ef1α gfp p2a puromycin selection cassette  (OriGene)

Journal: Biomarker Research

Article Title: Single-cell and spatial transcriptome analysis reveals the potential therapeutic targets for testicular sex cord-stromal cell tumor

doi: 10.1186/s40364-026-00924-0

Figure Lengend Snippet: Disordered cellular communication in TSCST. ( A ) Bar plot showing the number of inferred interactions in the leading edge and the tumor core. ( B ) Bar plot showing the interaction strength in the leading edge and the tumor core. ( C ) Circle plots display ligand-receptor interaction weights between distinct cellular components in the leading edge and the tumor core. Colors distinguish cell types. The circle size is proportional to the number of cells in each cluster, and the edge width represents communication probability. ( D ) Dot plots show the increased ligand-receptor interactions on the leading edge. ( E ) Dot plots showing the increased ligand-receptor interactions in the tumor core. ( F ) Dot plots show increased signaling in macrophages to tumor and Sertoli cells. ( G ) Feature plots of APP-CD74 by ST in Patient 3 and Patient 4. ( H ) Feature plots of MDK-NCL by ST in Patient 3 and Patient 4. ( I ) Mapped SPP and MK signaling pathways in ST, colored by Seurat clusters. ( J ) Violin plots showing the expression level of APP-CD74 genes across the cell types in the leading edge and the tumor core, respectively. The y-axis shows the normalized read count. ( K ) Representative immunofluorescence images showing expression of CTNNB1 and CD74 (n = 4). Nuclei were stained with DAPI. ( L ) Relative mRNA expression of MRC1 and ARG1 (M2-type macrophage markers) and IL1B (M1-type macrophage marker) in macrophages treated with APP. ( M ) Relative mRNA expression of MMP2 (a marker associated with phagocytic function in macrophages) in macrophages treated with APP. The P value was calculated by Welch’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: THP-1-induced macrophages treated with APP protein (MedChemExpress, Shanghai, Catalogue number: HY- P72834 ) were collected, and total RNA was extracted using TRIzol Reagent (Invitrogen, 15596018CN, USA).

Techniques: Protein-Protein interactions, Expressing, Immunofluorescence, Staining, Marker

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Amyloid precursor protein and C99 are subunits in human microglial Hv1 channels that enhance current and inflammatory mediator release

doi: 10.1073/pnas.2509903122

Figure Lengend Snippet: APP knockdown reduces Hv1 currents in human iMG. iMG were studied by whole-cell patch clamp using a holding voltage of −60 mV, 0.5 s test pulses, and a 10 s interpulse interval, with pH i = 6.0 and pH o = 7.5. Values are mean ± SEM; n = 5-9 cells for each condition. ( A ) Representative proton current traces in the absence (−C6) and presence (+C6) of 2 μM C6 peptide, a specific inhibitor of Hv1 channels. Inset : voltage-step protocol consisting of 20 mV increments from −60 mV to +60 mV. ( B ) Peak proton current densities at 0 mV in the absence (−C6) or presence (+C6) of 2 μM C6 peptide. ( C ) Dose–response relationships for C6 inhibition of proton current at 0 mV. K i for C6 was estimated to be 297 ± 7 nM from a Hill equation fit (h = 1.6 ± 0.1). ( D ) Conductance–voltage relationships (G-V) for proton currents in iMG and cloned Hv1 expressed in HEK293T cells (HEK). ( E ) Representative proton currents in iMG treated with Scr siRNA as a negative control and APP siRNA knockdown (20 mV steps from −60 to +60 mV). APP knockdown slowed current activation 2.9-fold (4,100 ± 550 ms vs. 11,800 ± 1,700 ms) and speeded deactivation 1.7-fold (310 ± 40 ms vs. 180 ± 30 ms) at 0 mV. ( F ) Peak current densities of proton currents at 0 mV for iMG treated with Scr siRNA or with APP siRNA knockdown. ( G ) G-V relationships for proton currents in iMG treated with Scr siRNA or APP siRNA. APP knockdown induces a significant depolarizing shift (+9 mV, P = 0.01) in the V 1/2 of the G-V relationship.

Article Snippet: APP knockdown was achieved using the APP Human siRNA Oligo Duplex (OriGene), following the manufacturer’s protocol.

Techniques: Knockdown, Patch Clamp, Inhibition, Clone Assay, Negative Control, Activation Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Amyloid precursor protein and C99 are subunits in human microglial Hv1 channels that enhance current and inflammatory mediator release

doi: 10.1073/pnas.2509903122

Figure Lengend Snippet: APP knockdown reduces release of cytokines and ROS from iMG. Cytokines were quantified using ELISA as described in Materials and Methods . Values are mean ± SEM; n = 6 for each condition. Control (CTL) represents no LPS stimulation ( A ) C6 (2 μM) treatment decreased LPS-stimulated release of TNF-α, IL-6, IL-1β, IFN-γ, IL-2, IL-4, IL-8, IL-10, IL-12p70, and IL-13 concentrations. **** P ≤ 0.0001. ( B ) TNF-α release in response to LPS in the absence or presence of 2 μM C6 peptide or KTx* peptide (a negative control peptide that does not block ion channels) from iMG treated with Scr siRNA or APP siRNA knockdown. ( C ) IL-6 release in response to LPS in the absence or presence of 2 μM C6 or KTx* peptide from iMG treated with Scr siRNA or APP siRNA knockdown. ( D ) ROS release in response to LPS in the absence or presence of 2 μM C6 or KTx* peptide from iMG treated with Scr siRNA or APP siRNA knockdown.

Article Snippet: APP knockdown was achieved using the APP Human siRNA Oligo Duplex (OriGene), following the manufacturer’s protocol.

Techniques: Knockdown, Enzyme-linked Immunosorbent Assay, Control, Negative Control, Blocking Assay

Journal: Neurotherapeutics

Article Title: Discovery of an APP-selective BACE1 inhibitor for Alzheimer's disease

doi: 10.1016/j.neurot.2025.e00610

Figure Lengend Snippet: Inhibitory activity and brain permeability of FAH65E(-) . Shown are (A) dose-response curves for FAH65 racemate and the FAH65E(+) and (-) enantiomers in the P5-P5′ assay and (B) sAPPβ and (C) Aβ1-42 in CHO-7W cells after treatment with increasing concentrations of FAH65 racemate and enantiomers. Legend in B also applies to C. Data graphed as the mean and SEM. (D) FAH65E(-) (black line) and sAPP β (blue dashed line) levels in brain from ApoE4TR-5XFAD mice after oral delivery of 30 ​mg/kg FAH65E(-) doses are shown. PK-PD study design did not include 0 h timepoint untreated mice but included time points 1, 2, 4 and 6 ​h after last dose on Day 2. N ​= ​3 mice per time point.

Article Snippet: Media was assayed using an AlphaLISA for Aβ1-42 (Perkin Elmer catalog # AL276C), sAPPα (R&D Systems catalog # AF1168 conjugated with Perkin Elmer acceptor beads catalog # 6772001 + 2B3 antibody from IBL catalog # 11088 biotinylated), and sAPPβ (Perkin Elmer AL276-acceptor + IBL catalog # 18957 biotinylated).

Techniques: Activity Assay, Permeability