Journal: The EMBO Journal

Article Title: Ubiquitin pathway blockade reveals endogenous ADP-ribosylation marking PARP7 and AHR for degradation

doi: 10.1038/s44318-025-00656-1

Figure Lengend Snippet: ( A ) Schematic overview of the ubiquitin-proteasome system, major PARPs and their inhibitors used in this study. Ub: ubiquitin, E1: ubiquitin-activating enzyme, E2: ubiquitin conjugating enzyme, E3: ubiquitin ligase (HECT or RING type). ( B ) HCC44 cells were treated with DMSO or 1 μM TAK243 for 4 h. ( C ) Time-course experiment with TAK243 and MG132 at 1 and 10 μM, respectively, in HCC44 cells. ADPr was assessed by western blotting using the anti-poly/mono-ADPr antibody, with α-tubulin as a loading control. .

Article Snippet: Human HCC44 cell line was purchased from the German Collection of Microorganisms and Cell Cultures (#ACC 534, DSMZ, Braunschweig, Germany).

Techniques: Ubiquitin Proteomics, Western Blot, Control

Journal: The EMBO Journal

Article Title: Ubiquitin pathway blockade reveals endogenous ADP-ribosylation marking PARP7 and AHR for degradation

doi: 10.1038/s44318-025-00656-1

Figure Lengend Snippet: ( A ) HCC44 cells pre-treated with DMSO or PARP inhibitors for 20 h were further treated with 1 µM TAK243 for 4 h. ADPr was assessed by western blotting using the anti-poly/mono-ADPr antibody, with α-tubulin as a loading control. ( B ) HiBiT-PARP7 CT26 cells pre-treated with DMSO or PARP inhibitors for 20 h were further treated with 1 µM TAK243 for 4 h. ADPr was assessed by western blotting using the anti-poly/mono-ADPr antibody, with α-tubulin as a loading control. Overlap of the signals was obtained using fluorescently labelled anti-rabbit (poly/mono-ADPr, green) and anti-mouse (HiBiT, red) secondary antibodies. ( C ) HiBiT luminescence assay in HiBiT-PARP7 CT26 cells with 1 µM TAK243, 10 µM MG132, 100 nM RBN2397 (PARP7i) treatments for 4 h showing induction of PARP7 levels (HiBiT luminescence). Wild-type CT26 cells (non-HiBiT) were used as a negative control. Data are shown as mean ± s.e.m. of n = 3 technical replicates. ( D ) A549 cells were co-treated with PARP inhibitors and 1 µM TAK243 for 24 h. PARP inhibitor concentrations in panels (A– C ) are specified in the methods section. ADPr was assessed by western blotting using the anti-mono-ADPr antibody. α-tubulin was used as a loading control. The following PARP inhibitors were used: PARP1/2 (1 µM olaparib), PARP7 (100 nM RBN2397), PARP14 (RBN012759, 200 nM in Fig. 2A, B and 500 nM in Fig. 2D), TNKS (1 µM AZ6102). PARP14, PARP7, TNKS and PARP1 were detected with corresponding antibodies in relevant panels. .

Article Snippet: Human HCC44 cell line was purchased from the German Collection of Microorganisms and Cell Cultures (#ACC 534, DSMZ, Braunschweig, Germany).

Techniques: Western Blot, Control, Luminescence Assay, Negative Control

Journal: The EMBO Journal

Article Title: Ubiquitin pathway blockade reveals endogenous ADP-ribosylation marking PARP7 and AHR for degradation

doi: 10.1038/s44318-025-00656-1

Figure Lengend Snippet: ( A ) Schematic representation of AHR activation and degradation. Tapinarof is an agonist of AHR. ( B ) HCC44 cells were pre-treated with DMSO, 1 µM tapinarof and PARP inhibitors (PARP1/2 (1 µM olaparib), PARP7 (100 nM RBN2397), PARP14 (200 nM RBN012759), TNKS (1 µM AZ6102)) for 20 h followed by an additional 4 h of 1 µM TAK243 or 10 µM MG132 treatment. ( C ) MCF7 wild type (WT) and PARP7 knockout (KO) cells were treated as in ( B ). ADPr was assessed by western blotting using the anti-poly/mono-ADPr antibody. AHR, PARP7, TNKS and PARP14 were detected using corresponding antibodies with α-tubulin as a loading control. .

Article Snippet: Human HCC44 cell line was purchased from the German Collection of Microorganisms and Cell Cultures (#ACC 534, DSMZ, Braunschweig, Germany).

