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: HeLa Kyoto cells were transfected with 20 μg mCherry–EGFP–LC3 WT DNA (Addgene Plasmid #123230), using the Neon NxT Electroporation System (ThermoFisher) according to the manufacturer’s instructions.

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: HeLa Kyoto cells were transfected with 20 μg mCherry–EGFP–LC3 WT DNA (Addgene Plasmid #123230), using the Neon NxT Electroporation System (ThermoFisher) according to the manufacturer’s instructions.

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: HeLa Kyoto cells were transfected with 20 μg mCherry–EGFP–LC3 WT DNA (Addgene Plasmid #123230), using the Neon NxT Electroporation System (ThermoFisher) according to the manufacturer’s instructions.

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: HeLa Kyoto cells were transfected with 20 μg mCherry–EGFP–LC3 WT DNA (Addgene Plasmid #123230), using the Neon NxT Electroporation System (ThermoFisher) according to the manufacturer’s instructions.

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: HeLa Kyoto cells were transfected with 20 μg mCherry–EGFP–LC3 WT DNA (Addgene Plasmid #123230), using the Neon NxT Electroporation System (ThermoFisher) according to the manufacturer’s instructions.

Techniques: Expressing, 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: 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: Physiological cerebrospinal fluid like medium reveals autophagy dependency of leukaemia in the central nervous system

doi: 10.64898/2026.03.09.709824

Figure Lengend Snippet: A, Schematic diagram of autophagy pathway and the mechanism of action of bafilomycin A1 to inhibit autophagy. B, C, Relative GFP/RFP ratio of mean fluorescence intensity (MFI) in SEM (C) and REH (D) cells expressing GFP-LC3-RFP-LC3ΔG autophagic flux probe cultured in RPMI, Plasmax (PL), and CSFmax (CSF) for 24 h with or without 50 nM of bafilomycin A1 treatment (n=7 independent experiments). D, Immunoblot analysis showing changes in autophagy markers in human CD19 + (huCD19 + ) SEM cells isolated from the bone marrow (BM) and the brain (CNS) (n=2 biological replicates). E, Percentage of apoptosis in SEM cells cultured in RPMI, Plasmax (PL), and CSFmax (CSF) for 24 h in normoxia (21% O 2 ) with or without 1 μM of MRT403 treatment (n=3 independent experiments). F, Percentage of apoptosis in SEM cells cultured in RPMI, Plasmax (PL), and CSFmax (CSF) for 24 h in normoxia (21% O 2 ) or hypoxia (1% O 2 ) with or without 50 nM of bafilomycin A1 treatment (n=3 independent experiments). G, Immunoblot analysis showing deletion of ULK1 and changes in autophagy markers in SEM cells cultured in RPMI. H, Relative GFP/RFP ratio of mean fluorescence intensity (MFI) in SEM vector control (CTRL) and ULK1 knockout (KO) single clone 1 and 2 cells expressing GFP-LC3-RFP-LC3ΔG autophagic flux probe cultured in RPMI, Plasmax (PL), and CSFmax (CSF) for 24 h (n=3 independent experiments). I, Immunoblot analysis showing deletion of ATG7 and changes in autophagy markers in SEM cells cultured in RPMI. J, Relative GFP/RFP ratio of mean fluorescence intensity (MFI) in SEM CTRL and ATG7 KO single clone 1 and 2 cells expressing GFP-LC3-RFP-LC3ΔG autophagic flux probe cultured in RPMI, Plasmax (PL), and CSFmax (CSF) for 24 h (n=3 independent experiments). Data are shown as the mean ± s.e.m. P-values were calculated with a repeated measure one-way ANOVA with Tukey’s multiple comparison test (B, C, E, F, H, J).

Article Snippet: Similarly, to generate stable ALL cell lines expressing the fluorescent probe GFP-LC3-RFP-LC3ΔG we used PMRX-IP-GFP-LC3-RFP which was a gift from Noboru Mizushima (Addgene Plasmid #84573) [ ].

Techniques: Fluorescence, Expressing, Cell Culture, Western Blot, Isolation, Plasmid Preparation, Control, Knock-Out, Comparison