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-β-tubulin (E7) (mouse), 1:3000 , Developmental studies hybridoma bank , clone E7, RRID: AB_2315513.

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-β-tubulin (E7) (mouse), 1:3000 , Developmental studies hybridoma bank , clone E7, RRID: AB_2315513.

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-β-tubulin (E7) (mouse), 1:3000 , Developmental studies hybridoma bank , clone E7, RRID: AB_2315513.

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 ) 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-β-tubulin (E7) (mouse), 1:3000 , Developmental studies hybridoma bank , clone E7, RRID: AB_2315513.

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 ) Experimental scheme to identify cellular targets of CCW16. Whole HeLa WT cell lysate was incubated with biotin [10 µM] or biotin-CCW16 [10 µM], followed by streptavidin pulldown to enrich for biotin-CCW16 modified proteins, on-bead digestion and quantitative MS analysis. ( B ) All detectable proteins from the mass spectrometry experiment in ( A ) which were modified by biotin-CCW16. Gene names and respective modified cysteine residues are indicated. CCW16 target proteins were defined by two-sided Student’s t -test analysis comparing LFQ intensities of biotin-CCW16 pulldown with the respective biotin control pulldown. Experiments were performed as biological triplicates. ( C ) MS Spectrum of biotin-CCW16 modified PRDX1. ( D ) Validation of streptavidin pulldown results by immunoblotting in HeLa WT cells. The same treatment procedure as in ( A ). Tubulin was used as loading control. FT flow through; PD pulldown. .

Article Snippet: anti-β-tubulin (E7) (mouse), 1:3000 , Developmental studies hybridoma bank , clone E7, RRID: AB_2315513.

Techniques: Incubation, Modification, Mass Spectrometry, Control, Biomarker Discovery, Western Blot

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-β-tubulin (E7) (mouse), 1:3000 , Developmental studies hybridoma bank , clone E7, RRID: AB_2315513.

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

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 OCI-AML2 with ferrostatin-1 for 1 h [10 µM], followed by treatment with EN219 [1 µM], CCW16 [1 µM], or RSL3 [10 µM] for 6 h and measurement of cell viability by CellTiterGlo. Error bars show the standard deviation of the mean, n = 3 (biological replicates). ( B ) HeLa WT cells were treated either with EN219 [5 µM] or CCW16 [5 µM] for 6 h and analyzed by immunoblotting. The experiment was performed with three biological replicates. Tubulin was used as loading control. ( C ) Quantification of HMOX1 levels shown in ( B ) normalized to tubulin loading control and DMSO control treatment. 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). CCW16: p = 0.0102, EN219: p = 0.0018. ( D ) HT-1080 cells stably overexpressing an empty vector or GPX4 WT (GPX4 OE) were treated for 6 h with CCW16/CCW16-based PROTACs [5 µM] or RSL3 as a positive control [1 µM] after treatment with ferrostatin-1 [10 µM] for 1 h. Cell viability was measured by CellTiterGlo. Error bars show the standard deviation of the mean, n = 3 (biological replicates). OE overexpression. .

Article Snippet: anti-β-tubulin (E7) (mouse), 1:3000 , Developmental studies hybridoma bank , clone E7, RRID: AB_2315513.

Techniques: Standard Deviation, Western Blot, Control, Two Tailed Test, Stable Transfection, Plasmid Preparation, Positive Control, Over Expression

Journal: bioRxiv

Article Title: Separable roles for Microprocessor and its cofactors ERH and SAFB1/2 during microRNA cluster assistance

doi: 10.1101/2025.09.09.675111

Figure Lengend Snippet: (A) Deletion and/or depletion of ERH and SAFB1/2 in HEK293T cells. ERH mutant cells were generated by shRNA-mediated knockdown in an ERH[+/-] heterozygous cell line. SAFB1/2 double mutant cells were generated by knockdown of SAFB1 in a SAFB1[+/-], SAFB2[-/-] cell line. ERH+SAFB1/2 triple mutant cells were generated by knockdown of ERH and SAFB1 in an ERH[+/-]; SAFB1[+/-], SAFB2[-/-] cell line. Western blot validates strong loss of ERH and SAFB1 proteins and absence of SAFB2 in the respective mutant genotypes. β-tubulin was probed as control. (B) Northern blotting to assay processing of mir-144/451 cluster or solo mir-451 in wildtype or cofactor depleted HEK293T cells. Co-transfected mir-375 and endogenous let-7a and U6 snRNAs were probed as controls. RNA size markers (nt) are shown at left. Microprocessing of suboptimal pri-mir-451 , but not optimal pri-mir-144 and pri-mir-375 , is significantly impaired in ERH and/or SAFB1/2 mutant cells. However, lack of the neighboring optimal mir-144 hairpin ablated pri-mir-451 processing, even with all the cofactors present. (C) Rescue of pri-mir-451 processing in mutant cells. While ERH overexpression can restore mir-451 biogenesis in ERH mutant cells, only SAFB2 overexpression restored mir-451 biogenesis in SAFB1/2 and ERH+SAFB1/2 mutant cells.

Article Snippet: The blots were probed for 2 hrs at room temperature or overnight at 4°C with Rabbit polyclonal anti-SAFB1 antibody (Cat #A300-812A) diluted to 1:2000, or Rabbit polyclonal anti-SAFB2 antibody (Cat #A301-112A) diluted to 1:2000, or Rabbit polyclonal anti-ERH antibody (Cat #PA5-21388) diluted to 1:2000, or mouse monoclonal anti-β-tubulin antibodies (DSHB) diluted to 1:2000, and then incubated with a secondary antibody conjugated to horseradish peroxidase diluted to 1:5000.

Techniques: Mutagenesis, Generated, shRNA, Knockdown, Western Blot, Control, Northern Blot, Transfection, Over Expression