gfp reporter plasmid pst5552  (Addgene inc)

Journal: Nucleic Acids Research

Article Title: MDS/AML-associated DDX41 helicase facilitates homologous recombination repair by potentially resolving R-loops

doi: 10.1093/nar/gkag219

Figure Lengend Snippet: DDX41 loss diminishes HR repair. ( A ) Western blot assays of DDX41 siRNA and siRNA control in U2OS GFP reporter cell lines with indicated antibodies. β-Actin serves as a loading control. Quantification of relative protein level for DDX41 and I-SecI is shown on the right. ( B ) Percentages of GFP positive cells as assessed by flow cytometry 36 h after U2OS GFP reporter cell lines treated with siRNA control, siRNA control + I-SceI plasmid, or DDX41 siRNA + I-SceI plasmid. ( C ) Cell survival assays of WT and DDX41–KO HT1080 cells treated with Olaparib. ( D and E ) Immunofluorescence staining of WT and DDX41–KO HT1080 cells with γH2AX, RPA32 (D) or RAD51 (E), and DAPI without bleomycin treatment (UT) or 4 h post BLM treatment (30 μg/ml for 1 h). Quantification of RPA32 and RAD51 foci is shown in the middle. Data represent the mean ± SEM of three independent experiments. * P < 0.05, *** P < 0.001, and **** P < 0.0001.

Article Snippet: U2OS (HTB-96, ATCC) and U2OS GFP reporter cells [ ] (a gift from Jeremy Stark, City of Hope) were grown in Mccoy 5A media (16600082, Thermo Fisher) supplemented with 10% fetal bovine serum.

Techniques: Western Blot, Control, Flow Cytometry, Plasmid Preparation, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Identity, ontogeny, and age-related changes in splenic white pulp macrophages in mouse and human spleen

doi: 10.64898/2026.04.02.716095

Figure Lengend Snippet: Identification and characterization of macrophages in mouse spleen. a. Immunofluorescence showing GFP fluorescence (green, top left), MARCO staining for MZM (cyan, top right), CD169 staining for MMM (magenta, bottom left), and merge of all three channels (bottom right) from Cx3cr1 gfp/wt mouse spleen. Scale bars 200 μm. b. Gating strategy for flow cytometry cell sorting used to isolate cells for cytospin analysis, starting with live singlets. c. Representative cytospin images. Scale bars 10 μm. d. UMAP plot showing integrated analysis of 12,114 myeloid cells from 3–4-month-old Cx3cr1 gfp/wt mice, 2 male and 2 female. e. Expression of Cd163 , Marco , Siglec1 , and Gpnmb in the myeloid cells on the UMAP in d . f. Heatmap showing expression of selected genes within splenic macrophage clusters. Each row represents a single mouse. g. Immunofluorescent microscopy showing endogenous ZsGreen signal (green, top left), MARCO staining for MZM (cyan, top right), Tim-4 staining of MMM, MZM, and TBM (magenta; bottom left), and merge (bottom right) from Cx3cr1 ERCre x ZsGreen mouse spleen. Scale bars 150 μm.

Article Snippet: The Cx3cr1-Cre ERT2 mice , zs-Green reporter mice , and Cx3cr1 gfp reporter mice were obtained from Jackson Laboratories (Jax stocks 020940, 007906, and 005582, respectively).

Techniques: Immunofluorescence, Fluorescence, Staining, Flow Cytometry, FACS, Expressing, Microscopy

Journal: bioRxiv

Article Title: Identity, ontogeny, and age-related changes in splenic white pulp macrophages in mouse and human spleen

doi: 10.64898/2026.04.02.716095

Figure Lengend Snippet: a. UMAP plot showing integrated analysis of 14,574 cells from 3–4-month-old Cx3cr1 gfp/wt mice, 2 male and 2 female. b. Barplot illustrating composition of the clusters from UMAP on a . c. Dot plot illustrating expression of the top 5 cluster marker genes from a . d. Dot plot illustrating expression of selected genes within monocytes, macrophages, and dendritic cells from . Legend is the same as for panel c . e. Histograms illustrating the expression of selected proteins on monocytes, macrophages, and dendritic cells from as detected via the CITE-seq assay f. Heatmap illustrating the expression of selected proteins on monocytes, macrophages, and dendritic cells from as detected via the CITE-seq assay. Each column represents a single mouse. g. Cells with high GFP expression in Cx3cr1 gfp/wt mice uniformly express high levels of Tim-4 protein detected via flow cytometry. Representative flow plots, gated on live singlets, showing fluorescence minus one control (FMO, left) and Tim-4 staining (right). Numbers indicate the percent of cells in the gate. h. Heatmap illustrating expression of selected transcription factors in monocytes and macrophages. Each column represents a single mouse.

