Fig. 2: Silencing Cnnm4 protects against APAP toxicity in primary and human hepatocytes. | Nature Communications

Fig. 2: Silencing Cnnm4 protects against APAP toxicity in primary and human hepatocytes.

From: Restoring cellular magnesium balance through Cyclin M4 protects against acetaminophen-induced liver damage

Fig. 2

a Cell death was evaluated using TUNEL in WT hepatocytes under APAP overdose for 3 h and treated with a siRNA Cnnm1, Cnnm2, Cnnm3, and Cnnm4 or an unrelated control (siCtrl) compared to a control group (Ctrl). Scale bar correspond to 50 µm. Values are represented as mean ± SEM. **P < 0.01 (P = 0.002 APAP vs Ctrl; P = 0.005 APAP + siCnnm2 vs APAP; P = 0.01 APAP + siCnnm3 vs APAP; P = 0.000074 APAP + siCnnm4 vs APAP) (Student’s test, two-sided) b Cell death, apoptosis and necrosis reponse were evaluated using TUNEL and Annexin V Apoptosis and necrosis assay respectively in THLE2 cell line under APAP overdose for 3 h and treated with two different siRNA sequences of Cnnm4 and compared to a control group (Ctrl). Values are represented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (TUNEL P = 0.00001 APAP vs Ctrl; P = 0.00000067 APAP + siCnnm4#1 vs APAP; P = 0.000019 APAP + siCnnm4#2 vs APAP) (mRNA P = 0.00069 APAP vs Ctrl; P = 0.00022 APAP + siCnnm4#1 vs APAP; P = 0.0042 APAP + siCnnm4#2 vs APAP) (Apoptosis P = 0.00000048 APAP vs Ctrl; P = 0.00000002 APAP + siCnnm4#1 vs APAP; P = 0.0000028 APAP + siCnnm4#2 vs APAP) (Necrosis P = 0.0000012 APAP vs Ctrl; P = 0.000014 APAP + siCnnm4#1 vs APAP; P = 0.000023 APAP + siCnnm4#2 vs APAP) (Student’s test, two-sided). c Cell death, apoptosis, and necrosis reponse were evaluated using TUNEL and Annexin V Apoptosis and necrosis assay respectively were evaluated in primary murine hepatocytes treated with different GalNAc Cnnm4 siRNAs (GalNAc#1 and GalNAc#2) at different concentrations 1 nM and 10 nM and exposed to APAP for 3 h compared to untreated control group (Ctrl). Values are represented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (TUNEL P = 0.000055 APAP vs Ctrl; P = 0.000055 APAP + GalNAc#1 1 nM vs APAP; P = 0.000055 APAP + GalNAc#1 10 nM vs APAP; P = 0.000055 APAP + GalNAc#2 1 nM vs APAP; P = 0.000055 APAP + GalNAc#2 10 nM vs APAP) (mRNA P = 0.0039 APAP vs Ctrl; P = 0.00055 APAP + GalNAc#1 1 nM vs APAP; P = 0.00051 APAP + GalNAc#1 10 nM vs APAP; P = 0.0081 APAP + GalNAc#2 1 nM vs APAP; P = 0.00042 APAP + GalNAc#2 10 nM vs APAP) (Apoptosis P = 0.0004 APAP vs Ctrl; P = 0.005 APAP + GalNAc#1 1 nM vs APAP; P = 0.01 APAP + GalNAc#2 1 nM vs APAP) (Necrosis P = 0.027 APAP vs Ctrl; P = 0.0021 APAP + GalNAc#1 1 nM vs APAP; P = 0.0014 APAP + GalNAc#2 1 nM vs APAP) (Student’s test, two-sided). d Cell death, apoptosis, and necrosis response were evaluated using TUNEL and Annexin V Apoptosis and necrosis assay in WT hepatocytes under APAP overdose for 3 h with and without 5 mM of Mg2+, overexpressing Cnnm4 and overexpressing Cnnm4 with Mg2+ compared to a control group (Ctrl). Values are represented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (TUNEL P = 0.000055 APAP vs Ctrl; P = 0.0038 APAP + Cnnm4 vs APAP; P = 0.012 APAP + Cnnm4 + Mg2+ vs APAP) (Apoptosis P = 0.00000088 APAP vs Ctrl) (Necrosis P = 0.0062 APAP vs Ctrl) (Student’s test, two-sided). e Cell death, apoptosis, and necrosis response were evaluated using TUNEL and Annexin V Apoptosis and necrosis assay in WT hepatocytes under APAP overdose for overexpressing Cnnm4 and overexpressing the mutant Cnnm4 T495I and compared to a control group (Ctrl). Values are represented as mean ± SEM. *P < 0.05***P < 0.001 (TUNEL P = 0.000046 APAP vs Ctrl; P = 0.00007 APAP + Cnnm4 vs APAP; P = 0.044 APAP + Cnnm4 T495I vs APAP) (Student’s test, two-sided). Quadrupled were used for experimental conditions. Source data are provided as a Source Data file.

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