Journal: bioRxiv

Article Title: Cross-species single-cell atlases chart progression, therapy-driven remodelling and immune evasion in pancreatic cancer

doi: 10.64898/2026.03.19.712924

Figure Lengend Snippet: a) Schematic overview of the experimental design. Mouse PDAC cell lines were clonally barcoded using a lentiviral library (1), expanded and orthotopically transplanted into syngeneic immunocompetent mice (2–3), followed by scRNA-seq profiling of resultant tumours (4). Expressed barcode tracing enabled unambiguous separation of malignant (TAG⁺) from host-derived non-malignant cells (right panel, UMAPs depicting barcoded (TAG + ) (top) and malignant (bottom) cells). b-e) UMAPs of the integrated Mouse PDAC Atlas coloured by dataset (b), sex (c), treatment type (d), and model (orthotopic syngeneic immunocompetent allografts vs. autochthonous GEMMs (e). f) Sample-wise cell-type composition across treatment types, datasets, and models. Autochthonous tumours were dominated by classical epithelial-like malignant states, whereas orthotopic allografts displayed greater heterogeneity with an enrichment of EMT, hypoxic, and mesenchymal programs. g ) Level 3 hierarchical annotation of the Mouse Atlas using the same multi-tiered scheme as the Human Atlas, resolving lymphoid, myeloid, stromal, endocrine, exocrine, endothelial, and malignant compartments. h-j) Substate resolution of major immune and stromal lineages: CD4⁺ T cells (h), CD8⁺ T cells (i), and macrophages (j), showing distinct regulatory, effector, angiogenic, and lipid-processing programs. k) Validation of double-positive (DP) CD4⁺CD8⁺ T cells at the transcriptomics level (transcription density plots, left panel) and at the protein level by flow cytometry (right panel). l) UMAP showing DP T cells coloured by species (left) and Pearson correlation of mouse DP T cell gene expression against the human DP T cell archetype (right).

Article Snippet: For clonal and state-fate analysis by single-cell RNA-seq, primary mouse PDAC cells were clonally tagged with expressed DNA barcodes using the LARRY Barcode Library (Addgene #140024; RRID: Addgene 140024) .

Techniques: Derivative Assay, Biomarker Discovery, Transcriptomics, Flow Cytometry, Gene Expression

Journal: The FASEB Journal

Article Title: Klotho regulation by albuminuria is dependent on ATF3 and endoplasmic reticulum stress

doi: 10.1096/fj.201900893r

Figure Lengend Snippet: FIGURE 5 Albumin induces ER stress in vitro and in vivo. Relative mRNA and protein levels of ER stress response genes as measured by qPCR (N = 10), (A) and western blot (N = 8), (B), respectively, in HEK-293 overexpressing cells exposed to 10 mg/mL BSA for 24 hours. Relative mRNA and protein levels of ER stress response genes as measured by qPCR (N = 10), (C) and western blot (N = 8), (D), respectively, in the HK-2 cells exposed to 10 mg/mL BSA for 24 hours. Relative mRNA levels of ER stress response genes in the cortex of control (N = 13) and proteinuric mice (N = 16) 7 days post-injection (E). Representative immunohistochemical staining of ATF4 and ATF3 transcription factors in kidney sections (F). The mRNA levels are normalized to RPLP0 gene expression and the protein levels to β-actin. Mean ± SD, multiple t test without correction *P < .05

Article Snippet: HK-2 or FNL-7 cells were transiently transfected with plasmids expressing the human ATF4 (pRK-FLAG-ATF4, #26114; Addgene, Cambridge, MA) and human ATF3 (pRK-FLAG-ATF3, #26115; Addgene) transcription factors with the use of FuGENE HD as described before.

Techniques: In Vitro, In Vivo, Western Blot, Control, Injection, Immunohistochemical staining, Staining, Gene Expression

Journal: The FASEB Journal

Article Title: Klotho regulation by albuminuria is dependent on ATF3 and endoplasmic reticulum stress

doi: 10.1096/fj.201900893r

Figure Lengend Snippet: FIGURE 6 ATF4 and ATF3 regulate Klotho expression. Relative mRNA and protein levels of Klotho, ATF4 and ATF3 as measured by qPCR and western blot, respectively, in HK-2 cells transiently overexpressing the human ATF4 transcription factor (two experiments), (A) or the human ATF3 transcription factor (three experiments), (B). Relative luminescence in the FNL-7 cell line expressing the human Klotho promoter driving firefly luciferase expression in control and cells overexpressing human ATF4, ATF3, or both factors (N = 6), (C). The HK-2 cells were also transduced with shRNA lentiviral vectors targeting ATF4 or ATF3 and the relative mRNA levels of Klotho as well as the protein levels were assessed by qPCR (N = 8/8/4), (D) or western blot analysis (N = 3), (E), respectively, in the presence or absence of albumin in comparison to the scrambled shRNA vector. The silencing efficiency was verified by western blot analysis (N = 4/3), (F). The mRNA levels are normalized to RPLP0 gene expression and the protein levels to β-actin or Ponceau staining. Mean ± SD, unpaired two-tailed t test or multiple t test without correction or one-way ANOVA *P < .05

