Fig 2: Differential protein and transcript expression following FASN inhibition in vitro. (A) Cytoplasmic FASN expression (Alexa Fluor 555, red) was reduced in C75-treated cells relative to vehicle control (DMSO) across all cell lines. Phalloidin: Alexa Fluor 488, green. DAPI: blue. Scale bar: 10 µm for all panels. (B) Semi-quantitative evaluation of FASN expression by H-score determination revealed significant downregulation in response to C75 treatment in all cell lines. **** p ≤ 0.0001. (C) Relative FASN expression was significantly upregulated in all cell lines following C75 treatment. * p < 0.05, ** p < 0.01, *** p < 0.001. (D) Assessment of c-MYC protein concentration by ELISA showed significant decreases in MYC concentration in all SebCA lines treated with C75 relative to vehicle control. * p < 0.05, ** p < 0.01, *** p < 0.001. (E) Relative expression of MYC was significantly induced in response to C75 treatment in SebCA02 and SebCA03. ** p < 0.01. 2−ΔΔcT was utilized to normalize target transcript expression to polR2α.

Fig 3: MYC and FASN-mediated differential transcript expression in vivo. (A) Relative MYC and (B) FASN expression were significantly upregulated in vehicle-treated TG mice relative to other groups. (C) Relative PLIN2 expression was significantly downregulated in TG mice relative to wt controls and in C75-treated eyelids relative to contralateral vehicle treatment. ** p < 0.01, *** p < 0.001, **** p ≤ 0.0001. 2−ΔΔcT was utilized to normalize target transcript expression to polR2α.

Fig 4: MYC modulatory, differentiative and apoptotic effects of FASN inhibition in vivo. (A) Representative images of chromogenic and fluorescently immunolabeled FFPE sections of murine tissues. Vehicle-treated MYC-overexpressing transgenic (TG) Meibomian glands exhibited pronounced upregulation of MYC (DAB: brown) relative to wildtype (wt) littermates and compared to contralateral C75-treated eyelids. Scale bar: 50 µm. (B) Adipophilin (DAB: brown) expression was diminished in TG mice relative to wt littermates and in C75-treated eyelids relative to vehicle controls. Scale bar: 50 µm. (C) MYC-overexpressing TG glands exhibited upregulated FASN expression (Alexa Fluor 555, red) relative to wt littermates and when compared to C75-treated eyelids. C75-treated eyelids exhibited increased distribution of apoptotic cells (TUNEL: Alexa Fluor 488, green) when compared to vehicle controls. DAPI: blue. Scale bar: 50 µm. (D) FASN immunolabeling was assessed by H scoring, demonstrating significant downregulation in response to C75 treatment in both wt and TG mice and in wt when compared to TG littermates. **** p ≤ 0.0001. (E) Quantification of TUNEL-positive meibocytes demonstrated a significant increase in mean apoptotic rates in C75-treated eyelids relative to contralateral controls and in wt mice relative to TG. * p < 0.05; **** p ≤ 0.0001.

Fig 5: Differential transcript expression in the IIS-modulated murine Meibomian gland. Adult mice (n = 6) were topically treated with trofinetide, PPP, or vehicle, and eyelids were homogenized. There were no significant differences in relative (A) IGF1R or (B) Akt expression between treatment groups. (C) MYC expression was upregulated in trofinetide-treated murine MGs and downregulated in PPP-treated animals relative to respective contralateral vehicle-treated controls. ** p ≤ 0.01. 2−Δ∆cT was utilized to normalize target transcript expression to polR2α.