pkcγ sirna  (Santa Cruz Biotechnology)

Journal: iScience

Article Title: Downregulation of protein kinase C gamma reduces epithelial property and enhances malignant phenotypes in colorectal cancer cells

doi: 10.1016/j.isci.2022.105501

Figure Lengend Snippet: PKCγ stabilizes ARHGEF18 and maintains epithelial integrity (A) DLD-1 cells were transfected with Halo-ARHGEF18 and the indicated siRNAs. After 3 days, the cells were treated with cycloheximide (200 μg/mL) and then harvested at the indicated time points. The relative levels of Halo-ARHGEF18 normalized to ACTB were analyzed using western blotting (n = 3). (B) DLD-1 cells were transfected with indicated siRNAs. After 3 days, the cells were treated with cycloheximide (200 μg/mL) and then harvested at the indicated time points. The relative levels of ARHGEF18 normalized to ACTB were analyzed using western blotting (n = 3). (C) DLD-1 or SW480 cells were transfected with Halo-ARHGEF18 (WT or S581A), following which the cells were treated with cycloheximide (200 μg/mL) and harvested at the indicated time points. The relative levels of Halo-ARHGEF18 normalized to ACTB were analyzed using western blotting (n = 3). (D) DLD-1 cells were transfected with the indicated siRNAs. After 4 days, endogenous ARHGEF18 was detected using western blotting (n = 4). (E) DLD-1 cells were transfected with the indicated siRNAs. After 5 days, the cells were fixed with methanol and subjected to immunofluorescence staining with E-cadherin and ARHGEF18 antibodies (upper panels) or F-actin (Acti-stain 488 phalloidin; green) and ARHGEF18 antibodies (red) (lower panels). Scale bar = 10 μm. (F) DLD-1 cells were transfected with the indicated siRNAs. After 6 days, the cells were fixed with methanol and subjected to immunofluorescence staining with F-actin. Scale bar = 50 μm. (G) Caco-2 cells were transfected with the indicated siRNA, mounted in Matrigel, and then examined using immunostaining with Hoechst (blue) and phalloidin (red). The percentage of normally polarized cysts per total cell cluster containing 3–10 cells is shown in the bar graph (more than 85 clusters were counted from three experimental replicates). Scale bar = 50 μm. (H) A putative mechanism of epithelial maintenance by PKCγ. PKCγ phosphorylates and stabilizes ARHGEF18, which activates Rho A at cell-cell junctions to maintain epithelial polarity. (A–D and G) Data are presented as means ± SD. Statistical analysis was performed using Dunnett’s multiple comparison of means test (A and D) or Student’s t test (B, C, and G). ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. See also Figure S4 .

Article Snippet: Negative control siRNA (siNeg) and siRNAs for PKC γ (Qiagen, Hilden, Germany) were used at final concentrations of 20 nM.

Techniques: Transfection, Western Blot, Immunofluorescence, Staining, Immunostaining, Comparison

Journal: Scientific Reports

Article Title: Protocatechuic acid attenuates isoproterenol-induced cardiac hypertrophy via downregulation of ROCK1–Sp1–PKCγ axis

doi: 10.1038/s41598-021-96761-2

Figure Lengend Snippet: PKCγ knockdown reduces isoproterenol-induced cardiac hypertrophy in H9c2 cells. ( A‒E ) H9c2 cells were transfected with empty vector or pCMV3-N-GFPSpark-PKCγ and then treated with protocatechuic acid (10 μM). ( A ) Representative western blot images showing PKCγ and BNP protein levels in pCMV3-N-GFPSpark-PKCγ -transfected cells. β-actin was used as a loading control. ( B , C ) Quantification of PKCγ and BNP protein levels (n = 6). ***P < 0.001; ### P < 0.001. ( D , E ) Representative images of cells transfected with pCMV3-N vector or pCMV3-N-GFPSpark-PKCγ , incubated with or without protocatechuic acid (10 μM), and then stained with anti-PKCγ antibody, Alexa Fluor 488 phalloidin, and DAPI. PKCγ-positive cells (red); nuclei (blue); actin filaments (green). Scale bar = 50 μm. ( F , G ) H9c2 cells were transfected with control or PKCγ siRNA, serum starved overnight, and then treated with isoproterenol (10 μM). The mRNA levels of Prkcg and Nppb were determined by RT-PCR (n = 6). **P < 0.01 and ***P < 0.001; ### P < 0.001; @@@ P < 0.001. ( I ) Merged images of H9c2 cells transfected with PKCγ siRNA, incubated with or without isoproterenol (10 μM) for 24 h, and then stained with Alexa Fluor 488 phalloidin (green) and DAPI (blue). ( J ) Cell size was quantified by measuring the cell surface area of Alexa Fluor 488 phalloidin-stained cells (n = 40 cells). Scale bar = 50 μm. ### P < 0.001; @@@ P < 0.001; NS not significant. Data are presented as the mean ± S.E. Statistical analysis: one-way ANOVA followed by Bonferroni post hoc tests. Graphs were prepared using GraphPad 5.0.

Article Snippet: H9c2 cells were transfected with 100 nM control siRNA (cat no. SN-1003, Bioneer, South Korea), Sp1 siRNA (product name 1762865, Bioneer, South Korea), PKCγ siRNA (product name 24681, Bioneer, South Korea), or ROCK1 siRNA (cat no. L-095591-02-0005, Dharmacon Inc., Lafayette, CO, USA) using RNAiMAX reagent according to the manufacturer’s instructions. siRNA control sense: 5′-CCU ACG CCA CCA AUU UCG U-3′, siRNA control antisense: 5′-ACG AAA UUG GUG GCG UAG G-3′; siRNA Sp1 sense: 5′-GUG CAA AUC AAC AGA UCA U-3′, siRNA Sp1 antisense: 5′-AUG AUC UGU UGA UUU GCA C-3′; siRNA PKCγ sense: 5′-GUC CGA AUU UUA GGU CUC U-3′, siRNA PKCγ antisense: 5′-AGA GAC CUA AAA UUC GGA C-3′.

Techniques: Transfection, Plasmid Preparation, Western Blot, Incubation, Staining, Reverse Transcription Polymerase Chain Reaction