Ab79823

The Opal 7 multiplexed assay (PerkinElmer, MA, USA) was used to generate multiple immunostainings. The best concentration of antibodies was determined before multiple immunostainings, according to the instructions. The primary antibodies used were as follows: anti-CD45 (1:200, ab10558, Abcam), anti-α-SMA (1:2000, ab12964, Abcam), anti-VIM (1:150, ab8978, Abcam), anti-CD31 (1:50,ab9498,Abcam), anti-NINJ1 (1:200, GTX31596, GeneTex), anti-TPPP3 (1:200, GTX33554, GeneTex), anti-CD3 (1:200, 17,617–1-P,Thermo), anti-MMP9 (1:1000, ab74003,Abcam), anti-CD11b (1:3000, ab133357, Abcam), anti-CD31 (1:50, ab28364,Abcam), anti-GSDMD (1:800, 36,425, Cell signaling), anti-HMGB1(1:500, ab79823, Abcam). The stained slides were analyzed by Vectra Polaris Quantitative Pathology Imaging Systems (PerkinElmer).

Briefly, B16/F10 tumor cells were treated with AuNP@mSiO₂, AuNP@mSiO₂-ZnO or AuNP@mSiO₂@DOX-ZnO 50 μg/mL for 6 h and irradiated with 655 nm laser (1.0 W/cm²) for 5 min, after which the tumor cells were further cultured for 24 h or 48 h and the cell lysates were extracted. The following primary antibodies were used: Anti-HMGB1 (ab79823, Abcam), Anti-LC3B (ab51520, Abcam), Anti-p62 (ab56416, Abcam), anti-Baclin-1 (ab62557, Abcam), anti-GAPDH (ab181602, Abcam) and β-actin (C4, Santa Cruz).

Western blot analysis was performed as previously described.³⁵ The following antibodies were used: anti‐HMGB1 (1:1000, ab79823; Abcam, Cambridge, UK), anti‐HSP70 (1:1000, ab181606; Abcam), anti‐calreticulin (1:1000, ab92516; Abcam), and anti‐GAPDH (1:1000, 60004‐1‐Ig; Proteintech，Chicago, Illinois, 60601, USA). Proteins were visualized by using an ECL kit (4AW011; purchased from 4A Biotech Co., Ltd, Beijing, China). The integrated optical density of the protein bands was determined using Image Lab 4.0 image acquisition and analysis system software. Simultaneous detection of GAPDH protein bands was used as the internal reference.

4-6-week-old female BALB/c mice were purchased from Beijing HFK Bioscience Co. Ltd. The mice were housed in a specific pathogen-free (SPF) environment at Laboratory Animal Care Center of Tongji Hospital, and allowed to recover and were monitored closely for one week before any treatment. Then 4T1 breast cancer cells (1×105) were subcutaneously injected into the right posterior limb. For treatment, mice were randomized into two groups (n=5 per group), vehicle and Dinaciclib, since tumor volumes reaches 50 mm3. Mice were treated 3 times per week with Dinaciclib 30mg/kg administered via i.p injection. The tumor size was monitored every 3 days. Tumor length and width were measured using electronic calipers. The tumor volume was calculated as follows: volume = 0.5 × length × width2. At sacrifice, portions of tumors were stored in liquid nitrogen for follow-up western blot test or were fixed in 4% Polyformaldehyde for routine histopathologic processing. And the following antibodies were used: anti- HMGB1 (1:400; catalog: ab79823; Abcam), anti-CD8 (1:2,000; catalog: ab209775; Abcam), and anti-Granzyme B (1:3,000; catalog: ab255598; Abcam). All animal procedures were performed in accordance with the approved Guide for the Care and Treatment of Laboratory Animals of Tongji Hospital and approved by the Ethics Committees of Tongji Hospital.