Rgfp109

Primary antibodies were: mouse anti-human HSP70, specific for stress-inducible HSPA1A (StressMarq, Canada, SMC-100B; 1:100 ICC; 1:1000 WB), mouse anti-Flag M2 (Sigma-Aldrich, #F1804; 1:400 ICC); rabbit anti-FUS (Proteintech, USA, 11570-1-AP; 1:400 ICC), mouse anti-human SOD1 (Sigma-Aldrich Canada, SD-G6; 1:100 ICC), rabbit anti-acetyl-histone H3K9/K14 (Cell Signaling, #9677; 1:400 ICC; 1:1000 WB), mouse anti-GAPDH (MediMabs, Canada, #MM-0163; 1:1000 WB), moue monoclonal anti-acetylated tubulin (Sigma-Aldrich #T6793; 1:1000 WB) and rabbit anti-α-tubulin (Abcam #ab15246; 1:1000 WB).
Secondary antibodies (Jackson Immunoresearch: Cedarlane, Canada): Alexa Fluor 488-conjugated Affinipure donkey anti-mouse IgG (1:300); Cy3-conjugated donkey anti-mouse IgG 1:300); Cy5-conjugated donkey anti-rabbit IgG (1:300), and HRP-conjugated goat anti-mouse (1/5000 for WB, 1/500 for IC) or donkey anti-rabbit IgG (Jackson Immunoresearch (1:2500).
HDAC inhibitors and HSP-inducing drugs: SAHA (suberoylanilide hydroxamic acid) and tubastatin A (Cayman Chemical: Cedarlane, Canada); tacedinaline, RGFP109 and RGFP966 (Selleckchem: Cedarlane, Canada); sodium phenylbutyrate (Selleckchem); arimoclomol (Toronto Research Chemicals, Canada); NXD30001 was previously supplied by NexGenix Pharmaceuticals (Cha et al. 2014 (link)).

RGFP109 (RGFP109, Selleck Chemicals) was dissolved in DMSO and diluted in 30% (w/v) hydroxypropyl-β-cyclodextrin (#HK388-5g, Bio Basic Inc., Toronto, Canada) with 100 mM sodium acetate (pH 5.4). Then, RGFP109 was administered subcutaneously once per day for 2 weeks with 60 mg/kg dose.

Animal studies were approved by the Animal Ethical and Welfare Committee of Sun Yat-sen University. The hind flanks of 4-week-old female BALB/c-nu/nu mice were subcutaneously inoculated with 2 × 10⁵ GSC-1 cells. The mice were randomly divided into 6 groups after tumor cell injection and were injected intraperitoneally once per day with vehicle, 20 mg/kg dbcAMP (Sigma-Aldrich, USA), 40 mg/kg luteolin (Selleck, USA), 20 mg/kg MS275 (Selleck), 100 mg/kg temozolomide (Topscience, USA) or a combination of these drugs. Tumor diameters were measured every other day with a caliper, and tumor volumes were estimated using the following formula: width² × length/2 = V (mm³).
The orthotopic implantation of glioma cells was performed using 1 × 10⁵ GSCs. In brief, cells were injected 2 mm lateral and 0.5 mm anterior to the bregma and 2.5 mm below the skull of 4-week-old athymic nude mice. The mice were randomly divided into four groups and injected intraperitoneally with vehicle, 100 mg/kg RGFP109 (Selleck) plus 40 mg/kg luteolin, 100 mg/kg temozolomide or a combination once per day for 14 days (n = 8 animals for each group). The mice were monitored daily and killed when neurological symptoms were observed. Their brains were then dissected and fixed in formalin for H&E staining.