*4.8. RT-PCR*

Total RNA was extracted from cell lysates using the Nucleo Spin® RNA kit (Macherey-Nagel, Bethlehem, PA, USA) according to the manufacturer's instructions. For first-strand cDNA synthesis, 1 μg of total RNA from each sample was reverse-transcribed. Primers were designed based on the mouse GenBank sequences for HO-1, IL-10, IL-6, IL1-β, COX-2, iNOS, TNF-α, SOD2, NLRP3, and Arg1, and are reported in Table 2. Ribosomal protein large P1 (RPLP-1) was co-amplified as the reference [18]. For each target gene, the relative amount of mRNA in the samples was calculated as the ratio to RPLP-1 mRNA [19].

#### **Table 2.** Primer sequences.


#### *4.9. Real-Time PCR for mir-146b and mir-223 Expression Analysis*

For miRNA expression analysis, the total RNA was extracted from cell culture media by using TRIzol (Invitrogen, Carlsbad, CA, USA). Reverse-transcription of RNA was performed using the miRCURY LNA RT Kit according to the manufacturer's instructions (Qiagen). qRT-PCR assays were carried out in a Rotor-Gene®Q PCR System (Qiagen) using a miRCURY LNA SYBR® Green PCR Kit and miRCURY LNA miRNA PCR Assay according to the manufacturer's instructions (Qiagen). Briefly, each reaction was performed in a final volume of 10 μL containing two μL of the cDNA, a master mix containing 5 μL of 2× miRCURY SYBR Green PCR Master Mix, 1 μL of miRCURY LNA miRNA PCR Assay, and RNase-free water. The amplification profile was: PCR initial heat activation at 95 ◦C for 2 min, followed by 40 cycles of denaturation at 95 ◦C for 10 s and combined annealing/extension at 56 ◦C for 60 s. The expression of mir-146b and mir-223 was normalized to RNU6B and calculated as 2-ΔΔCt.

### *4.10. Dot-Blotting for COX-2 Protein Expression*

Cells were lysed in a 300 μL radioimmunoprecipitation assay buffer (RIPA) (Sigma-Aldrich, Milan, Italy). Total protein content was measured by using the Bio-Rad DC protein assay kit (Bio-Rad, Milan, Italy). Equal aliquots (30 μg) of proteins were applied to a nitrocellulose membrane (Millipore, Burlington, VT, USA) and allowed to dry for 30 min at RT. After blocking with 6% nonfat dry milk for 1 h at RT, the membranes were incubated overnight at RT with the Rabbit anti-COX-2 polyclonal antibody (1:200) (Cayman Chemical, MI, USA, catalog number 160126) followed by incubation with anti-rabbit IgG horseradish peroxidase-linked antibody (Cell Signaling, Danvers, MA, USA), 1:4000 for 1 h at RT. Chemiluminescence was developed by using the Immobilon Horseradish

Peroxidase Substrate (Merck Millipore, Darmstadt, Germany), and immunoreactive spots were quantified using Quantity-One software (Bio-Rad Laboratories S.r.l., Milan, Italy).

#### *4.11. Immunocytochemistry for COX-2 Protein Expression*

RAW 264.7 cells were grown in Poly-D-lysine-coated glass dishes for 24 h then treated with LPS and compounds and extracts tested as described above. After treatment, cells were fixed with cold 4% (*w/v*) paraformaldehyde for 20 min, washed in PBS, and then incubated for 15 min with 0.1% (*w/v*) TritonX-100 and 3% Bovine Serum Albumin (BSA). Thereafter, the cells were incubated with Rabbit anti-COX-2 polyclonal antibody (1:200) (Cayman, Ann Arbor, MI, USA, catalog number 160126) at 4 ◦C overnight, followed by the fluorescent secondary antibody: AlexaFluor 586 goat anti-rabbit (1:333) (Invitrogen, Carlsbad, CA, USA). Nuclei were also counterstained with DAPI dye (Sigma-Aldrich, Milan, Italy). Microscopic analysis was performed with an Olympus BX63 microscope equipped with a Metal Halide Lamp (Prior Scientific Instruments Ltd., Cambridge, UK) and a digital camera, Olympus XM 10 (Olympus, Milan, Italy).

#### *4.12. Statistical Analysis*

Data were analyzed by ANOVA test and Dunnett's Multiple Comparison test and expressed as the means ± standard error (SEM) of four independent experiments. All analyses were carried out using GraphPad Prism 7.0 (GraphPad Software, San Diego, CA, USA). *p* values less than 0.05 were considered significant.

**Author Contributions:** Conceptualization, all authors; methodology, E.B., M.D., L.C. and C.L.; validation, E.B., M.D. and L.C.; formal analysis, M.D., E.B. and C.L.; investigation, M.D., L.C., A.N. and L.B.D.S.N.; resources, C.L., M.R.T., N.B. and L.R.; writing—original draft preparation E.B., A.N. and C.L.; writing—review and editing, all authors; supervision, C.L.; funding acquisition, C.L. and M.R.T. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was co-funded by Ente Cassa di Risparmio Firenze grant numbers: 2015.0919 and 2018.1002 and by Regione Toscana (Italy) under Par-FAS 2007–2013 Projects (Centro di Competenza VALORE). A.N. holds a fellowship funded by the POR FSE 2014–2020—Progetto Strategico "STREAMING" sottoprogetto PhotoWING (grant number: UNIFI\_FSE2017, Regione Toscana, Italy).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data that support the findings of this study are available on request from the corresponding author C.L. (cristina.luceri@unifi.it).

**Acknowledgments:** The authors wish to thank Massimo D'Ottavio for fucoxanthin determination.

**Conflicts of Interest:** *T. lutea* F&M-M36 belongs to the Culture Collection F&M S.r.l. culture collection, where M.R.T. and L.R. have a financial interest. The other authors have no conflicts of interest.

#### **References**

