Journal: bioRxiv

Article Title: Regulation of intracellular cAMP levels in osteocytes by mechano-sensitive focal adhesion kinase via PDE8A

doi: 10.1101/2024.06.28.601153

Figure Lengend Snippet: Gsα, PTHR1 and PDE8A tyrosine phosphorylation sites identified by phosphoproteomics to be reduced by FAK inhibition or deletion.

Article Snippet: Recombinant human PDE8A (Sigma SRP0273) was incubated with 100 ng FAK tyrosine kinase (Promega #V1971) 50μM DTT, 2mM MnCl 2 , 50μM ATP and 1x kinase buffer for 15 min at 37°C in a reaction of total volume 25 µL.

Techniques: Inhibition

Journal: bioRxiv

Article Title: Regulation of intracellular cAMP levels in osteocytes by mechano-sensitive focal adhesion kinase via PDE8A

doi: 10.1101/2024.06.28.601153

Figure Lengend Snippet: ( a ) Left: GloSensor cAMP assay in Saos2 cells following PDE8A inhibitor treatment (PF04957325, 10 μM). Right: Dose response of the PDE8A inhibitor PF04957325 in cAMP in GloSensor- expressing Saos2 cells. cAMP luminescence over time is shown. ( b ) Left: PDE8A immunoblotting in PDE8A KO cells. Right : GloSensor assay revealed increased cAMP luminescence in PDE8A KO cells compared to control cells upon treatment with isoproterenol (10 -6 M). Control cells were transfected with the empty vector PX459. ( c ) qRT-PCR showing relative expression of Sost ± treatment with PDE8A inhibitor 10 μM (left) and dose response of PDE8A inhibitor (right). Cells were treated for 4 hours prior to RNA isolation. For each group in control vs PDE8A inhibitor treated cells n = 6 biologic replicates for RNA and two-sided unpaired t test were used. N=2 biologic replicates were used for the dose response experiment.

Article Snippet: Recombinant human PDE8A (Sigma SRP0273) was incubated with 100 ng FAK tyrosine kinase (Promega #V1971) 50μM DTT, 2mM MnCl 2 , 50μM ATP and 1x kinase buffer for 15 min at 37°C in a reaction of total volume 25 µL.

Techniques: cAMP Assay, Expressing, Western Blot, Control, Transfection, Plasmid Preparation, Quantitative RT-PCR, Isolation

Journal: bioRxiv

Article Title: Regulation of intracellular cAMP levels in osteocytes by mechano-sensitive focal adhesion kinase via PDE8A

doi: 10.1101/2024.06.28.601153

Figure Lengend Snippet: ( a ) Immunoblotting for phospho-tyrosine (pY) and PDE8A in vitro FAK kinase assay with recombinant human PDE8A. pY bands are located at the expected size of FAK (auto-phosphorylation) and PDE8A. ( b ) Immunoblotting for FLAG and V5 in lysates from cells transfected with plasmids expressing FAK and PDE8A tagged with FLAG and V5 respectively after immunoprecipitation with anti-FLAG affinity gel. ( c ) Immunoblotting for FLAG and V5 in protein lysates from cells transfected with plasmids expressing FAK and PDE8A tagged with FLAG and V5 respectively after immunoprecipitation with anti-V5 affinity gel.

Article Snippet: Recombinant human PDE8A (Sigma SRP0273) was incubated with 100 ng FAK tyrosine kinase (Promega #V1971) 50μM DTT, 2mM MnCl 2 , 50μM ATP and 1x kinase buffer for 15 min at 37°C in a reaction of total volume 25 µL.

Techniques: Western Blot, In Vitro, Kinase Assay, Recombinant, Transfection, Expressing, Immunoprecipitation

Journal: bioRxiv

Article Title: Regulation of intracellular cAMP levels in osteocytes by mechano-sensitive focal adhesion kinase via PDE8A

doi: 10.1101/2024.06.28.601153

Figure Lengend Snippet: ( a ) PDE8A dose response. X axis: recombinant PDE8A dose in ng/μl y axis: 665/620 TR-FRET signal ratio (inversely proportional to cAMP levels) ( b ) 665/620 ratio after incubation of recombinant PDE8A with different amounts of recombinant FAK (shown on axis x). All reactions occurred in presence of 6 nM cAMP. TR-FRET signal at 665 nm was normalized to the signal of the donor-channel at 620 nm to correct for well-to-well variability of the signal

Article Snippet: Recombinant human PDE8A (Sigma SRP0273) was incubated with 100 ng FAK tyrosine kinase (Promega #V1971) 50μM DTT, 2mM MnCl 2 , 50μM ATP and 1x kinase buffer for 15 min at 37°C in a reaction of total volume 25 µL.

