Journal: EMBO Reports

Article Title: Cysteine-reactive covalent chloro- N -acetamide ligands induce ferroptosis mediated cell death

doi: 10.1038/s44319-025-00593-4

Figure Lengend Snippet: ( A ) AML cells exhibit an elevated expression of RNF4 (derived from TNMplot.com; based on TGCA data). P value of Mann–Whitney U -test is indicated ( p = 2.22e-21); n = 407 (normal), n = 151 (tumor). Normal: Minima = 77, Maxima = 10,063, Median = 1009, Q1 = 675, Q3 = 1533, Upper whisker = 2726; Tumor: Minima = 691, Maxima = 10,010, Median = 1640, Q1 = 1283, Q3 = 2317.5, Upper whisker = 3750. Dashed lines: 25th and 75th percentile; solid line: 50th percentile (median); Whiskers: range within 1.5×IQR from Q1/Q3. ( B ) Effect of RNF4 expression level on AML patient survival. High expression of RNF4 is associated with a poor prognosis (analyzed using UALCAN). P value of Kaplan–Meier analysis is indicated ( p = 0.041); n = 42 (high expression), n = 121 (low expression). ( C , D ) RNF4 CRISPR dropout experiment in Cas9-expressing OCI-AML3 ( C ) or MV4-11 ( D ) cells. The transduction efficiency of PE-positive cells was around 50%. Survival rate was measured by flow cytometry and normalized to the empty vector control on day 3. Error bars show the standard deviation of the mean, n = 3 (biological replicates). ( E ) Cell viability assay of OCI-AML2 cells 6 days after RNF4 KD with siRNA in combination with decitabine (5-azadC) [1 μM] or aphidicolin [0.075 μg/ml]. P values of two-tailed, unpaired Student’s t -tests are indicated; error bars show the standard deviation of the mean, n = 8 (biological replicates, each three technical replicates). DMSO: p = 0.1754, Decitabine: p < 0.0001, Aphidicolin: p = 0.0532. ( F ) Viability assay of OCI-AML2 cells after RNF4 KD with siRNA in combination with different decitabine concentrations to measure dose-response dependency. IC 50 values were determined by a nonlinear dose-response-inhibition fit, n = 2 (biological replicates). .

Article Snippet: anti-RNF4 (rabbit), polyclonal, 1:2000 , Proteintech , Cat# 17810-1-AP; RRID: AB_2878443.

Techniques: Expressing, Derivative Assay, MANN-WHITNEY, Whisker Assay, CRISPR, Transduction, Flow Cytometry, Plasmid Preparation, Control, Standard Deviation, Viability Assay, Two Tailed Test, Inhibition

Journal: EMBO Reports

Article Title: Cysteine-reactive covalent chloro- N -acetamide ligands induce ferroptosis mediated cell death

doi: 10.1038/s44319-025-00593-4

Figure Lengend Snippet: ( A , B ) RNF4 CRISPR dropout experiment in Cas9-expressing OCI-AML3 ( A ) or MV4-11 ( B ) cells. The transduction efficiency of PE-positive cells was around 50%. Survival rate was measured by flow cytometry and normalized to the empty vector control on day 3. Error bars show the standard deviation of the mean, n = 3 (biological replicates). ( C , D ) Confirmation of RNF4 KO in OCI-AML3 ( C ) and MV4-11 ( D ) Cas9-expressing cells after transduction with three different guideRNAs by immunoblotting. Cells were transduced and medium was exchanged after 1 day, and supplemented with 2.5 µg/ml puromycin. After 3 days (OCI-AML3) and 4 days (MV4-11), cells were harvested. Tubulin was used as loading control. ( E ) Validation of RNF4 KD corresponding to Fig. (Rep1–Rep8) and 1 F (Rep1–Rep2) by immunoblotting. Cells were harvested 3 days after the performance of KD. Tubulin was used as loading control. Quantification of the RNF4 signal, normalized to tubulin, is indicated.

Article Snippet: anti-RNF4 (rabbit), polyclonal, 1:2000 , Proteintech , Cat# 17810-1-AP; RRID: AB_2878443.

