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: ACS Pharmacology & Translational Science

Article Title: In Vitro Evaluation of Mangostanin as an Antimicrobial and Biocompatible Topical Antiseptic for Skin and Oral Tissues

doi: 10.1021/acsptsci.4c00082

Figure Lengend Snippet: Biocompatibility and antimicrobial test of periodontal gels. (A) MTT assay in HOE tissue after the application of periodontal gels for 3 h. Results are expressed as % vs C- that was set to 100%. C+ represents 5% SDS. (B) P. gingivalis growth rate cultured at 10 h with different periodontal gels and P. gingivalis LIVE/DEAD ratio cultured for 10 h with different periodontal gels. (C) Histological characterization of HOE. Representative images were present for each treatment—detail of hematoxylin and eosin (H&E) staining of HOE at 400×. Total tissue disintegration was observed in C+ after the treatment with SDS 5% and they were not further processed for histological analysis. Values represent the mean ± SEM ( n = 3). (D) Effect of different periodontal gels on IL-1α release by HOE after 3 h of treatment. Results were statistically compared by ANOVA and Bonferroni as post hoc for MMT assay and by Kruskal–Wallis for P. gingivalis growth rate, P. gingivalis LIVE/DEAD ratio, and IL-1α levels: * p < 0.05 treatment vs C–. # p < 0.05 treatment vs C+. & p < 0.05 treatment vs Periokin HA 0.2% CHX. $ p < 0.05 treatment vs Perio-Aid HA 0.2% CHX. + p < 0.05 treatment vs Bexident Chitosan 0.2% CHX. @ pp < 0.05 treatment vs Mucorepair 0.2% Enoxolone.

Article Snippet: 58 , 59 Furthermore, histological images revealed a damaged layer of cells on the tissue surface-treated with Periokin HA 0.2% CHX compared to other treatments containing 0.2% CHX, such as Perio-Aid HA 0.2% CHX.

Techniques: MTT Assay, Cell Culture, Staining

Journal: The EMBO Journal

Article Title: Mitotic checkpoint gene expression is tuned by codon usage bias

doi: 10.15252/embj.2021107896

Figure Lengend Snippet: A Cellular protein noise (coefficient of variation, CV = std / mean) in live‐cell microscopy images of S. pombe ; n = 7 images (Nmt1‐GFP), 11 (Mad1‐GFP), 19 (Mad2‐GFP), 10 (Mad3‐GFP); single images had 16–79 GFP‐positive and 6–94 GFP‐negative (control) cells. Boxplots show median and interquartile range (IQR); whiskers extend to values no further than 1.5 times the IQR from the first and third quartile, respectively. Mad1, Mad2, and Mad3 all showed significantly lower noise than Nmt1 (Wilcoxon rank sum test; all P < 0.001). B Simulations of stochastic gene expression noise from selected mRNA/protein half‐life combinations assuming a constantly active promoter (see Methods). Synthesis rates were set to obtain a mean mRNA number of 4 per cell, and a mean protein number of 6,000 per cell. The x‐axis of each graph shows time, the y‐axis shows mRNA number per cell (blue) or protein number per cell (black). C Theoretical prediction for the coefficient of variation (CV = std/mean) of the protein number per cell, assuming different mRNA and protein half‐lives, using the same underlying model as in B. Synthesis rates were adjusted to maintain a mean mRNA number per cell of 3.5, and a mean protein number per cell of 6,000 (approx. 100 nM). D mRNA abundances by qPCR following metabolic labeling and removal of the labeled pool (two independent experiments). Lines are regression curves from generalized linear mixed model fits, excluding the measurements at t = 0 in order to accommodate for noninstantaneous labeling by 4tU. Act1 + and ecm33 + were used as long and short half‐life controls, respectively; qPCR was performed for the endogenous mRNAs. Half‐lives (95% confidence interval): mad1 + 5.6 min (4.3–8.4), mad2 + 7.7 min (6.2–10.4), mad3 + 5.2 min (4.3–6.9), act1 + 61.8 min (37.2–172.3), ecm33 + 5.0 min (4.5–5.7). E Protein abundances after translation shut‐off with cycloheximide (CHX); n = 3 experiments, error bars = std. Lines indicate fit to a one‐phase exponential decay. Cdc2 and Cdc13 were used as long and short half‐life controls, respectively. Immunoblots for the endogenous proteins (no tag). A representative experiment shown in Appendix Fig . Source data are available online for this figure.

Article Snippet: Cycloheximide (from Streptomyces griseus) , Chem Impex , Cat # 00083.

Techniques: Microscopy, Negative Control, Expressing, Labeling, Western Blot

Journal: The EMBO Journal

Article Title: Mitotic checkpoint gene expression is tuned by codon usage bias

doi: 10.15252/embj.2021107896

Figure Lengend Snippet:

Article Snippet: Cycloheximide (from Streptomyces griseus) , Chem Impex , Cat # 00083.

Techniques: Recombinant, In Vitro, Sequencing, Labeling, Protease Inhibitor, Isolation, Magnetic Beads, Bicinchoninic Acid Protein Assay, Software, Membrane