pan protein kinase c pkc inhibitor go6983  (Tocris)

Journal: bioRxiv

Article Title: Extra-ciliary role for polycystins in regulation of Ezrin and renal tubular morphology

doi: 10.1101/2025.11.05.684799

Figure Lengend Snippet: (A) Immortalized mouse Pkd1 fl/fl Pax8rtTA, TetO-Cre medullary renal epithelial cells = Pkd1 (M) (clone #313) treated with or without doxycycline then cultured in Matrigel for 14 days, with additional cultures of wildtype Pkd1 + (M) cells treated with 2μM of pan PKC inhibitor GO6983. (B-E) Semi-automated quantification from three separate experiments of tubuloid circularity (B) and (C) size (DMSO= Pkd1 +(M) n=273; PKC-inhib = Pkd1 +(M)+ 2μM GO6983 n=242) found significant differences in circularity and area in the structures treated with GO6983; +/− SEM, P<0.05, two tailed Student’s T-Test. (D,E) Comparison of the largest structures (e.g. structures indicated with black box) reveals a further enhanced difference in circularity (D) for the structures treated with GO6983; DMSO= Pkd1 +(M) n=88; PKC-inhib = Pkd1 +(M)+ 2μM GO6983 n=103; +/− SEM, P<0.05, two tailed Student’s T-Test. (F) The relationship between size and circularity in the largest structures ( Pkd1 +(M) and Pkd1 +(M) treated with 2μM GO6983), were analyzed with linear regression. Treatment with PKC-inhibitor abolished the relationship between circularity and size (PKC-inhib = Pkd1 +(M)+ 2μM GO6983 n=103; p<0.1517, R2=0.02024) from tubuloids similar to what was previously observed after Doxycycline treatment and treatment with Ezrin inhibitor NSC supporting a common targeted pathway. (G) At the completion of the 14 day experiment structures were removed from the Matrigel and Western blot was performed to confirm the effectiveness of the GO6983 to reduce the amount of phosphorylated PKC. Blot representative of three separate experiments confirmed a significant decrease in the levels of phospho-PKC in the 3D structures after treatment with GO6983. L.C. = loading control. (H-K) Representative merged images or separated montage of Immortalized mouse Pkd1 fl/fl Pax8rtTA, TetO-Cre renal epithelial cells, clone #313 ( Pkd1 (M)), cultured with vehicle (H, DMSO = Pkd1 +), doxycycline (K; DOX = Pkd1 -), or 2μM GO6983 (I,J; PKC-inhib = Pkd1 + 2μM GO6983) labeled with Ezrin 3145 (active form, green), Ezrin 3c12 (inactive, red), and ZO1 (purple). Repeated in three separate experiments, scale bar 50μm. Treatment with the PKC-inhibitor reduces apical active ezrin like treatment with DOX, but unlike DOX, the PKC-inhibitor causes accumulation of the active Ezrin near the junctions (K, inset, enlarged in J, white arrow), mirroring the inactive Ezrin and ZO1 localizations.

Article Snippet: In some experiments we used the ERM phosphorylation inhibitor compound NSC668394 (Sigma 341216) and the Pan PKC inhibitor GO6983 (Med Chem Express HY-13689) which were added directly to the media.

Techniques: Cell Culture, Inhibition, Two Tailed Test, Comparison, Western Blot, Control, Labeling

Journal: International Journal of Molecular Sciences

Article Title: LPCAT4 Knockdown Alters Barrier Integrity and Cellular Bioenergetics in Human Urothelium

