gfp rab5  (Addgene inc)

Journal: The Journal of Biological Chemistry

Article Title: Rab5 nucleotide binding promotes oxidative metabolism to fuel hepatocellular carcinoma cell proliferation

doi: 10.1016/j.jbc.2026.111321

Figure Lengend Snippet: Rab5 is recruited to lipid droplets (LDs) in a GTPase-dependent manner in Hep3B cells . Super-resolution confocal micrographs of Hep3B human hepatoma cells expressing ( A ) WT Rab5 (GFP-Rab5 WT), ( B ) constitutively active mutant (GFP-Rab5 Q79L), and ( C ) dominant-negative mutant (GFP-Rab5 S34N). LDs were stained with Oil Red O (ORO, red ) to visualize neutral lipid stores. GFP-Rab5 fluorescence (green ) marks Rab5 localization. Inlays depict Rab5 localization around LD borders, indicated further by yellow arrows . D , quantification of Rab5–LD colocalization by the ratio of LD:cytoplasm GFP-Rab5 intensity. Graphs represent mean ± SD from n = 3 independent experiments. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test. ∗∗ p < 0.01 and ∗∗∗ p < 0.001.

Article Snippet: The GFP-Rab5 was a gift from Marci Scidmore (Addgene plasmid #49888).

Techniques: Expressing, Mutagenesis, Dominant Negative Mutation, Staining, Fluorescence

Journal: bioRxiv

Article Title: Nup358 Sustains Intestinal Epithelial Homeostasis by Preventing Dvl1 Condensate Formation to Restrain Wnt Signaling

doi: 10.64898/2026.03.25.714063

Figure Lengend Snippet: a. Levels of the 7TGP fluorescent Wnt reporter in HEK293T cells transfected with control siRNA, Dvl1-specific siRNA, Nup358-specific siRNA, or a combination of Nup358-specific siRNA and Dvl1-specific siRNA and treated with either Wnt3a or vehicle. b. Western blot analysis and relative quantification of protein levels of Dvl1 and Axin1 in HEK293T cells transfected with either control or Nup358-specific siRNA. α-Tubulin and GAPDH were used as loading controls. Data are expressed as mean ± SD. **p ≤ 0.01. c. Immunofluorescence staining for Dvl1 and Nup358 in HEK293T cells transfected with either control or Nup358-specific siRNA. d. Immunofluorescence staining for Dvl1 and Nup358 in HEK293T cells transfected with control or Nup358-specific siRNA and treated with vehicle or 1,6-hexanediol 5% for 2 minutes. e. Fluorescence recovery after photobleaching (FRAP) and fusion analysis of Dvl1 condensates in HEK293T cells transfected with Nup358-specific siRNA and EGFP-Dvl1.

Article Snippet: Plasmids used in this study include β-catenin-EGFP (Addgene, #71367), EGFP-Dvl1 (Addgene, #194586) and plasmids HA-Nup358 1–2683 , HA-Nup358 1–2148 , HA-Nup358 1–1810 , HA-Nup358 1–1133 from Wälde et al 13 .

Techniques: Transfection, Control, Western Blot, Quantitative Proteomics, Immunofluorescence, Staining, Fluorescence

Journal: bioRxiv

Article Title: Nup358 Sustains Intestinal Epithelial Homeostasis by Preventing Dvl1 Condensate Formation to Restrain Wnt Signaling

doi: 10.64898/2026.03.25.714063

Figure Lengend Snippet: a. Schematic representing different functional domains of Nup358. b. Fluorescent Wnt reporter activity in HEK293T cells transfected with control siRNA, Nup358-specific siRNA, Ubc9-specific siRNA, or treated with the SUMOylation inhibitor 2-D08. c. Schematic representing different HA-tagged truncated forms of Nup358 that were transiently expressed in HEK293T cells. d. Fluorescent Wnt reporter activity in HEK293T cells transfected with either control or Nup358-specific siRNA alone or in combination with different HA-tagged truncated forms of Nup358 were analyzed by confocal microscopy (top) and quantified (bottom). Data are expressed as mean ± SD. ****p ≤ 0.0001 e. Western blot analysis of co-immunoprecipitation of HA-Nup358 and EGFP-Dvl1 transiently expressed in HEK293T cells. GAPDH was used as loading control. f. Immunofluorescence of HEK293T cells transfected with HA-Tagged Nup358 1–1133 and EGFP-Dvl1and stained with an anti-HA antibody. g. Immunofluorescence staining for endogenous Dvl1 and Nup358 in HEK293T cells transfected with either scramble control or Nup358-specific siRNA alone or in combination with HA-Tagged Nup358 1–1133 fragment were analyzed by confocal microscopy (top) and quantified (bottom). Data are expressed as mean ± SD. **p ≤ 0.01.

