Journal: Cell stress & chaperones
Article Title: Dynamics of heat shock protein 70 kDa in heat-shocked and hypoxic human endothelial cells.
doi: 10.1016/j.cstres.2025.100085
Figure Lengend Snippet: Figure 2. Morphological characterization of thermal stress in human endothelial cells. (A) Primary HUVECs were exposed to heat shock at 42 °C for 1 h and allowed to recover at 37 °C for 24 h. Images were acquired using optical microscopy (ZEISS Primovert) before heat shock (0 h), immediately after heat shock (1 h) and after recovery (24 h). Scale bar: 100 µm. Inset scale bar: 50 µm. Representative images of n=8 independent experiments using cells from different lots (19TL028325, 19TL023264, 19TL127368 and 21TL195720, Lonza). (B) Supernatants from these cells were collected, and heat shock cytotoxicity was measured by quantifying lactate dehydrogenase (LDH) activity. The data are shown as the means ± SEMs of 3 independent experiments (lot 19TL127368 and 19TL028325), ordinary one-way ANOVA, with Tukey’s multiple comparisons test, **p < 0.01, ***p < 0.001 versus positive control. HUVECs were treated with Triton X-100 to achieve maximum LDH release from the supernatant. (C) HSF1 (green, CST 4356S) and total HSP70 (red, Invitrogen MA3-006).
Article Snippet: Materials and reagents Material or Reagent Brand Catalog number PierceTM BCA Protein Assay Kits Thermo Fisher Scientific 23227 Acrylamide/Bis-acrylamide, 30% Sigma‒Aldrich A3574 Acrylamide/Bis-acrylamide, 40% Sigma‒Aldrich A7802 Lonza Endothelial Basal Medium-2 (EBM-2) Lonza CC-3156 BulletKit EGM-2 Lonza CC-3162 HaltTM Protease Inhibitor Cocktail (100X) Thermo Fisher Scientific 78429 HaltTM Phosphatase Inhibitor Cocktail (100X) Thermo Fisher Scientific 78420 75 cm2 U-Shaped Canted Neck Cell Culture Flask Corning 3290 100 mm Culture Dish Corning 430167 12-well Cell Culture Plate Corning 3513 Fetal Bovine Serum, Qualified, Brazil (SFB) Gibco 12657029 Matrigel® Matrix Corning 354230 PierceTM Protein A/G Agarose Thermo Scientific 20421 Millicell® Cell Culture Insert Sigma‒Aldrich PIHP01250 Target/Antibody Brand Catalog number Assay HSP70 Invitrogen MA3-006 WB 1:1000, IF 1:400 Inducible HSP70 Invitrogen MA3-009 NP 1:1000, WB 1:1000, IF 1:400 HSP90 Cell signaling 4877 NP 1:1000, WB 1:1000 HSP40 Cell signaling 4868 NP 1:500, WB 1:500 IF 1:200 HSC70 Invitrogen PA5-27337 NP 1:1000, WB 1:1000 IF 1:200 HSF1 Cell Signaling 4356S IF 1:200 HIF1α Cell signaling 48085 WB 1:1000 β-actin Sigma‒Aldrich A5441 WB 1:10.000 Anti-Mouse IgG Secondary Antibody LI-COR 926-32212 NP 1:4000, WB 1:10.000 Anti-Rabbit IgG Secondary Antibody LI-COR 926-68073 NP 1:4000, WB 1:10.000 Alexa Fluor 488 Invitrogen 2110499 IF 1:400 Alexa Fluor 546 Invitrogen A11030 IF 1:400 Jo ur al Pr epr oo f Cell Brand Lot number Assays HUVEC Lonza 19TL028324 Migration, IF HUVEC Lonza 19TL023264 Western blot Native PAGE Heat Shock, IF HUVEC Lonza 19TL127368 Western blot Native PAGE LDHGlo Heat Shock HUVEC Lonza 19TL028325 Western blot Native PAGE LDHGlo Heat Shock HUVEC Lonza 21TL195720 Migration Tube Formation Western blot Native PAGE Heat Shock HUVEC Lonza 23TL086130 Hypoxia Western blot HUVEC Lonza 21TL95720 23TL104314 23TL135742 WB HCAEC Lonza 20TL365545 Western blot Native PAGE Hypoxia Jo ur al Heat Shock HCAEC Lonza 20TL064651 Western blot Native PAGE Hypoxia Heat Shock HCAEC Lonza 21TL316169 Tube Formation
Techniques: Microscopy, Activity Assay, Positive Control
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