gene expression arrays  (fluidigm)

Journal: Neural Regeneration Research

Article Title: Overexpression of the inwardly rectifying potassium channel Kir4.1 or Kir4.1 Tyr 9 Asp in Müller cells exerts neuroprotective effects in an experimental glaucoma model

doi: 10.4103/NRR.NRR-D-24-00461

Figure Lengend Snippet: Identification and verification of potential functional phosphorylation sites in Kir4.1. (A) Schematic diagram illustrating the design of the Kir4.1 mutant lentiviral expression system. (B) Mutation sites in the kcnj10 gene. (C) Immunofluorescence staining showing primary cultured Müller cells infected with lentiviruses for overexpressing Kir4.1, the Kir4.1 mutants, and the eGFP control. All lentiviruses successfully infected purified cultured Müller cells. Scale bar: 20 μm. (D, E) Representative immunoblots (D) and quantification (E) demonstrating changes in total Kir4.1 protein expression in DHPG-treated cultured Müller cells infected with lentiviruses overexpressing Kir4.1 and seven different Kir4.1 mutants. All data are normalized to GAPDH levels and then to controls. One-way analysis of variance (Tukey–Kramer multiple comparisons test) was performed (* P < 0.05, *** P < 0.001, vs . Ctr group). (F, G) Representative immunoblots (F) and densitometric quantification (G) showing changes in membrane Kir4.1 protein expression in DHPG-treated cultured Müller cells infected with lentiviruses overexpressing Kir4.1, Kir4.1 Tyr 9 Asp, and Kir4.1 Ser 370 Arg. All data are normalized to Na-K-ATP levels and then to the Kir4.1 group. One-way analysis of variance (Tukey–Kramer multiple comparisons test) was used (** P < 0.01, vs . Kir4.1 group). CMV: Cytomegalovirus ; Ctr: control; DHPG: (S)-3,5-dihydroxyphenylglycine; eGFP: enhanced green fluorescent protein; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; SV40: simian vacuolating virus 40.

Article Snippet: Lentiviral expression vectors containing the stable selection marker puromycin, along with the kcnj10 gene (GeneCopoeia TM , Rockville, MD, USA), were constructed with seven different mutation sites in the kcnj10 gene using iGeneBio (Guangzhou, China).

Techniques: Functional Assay, Phospho-proteomics, Mutagenesis, Expressing, Immunofluorescence, Staining, Cell Culture, Infection, Control, Purification, Western Blot, Membrane, Virus

Journal: Molecular Therapy Oncology

Article Title: Survivin/BIRC5-derived peptide disrupts survivin dimerization and cell division and induces multifaceted anti-cancer effects

doi: 10.1016/j.omton.2025.201123

Figure Lengend Snippet: Cell death induction by 1H13-survivin/BIRC5-derived peptides targeted to the cytoplasm, mitochondria, or nucleus (A) The sequences added to the 1H13-BIRC5 peptide to target it to the: (1) cytoplasm, (2) mitochondria, or (3) nucleus. The underlined sequence represents the 1H13 peptide; in red, amino acids indicate D-amino acid substitutions. (B) A549 cells were incubated for 90 min with 5 μM FITC-labeled, nucleus-targeted peptide IH13-Nuc, and were also IF-stained with anti--SMAC, anti-IP3R or anti-GM130 antibodies, and stained with DAPI to visualize the mitochondria, ER, Golgi, and nucleus, respectively. Confocal microscope images are shown, with white arrows indicating peptide co-localization with the mitochondria (SMAC). Orange and yellow arrows indicate peptide presence in the nucleus and cytosol, respectively. (C) A549 cells were incubated with the mitochondria- or nucleus-targeted 1H13-BIRC5-derived peptide for 24 h in serum-free medium, followed by a cell proliferation assay using the SRB method. (D and E) Apoptotic cell death as induced in A549 cells following incubation for 24 h with the nucleus-targeted peptide (2/3D-1H13-Nuc) in the presence or absence of the indicated concentrations of the peptides in serum-free medium and subjected to FITC–annexin V/PI staining, followed by a flow cytometry analysis. Representative histograms for control and selected peptide concentration (D) and analysis of early and late apoptotic stages are shown (E). (F and G) Cell death as induced by 2/3D-1H13-Nuc in different cell lines, A549, SH-SY5Y, U-87MG, PC-3, and HUV-EC-C (F) or Jurkat, K562, and KMH2-LC (G) were treated with the indicated concentrations of the peptide for 24 h, then subjected to cell death analysis using propidium iodide (PI) staining and flow cytometry. (H and I) A549 cells were seeded at a density of 2 × 10 5 cells per well in a 12-well plate. After 24 h, the cells were transfected with 2 μg of a pCMV3-survivin expression plasmid (HG10356-UT, Sino Biological, China) or with an empty pCMV3 plasmid (control) using JetPrime transfection reagent (Polyplus, France), following the manufacturer’s instructions. Twenty-four hours post-transfection, the cells were re-seeded at 1 × 10 5 cells per well in a 12-well plate. After another 24 h, the culture medium was replaced with serum-free medium, and the cells were treated with the indicated concentrations of the 2/3D-1H13-Nuc peptide. Survivin overexpression levels were assessed by immunoblotting (H), and cell death was analyzed by propidium iodide (PI) staining followed by FACS analysis (I). Results represent the means ± SEM ( n = 3).

Article Snippet: After 24 h, the cells were transfected with 2 μg of a pCMV3-survivin expression plasmid (HG10356-UT, Sino Biological, China) or with an empty pCMV3 plasmid (control) using JetPrime transfection reagent (Polyplus, France), following the manufacturer’s instructions.

Techniques: Derivative Assay, Sequencing, Incubation, Labeling, Staining, Microscopy, Proliferation Assay, Flow Cytometry, Control, Concentration Assay, Transfection, Expressing, Plasmid Preparation, Over Expression, Western Blot