Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: KLF15 suppresses stemness of pancreatic cancer by decreasing USP21-mediated Nanog stability

doi: 10.1007/s00018-024-05442-6

Figure Lengend Snippet: Tazemetostat sensitizes PDAC cells to gemcitabine by upregulating the KLF15 expression ( A ) Cell viability analyses were performed in PANC-1-Vector, PANC-1-KLF15-OE, BxPC-3-Scramble and BxPC-3-KLF15-KD after Gemcitabine treatment. ( B ) Apoptotic cell analyses using flow cytometry were performed in PANC-1-Vector, PANC-1-KLF15-OE, BxPC-3-Scramble and BxPC-3-KLF15-KD after Gemcitabine treatment(left), and the proportion of apoptotic cells statistical analysis (right). * P < 0.05. (C-D) Western blot on the expression of H3K27me3, EZH2, KLF15 and Nanog after Tazemetostat treatment. (E-F) Q-PCR on the expression of KLF15 after treatment by different concentrations of Tazemetostat, GAPDH as the control. (G-H) ELDA for PANC-1 and SW1990 cells after Tazemetostat treatment, using the ELDA software ( http://bioinf.wehi.edu.au/software/elda ). All above experiments were repeated three times independently. Paired Student’s t-test was used for statistical analysis. * P < 0.05 ** P < 0.01*** P < 0.001. ( I ) The subcutaneous tumors formed by four groups: control, Tazemetostat, gemcitabine and the combination of Tazemetostat and gemcitabine, H&E staining and IHC staining of Ki67 in above four groups. (mean ± SD, n = 3), Scale bar (black), 200 μm. ( J ) The statistical analysis of Ki67 staining in the above four groups. (K-L) The volume ( K ) and the weight ( L ) of subcutaneous tumors in above four groups, (mean ± SD, n = 3), * P < 0.05 ** P < 0.01*** P < 0.001

Article Snippet: One week later, Gemcitabine (20 mg/kg/day, DMSO was used as control, MCE, HY-17026) was injected once a week, and Tazemetostat (100 mg/kg, DMSO was used as control, MCE, HY-147090) was injected three times a week.

Techniques: Expressing, Plasmid Preparation, Flow Cytometry, Western Blot, Control, Software, Staining, Immunohistochemistry

Journal: Nature Communications

Article Title: ATG8ylation-mediated tonoplast invagination mitigates vacuole damage

doi: 10.1038/s41467-025-62084-3

Figure Lengend Snippet: a Analysis of the subcellular location of GFP-FREE1 before and after monensin treatment. Scale bar, 20 μm. b Time-series analysis of the invagination of ATG8-positive vesicles in free1 (-/-) mutant. The magenta arrows indicated a vesicle that adhered inside the vacuolar membrane; white arrows indicated invaginated vesicles. Scale bar, 10 μm. c Confirmation of the validity of the dexamethasone (DEX)-inducible dominant-negative mutants, DEX:SNF7.1(L32W) and DEX:VPS4(E232Q). The transgenic seeds were germinated on agar plates containing 10 μM DEX. Representative images were captured after 5 days of growth under light. Scale bar, 1 cm. d A schematic diagram showing the treatment flow of DEX-inducible dominant-negative mutants. e , f Time-series analysis of the invaginated vesicles in YFP-ATG8e/DEX:SNF7.1(L32W) ( e ) and YFP-ATG8e/DEX:VPS4(E232Q) ( f ) plants after DEX induction. The magenta arrows indicated vesicles that adhered inside the tonoplast. Scale bars, 20 μm. g TEM analysis of vacuolar morphology in free1 (-/-) mutant and DEX:VPS4(E232Q) transgenic plants. The magenta arrows indicate invaginating vesicles. Scale bars, 1 μm. h , i Analysis of the effects of cytoskeletal disruption on the formation of ATG8-positive vesicles. Microtubules were visualized using a mCherry-TUA5 fusion protein ( h ), while microfilaments were labeled with ABD2-GFP ( i ). Oryzalin (10 μM) and latrunculin B (10 μM) were employed to disrupt microtubule and microfilament structures, respectively. The invaginated vesicles were denoted by yellow arrowheads. Scale bars, 20 μm. j Confocal time-lapse analysis the effects of microfilament disruption on the dynamics of ATG8-positive vesicle invagination. White arrowheads indicated the invaginated vesicles. Scale bar, 20 μm. Similar confocal imaging results were obtained in at least six individual roots with three replicates.

Article Snippet: ConcA (MCE, #HY-N1724) and wortmannin (MCE, #HY-10197), oryzalin (MCE, #HY-147092), and latrunculin B (MCE, #HY-101848) stock solutions were prepared at concentrations of 1, 16.5, 10, and 10 mM, respectively, in DMSO and stored at − 20 °C.

Techniques: Mutagenesis, Membrane, Dominant Negative Mutation, Transgenic Assay, Disruption, Labeling, Imaging