Techniques: CRISPR, Expressing, Transduction, Flow Cytometry, Plasmid Preparation, Control, Standard Deviation, Western Blot, Biomarker Discovery

Journal: EMBO Reports

Article Title: Cysteine-reactive covalent chloro- N -acetamide ligands induce ferroptosis mediated cell death

doi: 10.1038/s44319-025-00593-4

Figure Lengend Snippet: ( A ) The RNF4 binder CCW16 (left panel) was utilized for PROTAC development using diverse linkers (central panel) as well as VHL and CRBN E3 ligands (right panel). ( B ) NanoBRET assay for CRBN and VHL E3 ligand-based RNF4 PROTACs to determine cell membrane permeability in intact cells transiently expressing CRBN (left panel) or VHL (right panel) with an N-terminally tagged NanoLuc. Error bars show the standard deviation of the mean, n = 4 (biological replicates). ( C ) Treatment of HeLa WT cells with different RNF4 targeting PROTACs and evaluation of RNF4 degradation level by immunoblotting. Cells were treated 30 min before PROTAC treatment [5 µM] with MG-132 [20 µM], TAK-243 [1 µM], or MLN4924 [500 nM] and harvested after 6 h. Control cells were treated with DMSO. Tubulin was used as loading control. Same experiment is also shown in Fig. . ( D ) HeLa Flag-RNF4 endogenously tagged cells were transfected with His-Ub for 48 h and pretreated with TAK-243 [1 µM], followed by PROTAC 2c [5 µM] or CCW16 [5 µM] treatment for 6 h. Enrichment of ubiquitylated proteins was performed by denaturing Ni-NTA pulldown. Higher molecular weight bands show ubiquitylation signal. Control cells were treated with DMSO. HA-pulldown was performed as a negative control. ( E ) Whole cell proteome analysis by mass spectrometry. HeLa cells expressing RNF4 from a doxycycline-inducible promoter were treated either with CCW16 [5 µM] (upper panel) or 2c [5 µM] (lower panel) for 6 h. Results of the TMT-based MS analysis are visualized in a volcano plot comparing PROTAC treatment vs. DMSO control. Hits considered as significantly upregulated are highlighted in red (log 2 (ratio) ≥ 0.58, –log 10 ( p value) ≥ 1.3) and hits considered as significantly downregulated are highlighted in blue (log 2 (ratio) ≤ −0.58, −log 10 ( p value) ≥ 1.3). The identification of those candidates was based on a two-sided Student’s t -test analysis comparing the normalized TMT abundances of CCW16/ 2c treatment with DMSO control treatment. Experiments were performed with four biological replicates. ( F ) Validation of proteomic results by immunoblotting in HeLa WT and HeLa RNF4 KO cells. Same treatment procedure as in ( E ). Tubulin was used as loading control. .

Article Snippet: anti-RNF4 (rabbit), polyclonal, 1:2000 , Proteintech , Cat# 17810-1-AP; RRID: AB_2878443.

Techniques: Membrane, Permeability, Expressing, Standard Deviation, Western Blot, Control, Transfection, Molecular Weight, Negative Control, Mass Spectrometry, Biomarker Discovery

Journal: EMBO Reports

Article Title: Cysteine-reactive covalent chloro- N -acetamide ligands induce ferroptosis mediated cell death

doi: 10.1038/s44319-025-00593-4

Figure Lengend Snippet: ( A ) NanoBRET assay for PROTAC 2b to determine cell membrane permeability in intact and permeabilized cells transiently expressing VHL with an N-terminally tagged NanoLuc. Error bars show the standard deviation of the mean, n = 4 (biological replicates). ( B ) NanoBRET assay for CRBN and VHL E3 ligand-based RNF4 PROTACs to determine cell membrane permeability. IC 50 values of Figs. and EV2A are indicated. ( C ) HeLa WT cells were depleted of RNF4 (siRNF4) or SENP6 (siSENP6) for 72 h or were treated with CCW16-derived PROTACs [5 µM] for 6 h. Control cells were treated with DMSO. RNF4 and SENP6 levels were visualized by immunoblotting. Tubulin was used as loading control. ( D ) Treatment of HeLa FLAG-RNF4 (endogenously tagged) cells with different RNF4 targeting PROTACs and evaluation of RNF4 degradation level by immunoblotting. Different concentrations and time points are indicated. Control cells were treated with DMSO. Tubulin was used as loading control. ( E ) Evaluation of RNF4 degradation level by immunoblotting in NB-4 cells after treatment with different RNF4-targeting PROTACs. Control cells were treated with DMSO. Vinculin was used as loading control. ( F ) Treatment of OCI-AML2 cells with different RNF4 targeting PROTACs and evaluation of RNF4 degradation level by immunoblotting. Cells were pretreated with MG-132 [20 µM], TAK-243 [1 µM], or MLN4924 [500 nM] 30 min before PROTAC treatment [5 µM] and harvested after 6 h. Control cells were treated with DMSO. Tubulin was used as loading control. ( G ) Evaluation of CRBN and VHL levels in HeLa WT cells by immunoblotting after pretreating cells with MG-132 [20 µM], TAK-243 [1 µM], or MLN4924 [500 nM] 30 min before PROTAC treatment [5 µM], followed by harvesting after 6 h. DMSO was used as a control treatment, and tubulin as loading control. Same experiment is also shown in Fig. . ( H ) Whole cell proteome analysis by mass spectrometry. HeLa cells expressing RNF4 from a doxycycline-inducible promoter were treated with 1a [5 µM] for 6 h (same experiment as in Fig. ). Results of the TMT-based MS analysis are visualized in a volcano plot comparing PROTAC treatment vs. DMSO control. Hits considered as significantly upregulated are highlighted in red (log 2 (ratio) ≥ 0.58, –log 10 ( p value) ≥ 1.3) and hits considered as significant downregulated are highlighted in blue (log 2 (ratio) ≤ −0.58, −log 10 ( p value) ≥ 1.3). The identification of those candidates was based on two-sided Student’s t -test analysis comparing the normalized TMT abundances of 1a treatment with DMSO control treatment. Experiments were performed with four biological replicates.

