Fig 2: Knockdown or inhibition of GRK2 induces RIPK1/RIPK3-dependent necroptosis in HTR8/SVneo cells. (A) HTR8/SVneo cells are transfected with non-targeted siRNA (NC-siRNA) or siRNA targeted to GRK2 (GRK2-siRNA) for 48 h. Representative western blot analysis of cleaved-caspase 3, RIPK1, RIPK 3, phospho-MLKL (p-MLKL; S358), and ß-actin (loading control). (B) Cell viability is assessed using CCK8 assay after transfection with siRNA for 48 h. Values are expressed as Mean ± SEM. (C) Pharmacologic inhibition of GRK2 in HTR8/SVneo cells by GSK180736A at different concentrations (0.5, 1, 2.5, and 5 µM) for 12 h. Representative western blot analysis of cleaved-caspase 3, RIPK1, RIPK 3, phospho-MLKL (p-MLKL; S358), and ß-actin (loading control). (D) Cell viability is assessed using CCK8 assay after administration with GSK180736A for 12 h. Values are expressed as Mean ± SEM. (E) Flow cytometry with Annexin V/PI-combined staining shows early apoptosis (Ann V + /PI-), and late apoptosis/necrosis (Ann V + /PI +). The rate of late apoptotic/necrotic cells (Ann V + /PI +) is increased significantly after knockdown by GRK2-siRNA, ***P < 0.001. (F) To re-validate the necrosis rate induced by siRNA treatment, loss of mitochondrial membrane potential (??m) is measured by flow cytometry using JC-1 mitochondrial probes. (G) HTR8/SVneo cells transfected with GRK2-siRNA were examined for nuclear envelope integrity using Lamin B1 antibodies (red; nuclear envelope), or Hoechst (blue; nuclear context), at the indicated time points. Arrows indicate envelope breaches (Lamin B1 staining) and nuclear DNA leakage (Hoechst). Scale bar, 10 µM. (H) TEM images of EVTs treated with/without GSK180736A (cells from villi explants). EVTs on matrigel were necrotic after administration with GSK180736A. From upper to bottom: 8,000×, 20,000× magnification. (I,J) HTR8/SVneo cells treated with/without GSK180736A were collected and subjected to coimmunoprecipitation (Co-IP) with anti-RIPK1 antibody and anti-RIPK3 antibody. NS, not significant; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus blank group; ##P < 0.01, ####P < 0.0001 versus NC-siRNA group.

Fig 3: GRK knockout cells are a viable cellular platform to assess various features of b2AR–ß-arrestin complex formation.a Analysis of ß-arrestin2 (ßarr2) conformational changes. ?Q-GRK or Control cells were transfected with an untagged b2AR expression construct and the ßarr2-F5-NanoLuc conformational change biosensor (more details on the intramolecular BRET sensor will be published elsewhere), in the absence or presence of GRKs as noted and stimulated with isoprenaline (Iso). Conformational change data are shown as ? net BRET change in per cent, mean of n = 3 independent repetitions ± SEM. b–d The recruitment of ßarr2 to the b2AR following stimulation with Iso (b), Epinephrine (c) or Norepinephrine (d) in presence of all endogenous GRKs or individually overexpressed GRK2 or GRK6 in ?Q-GRK as indicated. Data are depicted as a mean of n = 3 independent experiments ± SEM and normalised to individual maxima. The tables below depict the EC50 ± SEM of the corresponding concentration-response curves. The EC50 of the indicated conditions were compared to the EC50 in Control using ANOVA and two-sided Dunnett’s test (*p < 0.05; **p < 0.01; ns not significant). e, f Utilisation of the ßarr recruitment assay for specificity determination of the GRK inhibitor cmpd101 in living cells. ?Q-GRK or Control cells were transfected with b2AR-NanoLuc, Halo-Tag-ßarr2 and either GRK2, 3, 5, 6 or EV as noted. The cells were incubated with different concentrations of cmpd101 (e) or the b2AR antagonist pindolol (f) for 10 min prior to stimulation with 1 µM Iso. The recruitment-induced BRET changes were measured and calculated as ? net BRET change in per cent, represented as the mean of n = 4 independent experiments ± SEM. All exact p values, test statistics, effect sizes, confidence intervals and degrees of freedom are provided in the Source Data files.

Fig 4: Grk3 is not expressed in NIH3T3 cellsRT–PCR assay of Grk2 and Grk3 expression in MEF and NIH 3T3 cells.

Fig 5: Suppression of GRK2 in placenta compromises placentation via necroptosis. (A) The 1st panel: Representative H&E staining images of E18.5 placentae from NC-LV and GRK2-LV groups. GRK2-LV placentae display a lack of vascularization, smaller placental size and a smaller labyrinth. Scale bar, 500 µm. Yellow lines, labyrinth and junction zone boundary; Green lines, junction zone and decidua boundary. The 2nd panel: Higher magnification of the upper white rectangle. Scale bar, 100 µm. 2nd and 3rd panels: The H&E staining and Von Kossa staining illustrate placental infarction and calcium deposition (red arrows) in GRK2-knockdown mice, respectively. (B) Volcano plots show differential expression for comparisons between GRK2-LV and NC-LV placentae (N = 3 per group. (C) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis for the differentially expressed genes in total RNA-seq: GRK2-LV vs. NC-LV groups (N = 3 per group). (D) TEM images show extensive necrosis in labyrinth layer of GRK2-LV group. Yellow dotted line, cell membranes; Yellow arrows swelling mitochondria. From upper to bottom, 500×, 1,500×, 2,500× magnification, respectively. (E,F) Western blot analysis of placental cleaved-caspase 3, RIPK1, RIPK 3, phospho-MLKL (p-MLKL; S358), and ß-actin (loading control). The DNA fragment analysis (G,H) and apoptosis rate analysis (I) in mice term placentae. Green signal indicates TUNEL-positive nuclei. Scale bar, 50 µm. (J,K) The DNA fragment in patients’ placentae. Scale bar, 100 µm. Dec, decidua; JZ, Junction Zone; Lab, labyrinth; H&E, hematoxylin and eosin staining; RBC, red blood cells; STB, syncytiotrophoblast. Data present as X ± SEM; NS, not significant; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus WT group; #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001 versus NC-LV group.