Article Snippet: The Cx3cr1-Cre ERT2 mice , zs-Green reporter mice , and Cx3cr1 gfp reporter mice were obtained from Jackson Laboratories (Jax stocks 020940, 007906, and 005582, respectively).

Techniques: Expressing, Marker, Flow Cytometry, Fluorescence, Control, Staining

Journal: bioRxiv

Article Title: Identity, ontogeny, and age-related changes in splenic white pulp macrophages in mouse and human spleen

doi: 10.64898/2026.04.02.716095

Figure Lengend Snippet: a. Schematic of the lineage-tracing experiment. Tam: tamoxifen. b. Scatterplots showing percentage of ZsGreen-positive Tim-4+ macrophages in Cx3cr1 ERCre x ZsGreen mice after tamoxifen treatment. Number of mice (from left to right): Female: 5, 5, 15, 6. Male: 13, 11, 17, 20. Results were compiled from 4 independent experiments. Significance was determined using one-way ANOVA, using a non-parametric Kruskal-Wallis test with Dunn’s multiple comparison test. The exact adjusted p-value is shown. c. Representative immunofluorescence microphotographs illustrating endogenous ZsGreen fluorescence (green), MARCO staining for MZM (cyan), CD169 staining for MMM (magenta), and merge of all three channels in the spleen of lineage-tracing Cx3cr1 ERCre x ZsGreen mouse after tamoxifen treatment. Scale bars 200 μm. d. Schematic of the spleen transplant experimental design. e. Bar plot showing percentage of donor-derived total immune cells (CD45 + cells), RPM, and Tim-4+ macrophages (Tim-4 macs) 3 months post-transplant. Data from 2 experiments, a total of 5 mice, male mice were used for both donors and recipients Kruskal-Wallis test followed by Dunn’s post-hoc testing was used to identify statistically significant differences between groups: adjusted p-value is 0.019 for CD45 + cells vs. RPM comparison, 0.029 for CD45 + cells vs. Tim-4 + macrophages, and >0.99 for RPM vs. Tim-4 + macrophages. ns: not significant. f. Schematic of the MMM depletion and repopulation experiment. Tam: tamoxifen. GM3-CL: GM3-modified clodronate-loaded liposomes. g. Representative immunofluorescence microphotographs illustrating endogenous ZsGreen fluorescence (green), MARCO staining for MZM (cyan), CD169 staining for MMM (magenta), and merge of all three channels in the spleen of lineage-tracing Cx3cr1 ERCre x ZsGreen mouse after treatment with GM3-CL. Scale bars 200 μm. h. Box plot illustrating the change in percentage of ZsGreen/CD169 double-positive area after GM3-CL treatment. One-way ANOVA followed by Dunnett’s multiple comparison test was used to identify statistically significant differences between all groups. i. Box plot illustrating the change in percentage of ZsGreen/MARCO double-positive area after GM3-CL treatment. One-way ANOVA followed by Dunnett’s multiple comparison test was used to identify statistically significant differences between all groups.

Article Snippet: The Cx3cr1-Cre ERT2 mice , zs-Green reporter mice , and Cx3cr1 gfp reporter mice were obtained from Jackson Laboratories (Jax stocks 020940, 007906, and 005582, respectively).

Techniques: Comparison, Immunofluorescence, Fluorescence, Staining, Derivative Assay, Modification, Liposomes

Journal: bioRxiv

Article Title: Identity, ontogeny, and age-related changes in splenic white pulp macrophages in mouse and human spleen

doi: 10.64898/2026.04.02.716095

Figure Lengend Snippet: a. Flow cytometry gating strategy for lineage-tracing experiments. Tim-4+ MP: Tim-4-positive macrophages. RPM: red pulp macrophages. b. Scatterplots showing percentage of ZsGreen-positive RPM in Cx3cr1 ERCre x ZsGreen mice after tamoxifen treatment. Number of mice (from left to right): Female: 5, 5, 15, 6. Male: 13, 11, 17, 20. Results were compiled from 4 independent experiments. Significance was determined using one-way ANOVA, using a non-parametric Kruskal-Wallis test with Dunn’s multiple comparison test. The exact adjusted p-value is shown. c. Representative microphotographs of spleens from mice treated with GM3-CL. Scale bar is 250 μm.

Article Snippet: The Cx3cr1-Cre ERT2 mice , zs-Green reporter mice , and Cx3cr1 gfp reporter mice were obtained from Jackson Laboratories (Jax stocks 020940, 007906, and 005582, respectively).

Techniques: Flow Cytometry, Comparison

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