Techniques: Activation Assay, Knock-Out, Western Blot, Control

Journal: The EMBO Journal

Article Title: Ubiquitin pathway blockade reveals endogenous ADP-ribosylation marking PARP7 and AHR for degradation

doi: 10.1038/s44318-025-00656-1

Figure Lengend Snippet: ( A ) HCC44 cells were transfected with siRNAs against DELTEX E3 ligases and other ADP-ribose-binding E3 ligases, treated with DMSO or 1 µM tapinarof for 24 h, and analysed by western blotting. ( B ) HCC44 cells transfected with two separate DTX2 siRNAs, treated with DMSO or 1 µM tapinarof for 24 h, and analysed by western blotting. ( C ) A549 cells transfected with two separate DTX2 siRNAs, treated with DMSO or 1 µM tapinarof for 24 h, and analysed by western blotting. ADPr was assessed with a poly-mono-ADPr antibody, DTX2 knockdown was confirmed with an anti-DTX2 antibody, α-tubulin was used as a loading control. Overlap of the signals was obtained using fluorescently labelled anti-rabbit (poly/mono-ADPr, green) and anti-mouse (AHR, red) secondary antibodies. .

Article Snippet: Human HCC44 cell line was purchased from the German Collection of Microorganisms and Cell Cultures (#ACC 534, DSMZ, Braunschweig, Germany).

Techniques: Transfection, Binding Assay, Western Blot, Knockdown, Control

Journal: The EMBO Journal

Article Title: Ubiquitin pathway blockade reveals endogenous ADP-ribosylation marking PARP7 and AHR for degradation

doi: 10.1038/s44318-025-00656-1

Figure Lengend Snippet: ( A ) Widefield images showing HCC44 cells treated with 1 µM tapinarof (24 h), 1 µM TAK243 (4 h) and 100 nM PARP7i (RBN2397, 24 h) alone and in combination as in Fig. . Cells were stained with Hoechst (blue), ADPr (Poly/mono-ADP-ribose, green) and AHR (magenta). ( B , C ) Quantification of nuclear ADPr ( B ) or AHR ( C ) from ( A ). Between 5156 and 6220 cells were measured in each condition. ( D ) Widefield images showing A549 WT or DTX2 knockout (KO) cells treated with 1 μM tapinarof (24 h), 1 μM TAK243 (4 h) and 100 nM PARP7i (RBN2397, 24 h) alone and in combination as in Fig. . Cells were stained with Hoechst (blue), ADPr (poly/mono-ADP-ribose, green) and AHR (magenta). ( E , F ) Quantification of nuclear ADPr ( E ) or AHR ( F ) from ( D ). Between 5716 and 13282 cells were measured in each condition. ( G ) Widefield images showing CT26 (HiBiT-PARP7) cells treated with 1 μM TAK243 (4 h) and 100 nM PARP7i (RBN2397, 24 h) alone and in combination, as in Fig. . Cells were stained with Hoechst (blue), ADPr (poly/mono-ADP-ribose, green) and HiBiT (magenta). ( H , I ) Quantification of nuclear ADPr ( B ) or HiBiT ( I ) from ( G ). Between 2991 and 6701 cells were measured in each condition. For all images, scale bar = 20 μm. Statistical analysis was performed using an ordinary one-way ANOVA. Asterisks indicate statistical significance (**** P < 0.0001). Red bars indicate the median for each condition. .

Article Snippet: Human HCC44 cell line was purchased from the German Collection of Microorganisms and Cell Cultures (#ACC 534, DSMZ, Braunschweig, Germany).

Techniques: Staining, Knock-Out

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: CRISPR screens and quantitative proteomics reveal remodeling of the aryl hydrocarbon receptor-driven proteome through PARP7 activity.

doi: 10.1073/pnas.2424985122

Figure Lengend Snippet: Fig. 1. A genome-wide CRISPR synthetic lethality screen with a panel of lung cancer cell lines identifies PARP7 inhibitor resistance hits. (A) Cell viability of selected cell lines treated with a range of concentrations of RBN2397 for 6 d measured in a CellTiter-Glo® assay. Data are shown as mean ± SEM of n = 3 biological replicates. (B) Schematic representation of the CRISPR screen workflow. (C) Overlap of resistance hits identified from the screen and compared to published studies (Dataset S4). The union of significant hits across the two timepoints (day 11 and 18) was used for each cell line of the screen. (D) Resistance hits identified from the genome-wide CRISPR screen in HCC44, SKMES1, and H838 cells with FDR < 0.1, shown as log2 fold-change of RBN2397 treatment relative to DMSO. PARP7 (TIPARP) is shown in green.

Article Snippet: HCC44 cell line was from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany).