Article Snippet: Plasmids used in this study include β-catenin-EGFP (Addgene, #71367), EGFP-Dvl1 (Addgene, #194586) and plasmids HA-Nup358 1–2683 , HA-Nup358 1–2148 , HA-Nup358 1–1810 , HA-Nup358 1–1133 from Wälde et al 13 .

Techniques: Functional Assay, Activity Assay, Transfection, Control, Confocal Microscopy, Western Blot, Immunoprecipitation, Immunofluorescence, 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: 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: Neurospheres from primary rodent brain cells to probe the 3D organization and function of synapses

doi: 10.64898/2026.03.19.712855

Figure Lengend Snippet: (A) Neurospheres were formed by mixing non-electroporated and electroporated cells before being plated in U-bottom ULA wells. (B) Maximum intensity projection image of a confocal stack of a DIV14 neurosphere in which neurons were separately electroporated with RFP (red) and Xph20-GFP (green). (C) Zoomed images on primary dendrites from neurons expressing intrabodies to PSD-95 (Xph20-GFP), or gephyrin (GPHN.FingR-GFP), revealing excitatory or inhibitory post-synapses, respectively. (D) Numbers of PSD-95 and gephyrin-positive puncta per electroporated neuron. Data represent the mean ± SEM of 14 and 10 neurospheres, respectively, and were compared by non-parametric Mann-Whitney test. Dots show individual neurospheres. (E) Maximum intensity projection of a confocal stack of a DIV14 neurosphere in which neurons were co-electroporated with GPHN.FingR-GFP (green) and Xph20-mRuby2 (magenta), allowing the detection of both excitatory and inhibitory post-synapses in the same cells. (F) Zoom on a dendritic segment corresponding to the rectangular area highlighted in (E). (G) Confocal image of a primary dendrite from a neuron expressing GFP-actin, further immunolabeled for GFP, showing numerous dendritic spines bulging out of the shaft.

Article Snippet: The intrabody to gephyrin, GPHN.FingR-GFP (Addgene # 46296 pCAG_GPHN.FingR-eGFP-CCR5TC), was a gift from D. Arnold (University of Southern California, Los Angeles, CA, USA) ( ).

Techniques: Expressing, MANN-WHITNEY, Immunolabeling

Journal: bioRxiv

Article Title: Neurospheres from primary rodent brain cells to probe the 3D organization and function of synapses

doi: 10.64898/2026.03.19.712855

Figure Lengend Snippet: (A) Neurospheres were formed by mixing non-electroporated and electroporated cells before being plated in U-bottom ULA wells. (B) Maximum intensity projection image of a confocal stack of a DIV14 neurosphere in which neurons were separately electroporated with RFP (red) and Xph20-GFP (green). (C) Zoomed images on primary dendrites from neurons expressing intrabodies to PSD-95 (Xph20-GFP), or gephyrin (GPHN.FingR-GFP), revealing excitatory or inhibitory post-synapses, respectively. (D) Numbers of PSD-95 and gephyrin-positive puncta per electroporated neuron. Data represent the mean ± SEM of 14 and 10 neurospheres, respectively, and were compared by non-parametric Mann-Whitney test. Dots show individual neurospheres. (E) Maximum intensity projection of a confocal stack of a DIV14 neurosphere in which neurons were co-electroporated with GPHN.FingR-GFP (green) and Xph20-mRuby2 (magenta), allowing the detection of both excitatory and inhibitory post-synapses in the same cells. (F) Zoom on a dendritic segment corresponding to the rectangular area highlighted in (E). (G) Confocal image of a primary dendrite from a neuron expressing GFP-actin, further immunolabeled for GFP, showing numerous dendritic spines bulging out of the shaft.

Article Snippet: The intrabody to PSD-95, Xph20-GFP (Addgene #135,530 pCAG_Xph20-eGFP-CCR5TC), was a gift from M. Sainlos (IINS, University of Bordeaux) ( ).

Techniques: Expressing, MANN-WHITNEY, Immunolabeling