Journal: Nature Communications

Article Title: Integrative transcriptome-wide association analyses reveal PRKCG-linked GABAergic dysfunction in Fragile X-associated tremor/ataxia syndrome

doi: 10.1038/s41467-025-68163-9

Figure Lengend Snippet: a Venn diagrams showing the number of TWAS candidate genes overlapping with DEGs from bulk RNA-Seq on the cortex and cerebellum of the FXTAS mouse model. Bar plot showing the normalized read counts of shared genes in FXTAS and control mice from bulk RNA-Seq. DEGs from 3 to 4 months were selected with P < 0.01; DEGs from 6 to 7 months were selected with FDR < 0.05. Data are presented as mean ± SD; n = 3–4 biological replicates per group. b Venn diagrams showing the number of TWAS candidate genes overlapping with DEGs from TRAP-Seq on GABAergic neurons from cortex and cerebellum of conditional FXTAS model. Bar plot showing the normalized read counts of shared genes in FXTAS and control mice from TRAP-Seq. DEGs from 3 to 4 months were selected with P < 0.01. Data are presented as mean ± SD; n = 2-3 biological replicates per group. Two-sided Fisher’s exact test demonstrated significant overlap between TWAS candidates and TRAP-seq DEGs in cerebellar GABAergic neurons at 6–7 months ( P = 0.018; Supplementary Data ). c Quantitative RT-PCR analysis of Prkcg expression relative to Gapdh in the cortex at two stages. Three biological replicates were used in each group. Data are presented as mean ± SD; n = 3 biological replicates per condition. Statistical significance was assessed using a two-sided unpaired t -test with Welch’s correction. Significance is defined as P < 0.05. d Quantitative RT-PCR analysis of PRKCG expression relative to GAPDH in NPCs from FXTAS patients and controls. Data are presented as mean ± SD; n = 3 biological replicates per condition. Statistical significance was assessed using a one-sided unpaired t -test. Significance is defined as P < 0.05. e Scheme of the overexpression of human PRKCG in the FXTAS fly model. f Overexpression of hPRKCG and aPKC resulted in enhancement of neurotoxicity in the FXTAS fly model. g Cell viability assays following shRNA-mediated knockdown of Prkcg (left) and hPRKCG overexpression (right) in N2a cells expressing expanded CGG repeats. Data are presented as mean ± SD and statistical significance was assessed using a two-sided unpaired t -test with Welch’s correction. Significance is defined as P < 0.05.

Article Snippet: Full-length human PRKCG cDNA was obtained from a tagged ORF clone (OriGene Technologies, PRKCG Human Tagged ORF Clone, RG208502).

Techniques: RNA Sequencing, Control, Quantitative RT-PCR, Expressing, Over Expression, shRNA, Knockdown

Journal: Frontiers in Cellular Neuroscience

Article Title: Long-Term Cultures of Spinal Cord Interneurons

doi: 10.3389/fncel.2022.827628

Figure Lengend Snippet: List of antibodies used for immunocytochemistry.

Article Snippet: PKC γ , , 1/100 , , Santa Cruz , sc-166385.

Techniques: Immunocytochemistry

Journal: The Journal of Neuroscience

Article Title: A New Mouse Model Related to SCA14 Carrying a Pseudosubstrate Domain Mutation in PKCγ Shows Perturbed Purkinje Cell Maturation and Ataxic Motor Behavior

