Fig 2: A Mann Whitney U test using PCC and MOC co-localisation values of TRPM3 with actin on NK cells of ME/CFS patients and HC. B Mann Whitney U test using PCC and MOC co-localisation values of TRPM3 with PIP2 on NK cells of ME/CFS patients and HC. Co-localisation values obtained following IL-2 stimulation (20 IU) (control) and pharmacological treatment using PregS (30 µM) and PregS + Ononetin (3 µM). Bar graphs represent correlation between target antigens using PCC represent degree of overlap between target antigens using MOC. Data presented as mean ± SEM and *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. HC, healthy control; ME/CFS, Myalgic encephalomyelitis/chronic fatigue syndrome; PregS, pregnenolone sulfate; Ono, ononetin

Fig 3: Co-localisation and immunofluorescent images of TRPM3 with PIP2 in NK cells of HC. A Example of immunostaining of PIP2 (red), nucleus (DAPI, blue), TRPM3 (green)) in an NK cell under control IL-2 conditions. Images taken using Nikon A1R + confocal microscopy. B Bar graphs represent correlation between target antigens using Pearson’s correlation coefficient and the degree of overlap between target antigens using Mander’s overlap coefficient. Co-localisation coefficients K1 (TRPM3) and K2 (PIP2) were used to determine contribution to co-localisation. Number of cells analysed are included within bar graphs: Control n = 287; PregS n = 292; and PregS + Ono n = 237. Data presented as mean ± SEM and *p < 0.05; **p < 0.01. TRPM3, transient receptor potential Melastatin; PIP2, phosphatidylinositol 4,5-bisphosphate; PregS, pregnenolone sulfate; Ono, ononetin; IL-2, interleukin 2

Fig 4: Heat stimulation stabilises TRPM3 in the DRG membrane.a The diagram shows the experimental procedure. The hindpaws of mice were soaked in a 43 °C water bath for 30 s, and the mice were placed at RT for 2, 5, 10, 20 or 40 min. Then, behavioural, co-IP and membrane protein immunoblotting experiments were performed. b, c ATF4/TRPM3 interactions (b) and TRPM3 membrane expression (c) in mice were evaluated at different time points after heat stimulation. The experiment was repeated four times in b. n = 6 mice per group in c. F(5,18) = 20.17, P = 0.0007 in 2 min, P < 0.0001 in 5 min, P < 0.0001 in 10 min, P = 0.0044 in 20 min in b. F(5,30) = 10.59, P = 0.0277 in 2 min, P = 0.0022 in 5 min, P < 0.0001 in 10 min, P = 0.0027 in 20 min in c. *P < 0.05, **P < 0.01 versus the RT group. d The diagram shows the experimental procedure. Isolated DRG neurons were placed in a 43 °C water bath for 30 s and placed at RT for 2, 5, 10, 20 or 40 min. The neurons were then lysed to detect the membrane abundance of TRPM3. e TRPM3 membrane expression in isolated DRG neurons was evaluated at different time points after direct heat stimulation. n = 3. F(5,12) = 12.3, P = 0.0358 in 2 min, P = 0.0100 in 5 min, P < 0.0001 in 10 min, P = 0.0085 in 20 min. *P < 0.05, **P < 0.01 versus the RT group. f–h Naïve (f), ATF4 siRNA-injected (g) and Atf4+/- (h) mice were subjected to the Hargreaves test at different time points after heat stimulation. n = 12 mice per group in f, g. n = 6 mice per group in h. F(5,66) = 14.36, P = 0.0004 in 2 min, P < 0.0001 in 5 min, P < 0.0001 in 10 min, P = 0.0057 in 20 min. **P < 0.01 versus RT group. One-way ANOVA followed by Tukey’s multiple comparisons test. The error bars indicate the SEMs.

Fig 5: Co-localisation and immunofluorescent images of TRPM3 with actin in NK cells of ME/CFS patients. A Example of immunostaining of actin (phalloidin, red), nucleus (DAPI, blue), TRPM3 (green) in NK cell under control IL-2 condition. Cells were stimulated overnight with IL-2 (20 IU) (control) and treated with PregS (30 µM) and ononetin (3 µM). Images taken using Nikon A1R + confocal microscopy. B Bar graphs represent correlation between target antigens using Pearson’s correlation coefficient and the degree of overlap between target antigens using Mander’s overlap coefficient. Co-localisation coefficients K1 (TRPM3) and K2 (actin) were used to determine contribution to co-localisation. Number of cells analysed are included within bar graphs: Control n = 185; PregS n = 193; and PregS + Ono n = 197. Data presented as mean ± SEM. TRPM3, transient receptor potential Melastatin; PregS, pregnenolone sulfate; Ono, ononetin; IL-2, interleukin 2