Tetramethylrhodamine methyl ester perchlorate

Immunofluorescence staining was performed as previously described¹¹, ¹², ¹⁸, ²³ and imaging was performed using a Zeiss LSM‐710 model, equipped with an Airyscan module (Carl Zeiss). Antibodies used were alpha smooth muscle actin (Abcam, ab5694), von Willebrand factor (Abcam, ab11713), CD4 (Invitrogen, LS14004182), Ki67 (Abcam, ab16667), nuclear factor of activated T‐cells c2 (Abcam, ab2722), hypoxia inducible factor 1α (Abcam, 51608), and ^Y705p‐STAT3 (Cell Signaling Technology, 9131). All antibodies used for immunofluorescence were diluted in 1:100 and all secondary antibodies in 1:1000. Apoptosis was measured using the Apoptag Apoptosis Detection Kit for TUNEL (Invitrogen) and the Dead Cell Apoptosis Kit with Annexin V FITC and Propidium iodide (Invitrogen). The mitochondrial membrane potential was measured by staining live PASMCs with 20 nmol/L tetramethyl‐rhodamine methyl‐ester perchlorate (Invitrogen) and 500 nmol/L Hoechst 33342 (Invitrogen) for 30 minutes at 37 °C. The mitochondrial reactive oxygen species were measured by staining live PASMCs with 5 μmol/L MitoSOX (Invitrogen) and 500 nmol/L Hoechst 33342 in nonserum media for 15 minutes at 37 °C, and then cells were washed and replaced with regular media.