Journal: Oncogenesis

Article Title: PKC-independent PI3K signalling diminishes PKC inhibitor sensitivity in uveal melanoma

doi: 10.1038/s41389-024-00511-8

Figure Lengend Snippet: A Accumulation of MAPK, PI3K/AKT, YAP and PKC signalling effectors including total and phosphorylated DUSP6, MARCKS, ERK, YAP, S6 and AKT 24 h after treatment with vehicle control (−) or 10 nM Trametinib (+). Western blot analyses were performed using three independent biological replicates ( n = 3). kD kilodalton. REVERT total protein loading stain is shown in Supplementary Fig. . B Expression levels of pS6 and DUSP6 (normalised to total S6 or REVERT staining, respectively) in vehicle control or 10 nM Trametinib-treated GNAQ/GNA11 -mutant (solid dot) or wild-type (crossed dot) UM cell lines. Data derived from three independent biological experiments ( n = 3), and P values were calculated using paired t tests. ns not significant. C Percentage of cells undergoing S-phase inhibition (dotted line set at 50% S-phase inhibition) and change in % sub-G1 (dotted line set at 30% sub-G1) in GNAQ/GNA11 -mutant (solid circle) or wild-type (crossed circle) UM cell lines treated with 5 µM IDE196 (data derived from ), 10 nM Trametinib or 2 µM BEZ235. Data derived from three independent biological experiments ( n = 3).

Article Snippet: Membranes were incubated at 4 °C overnight in primary antibodies diluted in Intercept Blocking Buffer (TBS) (Li-Cor, Lincoln, NE, USA) or Odyssey Blocking Buffer (Li-Cor) with Tween 20 (0.05%), as follows: total MARCKS (1:1000, 2C2, WH0004082M6, Sigma-Aldrich), phosphorylated MARCKS (pMARCKS; Ser 152/156 , 1:1000, 2741 S, Cell Signaling Technology, Danvers, MA, USA), DUSP6 (1:250 or 1:1000, EPR129Y, ab76310, Abcam, Cambridge, UK), total AKT (1:1000, 40D4, 2920S, Cell Signaling Technology), phosphorylated AKT (pAKT; Ser 473 , 1:100, D9E, 4060S, Cell Signaling Technology), pAKT (Ser 473 , 1:500, 736E11, 3787, Cell Signaling Technology), total ribosomal S6 (1:500, 54D2, 2317 S, Cell Signaling Technology), phosphorylated ribosomal S6 (pS6; Ser 235/236 , 1:1000, 2F9, 4856S, Cell Signaling Technology), total YAP (1:500, 1A12, 12395S, Cell Signaling Technology), phosphorylated YAP (pYAP; Ser 127 , 1:2000, 4911, Cell Signaling Technology), total ERK (1:2 000, 137F5, 4695S, Cell Signaling Technology), phosphorylated ERK (pERK; Tyr 204 , 1:250, E-4, SC-7383, Santa Cruz, Dallas, TX, USA), and MEK1/2 (1:500, L38C12, 4694S, Cell Signaling Technology).

Techniques: Control, Western Blot, Staining, Expressing, Mutagenesis, Derivative Assay, Inhibition

Journal: Oncogenesis

Article Title: PKC-independent PI3K signalling diminishes PKC inhibitor sensitivity in uveal melanoma

doi: 10.1038/s41389-024-00511-8

Figure Lengend Snippet: A Fold change in DUSP6 and pS6 expression (normalised log 2 protein expression in drug-treated – normalised log 2 protein expression in control-treated cells) in GNAQ/GNA11 -mutant (solid circle) and wild-type (crossed circle) UM cell lines treated with 5 µM IDE196, 10 nM Trametinib, or 2 µM BEZ235. Data compared using one-way ANOVA with the Geisser–Greenhouse correction and Tukey’s multiple comparison test, adjusted P values are shown. Data derived from three independent biological experiments ( n = 3, mean ± SD). B Accumulation of MAPK, PI3K, YAP and PKC signalling effectors, including total and phosphorylated DUSP6, MARCKS, ERK, YAP, S6 and AKT 24 h after treatment with vehicle control (−) or 2 µM BEZ235 (+). Western blot analyses were performed using three independent biological replicates ( n = 3). kD kilodalton. REVERT total protein loading stain is shown in Supplementary Fig. .

