Journal: Febs Letters

Article Title: Proteolysis of the low‐density lipoprotein receptor in hepatocytes is mediated by BMP1 but not by other astacin proteases

doi: 10.1002/1873-3468.14667

Figure Lengend Snippet: The expression profile of the astacin proteases in human cells. RNA samples from hepatocytes, HepG2, SW13 and HEK293 cells were analysed for mRNA expression of BMP1, mTLD, meprin α, meprin β, TLL1 and TLL2 using quantitative real‐time RT‐PCR. The mRNA levels were quantified relative to RPL13A. The expression of (A) BMP1 + mTLD, (B) mTLD, (C) meprin α, (D) meprin β, (E) TLL1 and (F) TLL2 are shown relative to the expression of each astacin protease in HEK293 cells. Data were analysed by using an ordinary one‐way ANOVA test with Tukey's multiple comparison test to compare between groups. * P < 0.05, *** P < 0.001, **** P < 0.0001, n = 3 experimental repeats for all analyses.

Article Snippet: Assay‐ready expanded hepatocytes (Axol #A3704, Axol Bioscience, Easter Bush, UK) were revived using hepatocyte thawing medium (Axol) and maintained using hepatocyte maintenance medium (Axol).

Techniques: Expressing, Quantitative RT-PCR, Comparison

Journal: Febs Letters

Article Title: Proteolysis of the low‐density lipoprotein receptor in hepatocytes is mediated by BMP1 but not by other astacin proteases

doi: 10.1002/1873-3468.14667

Figure Lengend Snippet: Pharmacological inhibition of BMP1, but not meprin α or meprin β, reduces cleavage of LDLR in HepG2 cells and primary hepatocytes. Human primary hepatocytes and HepG2 cells were cultured to confluency before treatment with the inhibitors UK383367 (10 μ m ), GM6001 (10 μ m ) or Actinonin (10 μ m ) for 24 h in OptiMEM. The cells and conditioned media were then harvested; lysates were prepared and the conditioned media concentrated. The LDLR profile in HepG2 cells was compared by western blot of (A) cell lysates and (B) conditioned media. Actin was used as a loading control. (C) The relative amounts of FL LDLR and the CTF were quantified by densitometry and expressed as a percentage of the total LDLR. (D) The relative amounts of the NTF were quantified by densitometry and are expressed as a percentage of the control (uninhibited) NTF level. The LDLR profile in primary hepatocytes was compared by western blot of (E) cell lysates and (F) conditioned media. (G) The relative amounts of FL LDLR and CTF were quantified by densitometry and expressed as a percentage of the total LDLR. (H) The relative amounts of NTF were quantified by densitometry and are expressed as a percentage of the control (uninhibited) NTF level. Data are shown as mean ± SEM. FL LDLR to CTF comparisons were made using a two‐way ANOVA with Dunnet's multiple comparison test to compare the data from each inhibitor to the uninhibited control. NTF comparisons for HepG2 data were made using a Welch's ANOVA with unpaired t ‐tests with Welch's correction (due to unequal standard deviations) to compare the data from each inhibitor to the control. NTF comparisons for hepatocyte data were made using an ordinary ANOVA with multiple comparisons made using a Fisher's LSD test to compare the data from each inhibitor to the control. * P < 0.05, ** P < 0.01, **** P < 0.0001, n = 4 experimental repeats for HepG2, n = 3 experimental repeats for hepatocytes using one cell preparation.

Article Snippet: Assay‐ready expanded hepatocytes (Axol #A3704, Axol Bioscience, Easter Bush, UK) were revived using hepatocyte thawing medium (Axol) and maintained using hepatocyte maintenance medium (Axol).

Techniques: Inhibition, Cell Culture, Western Blot, Control, Comparison

Journal: Febs Letters

Article Title: Proteolysis of the low‐density lipoprotein receptor in hepatocytes is mediated by BMP1 but not by other astacin proteases

doi: 10.1002/1873-3468.14667

Figure Lengend Snippet: siRNA knockdown of BMP1, but not other astacins, prevents LDLR cleavage in human hepatocytes. Human hepatocytes were cultured to 70% confluency and treated with either non‐targeting siRNA or siRNA (50 n m ) for BMP1, mTLD, meprin α, TLL1 or TLL2 for 24 h before incubation for a further 24 h in OptiMEM. The cells and conditioned media were then harvested; lysates and RNA samples were prepared and the conditioned media was concentrated. (A–E) Knockdown was confirmed by mRNA analysis using real‐time quantitative PCR. The mRNA expression for each gene was calculated relative to RPL13A and the expression shown relative to control. The LDLR profile in hepatocytes was compared by western blot of (F) cell lysates and (G) conditioned media. Total protein staining by amido black was used as a loading control. The relative amounts of (H) FL LDLR and CTF were quantified by densitometry and expressed as a percentage of the total LDLR. (I) The relative amounts of NTF were quantified by densitometry and are expressed as a percentage of the control (non‐targeting siRNA) NTF level. Data are shown as mean ± SEM, except for in (C) where data are shown as median and range. Comparisons for mRNA expression were made using an unpaired t ‐test with Welch's correction. FL LDLR to CTF comparisons were made using a two‐way ANOVA with Dunnett's multiple comparison test to compare the data from each knockdown to the control. NTF comparisons were made using an ordinary ANOVA test with multiple comparisons made using a Fisher's LSD comparison test to compare the data from each knockdown to the control. * P < 0.05, ** P < 0.01, **** P < 0.0001, n = 3 experimental repeats from one hepatocyte preparation for all analyses, except in (C) where n = 2.

Article Snippet: Assay‐ready expanded hepatocytes (Axol #A3704, Axol Bioscience, Easter Bush, UK) were revived using hepatocyte thawing medium (Axol) and maintained using hepatocyte maintenance medium (Axol).

Techniques: Knockdown, Cell Culture, Incubation, Real-time Polymerase Chain Reaction, Expressing, Control, Western Blot, Staining, Comparison

Journal: Cell Reports Medicine

Article Title: Cancer stem cell assay-guided chemotherapy improves survival of patients with recurrent glioblastoma in a randomized trial

doi: 10.1016/j.xcrm.2023.101025

Figure Lengend Snippet:

Article Snippet: Fetal Bovine Serum: Characterized, US-Sourced , HyClone Laboratories, Inc. , SH3087901.

Techniques: Recombinant, Injection, Software, Cell Counting, Hood, Microplate Reader Absorbance Measurement, Mass Measurement, Cell Culture, Light Microscopy