1. Introductory information 
Title of the dataset: Data underlying the publication: Activation of PAR-2/p-ERK/GPX4 axis is involved in MCT-induced PAH and hypoxia-induced PASMCs proliferation
For each file or group of similar files, a short description of what data it contains: there are 5 pictures and 37 files in pzfx format. And 37 files in pzfx format are the experimental data related to the content of the picture. For example, Fig1A. media to vessel diameter ratio.pzfx, contains the data of "media to vessel diameter ratio" showed in A in Fig 1.

Fig 1. Formation of MCT-induced rats PAH

To investigate the expression of PAR-2, p-ERK, and GPX4 in relation to PASMCs proliferation and survival, as well as PAH formation, we first established a PAH animal model. After successfully creating the rat PAH model, we examined the expression of PAR-2, p-ERK, and GPX4 in these rats. At the 4th week, H&E and IHC staining were used to evaluate medium-sized pulmonary arterioles' membrane proliferation and non-muscular arterioles' degree of muscularization. At the 4th week, the pulmonary arterioles in the PAH model group showed narrowed lumens and significantly thickened medium membrane. The diameter of medium-sized pulmonary arterioles' medium membrane (50-150μm) and the degree of muscularization of nonmuscular arterioles (<50μm) were increased in the PAH model (Fig. 1A).

The RV of the rat was punctured through the thorax at the 4th week after MCT administration to measure the systolic pressure as an indicator of PASP. This model was successful, showing an increase in the RV index in the rats model of PAH induced by MCT. To further validate the stability of the PAH model, H&E staining on the right ventricular wall revealed myocardial fibrosis, with a significant increase in fiber rupture and fibrosis compared with the control group, validating the stability of the PAH model (Fig. 1B). Additionally, the PASP of rats in the PAH group was significantly higher than that of rats in the control group (Fig. 1C). 

In MCT-induced PAH rats, PASP, media thickness to vessel diameter ratio in medium-sized pulmonary arterioles, and musculature in non-muscular vessels in small-sized pulmonary arterioles were all increased. Additionally, there was an increase in right ventricular index and myocardial fibrosis scores in the RV. These results provided evidence for the successful establishment of the PAH model.

Fig 2. An increase in the expression of protein PAR-2, p-ERK and GPX4 was observed in the lung tissue of rats with MCT-induced PAH.

According to the introduction, Immunohistochemical staining was used in the experiment at the 4th week to evaluate lung protein expression. PAR-2, p-ERK and GPX4 showed significant increases compared to the control group Fig. 2A.

WB method was used in experiment at the 4th week to assess lung protein expression. PAR-2、p-ERK and GPX4 were found to be significantly increased, exhibiting a notable distinction when compared with the control group Fig. 2B.

Based on successful establishment of the PAH model, we observe increased expression of PAR-2, p-ERK and GPX4, which are closely linked to the formation and exacerbation of PAH.

Fig 3. Hypoxia induces a time-dependent proliferation of PASMCs within 6 hours, accompanied by increased expression of PAR-2, p-ERK and GPX4.

The study found that PASMCs proliferated significantly within 6 hours under hypoxia condition (Li M, et al., 2022). We investigated the time course of hypoxia-induced proliferation and aimed to identify the most significant time point for cells proliferation. Different durations of hypoxia exposure were employed: 0 (normoxia), 1, 2, 4, and 6 hours under the condition of 95% N2-5% CO2-3% O2.

After different duration of hypoxia, rat PASMCs were used to investigate the impact of hypoxia duration on cells proliferation. The cells were exposed to varying lengths of hypoxia followed by culture in fresh 10% FBS medium under normoxia conditions for 5 days. After 72 hours, cells proliferation was assessed using the CCK-8 assay and BrdU-incorporation. Our findings showed that hypoxia induced a time-dependent proliferation of rat PASMCs, with modest increases after 2 hours of hypoxia and significant promotion of proliferation after 4-6 hours of hypoxia Fig. 3A.

