Fig 2: Antagonistic effects of the activators of TLRs, PI-3K/Akt, ERK1/2, p38/JNK, and NF-?B pathways on the anti-IAV activity of OMT. A549 cells were infected with IAV (MOI = 0.001), treated with or without ribavirin (25 µg/mL) and OMT (50 µg/mL), and simultaneously treated with or without TLR3 activator (polyI:C, 100 ng/mL), TLR4 activator LPS-B5 (1 µg/mL), TLR7/8 activator R-848 (10 µg/mL), PI-3K/Akt activator (IGF-1, 100 ng/mL), ERK activator (EGF, 100 ng/mL), p38/JNK activator (Anisomycin, 10 µM), and NF-?B activator (PMA, 1 µg/mL). The test doses of all activators were determined according to previous reports and our preliminary tests. After 48 h, cell viability was determined by the SRB method (A) and IAV vRNA levels were determined by a qRT-PCR assay (B). Data shown were mean ± SD of three independent experiments performed in triplicate. * p < 0.05, compared with the virus + drug control.

Fig 3: PEITC suppressed OS growth in vivo. MNNG/HOS cells were injected subcutaneously into the right flank of the male BALB/c nude mice. One week after the OS xenograft mouse model was established, the mice were randomly divided into two groups and, respectively, administrated with 10% sesame seed oil and 30 mg/kg PEITC once daily for 24 consecutive days. (a) Body weight change of the two groups. (b) Volume change of OS tissues of the two groups. Data were calculated by the formula: volume = length × width2 × 0.5. (c) Weight of OS tissues from the two groups. (d) H&E staining analysis of tumor tissues (200× and 400×). (e) The protein expression levels of TfR1, DMT1, FTH1, and FPN in tumor tissues. (f) The protein expression levels of LC3B, C-caspase3, GPx4, and Cdc2 in tumor tissues. (g) Phosphorylation levels of ERK, p38, and JNK in tumor tissues. All data were presented as the means ± SD (n = 7). *P < 0.05, **P < 0.01, and ***P < 0.001 versus control group.

Fig 4: PEITC activated the MAPK signaling pathway in human OS cells. (a–d) Phosphorylation levels of ERK, p38, and JNK in MNNG/HOS, U-2 OS, MG-63, and 143B cells treated with the indicated concentrations of PEITC for 20 h or 30 µM PEITC for 4 h, 12 h, 24 h, and 48 h.

Fig 5: Generation of myofibroblast specific transforming growth factor ß receptor II gene (Tgfbr2) (r2)–ablated mice. A, Schematic representation of the different mouse lines used. Mice with cardiomyocyte-specific mutant a-B-crystallin expression (CryABR 120G) were crossed with Tgfbr2-loxP–containing gene targeted lines and those double transgenic (Tg) lines further crossed with mice containing the periostin locus (Postn) with a tamoxifen (TAM) regulated Mer-Cre-Mer cassette (Postnmcm) inserted into exon (E1). B, Experimental scheme where mice were fed a TAM diet starting from 6 to 8 weeks until they were euthanitized. C and D, Western blot and quantification for Tgfßr2 protein in purified fibroblasts (n=6 separate, total hearts). E and F, Expression and quantification of SMAD3 (phospho [p]-SMAD3/SMAD3) and (G and H) p38 signaling (p-p38/p38) in purified fibroblasts isolated from hearts of 3-month-old experimental cohorts fed with TAM chow (TAM+) or normal chow (-) (n=4–6 separate, total hearts). *P<0.05 vs uninduced Cre Cry ABR 120G /Postnmcm/r2 f/f animals. GAPDH indicates glyceraldehyde 3-phosphate; a-MHC, a-myosin heavy chain; SMAD3, small mothers against decapentaplegic homolog 3.