Article Snippet: HK-2 or FNL-7 cells were transiently transfected with plasmids expressing the human ATF4 (pRK-FLAG-ATF4, #26114; Addgene, Cambridge, MA) and human ATF3 (pRK-FLAG-ATF3, #26115; Addgene) transcription factors with the use of FuGENE HD as described before.

Techniques: Expressing, Western Blot, Luciferase, Control, Transduction, shRNA, Comparison, Plasmid Preparation, Gene Expression, Staining, Two Tailed Test

Journal: Molecular Cancer Research

Article Title: Single-Cell Evolutionary Analysis Reveals Drivers of Plasticity and Mediators of Chemoresistance in Small Cell Lung Cancer

doi: 10.1158/1541-7786.mcr-22-0881

Figure Lengend Snippet: Figure 1. scRNA-seq reveals two distinct subpopulations in SCLC. A, Overview of the autochthonous barcoding model. Tumors in the RPR2-Cas9 model were initiated, and barcoded at 1-month intervals, up to 5 months after initiation. Tumors were harvested at 1-month intervals following barcoding, up to 6 months, and GFPþ cells were isolated and scRNA-seq was performed. B, Multiple cellular populations were detected in the scRNA-seq due to the presence of microdissected tumors in this analysis, including a large population of SCLC cells. C, Proportion of cells in each stage of the cell cycle at varying months of tumor development. D, UMAP plot of the SCLC cells labeled by months of tumor development. Black outlines describe the clusters identified in E. E, Unbiased clustering using Leiden community detection of the SCLC cells identifies two clusters. Because of the distribution of timepoints in D, clusters are labeled “early” or “late”. F, Proportion of cells in the early and late populations at each timepoint after tumor initiation. G, Gene modules that differentiate early and late populations. Significance determined by an unpaired Student t test where , P < 0.05; , P < 0.01; , P < 0.001.

Article Snippet: To clone the retroviral barcoding library, a CAG-GFP retroviral plasmid was used as a backbone (gift from Fred Gage, Addgene plasmid #16664; ref. 27).

Techniques: Isolation, Labeling

Journal: Molecular Cancer Research

Article Title: Single-Cell Evolutionary Analysis Reveals Drivers of Plasticity and Mediators of Chemoresistance in Small Cell Lung Cancer

doi: 10.1158/1541-7786.mcr-22-0881

Figure Lengend Snippet: Figure 3. Generation and validation of barcoded xenografts and PCR error rate correction. A, The xenograft barcoding system. The barcode is inserted retrovirally, and contains a CAG promoter, GFP, the LBC, and a polyA tail. B, Overview of the lineage tracing process. Cells are barcoded in culture and a portion taken for scRNA-seq. The remainder of the cells are injected as a xenograft. Half of this xenograft undergoes scRNA-seq, and the other half is injected as a serial xenograft into a new mouse, which is treated with chemotherapy and then undergoes scRNA-seq. C, Doubling time of the two SCLC cell lines used for making xenografts, NCI-H209 and NCI-H82. D, Process of sequencing LBCs for diversity validation. E, PCR purification of the LBC and subsequent sequencing. Diversity estimates from each read direction of the PCR products are shown. F, Radial plot showing the unbiased clustering of LBC similarity for a single sample. A single A>G transition is observed which is interpreted as a PCR error as its relative occurrence in the population is less than the calculated PCR error rate determined by variants in the LBC constant regions. G, Representation of the degree of overlap between two subdivided samples split at various cellular doublings measuring in C. H, Degree of overlap between a single-sample split in half to represent the labeled starting cell line and the cells to be divided into four subpopulations for xenograft injection.

Article Snippet: To clone the retroviral barcoding library, a CAG-GFP retroviral plasmid was used as a backbone (gift from Fred Gage, Addgene plasmid #16664; ref. 27).

Techniques: Biomarker Discovery, Injection, Sequencing, Labeling