After hypoxia for different duration, rat PASMCs were lysed using cells lysis buffer to explore the expression of interest proteins. We observed a time-dependent elevation in the expression of PAR-2, p-ERK and GPX4. Specifically, after 6 hours of hypoxia, Western blot analysis showed a significant increase in the expression of PAR-2, p-ERK and GPX4 Fig. 3B. This result was further confirmed by immunofluorescence staining Fig. 3C.

Fig 4. FSLL, a PAR-2 inhibitor, inhibited hypoxia-induced proliferation and regulated the expression of PAR-2, p-ERK and GPX4 in rat PASMCs.

The above studies found that 6 hours of hypoxia exposure increased the expression of PAR-2, p-ERK and GPX4, leading to cells proliferation. It is reported that activation of the PAR-2 receptor and up-regulation of p-ERK played a significant role in PASMCs proliferation. To further explore PAR-2/p-ERK/GPX4 pathway's involvement in hypoxia-induced rat PASMCs proliferation, we pre-incubated with the PAR-2 receptor inhibitor FSLL before subjecting rat PASMCs to hypoxia.

After 6 hours of hypoxia exposure, the cells were cultured under normoxia conditions with fresh 10% FBS media until day 5. Cells proliferation was then assessed, and our results demonstrated that FSLL effectively inhibited hypoxia-induced cell proliferation in a dose-dependent manner Fig.4A. The working concentration of FSLL was determined based on the maximum permitted concentration in the literature (Joseph C, et al., 2022).

The above study showed the activation of PAR-2 was involved in hypoxia-induced proliferation of rat PASMCs. We further investigated whether the inhibitor FSLL could regulate the expression of p-ERK and GPX4, known to be closely associated with PASMCs proliferation. We found that FSLL dose-dependently inhibited the hypoxia-induced increase in p-ERK and GPX4 expression, as well as PAR-2 expression, as shown by immunofluorescence staining Fig. 4B. This result was also confirmed by Western blot analysis Fig. 4C. 

The results suggest that the activation of PAR-2 stimulates p-ERK and GPX4, leading to their up-regulation. Additionally, activation of the PAR-2 receptor could promote the expression of intermediate factors, which in turn stimulates its own expression.

Fig 5.Inhibition of MEK1/2/p-ERK with U0126 suppressed hypoxia-induced proliferation in rat PASMCs and regulated the expression of PAR-2, p-ERK, and GPX4.

After confirming that PAR-2 activation is upstream of p-ERK and GPX in the hypoxia-induced proliferation of rat PASMCs, we investigated the intermediate link in the PAR-2/p-ERK/GPX4 pathway. Our findings show that U0126 effectively inhibits hypoxia-induced PASMC proliferation in a significant dose-dependent manner Fig.5A.

The results show that MEK1/2/p-ERK activation contributes to rat PASMC proliferation, consistent with previous literature (Wei L, et al., 2014). We also investigated the effect of U0126 on hypoxia-induced expression of p-ERK and GPX4. Our findings demonstrated that U0126 dose-dependently inhibited the increase in expression of p-ERK and GPX4, as well as PAR-2 expression, as shown by immunofluorescence staining and confirmed by Western blot analysis. Fig. 5,B C.

These findings indicate that p-ERK can regulate GPX4, which prevents ferroptosis and promotes cell survival. Interestingly, U0126 also down-regulates PAR-2, suggesting that p-ERK may in turn regulate the expression of PAR-2, which was consistent with existing literature (Wei L, et al., 2014).

2. Methodological information
The data were presented as means ± SEM. Statistical analysis was performed using T test and One-way analysis of variance (ANOVA) followed by Dunnett's test, where appropriate, with GraphPad Prism 5.0 software. Multiple comparisons between the groups were conducted using Student-Newman-Keul's post hoc test. A P-value less than 0.05 was considered statistically significant.