Journal: bioRxiv

Article Title: A live-cell autophagy reporter reveals reversible vacuolation in naked mole-rat skin fibroblasts under lysosomal stress

doi: 10.64898/2026.03.18.712644

Figure Lengend Snippet: Western blot validation of mCherry–EGFP–LC3 NMR stable over-expression in NMR skin fibroblast lines. Cell lysates from single-cell–derived colonies were probed with antibodies against LC3 ( A ) and EGFP ( B ). Both antibodies detected doublet bands at approximately 80–82 kDa, consistent with the predicted molecular mass of the mCherry–EGFP–LC3 NMR fusion protein in the non-lipidated (LC3-I; top band) and lipidated (LC3-II, bottom band) forms. Markers are shown in lane M and individual colonies are labelled C1-C6. C ) mTOR inhibition by PP242 enhances autophagic flux in NMR skin fibroblasts. Representative live-cell confocal images of mCherry–EGFP–LC3 NMR expressing cells, either untreated or treated with PP242 (2 μM or 4 μM) for 24 h. PP242 treatment induces a shift from the appearance of autophagosomes (mCherry⁺/EGFP⁺ (yellow)) to autolysosomes (mCherry⁺/EGFP⁻ (red)). Scale bar is 10 μm. D) Bafilomycin A1 (Baf A1) impairs autophagic flux in NMR skin fibroblasts. Representative live-cell confocal images of mCherry–EGFP–LC3 NMR expressing cells, either untreated or treated with Baf A1 (166 nM, 250 nM, or 333 nM) for 24 h. Baf A1 treatment results in accumulation of mCherry⁺/EGFP⁺ (yellow) puncta, consistent with impaired autophagic flux. In C) and D) the nucleus is stained with Hoescht (blue).

Article Snippet: Site-directed mutagenesis was used to change three amino acids in the mammalian cell reporter plasmid expressing mCherry–EGFP–LC3 (Addgene #123230) to create the NMR version of LC3 (E105G, M121R, K122G) (mCherry–EGFP–LC3 NMR ).

Techniques: Western Blot, Biomarker Discovery, Over Expression, Single Cell, Derivative Assay, Inhibition, Expressing, Staining

Journal: bioRxiv

Article Title: A live-cell autophagy reporter reveals reversible vacuolation in naked mole-rat skin fibroblasts under lysosomal stress

doi: 10.64898/2026.03.18.712644

Figure Lengend Snippet: A ) Representative live-cell confocal images of HeLa cells and NMR skin fibroblasts stably expressing mCherry–EGFP–LC3 under basal-level culture conditions. The nucleus is stained with Hoescht (blue). Few puncta are present in the HeLa cells, and those that are correspond to mCherry + /EGFP - (red). In contrast, NMR skin fibroblasts display more puncta with a mixture of mCherry + /EGFP - (red) and mCherry + /EGFP + (yellow). B ) Quantification of LC3 puncta density in HeLa and NMR skin fibroblasts, normalised to cell area (puncta per μm²). Ten individual cells per cell line were analysed, sampled from four independent fields of view. Statistical significance was assessed using a two-tailed unpaired t-test. ****P < 0.0001. C ) Quantification of LC3 puncta diameter in HeLa cells and NMR skin fibroblasts. Twenty individual LC3 puncta per cell line were analysed, sampled from four independent fields of view. Statistical analysis was performed using a two-tailed unpaired t-test. n.s. - not significant.

Article Snippet: Site-directed mutagenesis was used to change three amino acids in the mammalian cell reporter plasmid expressing mCherry–EGFP–LC3 (Addgene #123230) to create the NMR version of LC3 (E105G, M121R, K122G) (mCherry–EGFP–LC3 NMR ).

Techniques: Stable Transfection, Expressing, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: A live-cell autophagy reporter reveals reversible vacuolation in naked mole-rat skin fibroblasts under lysosomal stress

doi: 10.64898/2026.03.18.712644

Figure Lengend Snippet: A) Representative live-cell confocal images of mCherry–EGFP–LC3 NMR expression in cells either untreated or treated with increasing concentrations of CQ (40, 60 and 100 μM) for 4 h. The nucleus is stained with Hoescht (blue). CQ-treated cells exhibited predominantly mCherry⁺/EGFP⁺ (yellow) puncta, consistent with impaired lysosomal degradation. Brightfield panels show the appearance of the cytoplasmic vacuolation with increasing CQ concentrations. B ) WB analysis of mCherry–EGFP–LC3 NMR protein levels from cells either untreated or treated with increasing concentrations of CQ (10 μM, 20 μM, 40 μM, or 60 μM) for 24 h. CQ treatment resulted in an accumulation of LC3-II relative to LC3-I. ( C ) Quantification of LC3-II/LC3-I ratios derived from densitometric analysis. CQ treatment significantly increases LC3-II accumulation relative to controls. **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Site-directed mutagenesis was used to change three amino acids in the mammalian cell reporter plasmid expressing mCherry–EGFP–LC3 (Addgene #123230) to create the NMR version of LC3 (E105G, M121R, K122G) (mCherry–EGFP–LC3 NMR ).

