To prepare tissue sections of 40 melanoma and 18 nevus from patients for immunohistochemistry, sections from each patient were deparaffinized with xylene (3×5 min) followed by treatment with serial dilutions of ethanol (100, 100, 95 and 95%, 10 min each) and by two changes of ddH₂O. Antigen unmasking was conducted by boiling the slides (95–99°C) for 10 min. Sections were rinsed three times with ddH₂O, immersed in 3% H₂O₂ for 20 min, washed twice with ddH₂O and once with TBS-T (TBS, 0.1% Tween-20) and blocked for 1 h with blocking solution (5% normal goat serum in TBS-T). Antibody of RGS1 (cat. no. PA5-29579; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was diluted according to the manufacturer instructions and the sections were incubated overnight at 4°C. Then, the sections were washed three times, 5 min each, with TBS-T and incubated for 1 h at room temperature with Signal Stain Boost (Cell Signaling Technology, Inc., Danvers, MA, USA). The negative control used for immunohistochemistry included the use of phosphate-buffered saline instead of the primary antibody. Finally, tissues were dehydrated. Images were captured with an Olympus microscope (Olympus DP80; Olympus Corp., Tokyo, Japan). All images were captured and processed using identical settings. During evaluation, for each sample, five horizons were randomly chosen to calculate the average positive ratio.

The immunostaining scores were calculated using an approved standard (13). The regions of most uniform staining were scored for each tissue array core, which included the entire midportion of the core, to exclude any ‘edge effect’ of increased staining. Expression of RGS1 protein was graded combining two factors. One factor was the staining intensity: 0, no staining; 1, weak staining; 2, moderate staining; and 3, intense staining. The other factor included the proportion of positive-staining cells. In all target cells of one region, the proportion of ‘no staining’ cells was considered ‘A’, and ‘weak staining’ was ‘B’, and by this analogy, the final score of this region was equal to: (0 × A) + (1 × B) + (2 × C) + (3 × D). This score was categorized into 3 grades: ≤1.0 (+); >1.0 but ≤1.5 (++); >1.5 (+++). The arrays were scored by a pathologist blinded to the identity of the patients, and each score was replicated by a separate, independent scoring trial by the study pathologist. For the melanoma patients, we divided them into high and low expression groups, and were followed up to determine their disease-related survival, and analyzed it using the Kaplan-Meier curve.

The A375 human melanoma cell line (Cell Bank in Shanghai, Chinese Academy of Sciences), RGS1-knockdown (KD) A375 cells and RGS1-overexpression cells were incubated at 37°C in a humidified 5% CO₂ enriched atmosphere. These cells were cultured with Dulbecco's modified Eagle's medium with high glucose (DMEM; Gibco; Thermo Fisher Scientific, Inc.), supplemented with 10% heat inactivated fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 1% fungi zone (Invitrogen; Thermo Fisher Scientific, Inc.), and 1% penicillin, twice weekly, at every change in media, for normal growth by phase contrast microscopy. The cultures were grown to confluence and passaged by treatment with 0.25% trypsin-EDTA (Gibco; Thermo Fisher Scientific, Inc.) at 37°C and washed in 7 ml DMEM media before being centrifuged at 120 × g for 10 min to form a pellet. The lentivirus base RGS1 overexpression system and RGS1 knockdown system (sequence of shRGS1, 5′-GATCCGCCCTGTAAAGCAGAAGAGATTTCAAGAGAATCTCTTCTGCTTTACAGGGCTTTTTTG-3′) were purchased from Hanyin Biotechology (Shanghai, China) and used to infect cells as described in a previous study (21).

Cells were seeded into 96-well plates (Corning Inc., Corning, NY, USA) at a density of 2×10³ cells/well. Cell viability was assessed using Cell Counting Kit-8 assay (CCK-8; Dojindo Molecular Technologies, Inc., Rockville, MD, USA). The absorbance of each well was read on a spectrophotometer (Thermo Fisher Scientific, Inc.) at 450 nm (OD450). Three independent experiments were performed in quintuplicate.

For the determination of cell invasion, Transwell chambers were coated with 30 µl Matrigel (Merck KGaA, Darmstadt, Germany), and incubated at 37°C for 40 min. In the Transwell assays with and without Matrigel, the cells were trypsinized and then seeded in chambers at a density of 1×10⁴ cells/well at 48 h after transfection. The cells were then cultured in DMEM with 2% serum. Meanwhile 600 µl of medium supplemented with 10% FBS was injected into the lower chambers. After cell harvest, the inserts were fixed and stained in a dye solution containing 1% crystal violet and 20% methanol. Cells adhering to the lower membrane of the inserts were imaged with a microscope (Olympus DP80; Olympus Corp.). Six views are randomly picked for each well.

