Journal: iScience

Article Title: Peroxisome-dependent and -independent functions of PEX19 in mouse embryonic stem cells

doi: 10.1016/j.isci.2026.117658

Figure Lengend Snippet: Deletion of Pex19 induces mitotic block in mESCs (A) Deletion of Pex19 impairs cell cycle progression and induces polynuclear mESCs. WT, Pex19 KO, and Pex19 KO + PEX19 46C mESCs were stained with PI for DNA content, then subjected to FACS analysis ( n = 3 biological replicates/group; data are presented as mean ± SEM). (B) Deletion of Pex5 in mESCs fails to induce detectable defects in the cell cycle. WT, Pex5 KO, and Pex19 KO 46C mESCs were stained with DAPI for DNA content, then subjected to FACS analysis. (C) Deletion of Pex19 but not Pex5 induces a new fraction of polynuclear mitotic cells. WT, Pex5 KO, and Pex19 KO 46C mESCs treated with or without 50 ng/mL nocodazole were stained with an anti-H3pS10 antibody for detection of mitotic cells and DAPI for DNA content, then subjected to FACS analysis. The M-B population represents polynuclear mitotic cells arrested by deletion of Pex19 or treatment with nocodazole. (D and E) Deletion of Pex19 delays cell cycle progression of mESCs. WT and Pex19 KO 46C mESCs treated with 50 ng/mL nocodazole for 5 h were transferred into normal culture medium without nocodazole, and cell cycle progression was analyzed 15 and 25 min later (three groups of cells (80–250 cells/group) from randomly selected fields were analyzed for each time point for both WT and Pex19 KO mESCs; data are presented as mean ± SEM). Scale bars, 10 μm. See also .

Article Snippet: After fixation with 4% paraformaldehyde, cells were processed for immunofluorescence assay with anti-H3pS10 antibody and phalloidin (Cytoskeleton, PHDH1) staining.

Techniques: Blocking Assay, Staining, Analysis

Journal: iScience

Article Title: Peroxisome-dependent and -independent functions of PEX19 in mouse embryonic stem cells

doi: 10.1016/j.isci.2026.117658

Figure Lengend Snippet: Pex19 KO mESCs have reduced exit from pluripotency during the early stage of neural differentiation (A) Morphology of WT and Pex19 KO 46C mESCs after three days of in vitro neural differentiation. Scale barss, 100 μm. (B) Deletion of Pex19 delays the reduction of pluripotency genes during early neural differentiation. The expression of indicated genes was analyzed before and after three days of in vitro neural differentiation by qPCR ( n = 3 biological replicates/group; data are presented as mean ± SEM; two-way ANOVA, ∗∗∗q < 0.001, ∗∗∗∗q < 0.0001, n.s., not significant). (C) The protein level of Nanog in WT and Pex19 KO cells after three days of in vitro neural differentiation. Scale bars, 20 μm. (D) Adding back PEX19 restores the differentiation-induced reduction of pluripotency protein in Pex19 KO cells. The levels of indicated proteins were analyzed in WT, Pex19 KO (KO), and Pex19 KO + PEX19 (KO + PEX19) 46C mESCs before and after three days of in vitro neural differentiation by immunoblotting. (E) Treatment with DMSO partially increases G1 phase in Pex19 KO cells after three days of in vitro neural differentiation. WT and Pex19 KO 46C mESCs after three days of in vitro neural differentiation with or without 0.1% DMSO were stained with an anti-H3pS10 antibody for detection of mitotic cells and DAPI for DNA content, then subjected to FACS analysis ( n = 3 biological replicates/group; data are presented as mean ± SEM, two-way ANOVA, ∗q < 0.05, ∗∗q < 0.01, n.s., not significant). (F) Adding back PEX19 or treatment with DMSO partially rescues the differentiation defects in Pex19 KO cells. The levels of indicated genes were analyzed in WT, Pex19 KO (KO), or Pex19 KO + PEX19 (KO + P19) 46C mESCs after three days of in vitro neural differentiation with or without 0.1% DMSO by qPCR ( n = 3 biological replicates/group, data are presented as mean ± SEM, two-way ANOVA, ∗q < 0.05, ∗∗q < 0.01, ∗∗∗q < 0.001, and ∗∗∗∗q < 0.0001). (G) Treatment with DMSO partially restores the pluripotency exit defect in Pex19 KO cells. (H) Treatment with a low dose of nocodazole decreases the G1 population in WT cells after three days of in vitro neural differentiation. WT 46C mESCs before (D0) or after three days of in vitro neural differentiation with or without 12.5 ng/mL nocodazole were analyzed for G1 population as in (E) ( n = 3 biological replicates/group; data are presented as mean ± SEM; one-way ANOVA; ∗q < 0.05, ∗∗q < 0.01, n.s., not significant). (I) Treatment with a low dose of nocodazole delays the reduction of pluripotency genes in WT cells after three days of in vitro neural differentiation. The levels of indicated pluripotency genes were analyzed in WT 46C mESCs treated as in (H) ( n = 3 biological replicates/group; data are presented as mean ± SEM, one-way ANOVA, ∗ q < 0.05, ∗∗ q < 0.01, ∗∗∗ q < 0.001, ∗∗∗∗ q < 0.0001, n.s., not significant). See also .

