hcc44  (DSMZ)

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