Article Snippet: anti-RNF4 (rabbit), polyclonal, 1:2000 , Proteintech , Cat# 17810-1-AP; RRID: AB_2878443.

Techniques: Membrane, Permeability, Expressing, Standard Deviation, Derivative Assay, Control, Western Blot, Mass Spectrometry

Journal: EMBO Reports

Article Title: Cysteine-reactive covalent chloro- N -acetamide ligands induce ferroptosis mediated cell death

doi: 10.1038/s44319-025-00593-4

Figure Lengend Snippet: ( A ) Scheme outlining in vitro interaction studies of biotin-CCW16 and GST-based purified RNF4. Biotin-CCW16 modification of RNF4 was visualized by immunoblotting with a streptavidin protein linked to a fluorophore. ( B ) Biotin or biotinylated CCW16 was incubated with wild-type GST-RNF4 or mutants with cysteine residues changed to serines as indicated. Covalent biotin-CCW16-RNF4 conjugates were detected by streptavidin immunoblotting. ( C ) Experimental procedure of in vitro interaction studies of CCW16 or PROTAC 2a with GST-RNF4 WT to identify modified cysteine residues in RNF4 by mass spectrometry. The experiment was performed with three replicates. ( D ) AlphaFold2 (Jumper et al, ; Varadi et al, , ) model of human RNF4, highlighting the CCW16 modified cysteine residue identified in the mass spectrometry experiment in ( C ) in pink, the two catalytic, but unmodified cysteine residues are shown in blue, the not detectable cysteine residue 51 is shown in gray and all remaining cysteine residues are highlighted in green. ( E , F ) Same procedure as in ( B ). GST-RNF4 cysteine to serine mutations are indicated. Covalent biotin-CCW16-RNF4 conjugates were detected by streptavidin immunoblotting. .

Article Snippet: anti-RNF4 (rabbit), polyclonal, 1:2000 , Proteintech , Cat# 17810-1-AP; RRID: AB_2878443.

Techniques: In Vitro, Purification, Modification, Western Blot, Incubation, Mass Spectrometry, Residue

Journal: EMBO Reports

Article Title: Cysteine-reactive covalent chloro- N -acetamide ligands induce ferroptosis mediated cell death

doi: 10.1038/s44319-025-00593-4

Figure Lengend Snippet: ( A ) MS spectrum of CCW16 modified GST-RNF4 on cysteine residue 91, corresponding to Fig. C, . ( B ) Detected RNF4 peptides including the respective modifications on different cysteine residues (as indicated), corresponding to Fig. C, . ( C ) MS spectrum of 2a modified GST-RNF4 on cysteine residue 91, corresponding to Fig. C, .

Article Snippet: anti-RNF4 (rabbit), polyclonal, 1:2000 , Proteintech , Cat# 17810-1-AP; RRID: AB_2878443.

