Additional file 1 of MiR-15b-5p and PCSK9 inhibition reduces lipopolysaccharide-induced endothelial dysfunction by targeting SIRT4
Creators: name: Martino, Elisa
name: D’Onofrio, Nunzia
name: Balestrieri, Anna
name: Mele, Luigi
name: Sardu, Celestino
name: Marfella, Raffaele
name: Campanile, Giuseppe
name: Balestrieri, Maria Luisa
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Physiology
subject: Chemical Sciences not elsewhere classified
subject: Immunology
subject: FOS: Clinical medicine
subject: Biological Sciences not elsewhere classified
subject: Cancer
subject: Infectious Diseases
subject: FOS: Health sciences

description: Additional file 1: Figure S1. LPS-mediated inflammation on HUVEC cells. A Cell viability on HUVEC exposed to LPS. Detection of B LDH, C NO, D VCAM1 E ICAM1, F MCP-1, G IL-1β and H IL-18 levels. Caspase-4 I activity and J levels and K LOX-1 evaluation by ELISA. Representation of L hsamiR-15b-5p, M hsa-miR-16-5p and N hsa-miR-195-5p levels measured by qRT-PCR. SIRT4 levels assessed by O ELISA and P immunoblotting. Mean ± SD, n = 3. M, molecular weight markers; lane 1, Ctr; lane 2 = LPS. *p &lt; 0.05 vs. 0 µg/mL or Ctr; ‡p &lt; 0.01 vs. 0 µg/mL or Ctr; ¶p &lt; 0.001 vs. Ctr; n.s., non-significant. Statistical analysis of data was performed using Student’s t-test. Figure S2. LPS modulation of PCSK9 protein on HUVEC cells. A Representative intracellular PCSK9 protein content on TeloHAEC detected by FACS analysis. Detection of PCSK9 by B ELISA, C mRNA levels by qRT-PCR and D immunoblotting analysis on HUVEC. Mean ± SD, n = 3. M, molecular weight markers; lane 1, Ctr; lane 2, LPS. *p &lt; 0.05 vs. Ctr; ‡p &lt; 0.01 vs. Ctr. Statistical analysis of data was performed using Student’s t-test. Figure S3. LPS-induced pyroptosis on HUVEC cells. Representative FACS analysis of A pyroptosis, B intracellular NLRP3 levels, C lysosomes, D autophagy, E mitochondrial ROS levels and F annexin V-FITC and PI-staining on TeloHAEC. Q1: necrotic cells; Q2: late apoptotic cells; Q3: early apoptotic cells; Q4: viable cells. G Images and H, I cytometer analysis of pyroptosis on HUVEC. Mean ± SD, n = 3. Scale bars = 100 μm. ‡p &lt; 0.01 vs. Ctr. Statistical analysis of data was performed using Student’s t-test. Figure S4. Transfection with i-miR-15b. TeloHAEC viability evaluated A after antagomir Negative Control (NC) and antagomiR hsa-miR-15b-5p (i-miR-15b) transfection and B after exposure to LPS on NC-transfected cells. Representative FACS analysis of C pyroptosis and D intracellular NLRP3 levels detected on TeloHAEC. Mean ± SD, n = 3. *p &lt; 0.05 vs. NC; ‡p &lt; 0.01 vs. NC; §p &lt; 0.001 vs. NC. Statistical analysis of data was performed using Student’s t-test. Figure S5. FACS analyses. Representative FACS analysis of A lysosomes, B autophagy, C mitochondrial ROS levels and D annexin V-FITC and PI-staining performed on TeloHAEC. Q1: necrotic cells; Q2: late apoptotic cells; Q3: early apoptotic cells; Q4: viable cells. Figure S6. i-PCSK9 effects on LPS-induced pyroptosis and autophagy. A TeloHAEC viability after treatment with LPS, i-PCSK9 or transfection with NC before i-PCSK9 and/or LPS stimulation. Representative images and FACS analysis of B, C pyroptosis and D, E autophagy performed on TeloHAEC. Mean ± SD, n = 3. Scale bars = 100 μm. ¶p &lt; 0.001 vs. Ctr; §p &lt; 0.001 vs. NC; •p &lt; 0.05 vs. NC + LPS; &amp;p &lt; 0.05 vs. LPS. Statistical analysis of data was performed using one-way ANOVAs.