*2.7. LnHS did not Modify Sirtuins and Cytokines Expression in both U-87 and HF Cells*

To verify LnHS ability to display cytotoxicity, interfering oxidative and inflammation processes, the expression of Sirt1 and Sirt2, as well as of some pro-inflammatory cytokines involved, was evaluated. In this context, cannabisin F was recently found to act as a modulator of SIRT1, whose activation is considered beneficial in attenuating neuro-inflammation and damage due to oxidative stress [21]. Thus, both U-87 and HF cells were treated with three LnHS doses (2.5, 25 and 50 µg/mL) for 24 and 48 h, and the expression level of Sirt1, Sirt2, IL-6 and IL-10 was assessed at mRNA level. Indeed, no effects were found on these markers in both the cell types (Figure S22).

### *2.8. LnHS Blocks Autophagy While Inducing Apoptosis in U-87 Cells*

Autophagic cell death was hypothesized to be the mechanism through which cannabisin B exerts antiproliferative activity in human hepatocarcinoma HepG2 cells [11]. Autophagy is a cell catabolic program, and it is triggered following nutrient starvation, and requires for its initiation ULK kinases [52]. In particular, ULK-1 phosphorylates Beclin-1 is a multi-domain protein, which exerts a dual role in autophagy and apoptosis cellular processes [53]. LnHS treatment at dose levels equal to 5, 25 and 50 µg/mL, at 24 and 48 h exposure times, promoted the down-regulation of Beclin 1 and ULK-1 in U-87 cells, while in fibroblasts, both the enzymes were down-regulated only by 50 µg/mL dose (Figure 9). Simultaneously, it was found that Bcl-2 was also inhibited at the same incubation times and LnHS doses (Figure 9). Altogether, these results indicated a strong down-regulation of autophagy process in U-87 cells. These two processes are accompanied by reduction in AKT phosphorylation. E-cadherin expression was affected at 50 mL at 24 and 48 h in both cell types. This finding is in line with a potential double-edge behavior of LnHS, which, according to autophagy occurrence, at low doses appears to maintain E-cadherin expression, while, on the contrary, at the highest dose, LnHS may determine a cell-cell adhesion injury by the E-cadherin loss (Figure 9).

**Figure 9.** Levels of Bcl-2, Beclin-1, p-AKT, E-cadherin and ULK-1 in U-87 and HF cell types were detected using western blotting with respective antibodies. Graphical representation of pixel quantization of Bcl-2, Beclin-1, p-AKT, E-cadherin, and ULK-1 normalized to GAPDH (panels **A**, **C**). The intensities of signals were expressed as arbitrary units. \**p* < 0.05 vs. untreated cells (ctrl). Representative western blotting image of Bcl-2, Beclin-1, p-AKT, E-cadherin, ULK-1 and GAPDH are in panels **B** (U-87 cell type), and **D** (HF cell type).