Techniques: Genome Wide, CRISPR, Glo Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: CRISPR screens and quantitative proteomics reveal remodeling of the aryl hydrocarbon receptor-driven proteome through PARP7 activity.

doi: 10.1073/pnas.2424985122

Figure Lengend Snippet: Fig. 2. Whole-proteome changes induced by PARP7 inhibition and AHR agonist and antagonist. (A) Cell viability of HCC44 cells treated with a range of concentrations of RBN2397 with or without 1 μM CH223191 or 1 μM tapinarof for 6 d, measured in a CellTiter-Glo® assay. Data are shown as mean ± SEM of n = 3 biological replicates (2 technical replicates each). (B) TMT 18-plex quantitative proteomics workflow. (C–E) Quantitative proteomics (TMT 18-plex) volcano plots showing significantly upregulated (red) and downregulated (blue) proteins in HCC44 cells treated with 1 μM tapinarof, 1 μM RBN2397, and a combination of RBN2397 and tapinarof (both at 1 μM), respectively. (F) Western blot validation of up- and downregulated hits from proteomics experiments in panels C–E in HCC44 cells.

Article Snippet: HCC44 cell line was from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany).

Techniques: Inhibition, Glo Assay, Quantitative Proteomics, Western Blot, Biomarker Discovery

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: CRISPR screens and quantitative proteomics reveal remodeling of the aryl hydrocarbon receptor-driven proteome through PARP7 activity.

doi: 10.1073/pnas.2424985122

Figure Lengend Snippet: Fig. 3. A genome-wide CRISPR synthetic lethality screen identi- fies SOCS3 as a conserved PARP7 inhibitor sensitivity hit. (A) Overlap of sensitivity hits identified from the screen and compared to published studies (Dataset S4). The union of significant hits across the two time- points (day 11 and 18) was used for each cell line of the screen. (B) Sensitivity hits identified from the genome-wide CRISPR screen in HCC44, SKMES1, and H838 cells with FDR < 0.1, shown as log2 fold-change of RBN2397 treatment relative to DMSO. (C) Western blotting analy- sis of SOCS3 knockout HCC44 cell lines compared to wild-type, treat- ed with or without 1 µM RBN2397. STAT3 phosphorylation was detect- ed with a phospho-STAT3-specific antibody. Western blot signal was quantified using Li-COR Odyssey ImageStudio software and used to calculate the pSTAT3/STAT3 ratio. Data are shown as mean ± SEM of n = 3 biological replicates, **P < 0.01. α-tubulin was used as a load- ing control. (D) CellTiter-Glo® assay on WT and SOCS3 knockout HCC44 cells. Cells were treated with a range of concentrations of RBN2397 for 6 d. Data are shown as mean ± SEM of n = 4 biological replicates (with 2 technical replicates each).

Article Snippet: HCC44 cell line was from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany).

Techniques: Genome Wide, CRISPR, Western Blot, Knock-Out, Phospho-proteomics, Software, Control, Glo Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: CRISPR screens and quantitative proteomics reveal remodeling of the aryl hydrocarbon receptor-driven proteome through PARP7 activity.

doi: 10.1073/pnas.2424985122

Figure Lengend Snippet: Fig. 4. SOCS3 knockout boosts IFN- and AHR-regulated proteins upon PARP7 inhibition. (A) Vol- cano plot showing significantly upregulated (red) and downreg- ulated (blue) proteins in SOCS3 KO compared to wild-type HCC44 cells. (B) Heatmap of proteomic changes to IFN-regulated pro- teins in SOCS3 knockouts com- pared to wild-type HCC44 cells with and without 1 µM RBN2397 treatment for 24 h. (C) Western blotting analysis of AHR and SOCS3 levels in HCC44 cells treated with 1 µM RBN2397 with or without 1 µM CH223191 and 1 µM tapinarof. Western blot sig- nal was quantified using Li-COR Odyssey ImageStudio software. Data are shown as mean ± SEM of n = 3 biological replicates, *P < 0.05. α-tubulin was used as a loading control. (D) Western blotting analysis of AHR levels in wild-type and SOCS3 KO HCC44 cells treated with RBN2397 or DMSO control. Western blot sig- nal was quantified using Li-COR Odyssey ImageStudio software. Data are shown as mean ± SEM of n = 3 biological replicates, **P < 0.01. α-tubulin was used as a loading control. (E) Heatmap of proteomic changes to AHR- regulated proteins (induced by the tapinarof/RBN2397 combina- tion treatment in Fig. 2F) in SOCS3 knockouts compared to wild-type HCC44 cells with and without 1 µM RBN2397 treatment for 24 h.

Article Snippet: HCC44 cell line was from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany).