Techniques: Modification, Residue

Journal: EMBO Reports

Article Title: Cysteine-reactive covalent chloro- N -acetamide ligands induce ferroptosis mediated cell death

doi: 10.1038/s44319-025-00593-4

Figure Lengend Snippet: ( A ) Pretreatment of HeLa WT cells with MG-132 [20 µM] 30 min before treatment with CCW28-3 [10 µM] or dBET6 [500 nM] and incubation for 6 h (left immunoblot). Pretreatment of HeLa WT cells or HeLa RNF4 KO cells with MG-132 [20 µM] 30 min before treatment with CCW28-3 [10 µM] or dBET6 [500 nM] and incubation for 6 h (right immunoblot). Both blots show the same experiment. Tubulin was used as loading control. *Unspecific band. ( B ) Quantification of BRD4 levels (short and long isoform) after CCW28-3 treatment in HeLa WT and HeLa RNF4 KO cells. Experiment was performed in three independent replicates. P values of two-tailed, unpaired Student’s t -tests are indicated; error bars show the standard deviation of the mean, n = 3 (biological replicates). Short isoform: WT CCW28-3 vs. DMSO: p < 0.0001, RNF4 KO CCW28-3 vs. DMSO: p = 0.0235, CCW28-3 KO vs. WT: p = 0.1343. Long isoform: WT CCW28-3 vs. DMSO: p = 0.0019, RNF4 KO CCW28-3 vs. DMSO: p = 0.0202, CCW28-3 KO vs. WT: p = 0.4538. ( C ) Treatment of HeLa WT cells with MG-132 [20 µM] or MLN4924 [500 nM] for 30 min followed by treatment with CCW28-3 [10 µM] or dBET6 [500 nM] for 6 h and evaluation by immunoblotting. Tubulin was used as loading control. *Unspecific band. ( D , E ) Measurement of BRD4 levels based on luciferase. Control KD (siControl, ( D )) and RNF4 KD (siRNF4, ( E )) were performed in HEK BRD4-HiBiT cells for 72 h, followed by treatment with different concentrations of CCW16 (upper panel) or CCW28-3 (lower panel) for 4, 6, and 24 h. Luciferase activity was measured by the addition of the large luciferase fragment (largeBiT) and substrate. Error bars show the standard deviation of the mean, n = 4 (technical replicates). ( F ) Confirmation of KD efficiency 3 days after performance of KD of Fig. EV4D,E by immunoblotting. ( G ) Evaluation of cell viability after CCW28-3 treatment in HEK BRD4-HiBiT cell lines by CellTiterGlo assay ( n = 2, biological replicates). Concentrations and time points are indicated.

Article Snippet: anti-RNF4 (rabbit), polyclonal, 1:2000 , Proteintech , Cat# 17810-1-AP; RRID: AB_2878443.

Techniques: Incubation, Western Blot, Control, Two Tailed Test, Standard Deviation, Luciferase, Activity Assay

Journal: EMBO Reports

Article Title: Cysteine-reactive covalent chloro- N -acetamide ligands induce ferroptosis mediated cell death

doi: 10.1038/s44319-025-00593-4

Figure Lengend Snippet: ( A ) Volcano plot of quantitative MS analysis after biotin-CCW16 pulldown of HeLa WT cell lysates. Significantly enriched interactors are shown in red (log 2 (ratio) ≥ 1, –log 10 ( p value) ≥ 1.3). Identification of candidates is based on two-sided Student’s t -test analysis comparing LFQ intensities of biotin-CCW16 pulldown and biotin control pulldown. Experiment was performed in biological triplicates. Proteins involved in the reduction of peroxides are additionally highlighted. ( B ) Gene Ontology term enrichment analysis of biological processes (GOBP) of the 38 biotin-CCW16 modified proteins (from Fig. ) identified by MS (log 2 (ratio) ≥ 1, –log 10 ( p value) ≥ 1.3). Shown here are the top ten enriched biological processes. The enrichment analysis was done using the ShinyGO tool, applying an FDR cutoff of 0.05. ( C ) RNF4 immunoblotting of streptavidin pulldown in HeLa WT cells. Same experiment as in Fig. . Tubulin was used as loading control. FT flow through, PD pulldown, h.e. high exposure. ( D ) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of significantly upregulated proteins (from Fig. ) identified by MS (log 2 (ratio) ≥ 0.58, –log 10 ( p value) ≥ 1.3). Shown here are the top three enriched biological processes. The enrichment analysis was done using the ShinyGO tool, applying an FDR cutoff of 0.05. ( E ) HeLa WT or OCI-AML2 cells were pretreated with ferrostatin-1 [10 µM] followed by treatment with CCW28-3 (10 µM for HeLa WT, 1 µM for OCI-AML2) for 6 h. BRD4 levels were evaluated by immunoblotting. Control cells were treated with DMSO. Tubulin was used as loading control. *Unspecific band. ( F ) Validation of overexpression of human GPX4 WT (hGPX4) compared to empty vector (mock) in HT-1080 cells used in Fig. . β-actin was used as loading control.

Article Snippet: anti-RNF4 (rabbit), polyclonal, 1:2000 , Proteintech , Cat# 17810-1-AP; RRID: AB_2878443.