Techniques: Recombinant, Incubation

Journal: bioRxiv

Article Title: Regulation of intracellular cAMP levels in osteocytes by mechano-sensitive focal adhesion kinase via PDE8A

doi: 10.1101/2024.06.28.601153

Figure Lengend Snippet: Normally, active FAK phosphorylates PDE8A and maintains high basal PDE8A-mediated cAMP hydrolysis. FFSS causes a reduction of FAK activity leading to reduced FAK-mediated PDE8A phosphorylation and, in turn, reduced PDE8A-mediated cAMP breakdown. Thus, FAK inhibition leads to suppressed PDE8A function causing an accumulation of cAMP that is generated by upstream GPCR signaling.

Article Snippet: Recombinant human PDE8A (Sigma SRP0273) was incubated with 100 ng FAK tyrosine kinase (Promega #V1971) 50μM DTT, 2mM MnCl 2 , 50μM ATP and 1x kinase buffer for 15 min at 37°C in a reaction of total volume 25 µL.

Techniques: Activity Assay, Inhibition, Generated

Journal: Scientific Reports

Article Title: Disruption of the pro-oncogenic c-RAF–PDE8A complex represents a differentiated approach to treating KRAS–c-RAF dependent PDAC

doi: 10.1038/s41598-024-59451-3

Figure Lengend Snippet: Targeted c-RAF–PDE8A disruption. ( A ) Immunofluorescent co-staining of endogenous c-RAF (mouse α-c-RAF, green) and PDE8A (rabbit α–PDE8A, red) proteins in fixed PANC1 cells. Nuclei counterstained with DAPI (blue) and composite highlighting areas of c-RAF–PDE8A colocalisation (n > 40 cells, scale bar = 20 µm). ( B )(i), (ii) Proximity ligation assay (PLA) highlighting formation of c-RAF–PDE8A complex (red) within cytoplasm and nuclei of fixed PANC1 cells (scale bar = 20 µm). Disruption of c-RAF–PDE8A complex formation by (4 h) DRx-170, but not DRx-150 or vehicle (1% DMSO)–(ii) shown by dot plot (N = 3, n ≥ 90 cells per sample set). ( C ) RTCA (xCELLigence) analysis demonstrating how c-RAF–PDE8A disruption influences PANC1 cancer cell growth (N = 3). MEAN ± SEM, ns, not significant; **P < 0.01, ***P < 0.001; ****P < 0.0001.

Article Snippet: Primary antibodies included ERK1/2 (cell signaling, 4696), pERK1/2 (cell signaling, 9101), PDE8A (protein-tech, 13956–1-AP), c-RAF (cell signaling, 9422), c-RAF (sigma, R2404), c-RAF pS43 (Abcam, ab150365), c-RAF pS259 (cell signaling, 9421), KRAS (protein tech, 12063–1-AP), HSP90 (Santa Cruz, sc-7947).

Techniques: Disruption, Staining, Proximity Ligation Assay

Journal: Scientific Reports

Article Title: Disruption of the pro-oncogenic c-RAF–PDE8A complex represents a differentiated approach to treating KRAS–c-RAF dependent PDAC

doi: 10.1038/s41598-024-59451-3

Figure Lengend Snippet: PKA Associated c-RAF Inhibition. ( A ) Immunofluorescent staining of endogenous PDE8A (rabbit α–PDE8A, green) protein in fixed PANC1 cells following 4 h treatment with vehicle (DMSO), DRx-150 (10 µM), or DRx-170 (10 µM). Nuclei counterstained with DAPI (blue) and composite highlighting PDE8A localisation. Respective bar chart of PDE8A protein expression (bottom right, RFU, n ≥ 115 cells per condition, scale bar = 20 µm). ( B )(i) Representative immunoblots of [1st row] total c-RAF (normalised to HSP90), [2nd row] PKA-specific pS259 c-RAF (normalised to total c-RAF), [3rd row] PKA-specific pS43 c-RAF (normalised to total c-RAF) and [4th row] pT202/pY204 ERK1/2 (normalised to total ERK1/2) protein levels in PANC1 cells. Protein expression at 0 h (N = 3, lane 1) was compared with DRx-170 (4, 24 or 72 h; 0.3 or 3 µM, N = 3, lanes 2–7), DRx-150 (4 h, 3 µM, N = 2, lane 8). ( B )(ii)–(v) Data showing % change in protein expression vs. 0 h time-point represented as MEAN ± SEM (top), with N = 1–3 independent replicates presented as a heatmap (bottom). P, statistical significance; ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Primary antibodies included ERK1/2 (cell signaling, 4696), pERK1/2 (cell signaling, 9101), PDE8A (protein-tech, 13956–1-AP), c-RAF (cell signaling, 9422), c-RAF (sigma, R2404), c-RAF pS43 (Abcam, ab150365), c-RAF pS259 (cell signaling, 9421), KRAS (protein tech, 12063–1-AP), HSP90 (Santa Cruz, sc-7947).

