Fig 2: Apigenin and galangin reduced TRPV1 expression and its phosphorylation in blue light-irradiated HaCaT cells. (A,B) TRPV1 expression (A), and its phosphorylation levels (B) were determined by Western blotting. (A) Cells were incubated with apigenin (10, 20, 30 μM) and galangin (1, 10, 20 μM) for 24 h and were then irradiated with blue light (30 min, 76 W/m2). After 24 h incubation, the cells were subjected to the same process twice and were finally subjected to Western blot analysis. (B) Phosphorylation levels of TRPV1 were determined by Western blotting at 30-, 60-, and 90-min incubation after pretreatment with apigenin (30 μM) or galangin (20 μM) for 1 h and subsequent blue light-irradiation (30 min, 76 W/m2). The total proteins were extracted from the cells immediately after experimental conditions, and β-actin was used as a loading control. (C,D) Apigenin and galangin inhibited blue light-induced calcium influx in HaCaT cells. (C) Ca2+ influx changes of blue light and with apigenin (10, 20, 30 μM) pretreatment, (D) with galangin (1, 10, 20 μM) pretreatment by Fluo-4 NW assay. Cells were pretreated with apigenin (10, 20, 30 μM) and galangin (1, 10, 20 μM) for 1 h and irradiated with blue light (10 min, 76 W/m2). After the irradiation, fluorescence intensities were measured immediately over a certain period.

Fig 3: Action mechanism of porphyra 334 in the AhR and TRPV1-mediated signaling. Porphyra 334 inhibits UPM effects by inhibiting both AhR and TRPV1 signaling. Red line: action step of porphyra 334. A red arrow pointing up indicates activation, and a red arrow pointing down indicates inhibition.

Fig 4: Effect of porphyra 334 on TRPV1 signaling induced by UPM. (a) In experiments in which HaCaT cells were treated with UPM (50 μg/mL) for 90 min, porphyra 334 (100 μm) was co-treated for 30 min, 60 min, and 90 min, respectively. After 90 min, the cells were harvested and the phosphorylated form of TRPV1 was detected using Western blot analysis. (b) The Fluo-4 NW assay was used to assess calcium influx into HaCaT cells. Calcium influx was measured after 20 min incubation of treated samples. (## p < 0.01: vs. control, ### p < 0.001: vs. control, ** p < 0.01: vs. UPM, *** p < 0.001: vs. UPM, NS: not significant vs. control).

Fig 5: Tumor-infiltrating neurons become transcriptionally and functionally altered.Trigeminal (TGM) ganglia from tumor bearing (red) or non-tumor bearing (blue) mice were analyzed by (A) quantitative PCR array. N=4 TGM/group from n=4 mice/group; n=4 arrays/group. Ct values for each gene normalized to that of a housekeeping gene. Statistical analysis by multiple students t-test. *, p<0.05; **, p<0.01; ***, p<0.001, ****, p<0.00001. Data are expressed as means ± standard deviation. (B) Representative fluorescent images of dissociated neurons from the ipsilateral or contralateral TGM ganglia from a MOC2–7 tumor-bearing mouse. N=6 of each ganglia harvested from 6 tumor-bearing animals with n= 4–8 neurons analyzed/ganglia. The color palette reflects the strength of the calcium signal. Images are taken pre- and post-treatment with capsaicin. (C) Graph of average change in fluorescence for all neurons analyzed in each group (ipsilateral and contralateral). Statistical analysis by student’s t-test. Arrow, time of capsaicin (300 nM) stimulation. Graph of average peak of the curve (D) and area under the curve (E) from panel C. Statistical analysis by student’s t-test. **, p<0.01. (F) Quantification of Ca+2 responses to capsaicin (300 nM) from non-tumoral (Control) and tumor-infiltrating, tracer+ (Tumor-infiltrating) neurons. N= 4–8 neurons/group from n=4 control and n=7 tumor-bearing mice. Statistical analysis by paired students t-test, *, p<0.05. Data expressed as means ± SEM. (G) Representative western blot of trigeminal ganglia from non-tumor (N) or MOC2–7 tumor-bearing (T) animals for phosphorylated TRPV1 (pTRPV1, Ser502, Ser800), σ1 receptor (σ1R) and β-actin (loading control). Densitometric quantification of western blots for pTRPV1 (H) and σ1R (I). Statistical analysis by student’s t-test. *, p< 0.05. Data are expressed as means ± SEM. (n=3–4 mice/group).