Fig 2: Validation of conformation-sensitive antibodies to classical PKCs.a Schematic of conformational changes involved in generation of the active state of cPKC. Purple represents the C2 domain that becomes exposed after cPKC activation. This domain is recognized by two C2-Cat-cPKC antibodies: the C2-Cat-PKCß antibody (specific for PKCß), and the C2-Cat-cPKC antibody (recognizes all the classical cPKC isoenzymes). b Neuro2A and Neuro2A PKCß-KO cells were probed with anti-C2-Cat-PKCß (AVG707-B9) antibody. Images were acquired using the InCell microscope (×40) and fluorescence intensity measured by Cell Profiler software. Representative images with the antibody staining in green and DAPI in blue are shown. Volcano plot, each dot represents one cell. Lines are mean ± SD of two experiments, number of counted cells are indicated. RFU indicates the integrated fluorescence intensity units. ****p value < 0.0001; t-test. c Neuro2A and Neuro2A PKCa-KO cells were treated with PMA (100 nM) for 30 min and probed with anti-C2-Cat-cPKC antibody. Images were acquired and analyzed as previously described. ****p value < 0.0001; t-test. d Neuro2A cells were treated with PMA (100 nM) for 30 min and stained with anti-PKCa antibody (Santa Cruz; sc-8393). Images were acquired and analyzed as previously described. NS non-significant; p value > 0.99; t test. e SK-N-SH cells were treated with PMA (100 nM) with or without Gö6983 (1 µM) for 30 min and stained with anti-C2-Cat-cPKC antibody. Images were acquired and analyzed as previously described. ****p value < 0.0001; t test.

Fig 3: Expression of key proteins involved in Kyoto Encyclopedia of Genes and Genomes pathways identified. Representative Western blot (A) and quantitative evaluation (B) of glial fibrillary acidic protein, PKCa, phospho (p)-Akt, Akt, ERK1/2, and p-ERK1 from control (Ctrl), hypoxic-ischemic (HI), and ischemic melatonin-treated (HI+ MEL) rats killed 1 h after HI. ß-Actin was used as the loading control. To avoid experimental variability that may occur in two different litters, two series of independent experiments were performed (n = 12 animals).

Fig 4: Chordoid glioma of the third ventricle is defined by PRKCA D463H mutation that localizes in the active site of the kinase domain in the encoded protein kinase C alpha (PKCa). a T1-weighted post-contrast magnetic resonance image of a chordoid glioma. b Hematoxylin and eosin stained section of a chordoid glioma. Scale bar, 40 µm. c Diagram of human PKCa with the location of the D463H mutation identified in each of the 13 chordoid gliomas shown. d Amino acid sequence for PKCa in the region of D463 (highlighted in yellow) demonstrating a high degree of conservation from Homo sapiens to C. elegans. e Structural model of the active site of the kinase domain for PKCa showing how the side chain of amino acid Asp463 sits within the ATP binding pocket, where it functions as a proton acceptor during the ATP hydrolysis reaction. Substitution of Asp with His at this codon (D463H) is structurally predicted to result in a side chain with a proton acceptor at nearly the same location, which may potentially alter kinase function through steric differences or ability of the side chains to be protonated or deprotonated based on differential proton affinities

Fig 5: Meta-analysis of retinal cells with different donors and species.a Highlighted region of the eye was selected for single-cell analyses. b tSNE plot visualisation of cells obtained from human retina. Ten transcriptionally distinct clusters were observed in the neural retina of the eye. c RAB41 colocalisation with OPN1SW using RNA FISH. INL inner nuclear layer, ONL outer nuclear layer, GCL ganglion cell layer. Scale bar = 20 µm. n = 2 technical replicates. d, e Representative RNA FISH images of the novel markers TRDN (e) and NIF3L1 (d) for different regions of the eye. MALAT1 was used as an internal control, while PKCA was immuno-stained with NIF3L1 and TRDN with VIM. Scale bar = 20 µm. n = 2 technical replicates. f Patterns of gene expression as determined by scCoGAPS algorithm in retinal cell types of the human eye (see Supplementary Information). The correlation of each pattern to human retinal cell types was colour-coded. g Pattern 17 showed a high correlation to Muller glial cells across species. h Bubble plot showing expression levels of the top 20 genes by gene weight of pattern 17. The size of each circle is proportional to the percentage of cells expressing the gene, and its intensity depicts the average transcript count within expressing cells. i Correlation of human pattern 17 with resting Muller glial cells and Muller glial cells activated after injury in zebrafish. j The patterns of gene expression in zebrafish Muller glial cells which were activated after injury. k Respective GO of the patterns in Fig. 2j. Metascape calculated the statistical significance of each GO term enrichment (p-value) based on the accumulative hypergeometric distribution. The grey colour indicated a lack of significance. l List of genes that were common between human pattern 17 and zebrafish pattern 78.