Additional file 1 of IP3R1-mediated MAMs formation contributes to mechanical trauma-induced hepatic injury and the protective effect of melatonin
Creators: name: Shi, Rui
name: Liu, Zhenhua
name: Yue, Huan
name: Li, Man
name: Liu, Simin
name: De, Dema
name: Li, Runjing
name: Chen, Yunan
name: Cheng, Shuli
name: Gu, Xiaoming
name: Jia, Min
name: Li, Jun
name: Li, Juan
name: Zhang, Shumiao
name: Feng, Na
name: Fan, Rong
name: Fu, Feng
name: Liu, Yali
name: Ding, Mingge
name: Pei, Jianming
Publisher: name: figshare
Year: 2024
Subjects: subject: Biophysics
subject: Biochemistry
subject: Cell Biology
subject: Molecular Biology
subject: Physiology
subject: FOS: Biological sciences
subject: Environmental Sciences not elsewhere classified
subject: Chemical Sciences not elsewhere classified
subject: Astronomical and Space Sciences not elsewhere classified
subject: Biological Sciences not elsewhere classified
subject: Developmental Biology
subject: Cancer

description: Additional file 1: Figure S1 Mediators that cause mechanical trauma-induced hepatic injury mainly existed in the serum of MT rats. (A) Schematic representation of the experimental grouping. (B-C) Determination of hepatocytes apoptosis by flow cytometry with Annexin V and PI staining and the apoptotic statistical graph. NH: normal hepatocytes; TH: traumatic hepatocytes; SS: 20% diluted serum in Sham rats; TS: 20% diluted serum in MT (after 4 hour) rats. (D) Cell viability in the hepatocytes incubated with a series of diluted SS and TS. (E) Serum IL-1β level of MT rats. n=8 (F) Serum IL-6 level of MT rats. n=8. (G) Serum TGFα level of MT rats. n=8 (H) Cell viability (percentage of SS). (I) Serum IL-1β level. All of the values are shown as the means ± SEM. n=6 in each group. **P &lt; 0.01 vs NH + SS, Ctrl, Sham, or SS. *P &lt; 0.05 vs Sham. ##P &lt; 0.01 vs TH + SS or MT+V. #P &lt; 0.05 vs MT+V. Figure S2 Knockdown of IP3R1 alleviated TS-induced MAM formation and mitochondrial calcium overload and mitochondrial dysfunction. (A) Representative confocal images of primary hepatocytes double-stained by mitotracker (red) and ER-tracker (green) at ×600 magnification. (B-C) Statistical quantification of the colocalization area between mitochondria and ER. (D) Representative images and quantitative analysis of MitoSOX-stained mitochondria-derived superoxide production. (E) Quantification of fluorescence intensity normalized by SS + Scramble RNAi. (F) Representative traces of ionomycin-induced changes of mitochondrial Ca2+ concentration (n=3). (G) Quantification of the relative increment of fluorescence intensity in mitochondrial Ca2+ concentration after ionomycin stimulation normalized by SS + Scramble RNAi. (H-I) Representative flow cytometry results and statistical analysis of mitochondrial membrane potential by JC-1 stanning in primary hepatocytes. (J) Cell viability (percentage of SS + Scramble RNAi). (K-L) Oxygen consumption rate (OCR) measured by Seahorse and quantitative statistical analysis of OCR (n=3). All of the values are shown as the means ± SEM. n=6 in each group. **P&lt; 0.01 vs SS + Scramble RNAi; #P&lt; 0.05, ##P &lt; 0.01 vs TS + Scramble RNAi. Figure S3 Transcription factor FoxO1 rather than JUNB suppressed IP3R1-mediated MAMs. (A) FoxO1 and JUNB inhibited the luciferase activity of the IP3R1 promoter (n=3). (B-D) Representative blots and quantitative analysis of nuclear FoxO1 and JUNB (PD98059, a MEK/ERK inhibitor). (E-H) ERK1/2 was knocked down by siRNA, after which the cells were subjected to TS with or without melatonin. Representative blots and quantitative analysis of phosphorylated FoxO1 (p- FoxO1), total ERK1/2 and nuclear FoxO1. (I) Interaction between ERK1/2 and FoxO1 determined by co-immunoprecipitation (n=3). All of the values are shown as the means ± SEM. n=4 in each group. **P&lt; 0.01 vs Ctrl or SS or TS + Scramble RNAi; ##P &lt; 0.01 vs TS or TS + Scramble RNAi; &amp;&amp;P &lt; 0.01 vs TS + Mel. Figure S4 Mel increased ERK1/2 and FoxO1 phosphorylation and expression of nuclear FoxO1 in MT-treated livers in vivo. (A-G) Representative blots and quantitative analysis of phosphorylated ERK1/2 (p-ERK1/2), ERK1/2, phosphorylated FoxO1 (p-FoxO1), FoxO1, and nuclear FoxO1; Mel, melatonin at a dosage of 30 mg/kg. n = 4 in each group. All data are shown as means ± SEM. **P&lt; 0.01 vs Sham; ##P&lt; 0.01 vs MT + V.