Protein lysis buffer

The samples were extracted with protein lysis buffer (Beyotime, China) supplemented with protease inhibitor cocktail. Protein concentration was determined using the BCA Kit (Beyotime, China). Proteins (25‐35 µg) were separated on a 10% polyacrylamide precast SDS‐PAGE gel (Bio‐Rad) followed by blotting on PVDF membranes (Millipore Billerica, MA, USA). The membranes were probed with the following antibodies against: 12‐LOX (Santa Cruz, sc‐365194, 1:100), UCP1 (proteintech, 23673‐1‐AP, 1:2000), OXPHOS cocktail (Abcam, ab110413, 1:1000), FABP4 (Abclonal, A11481, 1:750), AMPK (Cell Signaling Technology, AB 10622186, 1:1000), pAMPK (Cell Signaling Technology, AB 331250, 1:1000), β‐actin (Abclonal, AC038, 1:10000); GAPDH (Proteintech, 60004‐1‐Ig, 1:10000), and Flag (Abclonal, ae063, 1:3000).

Protein extraction, protein concentration determination, and Western blot analysis were performed following previously established protocols^{46 (link)}. Briefly, tissues or cells were lysed on ice for 20 min using protein lysis buffer (Beyotime, P0013) supplemented with protease inhibitors (Roche). The lysates were then centrifuged at 14,000 g for 20 min at 4 °C, and the supernatants were collected. Protein concentration was determined using the BCA Protein Assay Kit (Beyotime, P0009) according to the manufacturer’s instructions. SDS-PAGE gels were prepared, and proteins were separated at a constant voltage of 100 V. Subsequently, proteins were transferred onto PVDF membranes (Millipore, USA) using a semi-dry transfer system, and the membranes were incubated with primary antibodies for KAT8 (Abcam, ab200660)(1:1000), H4K16ac (Millipore, 07-329) (1:1000), CDX2 (Abcam, ab76541) (1:1000), EOMES (Abcam, ab23345) (1:1000), α-Tubulin (Proteintech, 11224-1-AP) (1:10000) overnight at 4 °C. After washing, the membranes were incubated with HRP-conjugated goat anti-rabbit IgG (Sigma, A6154) (1:5000) or HRP-conjugated goat anti-mouse IgG (Sigma, A4416) (1:5000) at room temperature for 1 h. Protein bands were visualized using an ECL chemiluminescent substrate (AQ, AQ529) and captured on X-ray film. All uncropped and unprocessed scans of western blots were supplied in Supplementary Fig. 7 in the Supplementary Information.

Zhilong Huoxue Tongyu capsule (Med-drug permit no. Chuan Z20070528; Patent number 200810147774.1) was provided by the Preparation Room for TCM at the Affiliated Hospital of TCM, Southwest Medical University. The specific information of antibodies (p-PI3K, PI3K, p-AKT, AKT, Nrf2, HO-1, GPX4, ACSL4, GAPDH) is presented in S1 Table. RNA extraction, reverse transcription, and reaction reagents were purchased from TOYOBO (Shanghai) Biotech Co., Ltd. The BCA protein concentration assay kit and protein lysis buffer were purchased from Beyotime Biotechnology Co., Ltd. The lactate dehydrogenase (LDH) kit, Creatine kinase-MB isoenzyme (CK-MB) kit, malondialdehyde (MDA) kit, superoxide dismutase (SOD) kit, reactive oxygen species (ROS) kit, and ELISA kit were purchased from Jiancheng (Nanjing) Biotech Co., Ltd.

