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: HCC44 ( Homo sapiens ) , DSMZ , ACC 534.

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: HCC44 ( Homo sapiens ) , DSMZ , ACC 534.

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: HCC44 ( Homo sapiens ) , DSMZ , ACC 534.

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: HCC44 ( Homo sapiens ) , DSMZ , ACC 534.

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: HCC44 ( Homo sapiens ) , DSMZ , ACC 534.

Techniques: Staining, Knock-Out

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: bioRxiv

Article Title: Post-Translational Modifications Remodel Proteome-Wide Ligandability

doi: 10.1101/2025.07.31.667978

Figure Lengend Snippet: a , Proteomic profile of P2 and ( R / S )- P6 binding to KRAS G13D following STR treatment in MDA-MB-231 cells. b , Quantitation of immunoblot replicates in . c , ChP analysis of P3 binding in LX293T cells expressing HA-KRAS G13D or HA-KRAS G12C following treatment with STR (250 nM, 3 h). d , ChP analysis of P3 binding to KRAS G12C after treatment with STR (250 nM, 3 h) and SHP099 (500 nM, 0.5 h) in HCC44 cells. e , ChP analysis of P3 binding to KRAS Q61H after treatment with STR (250 nM, 3 h) and SHP099 (500 nM, 0.5 h) in H460 cells. f , ChP analysis of P3 binding to KRAS WT after treatment with STR (250 nM, 3 h) or SHP099 (500 nM, 0.5 h) in H1975 cells (left) or in LX293T cells expressing HA-KRAS WT (right). g - h , Superimposed structure of KRAS G13D docked with P3 and BI-2865 (PDB: 8B00) and KRAS G12C with P3 and AMG510 (PDB 6OIM). i , ChP analysis P3 binding to KRAS G12C when co-incubated with AMG510 at indicated concentrations in HCC4 cells. j , ChP analysis of P3 binding to KRAS G13D when co - incubated with increasing concentration of GppNHp (left) and GDP (right) in MDA-MB-231 cells. k , ChP analysis of P3 binding to HA-KRAS G12C Y32F and Y64F recombinantly expressed in LX293T cells incubated. l , ChP analysis of P3 binding to KRAS G13D in dasatinib-treated MDA-MB-231 cells and KRAS G12C in HCC44 cells at indicated concentrations. m - n , Cellular thermal shift assay (CETSA) temperature gradient immunoblots of KRAS G13D stabilization with BI-2865 (10 nM) in STR- and SHP099-treated cells ( m ) and in dasatinib treated cells ( n ). Proteomic data were obtained from two independent biological replicates and are presented as mean ± s.d. Immunoblots were quantified from two independent biological replicates and plotted as the mean. Gel images are representative of two independent experiments. Significance was determined using a two-tailed Student’s t -test. n.s., not significant. Associated datasets are provided in Supplementary Tables 1 and 4.

Article Snippet: MDA-MB-231, HEK293T, H460, MIA PaCa2 and H1975 cell lines were procured from ATCC, while HCC44 cells were obtained from DSMZ, and Lenti-XTM 293T (LX293T) cells from Takara Bio.

Techniques: Binding Assay, Quantitation Assay, Western Blot, Expressing, Incubation, Concentration Assay, Thermal Shift Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Post-Translational Modifications Remodel Proteome-Wide Ligandability

doi: 10.1101/2025.07.31.667978

Figure Lengend Snippet: a , Monitoring KRAS activity using the RAS-binding domain (RBD) pull-down assay in multiple cell lines. Cells treated with staurosporine (STR, 250 nM) or SHP099 (500 nM) were incubated with indicated KRAS inhibitors, the levels of active-state (GTP-bound) of KRAS were determined via pulldown of GST-RBD and immunoblotting. b , Immunoblot of GTP-bound KRAS by RBD pull-down assay in MDA-MB-231 cells. Cells were pre-treated with STR (250 nM, 3 h) and co-treated with BI-2865 and STR for indicated time. c , Immunoblot and quantification plot of mutant GTP-bound HA-KRAS G13D levels in LX293T cells expressing HA-tagged KRAS G13D constructs containing Y32 and Y64 site-specific mutations. Cells were treated with BI-2865 for 5 hours followed by immunoblot after HA-tag pulldown. d, Immunoblot of GTP-bound KRAS by RBD pull-down assay in dasatinib-treated (1 µM, 1 h) MDA-MB-231 cells with BI-2865 (1 h). e, Immunoblot of GTP-bound KRAS by RBD pull-down assay in dasatinib-treated (1 µM, 1 h) HCC44 cells with AMG-510 (1 h). f , Immunoblot of GTP-bound KRAS Q61H by RBD pull-down assay in STR (250 nM) or SHP099 (500 nM) treated in H460 cells. g , LX293T cells expressing HA-tagged KRAS G13D Y32F and Y64F mutants were treated with RMC-7977 (500 nM, 2 hours) with STR followed by HA-pulldown and CYPA immunoblot. h , LX293T cells expressing HA-tagged KRAS G13D were treated with STR (250 nM) or SHP099 (500 nM), and RMC-7977 with indicated concentration for 2 hours followed by HA-pulldown and CYPA immunoblot. i, Immunoblot of pERK signaling levels restored following dasatinib pre-treatment (1 µM, 1 h), and KRAS inhibitors co-treatment in indicated cell lines (1 h for MDA-MB-231, MIA PaCa-2; 0.5 h for H460). j , Per-residue root-mean-square-fluctuation (RMSF) plots quantitatively illustrate the differential flexibility of SW-I (red) and SW-II (blue) regions for KRAS G13D and KRAS G13D pY64. Representative structural ensembles extracted from clustering unbiased simulations are shown for each system, highlighting the conformational heterogeneity of these KRAS variants. k , FES for KRAS G13D and the two potential phosphorylated variants, Y32 and Y64. As a reference, the BI-2865 binding-competent conformation is shown as a white cross. For the phosphorylated variants, a single energy minimum exists and matches the BI-2865 conformation. Results are representative of two or three biological replicates.

Article Snippet: MDA-MB-231, HEK293T, H460, MIA PaCa2 and H1975 cell lines were procured from ATCC, while HCC44 cells were obtained from DSMZ, and Lenti-XTM 293T (LX293T) cells from Takara Bio.

Techniques: Activity Assay, Binding Assay, Pull Down Assay, Incubation, Western Blot, Mutagenesis, Expressing, Construct, Concentration Assay, Residue

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