Article Snippet: After fixation with 4% paraformaldehyde, cells were processed for immunofluorescence assay with anti-H3pS10 antibody and phalloidin (Cytoskeleton, PHDH1) staining.

Techniques: Morphology, In Vitro, Expressing, Western Blot, Staining, Analysis

Journal: Frontiers in Immunology

Article Title: An optimized spectral flow cytometry pipeline for longitudinal blood leukocyte immunophenotyping reveals stable, individually distinct immune signatures

doi: 10.3389/fimmu.2026.1959895

Figure Lengend Snippet: Two complementary QC measures confirm stable instrument performance and spectral unmixing across the study and distinguish an instrument fault from a reagent fault. Instrument and mAb fluorescence stability was tracked across 38 acquisition dates spanning approximately 32 months (20231016–20260619). Instrument QC monitored the %CV of each of the eight fluorescent peaks (RB-1 to RB-8) of rainbow calibration beads (RB), relative to the first acquisition of each RB lot (a lot-specific baseline). mAb fluorescence QC monitored the fluorescence intensity (FI) of experimental mAb bound to SpectraComp beads across all instrument detectors (the signatures used for spectral unmixing) relative to a fixed reference derived from multiple early dates to encompass all experimental fluorescence. Dissimilarity was quantified as 1 − cosine similarity between each date’s detector profile (FI or %CV) and the reference, capturing shape change independent of magnitude (0 = identical, 1 = orthogonal). (A) Dot-plot heatmaps of mAb spectral-signature dissimilarity (left; per fluorochrome) and rainbow-bead %CV dissimilarity (right; per bead peak, RB-1 to RB-8) for each QC date (rows); circle area and color both encode dissimilarity (scales at right). Rows and columns are ordered by hierarchical clustering. (B, C) Detector-level detail for the most deviated fluorochrome and bead peak identified by the clustering in (A) , on the two flagged dates (Bad date 1, 20240501; Bad date 2, 20250808), each compared with a representative good date (20260619) and the stable reference (20250620): (B) per-detector FI for PE and PE-Fire 810 single-stain controls, and (C) per-detector %CV of rainbow-bead peak RB-4. Median dissimilarity across the study was 0.0014 (mAb spectra) and 0.023 (bead %CV); the largest single values (0.200 and 0.419, respectively) both occurred on 20240501. Dates were flagged by inspection of the clustered dissimilarity profiles, with no automated numerical threshold applied; numeric labels are shown for dissimilarities ≥ 0.05 (mAb panel) and ≥ 0.25 (RB panel) to highlight the largest changes in each metric.

Article Snippet: Assignments placed co-assigned antigens in non-overlapping branches of the gating hierarchy and were validated using single-stain spectral reference controls (using SpectraComp mAb binding beads; SSB-05-B, SlingshotBio, and ViaComp vitality dye binding beads; SSB-07-A, SlingshotBio) and lineage-gated fully-stained samples.

Techniques: Fluorescence, Stability, Derivative Assay, Staining, Automated