TP (purity > 98%, HPLC, CAS number 38748–32-2) was bought from Sanling Biotechnology Company (Guilin, Guangxi, China). Lipopolysaccharide (LPS) (L2755), 4-phenylbutyric acid (4-PBA) (purity ≥ 99%, P21005), N-acetylcysteine (A7250, NAC) and butylated hydroxyanisole (B1253, BHA) were obtained from Sigma‒Aldrich (St. Louis, MO, USA). Suc-LLVY-AMC (CAS number 94367–21-2, HY-P1002) and MG-132 (CAS number 133407–82-6, HY-13259) were obtained from MedChemExpress Ltd. (Monmouth Junction, NJ, USA).
Serum Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured with kits from Nanjing Wittmann Biotechnology Co. Ltd. (Nanjing, China); reduced glutathione (GSH) assay kits, BCA Protein Assay Kits and Protein Lysis Buffer were purchased from Nanjing Beyotime Co. Ltd. (Nanjing, China); reagents (TRIzol, SYBR Green Master Mix reagent, and Reverse Transcription Kit) for qPCR were purchased from Vazyme Biotech Co., Ltd. (Nanjing, China); MEM was obtained from Gibco (Grand Island, NY, USA); CellROX Green Reagent and LipofectamineTM3000 Transfection Reagent were purchased from ThermoFisher Scientific (Waltham, MA, USA); CytoTox96® Non-Radioactive Cytotoxicity Assays (G1780) were obtained from Promega Corporation (Madison, Wisconsin, USA); siRNA Negative Control, siRNA-ATF4-688 and siRNA-ATF4-1132 were purchased from Shanghai Gemma Pharmaceuticals (Shanghai, China).
Antibodies against cleaved caspase-3 (9661), FLIP (56343s), ATF4 (11815s), eIF2α (9722.0), p-eIF2α (s51) (9721 s) and CHOP (2895) were obtained from Cell Signaling Technology (Danvers, MA, USA). Antibodies against GRP78 (n-20) (sc-1050) and GAPDH (sc-365062) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies against cleaved PARP (ab32064) were purchased from Abcam (Waltham, MA, USA). Antibodies against β-actin (T0022) were obtained from Affinity Biosciences (Cincinnati, OH, USA). Alexa Fluor 647-conjugated goat anti-rabbit IgGs (H + L) were purchased from JAX. Goat anti-rabbit-HRP secondary antibodies (31460) and goat anti-mouse-HRP secondary antibodies (31430) were bought from Thermo Fisher Scientific (Waltham, MA, USA).

The total carbohydrate content of H. pluvialis was determined using the conventional phenol–sulfuric acid method with slight modifications. Specifically, 100 mL of microalgae was collected and then transferred into a mixed solution consisting of 1 mL of distilled water and 1 mL of 5% phenol solution (w/v), followed by the dropwise addition of 5 mL of 98% sulfuric acid along the tube wall. The mixture was incubated at 25 °C for 20 min and subsequently analyzed at an absorbance wavelength of 483 nm using UV–Vis spectrophotometry. The concentration of intracellular carbohydrate in H. pluvialis was normalized with the standard curve of a range of glucose concentrations.
The total protein of H. pluvialis was isolated using protein lysis buffer (Beyotime, China) supplied with the protease inhibitor phenylmethylsulfonyl fluoride (Beyotime, China), as instructed by the manufacturer. The microalgal protein content was quantified using the BCA Protein Assay kit (Beyotime, China) following the standard protocols provided by the manufacturer.
The total lipid content of H. pluvialis was determined gravimetrically using the conventional methanol–chloroform method with slight modifications. In brief, 200 mL of microalgae was harvested for further lyophilization. The lyophilized precipitation was ground under liquid nitrogen and transferred into a falcon tube containing a mixture solution of methanol, chloroform, and water at a volume ratio of 2:1:0.8. The solution was then subjected to ultrasonication at 200 W for 15 min. Subsequently, 2 mL of mixture solution containing chloroform and water at a volume ratio of 1:1 was added to the falcon tubes, followed by the gentle vortex. The lower phase of the solution, obtained after centrifugation at 2,000 rpm for 5 min, was collected into a pre-weighed Eppendorf tube and dried using a nitrogen stream for gravimetric determination.
The fatty acids of H. pluvialis were then transesterified into fatty acid methyl esters following previous protocols and analyzed using gas chromatography–mass spectrophotometry equipped with a NIST 147 spectrum library for fatty acid quantification based on the normalization of peak area for each fatty acid [54 (link)].