A375 cells in the three groups were measured by FACS. Annexin V-PE/7-AAD (cat# 559763; eBioscience; Thermo Fisher Scientific, Inc.) double staining was used to identify the apoptosis rate of the A375 cells. The cells (1×10⁶ cells/ml) were harvested, washed twice with 4 centigrade PBS, and incubated for 15 min in 1X Annexin V binding buffer containing 10 µl 7-AAD and 5 µl Annexin V-PE. Finally, apoptosis was detected by FACS and analyzed using FlowJo software (Tree Star Inc., Ashland, OR, USA). Experiments were carried out in triplicate.

The FLAG-tag RGS1 and HA-Gαs plasmids were instantly transferred into the 293T cells in the three groups and named 293T-RGS1, 293T-Gαs-GDP, 293T-Gαs-GDP-AlF₄⁻, which was without tetrafluoroaluminate (AlF₄). The cells were collected and lysed with 200 µl cold RIPA buffer (RIPA buffer:PMSF = 100:1; Beyotime Institute of Biotechnology, Haimen, China) for 30 min, followed with centrifugation at 13,200 × g, at 4°C for 10 min. HA-G proteins, (Gαs-GDP, Gαs-GDP-AlF₄⁻) were added into the cell lysis supernatant liquor separately and mixed. Each blend was divided into ‘total’ and ‘co-IP’ parts. The protein A agarose was prepared and washed using Lysis buffer B (pH 7.6) 4 times, 2,000 g. This was diluted by half with Lysis buffer B (pH 7.6). Protein A agarose was added into each ‘co-IP’ portion and was agitated slowly at room temperature for 2 h. Then, 1 µg of the Gαs flag antibody was added into the ‘co-IP’ parts, and swayed slowly at 4°C overnight. Then centrifugation was carried out instantaneously at 3,000 rpm, and the precipitate was collected and washed with cold Lysis buffer B (pH 7.6) 3 times. The samples were boiled for 5 min at 100°C, for immunoprecipitation.

Spontaneously, a group of absolute exogenous co-IP was prepared. The FLAG-tag RGS1 and HA-Gαs proteins were expressed and purified, and the binding experiment procedure was carried out as in Watson et al (1). The reaction buffer consisted of a solution of 50 mM Tris-HCI, pH 8.0, 0.1 M NaCl, 1 mM MgS0₄, 20 mM imidazole, 0.025% polyoxyethylene 10-lauryl ether (C₁₂E₁₀), 10 mM β-mercaptoethanol and 10% glycerol. Protein immunoprecipitation (IP) was performed, respectively using Chromatin ChIP Kits (EMD Millipore, Billerica, MA, USA). Antibody of HA-Gαs and FLAG-tag RGS1 were used.

The HA-Gαi and HA-Gαs were purified and extracted using the method in the binding process. Then this was proceeded according to the ATPase/GTPase Activity Assay kit (MAK113; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) instructions. First, the phosphate standards were set as indicated in the kit instructions. Second, a series of dilutions of enzyme were performed in assay buffer. The sample reactions and the control well were set up according to the scheme. The reaction was incubated for the desired period of time (in our research, 1, 3, 5 and 10 min) at room temperature. Reagent (200 ml) was added to each well and incubation was carried out for an additional 30 min at room temperature to terminate the enzyme reaction and generate the colorimetric product separately. Absorbance at 600–660 nm [maximum absorbance at 620 nm (A620)] was read. We calculated the change in absorbance values (DA620) for the samples by subtracting the A620 of the control well (A620) control from the A620 of the sample well (A620) sample. The concentration (mM) of free phosphate [P_i] was computed in the sample from the standard curve. The formula was: Enzyme activity (units/l) = [P_i] (mM) × 40 ml ÷ [10 µl × reaction time (min)]. One unit is the amount of enzyme that catalyzes the production of 1 mmol of free phosphate per minute under the assay conditions.

Protein was extracted from the cultured cells and dissolved, homogenized, and quantified using the BCA Protein Assay kit (Pierce; Thermo Fisher Scientific, Inc.). Sample buffer was then added and the prepared samples were stored at −80°C after boiling. During the western blotting, protein samples were subjected to SDS-PAGE and transferred to polyvinylidene difluoride membranes and placed in 25 mM Tris and 192 mM glycine. The membranes were blocked with 5% non-fat dry milk in PBS, 0.05% Tween-20 and probed with P-AKT (CY6569; Abways, Shanghai, China), P-ERK (CY5277; Abways), AKT (CY5551; Abways), ERK (CY5487; Abways), Gas (ab83735; Abcam, Cambridge, UK according to the manufacturers' instructions. Blots were developed with ECL reagent (Thermo Fisher Scientific, Inc.) and exposed using the FC2 Image Station (Alpha, Bellingham, WA, USA).

All of the statistical analyses were performed by SPSS (version 19.0) software (IBM Corp., Armonk, NY, USA) and all of the data are represented as the mean ± standard deviations (SD). Student's t-tests and analysis of variance (ANOVA) were performed to compare the differences between groups. P<0.05 was considered to be indicative of statistical significance. Three and more independent experiments were performed in each group.

