Journal: The Journal of Cell Biology

Article Title: Unraveling the kinetochore nanostructure in Schizosaccharomyces pombe using multi-color SMLM imaging

doi: 10.1083/jcb.202209096

Figure Lengend Snippet: Cloning strategy, sample preparation, and health assessment of 3C strains. (A) Cloning strategy. DNA fragments containing the POI 3′-end (pink), the FP-resistance cassette (yellow-red-grey-green), and the POI 3′- UTR (purple) were amplified with the corresponding primers with ∼20 bp overlap to the future neighboring DNA fragments from either genomic WT DNA or a plasmid DNA. Pieces were then fused by overlap extension PCR and transformed into the dual-color reference strain (h+, leu1-32, ura4-D18, sad1:mScarlet-I:hphMX6, PAmCherry1:cnp1 CENP-A ) using homologous recombination to create a 3C strain library . (B) Sample preparation. Cell cultures were synchronized using lactose gradient centrifugation, which accumulates cells in early G2 phase in an upper band. The cells were then extracted from the gradient column, grown for another 1–1.5 h until mitosis, and chemically fixed, washed, and embedded in agarose gel with fiducial markers. (C and D) Assessment of strain health & mitotic defects. Temperature (C) or TBZ (D), which induces mitotic defects by microtubule depolymerization, sensitivities between the parental WT, the dual-color reference, and the individual 3C strains were assessed by spot tests. Here, a 10-fold dilution series of OD 600 from 1.0 to 0.001 of overnight cultures was grown on either YES media plates at 25, 32, and 37°C for 3 d or YES media plates containing 2.5, 5.0, 7.5, and 10.0 μg/ml TBZ and incubation at 25°C for 3 d. (E) Flow cytometry measurements to assess S. pombe strain health. FSC-W and SSC-W contour plots (each level within the contour plot consists of 10% measured cells) and their corresponding histograms of 3C (POI-3C) strains and the dual-color template strain compared to a fission yeast WT strain. A defect in cell division usually results in increased cell length and higher FSC-W and SSC-W values, which we observed for cnp3 CENP-C -3C (top row, left column) but none of the other strains tested. N = 10,000 for each POI (see Materials and methods).

Article Snippet: For this, S. pombe strains from cryostocks were streaked on YES agar plates and incubated at 32°C for 2 d. After growing the overnight cultures in YES medium at 25°C to an OD 600 of 1–2, all cultures were adjusted to an OD 600 of 1.0 and 2 μl each of a serial dilution of 1:1, 1:10, 1:100, and 1:1,000 were plated either on YES agar plates and incubated at 25, 32, or 37°C for 3 d for temperature sensitivity testing, or on YES agar plates containing 2.5, 5.0, 7.5, or 10.0 μg/ml TBZ (#23391; Cayman Chemical Company) and incubated at 25°C for 3 d.

Techniques: Cloning, Sample Prep, Amplification, Plasmid Preparation, Transformation Assay, Homologous Recombination, Gradient Centrifugation, Agarose Gel Electrophoresis, Incubation, Flow Cytometry

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: The anti-fibrotic efficacy of adelmidrol depends on hepatic PPARγ levels.

doi: 10.1016/j.biopha.2023.115051

Figure Lengend Snippet: Fig. 6. Adelmidrol up-regulated the PPARγ in HSC-T6, RAW264.7, and HepG2. (A) The mRNA levels of PPARγ, TGFβ1, α-SMA, and PDGFRα in HSC-T6 cells after 24 h of treatment (n = 3). (B-C) The protein levels of PPARγ, a-SMA and PDGFRα in HSC-T6 cell (n = 3). (D) The mRNA level of PPARγ, NF-κB, TNF-α, and CCl2 in RAW264.7 cells after 24 h of treatment (n = 3). (E-F) The protein levels of nucleus PPARγ, NF-κB p65, and cytoplasm NF-κB p65 in RAW264.7 cells (n = 3). (G) The mRNA levels of PPARγ, CD36, SCD1, and PPARα in hepG2 cells after 24 h of treatment (n = 3). (H-I) The protein levels of PPARγ, CD36, and SCD1 in hepG2 cells (n = 3). Significant differences between the GW9662 pretreatment group and the ADE group are indicated by letters. Significant differences between the model group and the ADE group are indicated by asterisks. The data are presented as means ± SEM. Con refers to the normal control group; FFA refers to the free fatty acids (1 mM) treated group; LPS refers to the lipopolysaccharide (1 µg/mL) treated group; ADE refers to the adelmidrol (10 µM). GW, GW9662 (5 µM); a*P < 0.05; b**P < 0.01; c***P < 0.001.

Article Snippet: After blocking, the membranes were incubated overnight at 4 ◦C with primary antibodies against PPARγ (#2435, 1:500, CST), CD36 (#D161529, 1:1000, Sangon Biotech), SCD1 (#23391, 1:1000, Proteintech), NF-κB P65 (#8242 T 1:1000, CST) PDGFRα (#ab203491, 1:1000, Abcam), TGFβ1 (#AF1027,1:2000, Affinity), and α-SMA (#ab66133, 1:1000, Abcam).

Techniques: Control