lipofectamine ltx and plustm reagent  (Thermo Fisher)

Journal: bioRxiv

Article Title: Inhibition of NF-κB signaling pathway in astrocytes facilitates amyloid-β clearance by kallikrein-related peptidase 7

doi: 10.1101/2025.03.02.641088

Figure Lengend Snippet: a, b The mRNA expression level of KLK7 in H4 cells treated with 30 µM memantine (Mem) (n=6, Student’s t -test, p=0.003) (a) , 100 µM NMDA, and 100 µM L-glutamate (L-Glu) (n=3, Dunnett’s test, p Ctrl vs NMDA <0.0001, p Ctrl vs L-Glu <0.0001) (b) , respectively. c Schematic representation of Aβ degradation assay in H4 cells. Synthetic Aβ40 was added to the conditioned medium of H4 cells treated with memantine. After 24 hours, remaining Aβ40 in conditioned medium was detected by immunoblotting. d The Aβ-degrading activity of H4 cells treated with 30 µM memantine. A representative immunoblot and quantified results are shown (Input=1.0, n=3, Student’s t -test, p=0.0205). e Cell viability of H4 cells treated with 30 µM memantine (n=3, Student’s t -test, p=0.3927). All data are shown as mean±SEM. *p<0.05, **p<0.01, ****p<0.0001; n.s., non-significant.

Article Snippet: For transient expression of the luciferase constructs in H4 cells, Lipofectamine ® LTX Reagent & PlusTM Reagent (Thermo Fisher Scientific, catalog #15338100) was used following the manufacturer’s protocol.

Techniques: Expressing, Degradation Assay, Western Blot, Activity Assay

Journal: bioRxiv

Article Title: Inhibition of NF-κB signaling pathway in astrocytes facilitates amyloid-β clearance by kallikrein-related peptidase 7

doi: 10.1101/2025.03.02.641088

Figure Lengend Snippet: a Schematic representation of the establishment of luciferase constructs (Empty and h238 WT) and H4 stable cell lines. b, c Luciferase activity of H4 h238 WT treated with 100 µM NMDA, 100 µM L-glutamate (n=4, Dunnett’s test, p Ctrl vs NMDA =0.0038, p Ctrl vs L-Glu =0.0057) (b) , and 30 µM memantine (n=5, Student’s t -test, p=0.0003) (c) , respectively. d Schematic representation of luciferase constructs containing a deletion mutant and a motif mutant of the κB motif in the 238-bp KLK7 promoter region (h238 ΔκB motif and h238 mut-κB motif). e Luciferase activity of each luciferase construct transiently expressed in H4 cells (n=4, Dunnett’s test, p h238 WT vs h238 ΔκB motif =0.0432, p h238 WT vs h238 mut-κB motif =0.0318). f, g Luciferase activity of H4 h238 ΔκB motif (n=4, Dunnett’s test, p Ctrl vs NMDA =0.4847, p Ctrl vs L-Glu =0.3969) (f) and H4 h238 mut-κB motif (n=4, Dunnett’s test, p Ctrl vs NMDA =0.3716, p Ctrl vs L-Glu =0.5012) (g) treated with 100 µM NMDA or 100 µM L-glutamate. h, i Luciferase activity of H4 h238 ΔκB motif (n=5, Student’s t -test, p=0.8164) (h) and H4 h238 mut-κB motif (n=5, Student’s t -test, p=0.5011) (i) treated with 30 µM memantine. All data are shown as mean±SEM. *p<0.05, **p<0.01, ***p<0.001; n.s., non-significant.

Article Snippet: For transient expression of the luciferase constructs in H4 cells, Lipofectamine ® LTX Reagent & PlusTM Reagent (Thermo Fisher Scientific, catalog #15338100) was used following the manufacturer’s protocol.

Techniques: Luciferase, Construct, Stable Transfection, Activity Assay, Mutagenesis

Journal: bioRxiv

Article Title: Inhibition of NF-κB signaling pathway in astrocytes facilitates amyloid-β clearance by kallikrein-related peptidase 7

doi: 10.1101/2025.03.02.641088

Figure Lengend Snippet: a Schematic representation of the canonical NF-κB activation pathway with the targets of the inhibitors IKK-16 and JSH-23 indicated. b The mRNA expression level of KLK7 in H4 cells treated with 2 µM IKK-16 or 30 µM JSH-23 (n=6, Student’s t -test, p Ctrl vs IKK-16 =0.0270, p Ctrl vs JSH-23 =0.0103). c The Aβ-degrading activity of H4 cells treated with 2 µM IKK-16 or 30 µM JSH-23. Representative immunoblots and quantified results are shown (Input=1.0, n=4, Student’s t -test, p Ctrl vs IKK-16 =0.0010, p Ctrl vs JSH-23 =0.0082). d Cell viability of H4 cells treated with 2 µM IKK-16 or 30 µM JSH-23 (n=4, Student’s t -test, p Ctrl vs IKK-16 =0.9618, p Ctrl vs JSH-23 =0.9043). e Luciferase activity of H4 h238 WT treated with 2 µM IKK-16 or 30 µM JSH-23 (n=4 (IKK-16), n=6 (JSH-23), Student’s t -test, p Ctrl vs IKK-16 <0.0001, p Ctrl vs JSH-23 <0.0001). All data are shown as mean±SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; n.s., non-significant.

Article Snippet: For transient expression of the luciferase constructs in H4 cells, Lipofectamine ® LTX Reagent & PlusTM Reagent (Thermo Fisher Scientific, catalog #15338100) was used following the manufacturer’s protocol.

Techniques: Activation Assay, Expressing, Activity Assay, Western Blot, Luciferase