Journal: Molecular and Cellular Biology

Article Title: p150 Sal2 Is a p53-Independent Regulator of p21 WAF1/CIP

doi: 10.1128/mcb.24.9.3885-3893.2004

Figure Lengend Snippet: FIG. 5. p150Sal2 directly interacts with p21 promoter and regulates p21 transcription through a cis-acting element. (A) Schematic representation of the 2.7-kb human p21 promoter region and its digestion fragments used in the protein DNA immunoprecipitation assay (25) (sizes in base pairs are given under each fragment). (B) Immunoprecipitation of AvaI-digested, StyI-digested, or StyI/MspI double-digested fragments of p21 promoter by p150 antibody (AntiP150) with preimmune serum (Pre-Imm) as a control. Wild-type (Wt) p150 or p150 with a deletion of the putative DNA binding domain (3) was from either P19 cell extract (Cell) or the in vitro translation (IVT) product of cloned human cDNA. Empty cloning vector was used as a negative control for in vitro-translated p150 in the immunoprecipitation of StyI/MspI fragments. (C) A p150-responsive cis-acting element is located 1.4 kb upstream of the human p21 promoter corresponding to the distal p150Sal2 binding region. A luciferase assay was conducted in SKOV-3 cells to assess the responsiveness of various p21 promoter deletion constructs. Fold induction is the corresponding luciferase activity with cotransfected p150Sal2 over that with cotransfected empty vector. The p53 binding sites are shown as landmarks. (D) p150 binds p21 promoter region in vivo. ChIP was performed using serum against p150 (Anti-p150) to precipitate chromatin cross-linked with p150 in P19 cells and amplified using p21 promoter-specific primers (p21) and primers for GAPD. The same sets of primers were also used for PCR amplification from preimmune serum-precipitated chromatin (PreImm) and total input chromatin extract (Total) as controls.

Article Snippet: SKOV-3 and P19 cells were obtained from the American Type Culture Collection.

Techniques: Immunoprecipitation, Control, Binding Assay, In Vitro, Clone Assay, Cloning, Plasmid Preparation, Negative Control, Luciferase, Construct, Activity Assay, In Vivo

Journal: bioRxiv

Article Title: Localization of planteose hydrolysis during seed germination of Orobanche minor

doi: 10.1101/2021.06.16.448768

Figure Lengend Snippet: Localization of mCherry fusion proteins transiently expressed in leaves of Nicotiana benthamiana . (A) OmAGAL2:mCherry, (E) ΔSP-OmAGAL2:mCherry, (I) SP:mCherry, and (M) mCherey were co-expressed with the apoplast marker At5g11420:pH-tdGFP (B, F, J, N). (C, D, G, H, K, L, O, P) Merged images of fluorescence from mCherry and GFP. Scale bars: 50 µm (A–C, E–G, I–K, M–O) and 20 µm (D, H, L, P).

Article Snippet: Transient expression was conducted by co-inoculation of Agrobacterium tumefaciens strain GV3101 cultures carrying each construct with those carrying the vector pMDC-At5g11420:pH-tdGFP as an apoplast marker , and pDGB3alph2_35S:P19:Tnos (GB1203, Addgene #68214) in leaves of Nicotiana benthamiana . mCherry and green fluorescent protein (GFP) were excited at 555 and 488 nm, and observed in the range of 570–600 and 490–520 nm, respectively, using a LSM700 laser scanning confocal microscope (Carl Zeiss, Jena, Germany).

Techniques: Marker, Fluorescence

Journal: Advanced Healthcare Materials

Article Title: A Human Kidney Tubuloid Model of Repeated Cisplatin‐Induced Cellular Senescence and Fibrosis for Drug Screening

doi: 10.1002/adhm.202501795

Figure Lengend Snippet: Tubuloids exhibit cellular senescence and release inflammatory cytokines following repeated cisplatin treatment. A) Experimental design for repeated cisplatin treatment. B) Representative images of SA‐β‐GAL staining of tubuloids exposed to cisplatin (0.2 µg mL −1 ) 5 times repeatedly or control. Scale bars: 100 µm. C) Immunostaining of p16 in tubuloids exposed to cisplatin (2.0 µg mL −1 ) 5 times repeatedly or control. Scale bars: 100 µm. D) Western blotting of p16 in tubuloids exposed to cisplatin (0.2 and 2.0 µg mL −1 ) 5 times repeatedly or control. E) Statistical analysis of p16 expression (n = 4). F) Western blotting of p21 in tubuloids exposed to cisplatin (0.2 µg mL −1 ) 5 times repeatedly or control. G) Statistical analysis of p21 expression (n = 5). H) Western blotting of p53 and Lamin B1 in tubuloids exposed to cisplatin (2.0 µg mL −1 ) 5 times repeatedly or control. I) Statistical analysis of p53 expression (n = 3). J) Statistical analysis of Lamin B1 expression (n = 3). K) Cell Cycle Analysis in tubuloids exposed to cisplatin (0.2 µg mL −1 ) 5 times repeatedly or control. L) Immunostaining of an inflammatory cytokine, IL‐1β, in tubuloids exposed to cisplatin (2.0 µg mL −1 ) 5 times repeatedly or control. Scale bars: 100 µm. M,N) Quantitative PCR of IL‐1β (M) and IL‐6 (N) in tubuloids exposed to cisplatin (0.2 and 2.0 µg mL −1 ) 5 times repeatedly or control (n = 3). Statical analysis was applied to delta‐delta Ct value, not fold increase. All data are presented as means ± standard errors (SEs). Statistical significance is presented by * p < 0.05, ** p < 0.01.

Article Snippet: Primary antibodies were also used, including rabbit anti‐p16‐INK4A polyclonal antibody (1:200) (Proteintech), mouse anti‐p21 antibody (1:100) (sc‐6247, Santa Cruz Biotechnology, TX, USA), rabbit anti‐Lamin B1 polyclonal antibody (1:1000) (12987‐1‐AP, Proteintech), rabbit anti‐p53 polyclonal antibody (1:1000) (10442‐1‐AP, Proteintech), rabbit anti‐KIM‐1 antibody (1:400) (PA5‐20244, Thermo‐Fisher Scientific, MA, USA), rabbit anti‐caspase‐3 antibody (1:200) (Cell Signaling Technology), and rabbit anti‐Vimentin antibody (1:1000) (#5741, Cell Signaling Technology, MA, USA).

Techniques: Staining, Control, Immunostaining, Western Blot, Expressing, Cell Cycle Assay, Real-time Polymerase Chain Reaction