Journal: bioRxiv

Article Title: Hepatic cytochrome P450 endoplasmic reticulum-associated degradation (ERAD): Topological determinants and cellular partnerships that dictate the preferential P450 proteolytic sorting into macroautophagy rather than UPS

doi: 10.1101/2025.09.25.678692

Figure Lengend Snippet: Relative UPD vs ALD proteolytic preferences of the mCherry-tagged parent CYPs 1A1 and 1A2 as probed with CHX-chase analyses and diagnostic UPD (BTZ) and ALD (3MA/NH4Cl) inhibitors. HepG2 cells were seeded in two 6-wells culture plates, and then each well was transfected with 2 μg CYP1A1-mCherry plasmid ( A ) or CYP1A2-mCherry plasmid ( D ). After a 48 h-transfection, HepG2 cells were treated with CHX (50 µg/ml) for indicated times, ± BTZ (10 μM) or ± 3-MA (5 mM)/NH 4 Cl (30 mM) for 8 h. Cells were harvested at indicated times after CHX-treatment and lysates (10 µg) were subjected to IB analyses with GAPDH as the loading control. Densitometrically quantified CYP1A1-mCherry ( B ) or CYP1A2-mCherry ( E ) levels normalized to their corresponding individual GAPDH levels at each harvest time point were quantified relative to their 0 h levels. Values from five experimental replicates were analyzed by Prism Graphpad version 9.5.0 to determine the lifespan [half-life (t 1/2 ); mean ± SD] of each mCherry-tagged protein based on a single exponential fit of the data. Upon CHX-chase ± BTZ, or ± 3-MA/NH 4 Cl at 8 h, densitometrically quantified CYP1A1-mCherry ( C ) or CYP1A2-mCherry ( F ) levels normalized to their corresponding individual GAPDH levels and quantified relative to their BTZ or 3-MA/NH 4 Cl levels at 8 h. Values were expressed as Mean ± SD from three experimental replicates relative to their 0 h levels, and the statistical significance was calculated by an ordinary one-way ANOVA analyses. Identical analyses and quantification were conducted upon plasmid transfection and CHX-chase in HepG2 cells of the N-terminal mCherry-tagged chimeras: 1A1( –109)1A2-mCherry ( G - I ), 1A2( –107)-1A1-mCherry ( J - L ) and 1A2( –205)-1A1-mCherry ( M - O ).

Article Snippet: To determine the degradation pathways, cells were exposed to diagnostic UPD or ALD inhibitor probes under the following conditions: (i) untreated control (CHX only, 50 μg/mL); (ii) proteasome inhibition, with CHX 50 μg/mL + bortezomib (BTZ, 10 μM; Cat. #C835F72, Thomas Scientific); and (iii) autophagic–lysosomal inhibition, with CHX 50 μg/mL + 3-methyladenine (3-MA, 5 mM; Cat. #C910A04, Thomas Scientific), and NH 4 Cl (30 mM; Cat. #C988H67, Thomas Scientific).

Techniques: Diagnostic Assay, Transfection, Plasmid Preparation, Control

Journal: bioRxiv

Article Title: Hepatic cytochrome P450 endoplasmic reticulum-associated degradation (ERAD): Topological determinants and cellular partnerships that dictate the preferential P450 proteolytic sorting into macroautophagy rather than UPS

doi: 10.1101/2025.09.25.678692

Figure Lengend Snippet: HepG2 cells were seeded into four 6-well culture plates, plasmids (2 μg DNA) for FL 1A1-mCherry, 1A1( – )-mCherry, FL 1A2-mCherry, and 1A2( – )-mCherry were transfected into each well. After 48 h transfection, HepG2 cells were subjected to CHX-chase ± BTZ, or ± 3-MA/NH 4 Cl for 8 h, and analyzed similarly to CYP1A1-mCherry ( C ) or CYP1A2-mCherry ( F ) . Values were expressed as Mean ± SD from three experimental replicates relative to their 0 h levels, and the statistical significance was calculated by an ordinary one-way ANOVA analysis. All analyses were performed by Prism Graphpad version 9.5.0. Data indicate as mean ± SD of N = 3. Note the NT-subdomains retain the proteolytic preferences of their parent CYP1A proteins .

Article Snippet: To determine the degradation pathways, cells were exposed to diagnostic UPD or ALD inhibitor probes under the following conditions: (i) untreated control (CHX only, 50 μg/mL); (ii) proteasome inhibition, with CHX 50 μg/mL + bortezomib (BTZ, 10 μM; Cat. #C835F72, Thomas Scientific); and (iii) autophagic–lysosomal inhibition, with CHX 50 μg/mL + 3-methyladenine (3-MA, 5 mM; Cat. #C910A04, Thomas Scientific), and NH 4 Cl (30 mM; Cat. #C988H67, Thomas Scientific).

