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: Physiological Reports

Article Title: Acute tuft cell ablation in mice induces malabsorption and alterations in secretory and immune cell lineages in the small intestine

doi: 10.14814/phy2.70264

Figure Lengend Snippet: Primary antibody list.

Article Snippet: GIP , Rabbit poly , Unconjugated , Novus , NBP3‐04865 , 1:100.

Techniques: Conjugation Assay

Journal: Nature Communications

Article Title: Synaptic proteome diversity is shaped by the levels of glutamate receptors and their regulatory proteins

doi: 10.1038/s41467-025-65490-9

Figure Lengend Snippet: a Workflow and bioinformatic tools used to establish the proteome of synapses from the trisynaptic loop. b . Overlap between proteins from the trisynaptic loop and two reference proteomes. Proteome I, this study. Proteome II, PSDII from Distler et al. . c Sunburst plot of SynGO Cellular Component terms enriched among synaptic proteins from the trisynaptic loop. d –f Relative abundances of GluA2 (d, left), Vamp1 ( d , right), Shisa6 ( e , left), Prkar2a ( e , right), mGluR2 ( f , left) and Ptprd ( f , right) in synaptic fractions from CA1, CA3 and DG. Representative immunoblots shown. Bars = mea n ± SEM. Statistical test, one-way ANOVA, post-hoc Fisher’s LSD test, sample size (n, biological replicates) for GluA2, 8; Vamp1, 8; Shisa6, 8; Prkar2a, 8; mGluR2, 12 and Ptprd 16. * p < 0.05, *** p < 0.001, **** p < 0.0001. Grm2 dimer band, the most abundant form, was analyzed. g –l Left: Representative double immunofluorescences of the postsynaptic marker Psd95 (in red) with Homer2 ( g ), Calcineurin (Ppp3ca, h ), Homer3 ( i ), Band 4.1-like protein 1 (Epb41l1, k ) and Mpp2 ( l ) and the presynaptic marker vGlut1 (in red) with Synaptoporin (Synpr, j ). Right: Quantification of overlapping signal (see also Suppl. Figs. – ): Homer2 (g), Ppp3ca ( h ), Homer3 ( i ), Synpr ( j ), Epb41l1 ( k ) and Mpp2 ( l ). Bars = mea n ± SEM. Statistical test, one-way ANOVA, post-hoc Fisher’s LSD test, sample size (n, biological replicates) for Homer2, 10; Ppp3ca, 22; Homer3, 22; Synpr, 9; Epb41l1, 10 and Mpp2, 20. **** p < 0.0001. m–o Percentages of differentially expressed (DE) proteins in CA3–CA1 ( m ), DG–CA3 ( n ), and EC–DG ( o ) synapses with concordant or discordant in situ hybridization (ISH) levels. p–r DE proteins with increased scRNA-seq levels: CA3–CA1 synapses, in CA3do or CA1do neurons ( p , blue); DG–CA3 synapses, in DG or CA3do neurons ( q , green); EC–DG synapses, in EC or DG neurons ( r , orange). Source data are provided as a Source Data file.

Article Snippet: Primary antibodies used: Psd95 (#3450; Cell Signaling, [RRID:AB_2292883]); Synaptophysin (Ab8049; Abcam [SY38], [RRID:AB_2198854]); GluA2 (MAB397; Millipore [RRID:AB_2113875]; Shisa6 (NBP2-85726; Novus Biologicals [RRID:AB_3427376]); mGluR2 (# 191 103; Synaptic Systems [RRID:AB_2232859]; Prkar2a (ab32514; Abcam [RRID:AB_777289]); Ptprd (NBP2-94767; Novus Biologicals [RRID:AB_3464681]) and Vamp1 (#13151; Cell Signaling [RRID:AB_2798132]).

Techniques: Western Blot, Marker, In Situ Hybridization