Techniques: Control, Modification, Western Blot, Biomarker Discovery, Over Expression, Plasmid Preparation

Journal: International journal of molecular sciences

Article Title: Activation of the NALP3-CASP1-IL-1 β Inflammatory Pathway by Pesticide Exposure in Human Umbilical Vein Endothelial Cells.

doi: 10.3390/ijms26104947

Figure Lengend Snippet: Figure 4. The bar graph shows the statistical analysis, where data are expressed as mean ± standard deviation (SD) from three independent experiments evidencing mRNA levels of CASP1 between ctrl cells (A) and each single pesticide b (B), py (C), pr (D), lc (E) with the other experimental points. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparison test. Significant differences compared to the control are indicated (** p < 0.01, *** p < 0.001). The experiment was conducted in triplicate.

Article Snippet: After this time, cells were washed 3 times with PB for 5 min and permeabilized with 0.5% Triton X-100 (9474680K, LKB-Produkter AB, Bromma, Sweden) in PB for 10 min; 5% skim milk in PB was added to block nonspecific for 1 h. Primary monoclonal antibodies against human NALP3 (dilution: 1:400) (sc-8422, Santa Cruz Biotechnology, Santa Cruz, CA, USA), CASP1 (dilution: 1:400) (sc-17810, Santa Cruz Biotechnology), and IL-1 β (1:400) (sc-32294, Santa Cruz Biotechnology) were diluted in 0.5% non-fat milk solution and the samples were incubated for 2 h in a humidified environment at RT in the dark.

Techniques: Standard Deviation, Comparison, Control

Journal: International journal of molecular sciences

Article Title: Activation of the NALP3-CASP1-IL-1 β Inflammatory Pathway by Pesticide Exposure in Human Umbilical Vein Endothelial Cells.

doi: 10.3390/ijms26104947

Figure Lengend Snippet: Figure 10. The figures show the expression of CASP1 analyzed by immunofluorescence microscopy per- formed in HUVEC cells cultured alone (A1), with Boscalid (A2), Pyraclostrobin (A3), Propamocarb (A4), Lamba-cyhalothrin (A5), Boscalid + Pyraclostrobin (A6), Propamocarb + Lamba-cyhalothrin (A7), Boscalid + Pyraclostrobin + Propamocarb (A8), and Boscalid + Pyraclostrobin + Lamba-cyhalothrin (A9) over a period of 10 days. The experiment was conducted three times, each independently.

Article Snippet: After this time, cells were washed 3 times with PB for 5 min and permeabilized with 0.5% Triton X-100 (9474680K, LKB-Produkter AB, Bromma, Sweden) in PB for 10 min; 5% skim milk in PB was added to block nonspecific for 1 h. Primary monoclonal antibodies against human NALP3 (dilution: 1:400) (sc-8422, Santa Cruz Biotechnology, Santa Cruz, CA, USA), CASP1 (dilution: 1:400) (sc-17810, Santa Cruz Biotechnology), and IL-1 β (1:400) (sc-32294, Santa Cruz Biotechnology) were diluted in 0.5% non-fat milk solution and the samples were incubated for 2 h in a humidified environment at RT in the dark.

Techniques: Expressing, Immunofluorescence, Microscopy, Cell Culture

Journal: International journal of molecular sciences

Article Title: Activation of the NALP3-CASP1-IL-1 β Inflammatory Pathway by Pesticide Exposure in Human Umbilical Vein Endothelial Cells.

doi: 10.3390/ijms26104947

Figure Lengend Snippet: Figure 13. The bar graph shows the statistical analysis, where data are expressed as mean ± standard deviation (SD) from three independent experiments evidencing the CASP1 fluorescence intensity between ctrl cells (A) and each single pesticide b (B), py (C), pr (D), lc (E) with the other experimental points. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparison test. Significant differences compared to the control are indicated (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: After this time, cells were washed 3 times with PB for 5 min and permeabilized with 0.5% Triton X-100 (9474680K, LKB-Produkter AB, Bromma, Sweden) in PB for 10 min; 5% skim milk in PB was added to block nonspecific for 1 h. Primary monoclonal antibodies against human NALP3 (dilution: 1:400) (sc-8422, Santa Cruz Biotechnology, Santa Cruz, CA, USA), CASP1 (dilution: 1:400) (sc-17810, Santa Cruz Biotechnology), and IL-1 β (1:400) (sc-32294, Santa Cruz Biotechnology) were diluted in 0.5% non-fat milk solution and the samples were incubated for 2 h in a humidified environment at RT in the dark.

Techniques: Standard Deviation, Fluorescence, Comparison, Control