation in siControl (Ctrl) were set to 100% and results are mean ± SEM, and each dot represents an independent experiment ( n = 3). p values compare siCtrl, two-way ANOVA, Dunnett’s test. e HEK WT or ZDHHC20-KO cells were incubated with 100 μM C16:0-azide for the indicated times or with palmitic acid for 8 h. Incorporated C16:0-azide was detected by click chemistry with alkyne-mPEG 5 K (200 μM), followed by SDS–PAGE and WB for GPX4. Results are representative of three independent experiments. f–h same as in ( a ), for ( f ), HEK WT (Ctrl) or ZDHHC20-KO cells, recomplemented with empty vector or short/long ZDHHC20 or ( h ) U2OS cells left untreated or treated for 4 h with 50 μM H₂O₂. For f Myc-ZDHHC20 expression was confirmed by WB. The levels of 3 H-palm incorporation were set to 100% in Control-WT cells for g, or untreated control cells, in ( h ). Results are mean ± SEM, and each dot represents an independent experiment ( g ) n = 4 and h n = 6). p values comparing to Ctrl or as indicated by, ( g ) two-way ANOVA, Tukey’s test, and ( h ) Two-tailed Student’s t-test. i Cellular glutathione measured in U2OS cells, siCtrl or siZDHHC20 (72 h) alone or recomplemented with short/long ZDHHC20 (24 h). Data are mean ± SEM from one of three independent experiments ( n = 6). p values by, two-way ANOVA, Tukey’s test j Viability assessed using Promega Glo ATB detection in U2OS cells as in i, treated as in ( h ). Data are mean ± SEM from one of three independent experiments ( n = 36 replicates). p values by, two-way ANOVA, Tukey’s test k Proposed Model: Under homeostasis, ATF2–SETDB1–KAP1–ZNF354A repress antioxidant genes. Lipid peroxide accumulation activates p38/JNK signaling, leading to ZNF354A dissociation and induction of antioxidant responses. Created in BioRender. abrami, I ( https://BioRender.com/gvmrwgl ).

Article Snippet: The antibodies and reagents were acquired from: ATF2 (Abcam: ab_131484; RRID: AB_11156678, rabbit: 1:2000x), ATF2 (Abcam: E243 ab32160, RRID: AB_2243137, rabbit: 1:2000x), phosphor-ATF2 (T69) (Abcam: 131106; RRID: AB_11157608;rabbit:1:1000x), GAPDH (Thermofisher: 398600; RRID: AB_2533438; mouse: 1:4000x), ZDHHC20 (Sigma: SAB4501054; RRID: AB_10744838; rabbit: 1:2000x), HA-HRP (Roche: 112013819001; RRID: AB_390917; rat: 1:5000x), KAP1 (Abcam: ab_109289; RRID: AB_10863057, rabbit: 1:1000x), phosphor-KAP1 S473 was generated in D. TRONO laboratory using the following peptide krsrSgegevsgl (rabbit: 1:500x), Nucleocapside N SARS-CoV-2 (Genetex: GTX135357; RRID: AB_2868464; rabbit 1:4000x), FLAG (Sigma: F3165; RRID: AB_259529; mouse: 1:2000x), phosphor-Threonine-Serine (BD: 612548; RRID: AB_399843; rabbit: 1:1000x), GPX4 for WB (Cell Signalling: 52455; RRID: AB_2924984, rabbit: 1:1000x), GPX4 for IP (MA5-49216, RRID: AB_3074874, mouse 1:2000x), SETDB1 (Novus Biologicals: NBP1_51677; RRID: AB_11002952, mouse: 1:2000x), NRF2 (Abnova: H00004780; RRID:566020, mouse: 1:2000x), V5-HRP (Sigma: V2260; RRID: AB_261857, mouse: 1:2000x), Actin (Millipore: MAB1501; RRID: AB_2223041, mouse: 1:4000x), MAT1A (Abcam: 129176; RRID: AB_11145300, rabbit: 1:2000x), ZNF354A (Santa Cruz: 81140; RRID: AB_1131557, mouse: 1:1000x), MAT1A (Abcam: ab1129176, RRID: AB_11145300, rabbit: 1:1000x), Myc (Millipore: 9E10, 05-419, RRID: AB_309725, mouse: 1:3000x), and HRP-conjugated secondary antibodies (GE-Healthcare).

