Journal: The Journal of Reproduction and Development

Article Title: Perilipin2 depletion causes lipid droplet enlargement in the ovarian corpus luteum in mice

doi: 10.1262/jrd.2024-023

Figure Lengend Snippet: Generation of Plin2-null mice using CRISPR/Cas9-mediated genome editing. (A) Schematic diagram of the Plin2 gene structure and sgRNA targeting site for the CRISPR/Cas9-mediated generation of Plin2-mutant alleles. Targeting sites of sgRNA are indicated in red. The PAM sequence (NGG) is labeled in green. The four deleted nucleotides (Δ4) are indicated with a red hyphen. Underline shows the recognition sequence for the restriction enzyme ( Van91l ). ATG, translation initiation site. Bottom: the Plin2 sequence for wild-type and mutant is indicated. (B) Representative genotyping of PCR and subsequent Van91l digestion in Plin2-deficient mice. Bands corresponding to Van91l -digested (369 bp and 132 bp for wild-type [ Plin2 +/+ ]) or non-digested (497 bp for mutant [ Plin2 +/− and Plin2 −/− ]) Plin2 gene are indicated. (C) Representative immunoblots against PLIN2 and PLIN3 in whole ovarian lysates from hormone (PMSG + hCG)-treated Plin2 +/+ , Plin2 +/− , and Plin2 −/− mice. Tubulin was used as a loading control. (D) Representative immunoblots against PLIN2 in whole ovarian lysates from Plin2 +/+ , Plin2 +/− , and Plin2 −/− mice treated with or without PMSG + hCG. Actin was used as a loading control. (E) Representative confocal images of frozen ovarian sections from Plin2 +/+ and Plin2 −/− mice immunolabeled with anti-PLIN2 antibody. Cell nuclei were counterstained with DRAQ5. Ovarian follicles are outlined by dashed lines. Bottom panels show enlarged images of the boxed area. Numbers indicate the follicle stage. Arrows, PLIN2-puncta in Plin2 +/+ GC. Scale bars, 50 μm and 10 μm (inset).

Article Snippet: Membranes were blocked with Blocking One (03953-95, Nacalai Tesque, Kyoto, Japan) for 1 h, incubated overnight at 4°C with anti-PLIN2 antibody (1:1,000; NB110-40877; Novus Biologicals) or anti-PLIN3 antibody (1:1,000; NB110-40764, Novus Biologicals), and then incubated with horseradish peroxidase (HRP)-linked anti-rabbit IgG (1:10,000; #7074, Cell Signaling Technology).

Techniques: CRISPR, Mutagenesis, Sequencing, Labeling, Western Blot, Control, Immunolabeling

Journal: The Journal of Reproduction and Development

Article Title: Perilipin2 depletion causes lipid droplet enlargement in the ovarian corpus luteum in mice

doi: 10.1262/jrd.2024-023

Figure Lengend Snippet: Distribution of PLIN1, 2, and 3 in mouse ovaries. Representative confocal images of frozen ovarian sections from PMSG- or hCG-treated mCherHPos mice immunolabeled with antibodies against PLIN1 (A), PLIN2 (B), or PLIN3 (C). (D) Schematic illustration of the characteristics of mCherry-HPos, which accumulates in nascent to growing LDs synthesized in the ER and allows visualization of LD synthesis. Cell nuclei were counterstained with DRAQ5. The image on the right of each panel is a higher-magnification image of the boxed region in the left image. GC, granulosa cells; TC, theca cells; IC, interstitial cells; CL, corpus luteum. The ovarian follicles and CL are outlined with dashed lines. Numbers indicate the follicle stage (see Materials and Methods for details). Arrows, PLIN2- or PLIN3-puncta in the GC; arrowheads, PLIN2- or PLIN3-granules in the CL. Scale bars, 50 μm and 10 μm (inset).

Article Snippet: Membranes were blocked with Blocking One (03953-95, Nacalai Tesque, Kyoto, Japan) for 1 h, incubated overnight at 4°C with anti-PLIN2 antibody (1:1,000; NB110-40877; Novus Biologicals) or anti-PLIN3 antibody (1:1,000; NB110-40764, Novus Biologicals), and then incubated with horseradish peroxidase (HRP)-linked anti-rabbit IgG (1:10,000; #7074, Cell Signaling Technology).

Techniques: Immunolabeling, Synthesized