Techniques: Transfection

Journal: bioRxiv

Article Title: Hepatic cytochrome P450 endoplasmic reticulum-associated degradation (ERAD): Topological determinants and cellular partnerships that dictate the preferential P450 proteolytic sorting into macroautophagy rather than UPS

doi: 10.1101/2025.09.25.678692

Figure Lengend Snippet: Erlin-1 siRNA-KD reverses CYP1A2 ER-topology from DRMs to non-DRMs and impairs CYP1A2-ALD, resulting in CYP1A2-aggregate build-up. Similar impairment of CYP1A2 ( – ) -ALD upon erlin-1 siRNA-KD. HepG2 cells were transfected with the CYP1A2-mCherry plasmid. After 12 h, the culture medium was replaced with fresh MEM containing either scrambled siRNAs (control) or erlin-1 siRNA1 plus siRNA2 (erlin siRNA1/2) for erlin-1 KD. After an additional 24 h, the medium was replaced with fresh MEM containing either the scrambled or erlin-1 1/2 siRNAs, and the cells were incubated for another 24 h before being harvested. The samples were lysed in hypotonic buffer and sonicated at low speed (600 g) to remove nuclei and cell debris. A . The lysates were then incubated in a membrane solubilization buffer containing 1% Triton X-100, cleared and subjected to a 5-40% sucrose gradient subfractionation by ultracentrifugation (210,000 g/19h) for DRM isolation. B . HepG2 cells were transfected with the CYP1A2-mCherry plasmid and treated with the scrambled or erlin-1 1/2 siRNAs exactly as described above. Cells were processed, and the 1% Triton-treated cell lysates were subjected to a 5-40% sucrose gradient subfractionation as described above. Subfractions 7-11 were collected, diluted with 1.15 M KCl and resedimented by ultracentrifugation at 105,000 g /1 h. The pellet was collected, resuspended and designated as the “soluble” fraction. Subfractions 4-6 were also collected, diluted with 1.15 M KCl and resedimented by ultracentrifugation at 105,000 g /1 h, The pellet was resuspended in TISO buffer and designated as the “pellet” fraction (see Materials & Methods for further experimental details). HepG2 cells were similarly transfected with plasmids for CYP1A1( – )-mCherry ( C ) or CYP1A2( – )-mCherry ( D ). Transfected cells were then treated with scrambled siRNAs or erlin-1 1/2 siRNAs for erlin-1 KD and processed exactly as described above. After 48 h of siRNA-treatment, CHX was added at 0 h and the degradation of CYP1A1 NT(1 33)-mCherry and CYP1A2 NT( – )-mCherry subdomains was monitored through CHX-chase analyses from 0-12 h and the effect of diagnostic UPD (BTZ, 10 μM) and ALD [3-MA (5 mM)/ NH 4 Cl (30 mM)] inhibitors monitored at 8 h. Cell lysates (10 μg) were subjected to IB analyses with H3-Histone as the loading control.

Article Snippet: To determine the degradation pathways, cells were exposed to diagnostic UPD or ALD inhibitor probes under the following conditions: (i) untreated control (CHX only, 50 μg/mL); (ii) proteasome inhibition, with CHX 50 μg/mL + bortezomib (BTZ, 10 μM; Cat. #C835F72, Thomas Scientific); and (iii) autophagic–lysosomal inhibition, with CHX 50 μg/mL + 3-methyladenine (3-MA, 5 mM; Cat. #C910A04, Thomas Scientific), and NH 4 Cl (30 mM; Cat. #C988H67, Thomas Scientific).

Techniques: Transfection, Plasmid Preparation, Control, Incubation, Sonication, Membrane, Isolation, Diagnostic Assay

Journal: bioRxiv

Article Title: Hepatic cytochrome P450 endoplasmic reticulum-associated degradation (ERAD): Topological determinants and cellular partnerships that dictate the preferential P450 proteolytic sorting into macroautophagy rather than UPS

