Fig 2: SERCA2 was correlated with the malignance of human cervical cancer. The protein level of SERCA2 in the tissue arrays of normal cervix tissues (normal), cancer adjacent normal cervix tissue (NAT), malignant cervical cancer tissues of different clinical stages (carcinoma, stages I–III) and lymph node metastasis from uterine cervix (metastasis) was detected by immunohistochemistry (IHC). Then, IHC scores were evaluated as shown in Supplementary Figure S10, and the variance was analyzed with one-way ANOVA. (a) IHC scores for normal tissues, NAT, carcinoma and metastasis. (b) IHC scores for different clinical stages of human cervical cancer. (c) Representative images of normal tissues, NAT, different stages of carcinomas and lymph metastasis. Scale bars, 40 µm. (d) Illustration for the mechanism underlying the anticervical cancer effects of SBF-1. When cells are exposed to SBF-1, SERCA activity is suppressed, inducing depletion of ER Ca2+ and increase of cytosolic Ca2+, which disturbs ER folding capacity and increases unfolded and misfolded proteins, activating the signaling pathways of UPR response and causing ER stress-associated cell death. ATF6N, N terminal of ATF6a

Fig 3: Effect of prolonged exposure to insulin on [Ca2+]i, total calcium current (ICa2+), and SERCA2 protein. (A and B) INS1E ß-cells cultured on glass coverslips were loaded with fura-2–PE3 (2 µmol/L) and Mag–fluo-4-AM (1 µM), respectively (Molecular Probes, Eugene, OR). Pluronic acid (0.01%) was added to the medium and cells were incubated for 30 min at 37°C. [Ca2+]i was measured using an Olympus IX-50 inverted epifluorescence microscope (Olympus, Tokyo, Japan). Absolute [Ca2+]i was determined from R of Ca2+-bound fura-2 (excited at 340 nm) to unbound fura-2 (excited at 380 nM). The Ca2+ levels were determined using a standard equation for calibration. Rmax and Rmin were obtained by adding ionomycin (10 µM) or EGTA (1 mM), respectively, to fura-2–loaded ß-cells at the end of each experiment. [Ca2+]i was obtained using an excitation wavelength of 488 nm and an emission band-pass filter of 515/15 nm. Means ± SEM are from four independent experiments. The ICa2+ (Ba2+) from control and insulin pretreated cells for 48 h were evoked by a 100 msec step depolarization under whole-cell voltage clamp configuration. *P < 0.05, chronic insulin (48 h) compared with chronic insulin (24 h); ***P < 0.0005, 15 mM glucose (10 and 60 min control) compared with 5 mM basal; ###P < 0.0005, chronic insulin compared with control nontreated cells. (C) Representative two typical traces at the maximal current (-20 mV) from control (gray) and insulin-pretreated cells (1 µM, 48 h) (black). (D) Average integrated current (C)–voltage (V) relationships. Data represent means ± SEM from control (?, n = 18) or insulin-treated cells (?, n=6). (E) Quantification of the magnitude of the peak of the calcium current and the end of the voltage step (95 msec) of the maximal current (determined by the correspondent Influx-Voltage relationship for each cell tested, usually about -20 mV). *P < 0.05, compared with control nontreated cells. (F) Islets were treated with the indicated concentrations of insulin for 24 h, and then the expression of the ER-Ca2+-ATPase SERCA2 was analyzed by Western blot using a specific antibody in islet whole-cell lysates. A representative autoradiogram of four independent experiments is shown.

Fig 4: Protein levels of SERCA2a at the rAd-SERCA2a MOI of 2, 4 and 10 pfu/cell. (A) Western blot bands corresponding to SERCA2a. (B) Bar graph showing the IODs of the SERCA2a bands from (A), quantified using ImageJ software, and normalized to GAPDH. The data are representative of three independent experiments (mean ± SD). (C) Cell viability at the rAd-SERCA2a MOI of 2 pfu/cell. The data represent four independent experiments (mean ± SD). *P<0.05 vs. control. SERCA2a, sarco/endoplasmic reticulum Ca2+-ATPase; MOI, multiplicity of infection; IOD, integrated optical density.

Fig 5: SERCA2a expression is reduced in SMA hearts. a qRT-PCR analysis of SERCA2 mRNA. Expression levels in SMN-deficient cardiomyocytes are represented relative to unaffected controls (n = 3). b Western blot analysis of SERCA2a protein levels. Quantification of SERCA protein level in SMA cells relative to controls at right. c qRT-PCR analysis of SERCA2a and SERCA2b mRNA. Expression levels in SMN-deficient (SMA) cardiomyocytes are represented relative to unaffected controls. Statistical analysis: p values shown for one-way ANOVA, Tukey’s post hoc test, n = 7