CAV2 protein, human

The Kaplan–Meier plotter was used to evaluate the prognosis value of EHD2, CAV1, and CAV2 mRNA expressions alone and in combination (Györffy et al., 2010 (link)). To analyze the survival probability alone and in combinations of EHD2, CAV1, and CAV2 mRNA, the patient cohorts were split on the basis of trichotomization (T1 vs T3). The EHD2 mRNA (probe set 221870_at), CAV1 mRNA (probe set 212097_at), and CAV2 mRNA (probe set 203323_at) were entered into the KM Plotter patient cohort basal-like (PAM50 subtype) patient cohort (n=953) and the relapse-free survival (RFS) was determined. The mean expression of EHD2, CAV1 and CAV2 were used to perform survival analysis of high EHD2, CAV1 and CAV2 vs. low EHD2, CAV1 and CAV2. The hazard ratio (HR) with 95% confidence and log rank p-values were obtained from KM plotter. Gene correlation targeted analysis was performed to assess the correlation between EHD2, CAV1 and CAV2 mRNA expression in basal-like (PAM50 subtype) breast cancer patients (n=783) and TNBC (IHC) cohort (n=293) using bc-GenExMiner v4.5 platforms. TCGA and SCAN-B RNAseq dataset were used. Correlation heatmap, correlation plots and Pearson’s correlation coefficients computation were performed using bc-GenExMiner v4.5.

Although previous studies have validated the use of the CAV2-PRS construct using a range of actuators, including excitatory and inhibitory opsins, as well as potassium channels (Howorth et al., 2009a (link); Howorth et al., 2009b (link); Hickey et al., 2014 (link); Li et al., 2016 (link)), we used a separate cohort of rats to verify the functional effect of our DREADDs construct in vivo by quantifying changes in the expression of c-Fos, a recognized marker of neuronal activation, upon administration of DCZ. In order to obtain a high baseline of c-Fos activation, rats underwent a stress procedure. As shown in Figure 4—figure supplement 1B, rats were administered i.p. with either vehicle or DCZ (0.1 mg/kg) 45 min before being placed in a Plexiglas shock chamber equipped with stainless steel rods on the floor and a circuit generator connected to a scrambler and a timing unit. Rats received five shocks (0.5 s, 0.8 mA) randomly interspersed over 10 min (stress condition). As a control of c-Fos activation, we included in the experimental design animals administered with vehicle, but left untouched in their home cage (no stress condition). Rats were perfused 90 min after the procedure and coronal slices collected as described in the Histology section. For hM4Di/c-Fos colocalization analysis, sections were taken (see Figure 4—figure supplement 1—source data 1) from antero-posterior levels of the LC from –9.50 to –10.0 mm from Bregma (vehicle/no stress, n=2; vehicle-stress, n=4; DCZ-stress, n=4). Quantification of the percentage of LC hM4Di-positive cells (mCherry, red) that co-express c-Fos (Alexa 488, green) was performed by a trained observer blind to the experimental conditions.

Venom fractions were screened for neuroactivity at human (h) Na_V, Ca_V1, Ca_V2 and the α7 subtype of the human nicotinic acetylcholine receptor (nAChR-α7) as previously described (Cardoso et al., 2015 (link)). Briefly, SH-SY5Y cells were plated at 40,000 cells per well in 384-well flat clear-bottom black plates (Corning, NY, United States) and cultured at 37°C in a humidified 5% CO₂ incubator for 48 h. Cells were loaded with 20 μL per well Calcium 4 dye (Molecular Devices) reconstituted in assay buffer containing (in mM) 140 NaCl, 11.5 glucose, 5.9 KCl, 1.4 MgCl₂, 1.2 NaH₂PO₄, 5 NaHCO₃, 1.8 CaCl₂ and 10 HEPES pH 7.4 and incubated for 30 min at 37°C in a humidified 5% CO₂ incubator. For the hCa_V1 assay, the dye was supplemented with 1 μM ω-conotoxin-CVIF (CVIF) to inhibit Ca_V2, and in the hCav2 assay the dye was supplemented with 10 μM nifedipine to inhibit Ca_V1. For the nAChR-α7 assay, the dye was supplemented with PNU-120596 (Sigma-Aldrich), a positive allosteric modulator of nAChR-α7. Venom fractions were assayed in singleton for each ion channel tested. Fluorescence responses were recorded using excitation at 470–495 nm and emission at 515–575 nm for 10 s to set the baseline, then 300 s after addition of 10% venom fraction serial diluted at 1, 1:10, and 1:100, and for a further 300 s after addition of 50 μM veratridine for hNa_V, 90 mM KCl and 5 mM CaCl₂ for hCa_V, and 30 μM choline for nAChR-α7.