Techniques: Knock-Out, Inhibition, Western Blot, Software, Control

Journal: Translational Lung Cancer Research

Article Title: Role of the epithelial-mesenchymal transition-related circular RNA, circ-10720, in non-small-cell lung cancer

doi: 10.21037/tlcr-20-920

Figure Lengend Snippet: Circ-10720 regulates VIM and affects cell morphology in NSCLC cell lines. (A) Circ-10720 and CUL2 mRNA expression in the NSCLC cell lines A549, HCC44, H23 and H1299 in comparison to the expression in the BEAS2B normal immortalized lung cell line. (B) Heat map showing the expression of 29 EMT genes (data obtained from Cancer Cell Line Encyclopedia) in the four NSCLC cell lines studied. (C) Circ-10720 and CUL2 mRNA expression in HCC44 and A549 cells transfected with a siRNA against circ-10720 in comparison with control cells. (D) Representative Western blot analysis image of VIM and CDH1 after transfecting with circ-10720 siRNA. (E) Quantification of VIM and CDH1 relative protein levels in three independent replicates after silencing with circ-10720 siRNA. (F) Expression levels of miR-1246 in cells transfected with siRNA circ-10720 in comparison with control siRNA. (G) Immunofluorescence using VIM antibody (green) in control and circ-10720 silenced cells. (H) Bright field images of control or circ-10720 silenced cells. All bar plot data represented are the average of at least three independent replicates and error bars represent SEM. *, P<0.05; **, P<0.01. NSCLC, non-small-cell lung cancer; EMT, epithelial-mesenchymal transition; VIM, Vimentin; CDH1, E-cadherin.

Article Snippet: Cryopreserved samples of the lung cancer cell lines HCC44 (ACC 534, DSMZ, Branuschweig, Germany), A549 (ACC 107, DSMZ), H23 (CRL-5800, ATTC, Manassas, VA, USA) and H1299 (CRL-5803, ATCC); and the normal immortalized lung cell line BEAS2B (95102433, ECACC, Sigma-Aldrich) were received in our laboratory and passaged for less than 6 months.

Techniques: Expressing, Comparison, Transfection, Control, Western Blot, Immunofluorescence

Journal: Translational Lung Cancer Research

Article Title: Role of the epithelial-mesenchymal transition-related circular RNA, circ-10720, in non-small-cell lung cancer

doi: 10.21037/tlcr-20-920

Figure Lengend Snippet: Circ-10720 regulates migration and invasion in NSCLC cell lines. (A) Images of one of the 3 replicates of the results of the wound healing assay for study of migration at 0, 12 and 24 hours after transfection with circ-10720 siRNA or control siRNA. The percentage included in each picture represents the wound closure percentage from 0 h. (B) Quantification of wound closure rates at different time points: 0, 6, 12 and 24 h. (C) Representative image of the matrigel-based invasion assay for HCC44 and A549 cells transfected with circ-10720 siRNA or control siRNA. (D) Quantitative analysis of invasion by dissolving crystal violet stained cells in 10% acetic acid and colorimetric reading of OD at 560 nm. Data is presented as ratio of control. All data represented in bar plots are the average of at least three independent replicates and error bars represents SEM. *, P<0.05; **, P<0.01. NSCLC, non-small cell lung cancer.

Article Snippet: Cryopreserved samples of the lung cancer cell lines HCC44 (ACC 534, DSMZ, Branuschweig, Germany), A549 (ACC 107, DSMZ), H23 (CRL-5800, ATTC, Manassas, VA, USA) and H1299 (CRL-5803, ATCC); and the normal immortalized lung cell line BEAS2B (95102433, ECACC, Sigma-Aldrich) were received in our laboratory and passaged for less than 6 months.

Techniques: Migration, Wound Healing Assay, Transfection, Control, Invasion Assay, Staining

Journal: Translational Lung Cancer Research

Article Title: Role of the epithelial-mesenchymal transition-related circular RNA, circ-10720, in non-small-cell lung cancer

doi: 10.21037/tlcr-20-920

Figure Lengend Snippet: Circ-10720 regulates apoptosis and proliferation in NSCLC cell lines. (A) Apoptosis rate in HCC44 and A549 cells transfected with circ-10720 siRNA in comparison with control cells. (B) HCC44 and (C) A549 proliferation analysis by MTS (absorbance at 490 nm) at 0, 24, 48 and 72 hours. All data represented are the average of at least three independent replicates and error bars represents SEM. *, P<0.05.

Article Snippet: Cryopreserved samples of the lung cancer cell lines HCC44 (ACC 534, DSMZ, Branuschweig, Germany), A549 (ACC 107, DSMZ), H23 (CRL-5800, ATTC, Manassas, VA, USA) and H1299 (CRL-5803, ATCC); and the normal immortalized lung cell line BEAS2B (95102433, ECACC, Sigma-Aldrich) were received in our laboratory and passaged for less than 6 months.

Techniques: Transfection, Comparison, Control