Article Snippet: Membranes were incubated at 4 °C overnight in primary antibodies diluted in Intercept Blocking Buffer (TBS) (Li-Cor, Lincoln, NE, USA) or Odyssey Blocking Buffer (Li-Cor) with Tween 20 (0.05%), as follows: total MARCKS (1:1000, 2C2, WH0004082M6, Sigma-Aldrich), phosphorylated MARCKS (pMARCKS; Ser 152/156 , 1:1000, 2741 S, Cell Signaling Technology, Danvers, MA, USA), DUSP6 (1:250 or 1:1000, EPR129Y, ab76310, Abcam, Cambridge, UK), total AKT (1:1000, 40D4, 2920S, Cell Signaling Technology), phosphorylated AKT (pAKT; Ser 473 , 1:100, D9E, 4060S, Cell Signaling Technology), pAKT (Ser 473 , 1:500, 736E11, 3787, Cell Signaling Technology), total ribosomal S6 (1:500, 54D2, 2317 S, Cell Signaling Technology), phosphorylated ribosomal S6 (pS6; Ser 235/236 , 1:1000, 2F9, 4856S, Cell Signaling Technology), total YAP (1:500, 1A12, 12395S, Cell Signaling Technology), phosphorylated YAP (pYAP; Ser 127 , 1:2000, 4911, Cell Signaling Technology), total ERK (1:2 000, 137F5, 4695S, Cell Signaling Technology), phosphorylated ERK (pERK; Tyr 204 , 1:250, E-4, SC-7383, Santa Cruz, Dallas, TX, USA), and MEK1/2 (1:500, L38C12, 4694S, Cell Signaling Technology).

Techniques: Expressing, Control, Mutagenesis, Comparison, Derivative Assay, Western Blot, Staining

Journal: Cancer science

Article Title: Myristoylated alanine-rich C kinase substrate phosphorylation promotes cholangiocarcinoma cell migration and metastasis via the protein kinase C-dependent pathway.

doi: 10.1111/j.1349-7006.2009.01427.x

Figure Lengend Snippet: Fig. 3. Immunohistochemistry of myristoylated alanine-rich C kinase substrate (MARCKS) (a) and phosphorylated MARCKS (pMARCKS) (c) in cholangiocarcinoma (CCA) tissues. Poorly (i), moderately (ii), papillary (iii), and well-differentiated (iv) CCA showed a strong expression of MARCKS. Some CCA cells in the bile duct showed a strong intensity of pMARCKS. Original magnification ·40. Survival curves calculated for MARCKS (b) and pMARCKS (d) according to the Kaplan–Meier log–rank test (P = 0.02 and P = 0.12, respectively). (––), high level of MARCKS or pMARCKS, (- - -), low level of MARCKS or pMARCKS.

Article Snippet: The sections were incubated with the 1:1000 anti-MARCKS or 1:200 anti-pMARCKS antibody (Santa Cruz, Santa Cruz, CA, USA) at 4 C overnight.

Techniques: Immunohistochemistry, Expressing

Journal: Cancer science

Article Title: Myristoylated alanine-rich C kinase substrate phosphorylation promotes cholangiocarcinoma cell migration and metastasis via the protein kinase C-dependent pathway.

doi: 10.1111/j.1349-7006.2009.01427.x

Figure Lengend Snippet: Fig. 4. Intracellular localization of myristoylated alanine-rich C kinase substrate (MARCKS) and phosphorylated MARCKS (pMARCKS) (a). Cholangiocarcinoma (CCA) cells were stimulated with 10 ng ⁄ mL 12-0-tetradecanoyl phorbol-13- acetate (TPA) or 5 lM GF109203x before TPA treatment. Confocal laser microscopy with an objective lens of ·40 was employed. In the control group, MARCKS was mostly located at the plasma membrane and pMARCKS was evenly distributed throughout the cytoplasm. Upon TPA treatment, MARCKS was translocated to the cytoplasm and the perinuclear area, and pMARCKS became highly detectable at the perinuclear level. Upon GF109203x + TPA treatment, MARCKS and pMARCKS distribution and abundance were similar to those observed in non-stimulated CCA cells. Western blotting was used for protein checking (b). Apparent intensity of bands on the membranes was estimated using a densitometer (c). Protein kinase C (PKC) activities were compared between the control and TPA-treated cells (d). Asterisk denotes a significant increase of PKC activity when compared with the control. *P < 0.05.

Article Snippet: The sections were incubated with the 1:1000 anti-MARCKS or 1:200 anti-pMARCKS antibody (Santa Cruz, Santa Cruz, CA, USA) at 4 C overnight.

Techniques: Microscopy, Control, Clinical Proteomics, Membrane, Western Blot, Activity Assay