Journal: bioRxiv

Article Title: A stem cell knockout village reveals lineage rewiring and a non-canonical islet cell fate in monogenic diabetes

doi: 10.64898/2025.12.23.696311

Figure Lengend Snippet: a. The expression dynamics of perturbed genes during hPSC-derived islet differentiation, based on in-house bulk RNA-seq. b. The list of perturbed genes, categorized by the function, mutant type, cell background and clone number in the knockout village. c. Left: quality control strategy on LARRY barcode libraries and transcriptome libraries. Right: The fraction of cells passing both filtering steps. d. Histogram showing the distribution of LARRY barcode read counts before filtering steps. e. Stacked line plot showing the clone representation during differentiation. Each line represents a clone. f. Cell numbers of individual clones during differentiation. The lines represent the median cell number. g. Histograms showing the contribution of read depth, cell background, and genotype to gene expression variance. h. Dot plot showing the expression of marker genes associated with each cell type. i. UMAP embedding of scRNA-seq dataset, annotated by cell cycle phase.

Article Snippet: Individual GFP-barcode plasmids were cloned from the LARRY barcode library (Addgene #140024) and then transfected to 293T cells to produce lentivirus.

Techniques: Expressing, Derivative Assay, RNA Sequencing, Mutagenesis, Knock-Out, Control, Clone Assay, Gene Expression, Marker

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