doi: 10.3390/ijms231911871

Figure Lengend Snippet: Phenocopying of the LPCAT4 knockdown by independent inhibition of PKC and TSPO activity. ( A ) Expression of PRKCD is most abundant PKC gene in differentiated NHU cells, and significantly (by fold change and statistically) upregulated upon differentiation (red text; blue text for downregulated upon differentiation; data as described in A). Classical PKC isozymes (DAG- and calcium-dependent) are less abundant. PKD (historically PKC-μ) shown for reference. ( B ) Inhibition of eitherpan-PKC (Go6983; significance indicated above trend lines) or TSPO (PK11195; significance indicated below trend lines) activity led to elevated urothelial barrier resistance. TEER values based on 6 technical replicates per condition per time point, with standard deviation represented as shaded areas around mean values. TSPO protein reduction in an LPCAT4 knockdown environment was validated by western blotting ( C ) at 24 and 72-h after differentiation induction. Inhibition of PKC delayed wound healing ( D ), but high variance was observed with PK11195-treated cultures. ( E ) In proliferative conditions (left of dashed line), PK11195 treatment reduced NHU endogenous ATP availability by 6.9% ( p = 1.82 × 10 −4 ), with ketamine a strong positive control, as reported previously . Upon differentiation (right of dashed line) endogenous ATP availability increased by 57.7% ( p = 2.00 × 10 −9 ). LPCAT4 knockdown significantly reduced ATP availability by 15.7% ( p = 3.82 × 10 −6 ). Endogenous ATP fluorescence values from 5 technical replicates normalised by background blank cultures and total ng of protein. Across the figure, significance values were generated by independent ‘ t ’ tests, with * indicating p < 0.05, ** indicating p < 0.01, and *** indicating p < 0.001.

Article Snippet: Cells from each culture were seeded onto 1.13 cm 2 ThinCert ® membranes (6 technical replicates per culture) as described, supplementing the differentiating medium with either 100 nM [ ] pan-Protein Kinase C (PKC) inhibitor Go6983 (Tocris Bioscience, Avon, UK), or 100 nM [ ] Translocator Protein (TSPO) inhibitor PK11195 (Tocris).

Techniques: Knockdown, Inhibition, Activity Assay, Expressing, Standard Deviation, Western Blot, Positive Control, Fluorescence, Generated

Journal: eLife

Article Title: Type XVII collagen coordinates proliferation in the interfollicular epidermis

doi: 10.7554/eLife.26635

Figure Lengend Snippet: ( a ) COL17 staining of whole IFE skin treated with 5 mM EDTA. The quantitative fluorescent intensity of COL17 in basal cells of IFE from control (PBS) and 5 mM EDTA treated (n = 4). Mann-Whitney test. Samples were taken from young adult WT mice at 6–10 weeks. Scale bar: 20 μm. ( b ) Phospho-aPKC labeling (indicated with arrows) and quantitative fluorescent intensity results from young and aged WT IFE skin (n = 4). Mann-Whitney test. Scale bar: 20 μm.( c ) Representative figures of asymmetric cell division (ACD; scored as perpendicular to basement membrane) and symmetric cell division (SCD; in parallel to basement membrane) in young IFE. Survivin staining indicates the direction of the cell division. Laminin β1 signifies basement membrane. Scale bar: 10 μm. Graph of percentage of ACD and SCD in young and aged IFE (n = 4). Student’s t-test. ( d–e ) The pharmacological inhibition of pan-aPKC (d, 1 μM of Go6983; 0.00002% DMSO as control) and aPKCλ/ζ (e, 10 μM of myr PSI; water as control) in whole IFE skin from young adult WT mice, followed by COL17 staining. The quantitative fluorescent intensity of COL17 in lateral membrane of basal cells from control and 1 μM Go6983 treated ( d ) and 1 μM myr PSI ( e ) (n = 4). Mann-Whitney test. Scale bar: 10 μm. BM, basement membrane. ( f–h ) EDTA treatment (5 mM; PBS as control, f ) and pharmacological inhibition of pan-aPKC (g, 1 μM of Go6983; 0.00002% DMSO as control) and aPKCλ/ζ (h, 10 μM of myr PSI; water as control) on 3D epidermis. The relative fluorescent intensity of COL17 in lateral membrane of basal cells was measured (n = 4). Mann-Whitney test. BM, basement membrane. Scale bar: 20 μm. The data are the means ± SE. *0.01

Article Snippet: Whole mice paw skin samples were treated with the following inhibitors for 1 hr at 4°C: EDTA, pan-PKC inhibitor Go6983 (Tocris Bio, Bristol, UK) and myristoylated pseudosubstrate Inhibitor (myr PSI; Calbiochem, Billerica, Massachusetts, USA) before staining.

Techniques: Staining, Control, MANN-WHITNEY, Labeling, Membrane, Inhibition