Fig 2: TANC2-PRKCA-expressing cells exhibit reduced levels of cleaved caspase-3 and decreased apoptosis.A, immunoblot analysis of lysates from parental, CRISPR-edited TANC2-PKCa fusion, and heterozygous PKCa knockout cells, probed for caspase-3, cleaved caspase-3, PKCa, and ß-actin. Data are representative of three independent experiments. B, apoptosis assay performed on MDA-MB-231 parental (gray), CRISPR-edited TANC2-PKCa fusion (purple), or heterozygous PKCa knockout (orange) cells. Data represent luminescence upon binding of annexin V-luciferase to cell-surface-exposed phosphatidylserine as a function of time following addition of reagents. Graph depicts mean ± SEM from three independent experiments. **p < 0.01, ***p < 0.001 (Student’s t-test). Asterisk indicates significance between parental and fusion-expressing cells, and # indicates significance between parental and heterozygous PKCa knockout cells. C, apoptosis assay described in (B) was performed on MDA-MB-231 parental (gray), CRISPR-edited TANC2-PKCa fusion (purple), or heterozygous PKCa knockout (orange) cells treated with etoposide (12.5 µM) or vehicle (DMSO) for 24 h. Graph depicts mean ± SEM from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t-test). Asterisk indicates significance between vehicle and etoposide-treated conditions for each cell line. # and % indicate significance between the parental and CRISPR-edited fusion or heterozygous PKCa knockout cells in basal and etoposide-treated conditions, respectively.

Fig 3: ß1-AR mainly attenuated phagocytosis through cAMP-EPAC pathway.a–c Effect of 6-h dobutamine (1 µM) stimulation on GPCR-related signaling pathways. b, c Western blot analysis revealed that phosphorylated CREB (pCREB) (Gai/Gas-related marker) was increased by dobutamine (t-test, **p < 0.01), but not phospho-CaMKII alpha (pCaMKII) (Gaq-related maker) and phospho-PKCa/ßII [pPKCa/ß] (Gai/Gaq). Data are normalized by unphosphorylated protein or total protein and are presented as bar graphs (mean ± SEM) with scatter dot plots of independent experiments (n = 4). d Prostaglandin E2a (PGE2a), activating Gs-coupled GPCR, dose-dependently prevented iHTMC phagocytosis (*p < 0.05, **p < 0.01 vs. DMSO, one-way ANOVA [p < 0.001], Dunnett’s multiple comparison). Data are presented as bar graphs (mean ± SEM) with scatter dot plots of independent experiments (n = 3). Phagocytosis activity was normalized to that of control DMSO. Dose-dependent intracellular cAMP accumulation by cAMP inducers forskolin (FSK) (e), and by L-NE and dobutamine (f) (*p < 0.05, **p < 0.01, ***p < 0.001 vs. DMSO, one-way ANOVA [p < 0.001], Dunnett’s multiple comparison). g betaxolol dose-dependently suppressed L-NE (10 µM)- or dobutamine (1 µM)-induced cAMP accumulation (*p < 0.05, **p < 0.01 vs. DMSO, one-way ANOVA [p < 0.001], Dunnett’s multiple comparison). Data e–g are presented as bar graphs (mean + SEM) with scatter dot plots of independent experiments (n = 4). h iHTMC phagocytic activity was slightly but significantly reduced by cAMP inducers (FSK and IBMX) and cAMP analogs, such as PKA activator Sp-cAMP and EPAC/PKA activator 8-CPT-cAMP (*p < 0.05, **p < 0.01, ***p < 0.001 vs. DMSO, one-way ANOVA [p < 0.001], Dunnett’s multiple comparison). Phagocytosis activity was normalized to that of control DMSO. i siRNA (PRKACA, RAPGEF3, and RAPGEF4) administration significantly rescued ß1-AR-mediated suppression of iHTMC phagocytosis (**p < 0.01, one-way ANOVA [p < 0.001], Tukey’s multiple comparison). j The recovery effect of PKA and EPAC1/2 antagonists (KT5720 and ESI09, respectively) simultaneously added with dobutamine (1 µM) dose-dependently rescued dobutamine-suppressed iHTMC phagocytosis (*p < 0.05, **p < 0.01, ***p < 0.001 vs. DMSO, one-way ANOVA [p < 0.001], Tukey’s multiple comparison). The antagonists were added at the same time as dobutamine. Data h–j are presented as bar graphs (mean ± SEM) with scatter dot plots of independent experiments (n = 3–4).

Fig 4: A subset of AML patients and cell lines expresses detectable phosphorylated PKC. (A) AML cells from patient samples were subjected to Western blotting with an antibody that recognizes phosphorylated PKCa and PKCßII (pPKC). Actin was used a loading control. (B) HL60, K562, MFL2, MR2, and OCI-AML3 cells were harvested and subjected to Western blotting with an antibody that recognizes phosphorylated PKCa/ßII (pPKC) as well as total PKCa (PKCa). Actin was used a loading control.

Fig 5: Detection of PICK1 and PKC signaling in brain tissues.The brain samples from WT and Tspan7-/- rats were, respectively, homogenized in RIPA lysis buffer, and the total proteins were obtained and analyzed by Western blot using primary antibodies of anti-p-ERK (4370; dilution of 1:1,000; CST), ERK (9102; dilution of 1:1,000; CST), p-PKCe (ab63387; dilution of 1:500; Abcam), PKC; (2683; dilution of 1:500; CST), p-PKCa (9375; dilution of 1:1,000; CST), PKCa (2056; dilution of 1:500; CST), PICK1 (10983-2-AP; dilution of 1:1,000; ProteinTech), NMDAR1 (ab17345; dilution of 1:500; Abcam), and secondary antibodies of HRP-labelled goat anti-mouse IgG, HRP-labelled goat anti-rabbit IgG. Proteins were visualized using a chemiluminescent detection system and analyzed using Quantity One software. (A) Phosphorylated and total ERK, PKCe, PKCa, PICK1, and NMDAR1 proteins in the brain tissues from WT and Tspan7-/- rats were detected with Western blot. (B, C, D, E, F) Phosphorylated and total ERK, PKCe, PKCa, PICK1, and NMDAR1 proteins in the brain tissues from WT and Tspan7-/- rats were quantified with ImageJ. (G) Body weights and birth rates were compared between WT and Tspan7-/- rats at 2, 3, 4, and 8 wk. The results showed that the PICK1, ERK, PKC, and NMDAR1 were not changed obviously in Tspan7-/- rats and suggesting PICK1 or ERK signal could be not the main pathway of Tspan7-/-. Data information: In (B, C, D, E, F), data were analyzed by two-tailed unpaired t tests. Error bars represent SEM; n.s, not significant.Source data are available for this figure.