Techniques: Inhibition, Staining, Expressing, Western Blot

Journal: Scientific Reports

Article Title: Disruption of the pro-oncogenic c-RAF–PDE8A complex represents a differentiated approach to treating KRAS–c-RAF dependent PDAC

doi: 10.1038/s41598-024-59451-3

Figure Lengend Snippet: c-RAF–PDE8A disruption suppresses PANC1 growth. ( A ) PANC1 (RTCA) growth following 60 h dose response [0.1–3 µM] with DRx-170 as ( A )(i) monotherapy and ( A )(ii) combination with [0.5 µM] afatinib. ( A )(iii) Respective Log(µM) IC50s (N = 3). ( B )(i) Day 0 brightfield images (scale bar = 100 µm) of 3D floating PANC1 spheroids and respective Day 10 images following treatment with vehicle [0.2% DMSO], DRx-150, DRx-170, afatinib or DRx-170 + afatinib. ( B )(ii) Spheroid area over 10 day period and respective bar chart (iii) depicting spheroid area fold-difference vs. Day 0 (n = 5). Red arrows indicate treatment time points. MEAN ± SEM, ns, not significant; **P < 0.01; ****P < 0.0001.

Article Snippet: Primary antibodies included ERK1/2 (cell signaling, 4696), pERK1/2 (cell signaling, 9101), PDE8A (protein-tech, 13956–1-AP), c-RAF (cell signaling, 9422), c-RAF (sigma, R2404), c-RAF pS43 (Abcam, ab150365), c-RAF pS259 (cell signaling, 9421), KRAS (protein tech, 12063–1-AP), HSP90 (Santa Cruz, sc-7947).

Techniques: Disruption

Journal: Scientific Reports

Article Title: Disruption of the pro-oncogenic c-RAF–PDE8A complex represents a differentiated approach to treating KRAS–c-RAF dependent PDAC

doi: 10.1038/s41598-024-59451-3

Figure Lengend Snippet: c-RAF–PDE8A disruption attenuates PANC1 adherence and migration. ( A ) Cell area (µm 2 ) in untreated vs. DMSO vs. DRx-170 (1 μM) treated (24 h) PANC1 cells (n ≥ 60 cells per group, N = 3, scale bar = 10 µm). ( B )(i) RTCA (xCELLigence) of PANC1 cell adherence following 8 h treatment with vehicle (1% DMSO), DRx-150 (0.14 µM) or DRx-170 (0.035–1.4 µM). ( B )(ii) Representative bar chart of relative PANC1 (slope of curve) adherence (N = 3). ns, not significant; #, P < 0.05 vs. DRx-150 and Vehicle. ( C )(i) PANC1 cell migration analysis (in vitro wound healing, scale bar = 500 µm) following 24 treatment with vehicle (1% DMSO), DRx-150 (1 µM), DRx-170 (0.1–10 µM). Blue outline highlights ‘wound’ at time point 0 h and 24 h. ( C )(ii) Representative bar chart of relative PANC1 migration (i.e., relative % wound gap closure) (N ≥ 3). ( D ) In vitro cell viability (endpoint) assessment of non-cancerous human cell lines HEK293, IMR-90 following 72 treatment with DRx-170 (0.001–10 µM, N ≥ 3). Horizontal line represents vehicle 100% viability control MEAN ± SEM, ns, not significant; *P < 0.05.

Article Snippet: Primary antibodies included ERK1/2 (cell signaling, 4696), pERK1/2 (cell signaling, 9101), PDE8A (protein-tech, 13956–1-AP), c-RAF (cell signaling, 9422), c-RAF (sigma, R2404), c-RAF pS43 (Abcam, ab150365), c-RAF pS259 (cell signaling, 9421), KRAS (protein tech, 12063–1-AP), HSP90 (Santa Cruz, sc-7947).

Techniques: Disruption, Migration, In Vitro, Control

Journal: Scientific Reports

Article Title: Disruption of the pro-oncogenic c-RAF–PDE8A complex represents a differentiated approach to treating KRAS–c-RAF dependent PDAC

doi: 10.1038/s41598-024-59451-3

Figure Lengend Snippet: Schematic illustrating how DRx-170 binds c-RAF, displaces PDE8A and exposes c-RAF to surrounding cAMP microenvironment in the context of KRAS MT cancer. De-protection negatively regulates c-RAF activity in a PKA-dependent manner (pS43/pS259 validated, pS233/pS621 untested), promoting c-RAF conformational closure and dissociation from upstream KRAS. This conservative model depicting DRx-170 mechanism of action highlights how DRx-170 attenuates tumourigenesis through facilitating the allosteric inhibition c-RAF. RBD ras binding domain; CRD cysteine rich domain; AC adenylate cyclase; cAMP cyclic adenosine monophosphate; ATP adenosine triphosphate; AMP adenosine monophosphate; PKA protein kinase A.

Article Snippet: Primary antibodies included ERK1/2 (cell signaling, 4696), pERK1/2 (cell signaling, 9101), PDE8A (protein-tech, 13956–1-AP), c-RAF (cell signaling, 9422), c-RAF (sigma, R2404), c-RAF pS43 (Abcam, ab150365), c-RAF pS259 (cell signaling, 9421), KRAS (protein tech, 12063–1-AP), HSP90 (Santa Cruz, sc-7947).

Techniques: Activity Assay, Inhibition, Binding Assay