Fig. 1: Modulation of cell death pathways by BCL2 inhibitors in PACs treated with cerulein or EtOH/POA. | Cell Death & Disease

Fig. 1: Modulation of cell death pathways by BCL2 inhibitors in PACs treated with cerulein or EtOH/POA.

From: Repurposing BCL2 inhibitors: Venetoclax protects against acinar cell necrosis in acute pancreatitis by promoting apoptosis

Fig. 1

A Schematic illustration depicting the selectivity and affinity of BCL2 inhibitors, Navitoclax and Venetoclax, toward anti-apoptotic BCL2 family proteins (BCL2, BCL-xL, BCL-w, and MCL-1) [8]. Created in BioRender. B Representative images of mouse PACs loaded with NucView 488 and RedDot 2, pre-incubated with Navitoclax or Venetoclax at 10 μM (or DMSO control) for 15 min and then treated with cerulein at 10 nM for 1 hour. Green fluorescence of NucView 488 indicates active caspase 3 and 7, signifying apoptosis; red fluorescence of RedDot 2 indicates necrosis due to damaged cell membranes. Images taken with a 40× magnification objective. C−E Quantitative results from mouse PAC cell death assessment following pre-stimulation with Navitoclax, Venetoclax (10 μM), or control (DMSO) for 15 min, and subsequent cerulein treatment (10 nM) for 1 hour, representative images are shown in (B). Bar chart in (C) shows the percentage of necrotic cells, (D) the percentage of apoptotic cells, and (E) live cells, relative to the total cell count. Data collected from 5 independent experiments using freshly isolated mouse PACs (N = 5), 15 images taken per condition, with total cell counts as follows: Ctrl: 5506, Navi: 5824, Ven: 5116, Cer: 3911, Cer Navi: 3879, Cer Ven: 3406. Results are presented as mean ± SD. F ATP measurements in mouse PACs after pre-stimulation with Navitoclax, Venetoclax (10 μM), or control (DMSO) for 15 min, followed by cerulein treatment (10 nM) for 1 hour. Performed using the CellTiter-Glo 3D Cell Viability Assay. Results are normalized to total protein concentration and expressed as a percentage relative to control (Ctrl). Data derived from 4 separate PAC isolations (N = 4). Results are presented as mean ± SD. G Representative images of mouse PACs loaded with NucView 488 and RedDot 2, pre-incubated with Navitoclax or Venetoclax at 10 μM (or DMSO control) for 15 min and then treated with EtOH 200 mM + POA 200 μM for 1 hour. Green fluorescence of NucView 488 indicates active caspase 3 and 7, signifying apoptosis; red fluorescence of RedDot 2 indicates necrosis due to damaged cell membranes. Images taken with a 40× magnification objective. H−J Quantitative results from mouse PAC cell death assessment following pre-stimulation with Navitoclax, Venetoclax (10 μM), or control (DMSO) for 15 min, and subsequent treatment with EtOH 200 mM + POA 200 μM for 1 hour, representative images are shown in (G). Bar chart in (H) shows the percentage of necrotic cells, (I) the percentage of apoptotic cells, and (J) live cells, relative to the total cell count. Data collected from 5 independent experiments using freshly isolated mouse PACs (N = 5), 15 images taken per condition, with total cell counts as follows: Ctrl: 5682, Navi: 5616, Ven: 5858, E/P: 5316, E/P Navi: 5521, E/P Ven: 5222. Results are presented as mean ± SD. K ATP measurements in mouse PACs after pre-stimulation with Navitoclax, Venetoclax (10 μM), or control (DMSO) for 15 min, followed by treatment with EtOH 200 mM + POA 200 μM for 1 hour. Conducted using the CellTiter-Glo 3D Cell Viability Assay. Results are normalized to total protein concentration and expressed as a percentage relative to control (Ctrl). Data were obtained from 4 separate PAC isolations (N = 4). Results are presented as mean ± SD. Statistical analyses: Data distribution was assessed for normality using the Shapiro-Wilk test, confirming that the data were normally distributed. Statistical analyses were performed using one-way ANOVA followed by Sidak’s multiple comparisons test to assess differences between groups. Figure abbreviation legend: Ctrl (NaHEPES), Navi (Navitoclax 10 μM), Ven (Venetoclax 10 μM), Cer (cerulein 10 nM), Cer Navi (cerulein 10 nM + Navitoclax 10 μM), Cer Ven (cerulein 10 nM + Venetoclax 10 μM), E/P (EtOH 200 mM + POA 200 μM), E/P Navi (EtOH 200 mM + POA 200 μM + Navitoclax 10 μM), E/P Ven (EtOH 200 mM + POA 200 μM + Venetoclax 10 μM).

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