Journal: Communications Biology

Article Title: Contrasting effects of intracellular and extracellular human PCSK9 on inflammation, lipid alteration and cell death

doi: 10.1038/s42003-024-06674-9

Figure Lengend Snippet: a PCSK9 was measured from bronchoalveolar lavage fluid (BALF) of both smokers with ( n = 18) or without chronic obstructive pulmonary disease (COPD) ( n = 17) and non-smokers ( n = 17). b – d Primary bronchial epithelial cells (PBEC) were cultured with PCSK9 or in untreated (UT) condition for 24 h. In response to stimulation with PCSK9, PBEC induced production of pro-inflammatory cytokines, including IL-6, TNF-α, and the chemokine IL-8 ( n = 6). e PCSK9d induced matrix metalloproteinase 9 (MMP9) expression, with a concentration-dependent effect ( n = 6). f PCSK9 was identified as an inducer of phosphorylation of MAPKp38 ( n = 3), with higher concentrations demonstrating a more pronounced effect. Error bars represent median interquartile range. p -value ≤ 0.05 was considered statistically significant.

Article Snippet: In addition, one batch of PBEC was used from commercially available from Lonza.

Techniques: Cell Culture, Expressing, Concentration Assay, Phospho-proteomics

Journal: Communications Biology

Article Title: Contrasting effects of intracellular and extracellular human PCSK9 on inflammation, lipid alteration and cell death

doi: 10.1038/s42003-024-06674-9

Figure Lengend Snippet: a PCSK9 was measured from bronchoalveolar lavage fluid (BALF) of both smokers with ( n = 18) or without chronic obstructive pulmonary disease (COPD) ( n = 17) and non-smokers ( n = 17). b – d Primary bronchial epithelial cells (PBEC) were cultured with PCSK9 or in untreated (UT) condition for 24 h. In response to stimulation with PCSK9, PBEC induced production of pro-inflammatory cytokines, including IL-6, TNF-α, and the chemokine IL-8 ( n = 6). e PCSK9d induced matrix metalloproteinase 9 (MMP9) expression, with a concentration-dependent effect ( n = 6). f PCSK9 was identified as an inducer of phosphorylation of MAPKp38 ( n = 3), with higher concentrations demonstrating a more pronounced effect. Error bars represent median interquartile range. p -value ≤ 0.05 was considered statistically significant.

Article Snippet: To suppress expression of PCSK9, PBEC were transfected with PCSK9 siRNA (Santa Cruz, Germany, cat# sc4548-2).

Techniques: Cell Culture, Expressing, Concentration Assay, Phospho-proteomics

Journal: Communications Biology

Article Title: Contrasting effects of intracellular and extracellular human PCSK9 on inflammation, lipid alteration and cell death

doi: 10.1038/s42003-024-06674-9

Figure Lengend Snippet: a PCSK9 levels were quantified from cell lysates of A549 cell ( n = 6) and PBEC ( n = 6) by ELISA. b Western blot from the cells lysates of A549 cells and PBEC. c In addition, immunostaining was performed to visualize the microscopic images for the PCSK9, scale bar 10 µm. Both PBEC and A549 cells expressed PCSK9 at the protein level. d Differentiated PBEC on air liquid interface (ALI) exhibited higher levels of PCSK9 protein compared to undifferentiated PBEC in submerged culture (SM). e In compared to untreated (UT) condition, treatment with cigarette smoke extract (CSE) resulted in a reduction of endogenous PCSK9 protein levels in undifferentiated PBEC. f , g Undifferentiated PBEC, upon CSE treatment, released PCSK9 into the cell culture ( n = 6). Notably, this CSE-induced PCSK9 release was inhibited by brefeldin ( n = 6). h In CSE-treated PBEC, PCSK9 was primarily located in the cytoplasm, 10 µm scale bar was used for microscopic images i Minimal increase in PCSK9 was observed in differentiated PBEC in response to CSE. Error bars represent median interquartile range. p -value ≤ 0.05 was considered statistically significant.

Article Snippet: To suppress expression of PCSK9, PBEC were transfected with PCSK9 siRNA (Santa Cruz, Germany, cat# sc4548-2).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Immunostaining, Cell Culture

Journal: Communications Biology

Article Title: Contrasting effects of intracellular and extracellular human PCSK9 on inflammation, lipid alteration and cell death

doi: 10.1038/s42003-024-06674-9

Figure Lengend Snippet: Bulk RNA sequencing analysis from control siRNA and PCSK9 siRNA transfected PBEC. a Endogenous inhibition of PCSK9 showed alterations in the expression of approximately 2000 genes, both upregulated and downregulated genes. b , c The impact of PCSK9 inhibition extended to crucial biological pathways and processes, demonstrating an influence on cellular functions. 10 upregulated and 10 downregulated crucial pathways or biological process were presented. d Gene co-expression network of affected genes encompassed pathways and processes vital to cellular function, such as cholesterol synthesis, COX, apoptosis, ARH (Aryl hydrocarbon receptor), and the CYP450 pathway.

Article Snippet: To suppress expression of PCSK9, PBEC were transfected with PCSK9 siRNA (Santa Cruz, Germany, cat# sc4548-2).

Techniques: RNA Sequencing, Control, Transfection, Inhibition, Expressing, Cell Function Assay

Journal: Communications Biology

Article Title: Contrasting effects of intracellular and extracellular human PCSK9 on inflammation, lipid alteration and cell death

doi: 10.1038/s42003-024-06674-9

Figure Lengend Snippet: a Lipid level was measure by lipid accumulation specific bodipy reagent. Inhibition of PCSK9 was associated with an increase in lipid accumulation. b , c Lipid peroxidation was measured by lipid peroxidation specific bodipy and lipid peroxidation product MDA ( n = 3). In compared to untreated (UT), cigarette smoke extract (CSE) induced a higher level of lipid peroxidation in cells in the PCSK9-suppressed PBEC. d Cell cycle was analyzed by propidium iodide staining. PCSK9 inhibition affected cell cycle, primarily affecting the G2/M phase. e LDH assay was performed to determine cell death. CSE-induced cell death was notably higher in PCSK9-silenced cells compared to control siRNA transfected cells ( n = 6). f , g In response to the staurosporine, PCSK9 inhibition induced apoptosis and an increase in caspase 3 activation ( n = 3). Error bars represent median interquartile range. p -value ≤ 0.05 was considered statistically significant.

Article Snippet: To suppress expression of PCSK9, PBEC were transfected with PCSK9 siRNA (Santa Cruz, Germany, cat# sc4548-2).

Techniques: Inhibition, Staining, Lactate Dehydrogenase Assay, Control, Transfection, Activation Assay