doi: 10.1101/2025.09.25.678692

Figure Lengend Snippet: ( – ) subdomain is sufficient to reverse the loss of CYP1A2-mCherry DRM-buoyancy and CYP1A2-mCherry ALD upon siRNA-elicited erlin-1-KD. HepG2 cells were transfected with the CYP1A2-mCherry plasmid exactly as described in . After 12 h, the culture medium was replaced with fresh MEM containing the scrambled siRNA (control) or siRNAs 1/2. Twelve h after siRNA treatment, the medium was replaced again, and fresh scrambled siRNA (control) or siRNAs 1/2 plasmids along with either erlin-1 (DNA mutant) or erlin-1 ( – )-mCherry plasmid was added and the cells incubated for another 24 h. The medium was again replaced with fresh MEM containing the same siRNAs, as well as erlin-1 (DNA mutant) or erlin-1 ( – )-mCherry plasmids and the cells were cultured for an additional 24 h. A . To determine the relative subfractionation of CYP1A2 in DRM-vs non-DRM-subfractions, cleared cell lysates were subjected to a discontinuous 5-40% sucrose-gradient ultracentrifugation exactly as described . Gradient fractions (1 mL) each were carefully collected starting from the top of the gradient and aliquots of these gradient subfractions subjected to IB analyses with mCherry-IgGs (CYP1A2) and GAPDH-IgG (controls). Note that the loss of CYP1A2-DRM localization upon erlin-1 siRNA, is reversed upon co-expression of the siRNA resistant erlin-1 DNA mutant plasmid or even just the erlin-1 N( – ) subdomain. B. Cells were treated with scrambled siRNA (control) or erlin-1 1/2 siRNAs, and subsequently with either erlin-1 (DNA mutant) or erlin-1 ( – )-mCherry plasmid, exactly as described above. After the second 24 h-incubation period, cells were treated with CHX (50 μg/mL) at 0 h, with or without BTZ (10 µM) or 3-MA (5 mM)/NH 4 Cl (30 mM) for 8 h. Cell lysates (10 µg) were subjected to mCherry or Ub-IB analyses, with GAPDH as a control. Note that the loss of CYP1A2-ALD upon erlin-1 siRNA 1/2-KD (Compare lanes 8 vs 4), is reversed upon coexpression of either erlin-1 DNA-mutant (Compare lanes 12 vs 8) or just erlin-1 N ( – ) subdomain (Compare lanes 16 vs 8) .

Article Snippet: To determine the degradation pathways, cells were exposed to diagnostic UPD or ALD inhibitor probes under the following conditions: (i) untreated control (CHX only, 50 μg/mL); (ii) proteasome inhibition, with CHX 50 μg/mL + bortezomib (BTZ, 10 μM; Cat. #C835F72, Thomas Scientific); and (iii) autophagic–lysosomal inhibition, with CHX 50 μg/mL + 3-methyladenine (3-MA, 5 mM; Cat. #C910A04, Thomas Scientific), and NH 4 Cl (30 mM; Cat. #C988H67, Thomas Scientific).

Techniques: Transfection, Plasmid Preparation, Control, Mutagenesis, Incubation, Cell Culture, Expressing

Journal: Science Advances

Article Title: Targeting oncoproteins with a positive selection assay for protein degraders

doi: 10.1126/sciadv.abd6263

Figure Lengend Snippet: ( A ) Immunoblot and ( B ) RT-qPCR analysis of the NCI-H1876 SCLC cell line that endogenously expresses ASCL1 infected to express the indicated sgRNAs. n = 3 biological replicates. ( C and E ) Immunoblot and ( D and F ) RT-qPCR analysis of NCI-H1876 human SCLC cells (C and D) and 97-2 mouse SCLC cells (E and F) after treatment with the CDK2 PROTAC degraders (TMX-2138 and TMX-2172) or the indicated negative controls, all used at 500 nM for either 36 hours (C and D) or 8 hours (E and F). Neg Deg, negative control degrader ZXH-7035. n = 3 biological replicates. ( G ) Immunoblot analysis and ( H ) quantification of ASCL1 protein levels in 97-2 cells first treated with the CDK2 PROTAC degrader or negative control (500 nM) for 4 hours and then treated with cycloheximide (CHX) (150 μg/ml) for the indicated times. S.E., short exposure; L.E., long exposure. n = 4 biological replicates. In all experiments, error bars represent SD except in (H), where error bars represent SEM. * P < 0.05; *** P < 0.001; **** P < 0.0001.

Article Snippet: The following compounds were purchased: POM (Selleck, #S1567), LEN (Selleck, #S1029), MG132 ( N -carbobenzyloxy- l -leucyl- l -leucyl- l -leucinal; Thermo Fisher Scientific, #47479020MG), MLN4924 (Active Biochem, #A-1139), MLN7243 (Thermo Fisher Scientific, #NC1129906), Spautin-1 (BioTechne; #5197/10), cycloheximide (VWR, #97064-724), BVdU (Chem-Impex International Inc., catalog no. 27735), actinomycin D (Thermo Fisher Scientific, #11805017), and dinaciclib (Selleck, #S2768).

Techniques: Western Blot, Quantitative RT-PCR, Infection, Negative Control