Techniques: Expressing, Staining, Derivative Assay

Journal: bioRxiv

Article Title: A live-cell autophagy reporter reveals reversible vacuolation in naked mole-rat skin fibroblasts under lysosomal stress

doi: 10.64898/2026.03.18.712644

Figure Lengend Snippet: Representative live-cell confocal images monitoring mCherry–EGFP–LC3 NMR following CQ treatment. The nucleus is stained with Hoescht (blue). Cells were treated with 40 μM CQ for 16 h or 24 h, as indicated. In the control (basal level), LC3-positive puncta are observed as mCherry⁺/EGFP⁻ (red) or mCherry⁺/EGFP⁺ (yellow) structures. After 16 h of CQ treatment, additional LC3-positive structures emerge, including mCherry⁺/EGFP⁺ ring-like structures associated with the surface of large cytoplasmic vacuoles (pink arrow) and mCherry⁺/EGFP⁺ ring-like structures enclosing red puncta (white arrow). Following 24 h of CQ treatment, the abundance of LC3-labelled vacuoles increases further.

Article Snippet: Site-directed mutagenesis was used to change three amino acids in the mammalian cell reporter plasmid expressing mCherry–EGFP–LC3 (Addgene #123230) to create the NMR version of LC3 (E105G, M121R, K122G) (mCherry–EGFP–LC3 NMR ).

Techniques: Staining, Control

Journal: bioRxiv

Article Title: A live-cell autophagy reporter reveals reversible vacuolation in naked mole-rat skin fibroblasts under lysosomal stress

doi: 10.64898/2026.03.18.712644

Figure Lengend Snippet: Representative live-cell confocal images of mCherry–EGFP–LC3 NMR expression in cells left untreated, treated with CQ (40 μM) for 24 h, or allowed to recover for 4 h or 24 h following CQ removal, as indicated. The nucleus is stained with Hoescht (blue). After 24 h of CQ treatment, LC3-positive structures predominantly appear as mCherry + /EGFP + (yellow) puncta and LC3-decorated ring-like vacuolar structures. Following the removal of CQ from the media, progressive reorganisation of LC3-labelled structures is observed. At 4 h of recovery, smaller mCherry + /EGFP - LC3 puncta and mCherry + /EGFP + structures are frequently observed, and ring-like structures are less apparent. By 24 h of recovery, LC3 labelling is no longer associated with vacuoles, and the majority of LC3 puncta exhibit a distribution comparable to untreated control cells.

Article Snippet: Site-directed mutagenesis was used to change three amino acids in the mammalian cell reporter plasmid expressing mCherry–EGFP–LC3 (Addgene #123230) to create the NMR version of LC3 (E105G, M121R, K122G) (mCherry–EGFP–LC3 NMR ).

Techniques: Expressing, Staining, Control

Journal: bioRxiv

Article Title: GJB3, a gap junction gene, supports cell growth by mediating cystine uptake and regulating cellular stress pathways in SLC7A11 low adenocarcinomas

doi: 10.1101/2024.12.03.626548

Figure Lengend Snippet: A) SW480 cells with GJB3 knockdown were analyzed by RNA sequencing. Differentially expressed genes (upregulated and downregulated) were identified and their normalized expression values (Z-score transformed Transcripts Per Million - TPM) are visualized as a heatmap. B) Gene ontology (GO) analysis was performed using Metascape software on the differentially expressed genes identified in GJB3 knockdown cells. The most significantly enriched GO terms are presented. C) GO analysis was performed specifically on the upregulated genes identified in GJB3 knockdown cells. The top enriched terms are displayed in a bubble plot. D) Protein lysates were isolated from control and GJB3 knockdown SW480 cells after 24 hrs of knockdown. Western blotting was performed to determine the expression levels of specific proteins of interest. E) Protein lysates were isolated from control and GJB3 knockdown SW480 cells after 48 hrs of knockdown. Western blotting was performed to determine the expression levels of specific proteins of interest. F) Expression level of ATF target as determine by RNA-seq data in SW480 cells after treatment with control or shGJB3 plasmids. G) SW480 cells stably expressing both GFP-LC3 and RFP-LC3ΔG were used to assess the effect of GJB3 knockdown on autophagy. Cells were transfected with two different shRNAs targeting GJB3 or a control vector. Knockdown efficiency was confirmed, and then cells were imaged for GFP and RFP fluorescence. H) SW480 cells were used for GJB3 knockdown using control and GJB3 shRNAs. After knockdown the cells were stained with a caspase-3 activity detection reagent, and fluorescence intensity was measured at given time points. The line plot shown to detect the level of apoptosis of different cells. I) Control and GJB3 knockdown SW480 cells were transfected with siATG5 or treated with 3-methyladenine (3-MA), an inhibitor of autophagy. Protein lysates were then extracted and analyzed by Western blotting to assess LC3 protein levels. J) Control and GJB3 knockdown SW480 cells were plated, and their proliferation was monitored using an Incucyte live-cell imaging system. K) Control and GJB3 knockdown SW480 cells were transfected with siATG5, and then plated for proliferation analysis using the Incucyte live-cell imaging system. L) Control and GJB3 knockdown SW480 cells were treated with 3-MA and plated. Proliferation was measured using the Incucyte live-cell imaging system.

Article Snippet: To study autophagy in GJB3-silenced cells, the autophagosome reporter plasmid DEST-CMV mCherry-GFP-LC3B WT (Addgene, 123230) and the autophagic flux reporter plasmid pMRX-IP-GFP-LC3-RFP-LC3ΔG (Addgene, 84572) were employed.

Techniques: Knockdown, RNA Sequencing Assay, Expressing, Transformation Assay, Software, Isolation, Control, Western Blot, Stable Transfection, Transfection, Plasmid Preparation, Fluorescence, Staining, Activity Assay, Live Cell Imaging