Techniques: Metabolic Labelling, Labeling, Immunoprecipitation, SDS Page, Western Blot, Autoradiography, Incubation, Plasmid Preparation, Expressing, Control, Two Tailed Test

Journal: Cancer Immunology Research

Article Title: The SETDB1–TRIM28 Complex Suppresses Antitumor Immunity

doi: 10.1158/2326-6066.cir-21-0754

Figure Lengend Snippet: Figure 1. Identification of Trim28 as a suppressor of PD-L1 expression. A, Schematic of the experimental design for the epigenome-wide CRISPR screen. NGS, next-generation sequencing. B, Volcano plot illustrating the genes whose knockout significantly upregulated (red) or downregulated (green) PD-L1 expression stimulated by IFNg, with top hits highlighted. C, Robust rank aggregation (RRA) of the top seven hits from the screen whose knockout increases PD-L1 expression. D, Enrichment of 10 individual sgRNAs against Trim28 in the PD-L1high population compared with controls. E, Expression of PD-L1 was determined by FACS analysis in ID8 cells expressing two individual shTRIM28 or control treated with or without 30 ng/mL IFNg for 24 hours. An isotype matched IgG was used as a negative control. F, Quantification of E; the mean fluorescence index of PD-L1 was determined from three biologically independent experiments. G, Significant correlation between TRIM28 and CD274 expression based on RNA-seq analysis from 24 TCGA cancer types with at least 100 patients. H, Expression of PD-L1 was determined by FACS analysis in control and Setdb1-knockout ID8 cells treated with or without 30 ng/mL IFNg for 24 hours. An isotype matched IgG was used as a negative control. I, Quantification of H; the mean fluorescence index of PD-L1 was determined from three biologically independent experiments. J, Correlation between SETDB1 and CD274 expression based on RNA- seq analysis in TCGA ovarian cancer dataset. K, Significant correlation between SETDB1 and CD274 expression based on RNA-seq analysis from 24 TCGA cancer types with at least 100 patients. Data represent mean SEM; n ¼ 3 biologically independent experiments unless otherwise stated. P values were calculated using a two- tailed Student t test except in B and D (MAGeCK analysis) and G, J, and K (Pearson r analysis).

Article Snippet: The sections were incubated with blocking buffer for 1 hour, primary antibody against TRIM28 (Abcam, #ab10484, RRID: AB_297223), SETDB1 (Biological Novus, #NBP2-20322) or PD-L1 (Cell Signaling Technology, #13684S, RRID:AB_2687655) at 4 C overnight and secondary antibody for 1 hour (Dako, #K4011).

Techniques: Expressing, CRISPR, Next-Generation Sequencing, Knock-Out, Control, Negative Control, RNA Sequencing, Two Tailed Test

Journal: Cancer Immunology Research

Article Title: The SETDB1–TRIM28 Complex Suppresses Antitumor Immunity

doi: 10.1158/2326-6066.cir-21-0754

Figure Lengend Snippet: Figure 2. SETDB1–TRIM28 negatively correlates with infiltration of effector CD8þ T cells. A, Representative staining of SETDB1, TRIM28, and PD-L1 in SETDB1–TRIM28-high or -low expressingtumorsfromaTMAwith131HGSOCcases.Scalebar¼ 100mm.B,NegativecorrelationbetweenSETDB1andPD-L1expression.C,Negativecorrelationbetween TRIM28 and PD-L1 expression. D, Positive correlation between SETDB1 and TRIM28 expression. High SETDB1 expression correlates with significantly lower tumor- infiltrated CD8þ T cells (E) and Granzyme Bþ/CD8þ T cells (F). Negative correlation between SETDB1 and CD8A (G) or GZMB (H) in RNA-seq analysis using TCGA ovarian cancerdataset.SignificantnegativecorrelationbetweenSETDB1andCD8A(I)orGZMB(J)expressionbasedonRNA-seqanalysisfrom24TCGAcancertypeswithatleast 100 patients. K, Overall survival of patients with high or low SETDB1 from the TMA based on Kaplan–Meier analysis. L, TRIM28 protein is expressed at significantly higher levels in patients with melanomawhofailed torespondtoanti–PD-1therapycomparedwiththose whoresponded tothetherapy basedona publicly available proteomics dataset (35). Data represent mean SEM. P values were calculated using a two-tailed Student t test except in B–D and G–J (Pearson r analysis) and K (log-rank test).