RGS1 expression has been detected throughout the cell (22). In the present study, we analyzed RGS1 expression by immunohistochemical staining in nevus and melanoma tissues. As shown in Fig. 1A, the RGS1 antibody staining intensity was darker and stained more target cells in the melanoma samples when compared to the nevus samples (Fig. 1A). Compared with the nevus tissue, RGS1 expression was significantly upregulated in the melanoma tissues (Fig. 1B, P=0.0023). Furthermore, we collected the DSS data and performed the Kaplan-Meier estimation. The results demonstrated that high RGS1 expression was inversely correlated with overall survival (Fig. 1C, P<0.0001). Collectively, RGS1 is highly expressed in melanoma and is inversely associated with DSS.

To study the function of RGS1 in the melanoma cell line A375, the plasmid-based RGS1 overexpression and knockdown systems (shRGS1) were used for transfection. A western blot assay showed that RGS1 expression was efficiently downregulated in the shRGS1-transfected A375 cells (Fig. 2A), while expression was significantly upregulated in the RGS1-transfected overexpressing A375 cells (Fig. 2B). Next, cell viability in the shRGS1-transfected, RGS1-transfected, and negative control (NC)-transfected A375 cells were determined using the CCK-8 assay. We found that knockdown of RGS1 significantly inhibited A375 cell proliferation (Fig. 2C), and overexpression of RGS1 significantly promoted A375 cell proliferation (Fig. 2D). Furthermore, a Matrigel-based invasion assay indicated that knockdown of RGS1 significantly inhibited A375 cell invasion (Fig. 2E) and overexpression of RGS1 significantly promoted A375 cell invasion (Fig. 2F). These results demonstrated the stimulatory role of RGS1 in melanoma proliferation and invasion.

Previous research has demonstrated that Gαs is a tumor suppressor in neural and epidermal progenitor-derived malignancies (18,19). In the present study, co-immunoprecipitation (Co-IP) was performed to determine the potential for direct targeting of RGS1 to Gαs. As shown in Fig. 3A, RGS1 was found to directly target Gαs in the RGS1-overexpressing A375 cells. Next, the binding of RGS1 protein and Gαs was assessed in the following three types of exogenous environments: Gαs-GDP, Gαs-GDP-AlF₄⁻, and nothing added to the buffer. The result indicated that RGS1 did not bind to Gαs in any state (Fig. 3B). In the circumstance of the 293T cell lysis with added GDP or GDP+AlF₄⁻, RGS1 binding to Gαs was detected in both environments (Fig. 3B). Binding was also detected in the endogenous experiment performed with A375 cells (Fig. 3B). The above results demonstrated the direct targeting of RGS1 and Gαs in the endogenous environment.

GTPase activity was evaluated using a specific kit testing GTPase activity in the exogenous environment (no other molecules added). The result showed that the GTPase activity was not significantly elevated for Gαs after the combination with RGS1 (Fig. 3C). This indicated that the binding of RGS1 to Gαs might not accelerate the GTP hydrolysis process (Fig. 3C). Further western blotting demonstrated the stimulatory role of RGS1 on AKT and ERK activation in A375 cells (Fig. 3D). Collectively, the above results suggest that RGS1-induced AKT and ERK phosphorylation is dependent on the non-GAP function of Gαs.

To confirm that Gαs regulates the RGS1-mediated promotion of AKT and ERK activation involved in A375 cell proliferation and invasion, Gαs overexpression and knockdown systems were utilized. As shown in Fig. 3D, the increased expression of RGS1 significantly promoted AKT and ERK phosphorylation. Using the Gαs overexpression system, the phosphorylation of AKT and ERK was reduced (Fig. 4A). In contrast to RGS1 overexpression, knockdown of RGS1 significantly decreased AKT and ERK phosphorylation (Fig. 3D). Using the knockdown system to reduce the expression of Gαs, the phosphorylation of AKT and ERK was enhanced (Fig. 4A). The above results demonstrated that Gαs plays a critical role during RGS1-mediated promotion of AKT and ERK phosphorylation in melanoma.

Our CCK-8 assay also demonstrated the stimulatory role of RGS1 in melanoma proliferation (Fig. 2C), but increased expression of Gαs reduced the cell proliferation (Fig. 4B). In contrast, knockdown of Gαs reversed the shRGS1-mediated inhibition of proliferation (Figs. 2D and 4B). Furthermore, RGS1 was found to function as a promoter of melanoma invasion (Fig. 2E and F). Overexpression of Gαs abrogated RGS1-mediated promotion of A375 invasion (Fig. 4C), while knockdown of Gαs promoted A375 invasion of the shRGS1-transfected cells (Fig. 4C). Collectively, the above results suggest that Gαs plays a critical role during RGS1-mediated promotion of melanoma proliferation and migration.