doi: 10.1523/jneurosci.1946-20.2021

Figure Lengend Snippet: Figure 1. Pseudosubstrate mutant PKCg protein is unstable and shows aggregation. A, Illustrations of PKCg protein do- main mutations and deletions found in SCA14 families. Most mutations are found in the C1B domain. B, The 5 mg of GFP- Control plasmid, GFP-PKCg-Wt, or GFP-PKCg-A24E was transfected to the HeLa cells; and after 24 h, cells were fixed with PFA following the immunostaining. GFP-PKCg-A24E showed aggregation and accumulated in HELA cells. Images were acquired with confocal microscopy (Carl Zeiss, LSM700) using a Plan-Apochromat 100/1.3 Oil DIC M27 objective (Carl Zeiss). Scale bar, 10mm. C, Pseudosubstrate domain mutant PKCg is unstable and is degraded after 35mg/ml cycloheximide treatment. After 48 h transfection, cycloheximide was applied to the cells. Samples were collected at 0 min, 30min, 90min, 240 min, and 24 h. Twenty-four hours after cycloheximide treatment, the PKCg protein expression level is GFP-PKCg- Wt = 96.68% and GFP-PKCg-A24E = 41.27% compared with each starting point, respectively. D, Degradation of pseudosub- strate domain mutant PKCg occurs via the proteasome pathway. Twenty-four hours after 5 mM of proteasome inhibitor MG132 treatment, HEK293T cells show more ubiquitinated proteins, and this treatment rescued GFP-PKCg-A24E protein lev- els (GFP-PKCg-Wt = 100.0%; GFP-PKCg-Wt 1 MG132 = 192.90% 6 25.35; GFP-PKCg-A24E = 32.26% 6 6.75; GFP- PKCg-A24E 1 MG132 = 114.60% 6 28.54; n = 6). Protein expression was analyzed using the two-tailed Mann–Whitney test (GFP-PKCg-Wt vs GFP-PKCg-A24E, p = 0.0022, GFP-PKCg-Wt vs GFP-PKCg-Wt 1 MG132, p = 0.0022; GFP-PKCg- A24E vs GFP-PKCg-A24E 1 MG132, p = 0.0087).

Article Snippet: PKCg -A24E overexpression in HeLa cells and HEK293T cells Human PRKCG gene was obtained from Origene in pCMV6-XL4 (pCMV6-XL4-PRKCG); 5 mg of pCMV-GFP control, pCMV-PKCg - Wt, or pCMV-PKCg -A24E was transfected into HeLa cells (ATCC, RRID: CVCL_0030) or HEK293T cells (ATCC, RRID: CVCL_0063) using X-fect Transfection Reagent (Takara).

Techniques: Mutagenesis, Control, Plasmid Preparation, Transfection, Immunostaining, Confocal Microscopy, Expressing, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Calcium transients regulate epithelial integration of multiciliated cells during Xenopus skin development

doi: 10.1101/2024.11.01.621480

Figure Lengend Snippet: A) Schematic depicting the series of events leading to calcium release from the ER through activation of the IP3 receptor. 1) PLC hydrolyses PIP2 to IP3 and DAG. 2)IP3 binds at the IP3 receptor located at the ER membrane. 3) Activation of the IP3 receptors leads to release of calcium from the ER to the cytoplasm. 2APB inhibits ER mediated calcium release by inhibiting IP3 receptor. B) Stills from a representative time lapse recording showing an intercalating MCC expressing the PLC activity sensor GFP-PKCγ-C1a. PLC displays a transient pulsed activation, as evident by the localized (green dots) at the apical surface of MCC. C)Quantification of GFP-PKCγ-C1 over time. D) Stills from time lapse recordings showing a region of the skin epithelium of control and PLC-inhibitor treated embryos expressing the calcium sensor GECO-RED. Inhibition of PLC suppresses calcium transients (green circles) generation. E) Kymographs from distinct regions of the skin epithelium of control and PLC inhibitor treated embryos. Transient increases in calcium levels in intercalating MCCs (green rectangles) are blocked in presence of the PLC inhibitor. F) Representative images of the skin neuroepithelium of stage 24 control and PLC inhibitor treated embryos. MCCs fail to enter the superficial epithelial (yellow arrows) or expand their surface area (yellow circles) when PLC activity is impaired. F’) Zoomed images of MCCs from a control and PLC inhibitor treated embryo. G) Quantification of MCCs apical surface area in control (n=100 MCCs) and PLC-inhibitor treated embryos (n=100 MCCs). Two-sided, unpaired Student’s t -test H) Representative images of the skin epithelium of stage 24 embryos expressing the PLC dominant negative construct PH-PLCD1. Expression of PLCD1 results in defective MCC apical emergence (yellow arrow). I) Quantification of successful MCC apical emergence of control (n=150 MCCs) and PH-PLCD1 (n=116 MCCs) expressing MCCs, χ 2 test.

Article Snippet: Par3GFP, GFP-PKC-γ-C1a(Addgene #21205), PH-PLCD1 (Addgene #21179) we injected 80pg of DNA per blastomere.

Techniques: Activation Assay, Membrane, Expressing, Activity Assay, Control, Inhibition, Dominant Negative Mutation, Construct