Fig 2: Expression of Ca2+ regulatory proteins. (A) Representative Western blot of sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA)2 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and (B) summary data of SERCA2a show no significant changes between transgenic (TG)/phospholamban (PLN) and TG/phospholamban knockout (PLNKO) groups. (C) Representative Western blot of phosphorylation PLN expression and phosphorylation at Ser16 (p-PLN Ser16) and GAPDH. Summary data show no significant changes in PLN abundance (D) and p-PLN Ser16 (E) between the non-transgenic (NTG)/PLN and TG/PLN groups. (F) Representative Western blot of PLN expression and phosphorylation at Thr17 (p-PLN Thr17) and GAPDH. Summary data show no significant changes in PLN expression (G) and p-PLN Thr17 (H) between the NTG/PLN and TG/PLN groups. (I) Representative Western blot of CaMKII abundance, phosphorylation (T287), and GAPDH. Summary data show no significant changes in CaMKII expression (J) but increased phosphorylation of CaMKII (K) in TG/PLN group. Data are presented as means ± SEM. N = 4–6. Differences among groups were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test. If only two groups were compared, unpaired t-test was used. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 3: Combined treatment of CaMKII and NK1R inhibitors synergistically suppressed expression of GSC markers. (A,B) U87MG- and U373MG-derived GSCs were treated with the indicated compounds for 12 h. Protein levels were detected by Western blot analysis using specific antibodies and were further quantified by densitometry. ß-actin levels were used as an internal control. Original Western blots are shown in Figure S1. SR, SR 140333; Apre, Aprepitant. * p < 0.05 vs. the compound alone.

Fig 4: Synaptic potentiation increase spine (synaptic) translocation of CaMKII. (A–D) Representative western blots and bar graphs demonstrating the distribution of CaMKII and T286 pCaMKIIa/T287 pCaMKIIß in synaptosomal and shaft-cytosol tissue extracts derived from WT hippocampus. (E) Representative immunoblots illustrating the comparative synaptic (spine) and shaft-cytosol expression of CaMKII and T286/T287 pCaMKIIa/ß. (F) Bar graph (T-test) illustrating the synaptic translocation of T286/T287 pCaMKIIa/ß post L-Glutamate treatment. (G) Schematic illustration of synaptic translocation of T286/T287 pCaMKIIa/ß. (1) NMDAR potentiation; (2) post-synaptic Ca++ transient activates SK2; (3) Ca++ transient facilitates T286/T287 CaMKII phosphorylation, and synaptic translocation; (4) increased SK2 activity facilitates removal of synaptic T286/T287 pCaMKIIa/ß; and (5) translocation of CaMKII from dendritic spine to shaft-cytosol. (B,D,F; ***p < 0.001).

Fig 5: Identification of synthetic lethal interaction between CaMKII and NK1R by RNA interference in GSCs. U87MG and U373MG cells were transfected with either NK1R siRNA or CaMKII? siRNA. Knockdown of (A) NK1R and (C) CaMKII? genes was confirmed by Western blot analysis. Original Western blots are shown in Figure S1. * p < 0.05 vs. the control siRNA. Following genetic knockdown, U87MG- and U373MG-derived GSCs were treated with the indicated concentrations of (B) CaMKII (HBC, Berbamine, KN93) inhibitors and (D) NK1R inhibitors (SR 140333, Aprepitant) for 7 days. Cell viability was measured using the CellTiter-Glo® luminescent assay system. The number of formed tumorspheres was counted under an optical microscope. (E) The effect of simultaneous knockdown of CaMKII? and NK1R genes on the cell viability and tumorsphere formation of both GSCs was also determined after incubation for 7 days. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the compound alone or the single gene knockdown.