Journal: Cell stem cell

Article Title: Glucose modulates IRF6 transcription factor dimerization to enable epidermal differentiation.

doi: 10.1016/j.stem.2025.02.017

Figure Lengend Snippet: Figure 1. Intracellular glucose accumulates in keratinocyte differentiation (A) Heatmap representing average fold change of 614 metabolites identified in metabolomics, in early (day 3) and late (day 6) differentiation compared with the undifferentiated state (n = 5 biological replicates). (B) Accumulation of 2-NBDG (green), a fluorescent glucose analog, in differentiated keratinocytes with Hoechst nuclear stain (blue). Progenitor (PG) represents undifferentiated cells versus days 3 and 6 of calcium-induced differentiation in vitro (representative image of 3 independent samples, scale bar, 10 mm). (C and D) Quantitation of glucose-14C (C6) (C) or glucose-14C (C1) (D) accumulation during differentiation in culture, after 7 day incubation, normalized to mg of protein per sample (n = 3 biological replicates, 3 technical replicates per experiment). (E) Quantitation of glucose concentrations in regenerated epidermal organoid tissue, and whole epidermis undergoing stratification through day 6 analyzed (n = 3 biological replicates). (F) Epidermal organoids expressing vector control or the Red Glifon 3000 glucose sensor, and the dsRed signal (red) indicates detection of free glucose, with nuclear DAPI (blue) and phalloidin (green) stains (representative image of 3 biological replicates, scale bar, 50 mm). (G) Quantitation of dsRed intensity in the basal and spinous layers of the tissue normalized to phalloidin (n = 8 fields of view across 2 biological replicates). (H) Epidermis from transgenic mice expressing Red Glifon 3000; detection of free glucose (red), nuclear DAPI stain (blue), and immunostaining of differentiation marker keratin 10 (green); and gray bar indicates region of interest (ROI) used for quantitation in (I) (representative image shown, n = 3 biological replicates, scale bar, 50 mm). (I) Average fluorescence intensity with standard error (shaded region) in the ROI of Red Glifon, DAPI, and K10 across the epidermis from the basal layer up to but excluding the stratum corneum (n = 6). Data are represented as mean ± SEM, two-tailed unpaired Student’s t test was used for statistical analysis, *p < 0.05, ****p < 0.001. See also Figure S1 and Table S1.

Article Snippet: The iGlucoSnFR sensor (a gift from J. Keller and L. Looger, Addgene plasmid # 164514) and Red Glifon 300/3000 (a gift from Takashi Tsuboi, Addgene plasmid # 163115; http://n2t.net/addgene:163115) glucose sensors were cloned into a lentiviral expression vector (pLEX) and transduced into human keratinocytes.

Techniques: Staining, In Vitro, Quantitation Assay, Incubation, Expressing, Plasmid Preparation, Control, Transgenic Assay, Immunostaining, Marker, Two Tailed Test

Journal: Development (Cambridge, England)

Article Title: A simple MiMIC-based approach for tagging endogenous genes to visualise live transcription in Drosophila

doi: 10.1242/dev.204294

Figure Lengend Snippet: Transcription live imaging of pxb tagged with MS2 and PP7 stem-loops in embryos. (Ai) Schematic showing the pxb gene locus with the 24×MS2V6 insertion. Males with the 24×MS2V6 loops inserted into pxb are crossed to females expressing HisRFP and nos-2×MCP2×mNeonGreen . Embryos have the pxb-24×MS2V6 TS visible as an intense mNeonGreen signal within the HisRFP nucleus; the imaging region is shown in yellow. (Aii) Top: still images from a time-lapse movie of pxb-24×MS2V6 TSs in two anterior stripes in the nc14 embryo. Scale bars: 20 µm. Bottom: higher magnification images from nuclei with the stripe. Nuclei are marked in magenta and pxb-MS2 TSs are in green. Scale bars: 5 µm. (Bi) As in Ai but with 24×PP7 loops inserted into pxb and females are expressing HiseBFP2 and nos-2×PCP2×mCherry . (Bii) Still from a movie of an embryo expressing HiseBFP2 and nos-2×PCP2×mCherry marking the nuclei in blue and the pxb-PP7 TSs in magenta. (Ci) Schematic showing pxb labelled to visualise each allele with a different loop type (MS2 or PP7). (Cii) Dual imaging of these embryos detects transcription of both alleles. pxb-PP7 TSs are in magenta, pxb-MS2 TSs are in green, nuclei are blue in the merge. See also Movies 1-3 . Fly cartoons created in BioRender. Ashe, H. (2024) https://BioRender.com/u96a435 .

Article Snippet: Plasmids pCR4-24×MS2SL-stable (Addgene, # 31865 ), pBlueScript-24×PP7 ( ) and pET264-pUC 24×MS2V6 Loxp KANr Loxp (Addgene, # 104393 ) were digested using BamHI and BglII to obtain 24×MS2-SL, 24×PP7 and 24×MS2V6, respectively.

Techniques: Imaging, Expressing