Article Snippet: The sections were incubated with blocking buffer for 1 hour, primary antibody against TRIM28 (Abcam, #ab10484, RRID: AB_297223), SETDB1 (Biological Novus, #NBP2-20322) or PD-L1 (Cell Signaling Technology, #13684S, RRID:AB_2687655) at 4 C overnight and secondary antibody for 1 hour (Dako, #K4011).

Techniques: Staining, Expressing, RNA Sequencing, Two Tailed Test

Journal: Cancer Immunology Research

Article Title: The SETDB1–TRIM28 Complex Suppresses Antitumor Immunity

doi: 10.1158/2326-6066.cir-21-0754

Figure Lengend Snippet: Figure 3. SETDB1–TRIM28 loss activates the cGAS–STING pathway. A, GSEA hallmark enrichment analysis of DEGs between control and Setdb1-knockout ID8 cells. NES, normalized enrichment score. B, Enrichment plot of GSEA for inflammatoryresponse hallmark. C, Heatmap of the inflammatoryresponsehallmark genes upregulated in Setdb1-knockout ID8 cells. KO, knockout. D, Expression of Ccl5 and Cxcl10 in control and the indicated Setdb1-knockout ID8 cells determined by qRT-PCR analysis. E, Immunofluorescence staining of cGas and g-H2AX in control and Setdb1-knockout ID8 cells. Arrows point to examples of cGas- and gH2AX-positive micronuclei. Scale bar ¼ 10 mm. F, Quantification of micronuclei-positive cells in control and indicated Setdb1-knockout ID8 cells. G and H, Same as E and F but for Trim28-knockout ID8 cells. I, Expression of Ccl5 and Cxcl10 in control and Setdb1-knockout ID8 cells treated with or without the cGAS inhibitor RU.521 (2.5 mg/mL for 48 hours), as determined by qRT-PCR analysis. J, Expression of Ccl5 and Cxcl10 in control, Setdb1-knockout, cGas-knockout and cGas/Setdb1 double-knockout ID8 cells determined by qRT-PCR analysis. K, Expression of Setdb1, cGas, Sting, phospho-Irf3, and loading control b-actin in the indicated cells determined by immunoblot. L, Percentage of PD-L1þ tumor cells in tumors formed by orthotopic injection of the indicated ID8 cell lines into C57BL/6J mice. Data represent mean SEM; n ¼ 3 biologically independent experiments unless otherwise stated. P values were calculated using a two-tailed Student t test except in B (GSEA).

Article Snippet: The sections were incubated with blocking buffer for 1 hour, primary antibody against TRIM28 (Abcam, #ab10484, RRID: AB_297223), SETDB1 (Biological Novus, #NBP2-20322) or PD-L1 (Cell Signaling Technology, #13684S, RRID:AB_2687655) at 4 C overnight and secondary antibody for 1 hour (Dako, #K4011).

Techniques: Control, Knock-Out, Expressing, Quantitative RT-PCR, Staining, Double Knockout, Western Blot, Injection, Two Tailed Test

Journal: Cancer Immunology Research

Article Title: The SETDB1–TRIM28 Complex Suppresses Antitumor Immunity

doi: 10.1158/2326-6066.cir-21-0754

Figure Lengend Snippet: Figure 4. Setdb1 loss synergizes with anti–PD-L1 in reducing tumor burden. A, Schematic of the experimental design using the ID8 syngeneic orthotopic mouse ovarian cancer model. B, Reproductive tracts with tumors from the indicated treatment groups were dissected at the end of treatment (n ¼ 5 mice per group). C, The weights of tumors dissected from the indicated groups were measured as a surrogate for tumor burden. P values were calculated using a two-tailed t test. CI for Setdb1 loss and anti–PD-L1 is 0.82 (<1), which indicates synergy between Setdb1 loss and anti–PD-1 combination. Infiltration of CD8þ T cells (D) and Granzyme Bþ/CD8þ T cells (E) in the tumors dissected from the indicated treatment groups were analyzed by flow cytometry (n ¼ 5 mice per group). Data represent mean SEM; P values were calculated using a two-tailed t test.

Article Snippet: The sections were incubated with blocking buffer for 1 hour, primary antibody against TRIM28 (Abcam, #ab10484, RRID: AB_297223), SETDB1 (Biological Novus, #NBP2-20322) or PD-L1 (Cell Signaling Technology, #13684S, RRID:AB_2687655) at 4 C overnight and secondary antibody for 1 hour (Dako, #K4011).

Techniques: Two Tailed Test, Cytometry