Journal: International Journal of Molecular Sciences

Article Title: Mesothelioma-Associated Fibroblasts Modulate the Response of Mesothelioma Patient-Derived Organoids to Chemotherapy via Interleukin-6

doi: 10.3390/ijms25105355

Figure Lengend Snippet: IL-6 was increased in the medium of mPDO + MAF co-cultures after C+P challenge and mediated increased OFA. ( A ) A Luminex-based assay for eight cytokines was used to detect inflammatory chemokines in the conditioned media of mPDO+MAF co-cultures. The histogram shows the average levels of the indicated cytokines from two independent experiments. ( B ) IL-6 is mainly secreted by MAFs after C+P. mPDOs, MAFs, and co-cultured mPDO+MAFs were assayed for IL-6 secretion 24 h after the C+P challenge by indirect ELISA. Results are expressed as fold-over controls, where controls are ctrl (vehicle)-treated cells. ( C ) Increased IL-6 may mediate the resistance of mPDOs to C+P. Y axis: OFA score (number of organoids formed/1000 live cells) was calculated in mPDO cultures challenged with ctrl (MAF-CM, not treated with C+P) or with MAF-CM (treated with C+P), in the presence of anti-IL-6 neutralising antibody (IL-6-IgG) or a control antibody (ctrl-IgG). Histograms represent the mean + SE of three independent experiments. Statistics: ** p < 0.01; ns = not significant.

Article Snippet: Neutralizing monoclonal antibody against human interleukin 6 and its biologically inactive isotype ctrl ab Mouse IgG1, kappa was from Invivogen (Invivogen, San Diego, CA, USA) and was used as indicated in the main text. mRNA extraction and expression analysis.

Techniques: Luminex, Cell Culture, Indirect ELISA, Control

Journal: Alcohol, clinical & experimental research

Article Title: Protein kinase C epsilon-mediated modulation of T-type calcium channels underlies alcohol withdrawal hyperexcitability in the midline thalamus

doi: 10.1111/acer.15342

Figure Lengend Snippet: PKC mediates a T channel gain of function during alcohol withdrawal. (A–C) Representative traces of T-type current inactivation curves for (A) Air, (B) WD, and (C) WD + PKC (−) groups, respectively, elicited at −50 mV from holding potentials ranging from −135 to −60 mV for 1 s (inset). (D, E) Alcohol WD produced a significant depolarizing shift in the inactivation curve of native T-type calcium current. The depolarizing shift was blocked by PKC peptide inhibitors, including PKC epsilon-specific inhibitors. (F) V 50 derived from the inactivation curve ~5 h. into the 4th alcohol WD in C57BL/6 mice was analyzed. The significant depolarizing shift for V 50 was blocked by the PKC peptide inhibitor (PKC (−), 10 μM). This inhibition was mimicked by specific PKCε isoform inhibitors (#530993, 539522; 5 μM) but not a PKCε translocation inhibitor inactive peptide (#539542; 5 μM). All drugs were delivered through the internal pipette solution. Statistics for V 50 showed F (6, 73) = 3.87, p = 0.0021 (One-Way ANOVA). In the plot * indicates p <0.05 and ** indicates p <0.01.

Article Snippet: #539542 (Calbiochem) , PKCε Inactive peptide, negative control , WD vs WD + PKC inactive peptide , ns (p = 0.529), n = 24 , 4.26 ± 7.899.

Techniques: Produced, Derivative Assay, Inhibition, Translocation Assay, Transferring

Journal: Alcohol, clinical & experimental research

Article Title: Protein kinase C epsilon-mediated modulation of T-type calcium channels underlies alcohol withdrawal hyperexcitability in the midline thalamus

doi: 10.1111/acer.15342

Figure Lengend Snippet: Summary of inhibitors and results ( n = cell numbers).

Article Snippet: #539542 (Calbiochem) , PKCε Inactive peptide, negative control , WD vs WD + PKC inactive peptide , ns (p = 0.529), n = 24 , 4.26 ± 7.899.

Techniques: Translocation Assay, Negative Control

Journal: Alcohol, clinical & experimental research

Article Title: Protein kinase C epsilon-mediated modulation of T-type calcium channels underlies alcohol withdrawal hyperexcitability in the midline thalamus

doi: 10.1111/acer.15342

Figure Lengend Snippet: Kinetic changes of T-channel during the application of PKC inhibitors recorded from the reuniens after 5 h into 4th alcohol withdrawal. (A, B) PKCε inhibitor (#530993, 5 μM) was applied through the bath solution, it significantly reduced rise time (A. paired t test, p = 0.0311, n = 6); increased rise slope (B. paired t test, p = 0.0266, n = 6); (C) Rise time did not show a significant main effect [ F (4, 53) = 2.205, p = 0.0809], although Dunnett’s post hoc test showed a significant difference between WD and WD plus PKCε (−), (#529522, p = 0.0223.* p <0.05). All drugs were delivered through the patch pipette internal solution, including the PKC peptide inhibitor (10 μM), and PKCε inhibitors (#530993, 5 μM; #539522, 5 μM).

Article Snippet: #539542 (Calbiochem) , PKCε Inactive peptide, negative control , WD vs WD + PKC inactive peptide , ns (p = 0.529), n = 24 , 4.26 ± 7.899.

Techniques: Transferring

Journal: Alcohol, clinical & experimental research

Article Title: Protein kinase C epsilon-mediated modulation of T-type calcium channels underlies alcohol withdrawal hyperexcitability in the midline thalamus

doi: 10.1111/acer.15342

Figure Lengend Snippet: Midline thalamic neurons exhibit more spikes per low-threshold Ca 2+ burst during alcohol withdrawal. PKCε translocation inhibitor peptide (#539522) reduced the number of spikes/burst after 4th WD. (A–E) representative traces for the bursts elicited near resting membrane potential by prepulse negative current injection of −200 pA, 800 ms. (F) Statistics for the bursts induced by prepulse negative current injection of −200 pA. One-way ANOVA: F (4, 56) = 6.321, p = 0.0003, Dunnett’s multiple comparisons test, WD and Control, p = 0.0013; WD and Air exposed, p = 0.0461; WD and WD + PKCε (−), p = 0.0469; WD and WD + PKCε inactive peptide, p = 0.9990. In the plots * indicates p <0.05, and ** indicates p <0.01.

Article Snippet: #539542 (Calbiochem) , PKCε Inactive peptide, negative control , WD vs WD + PKC inactive peptide , ns (p = 0.529), n = 24 , 4.26 ± 7.899.

Techniques: Translocation Assay, Membrane, Injection, Control

Journal: Alcohol, clinical & experimental research

Article Title: Protein kinase C epsilon-mediated modulation of T-type calcium channels underlies alcohol withdrawal hyperexcitability in the midline thalamus

doi: 10.1111/acer.15342

Figure Lengend Snippet: T-current-mediated bursting was elicited near resting membrane potential from a series of hyperpolarizing prepulse current injections. Using a procedure analogous to the voltage clamp inactivation protocol (current injections started from 0 to −400 pA, −50 pA step, 500 ms of duration), we measured T-current-mediated bursting. Control (A) and air-exposed (B) conditions elicit 1–3 action potentials/burst. After the 4th WD (C), the cells responded with increased numbers of spikes/burst, which were reduced by the PKC ε inhibitor (D) but not the PKC ε inhibitor inactive peptide (E).

Article Snippet: #539542 (Calbiochem) , PKCε Inactive peptide, negative control , WD vs WD + PKC inactive peptide , ns (p = 0.529), n = 24 , 4.26 ± 7.899.

Techniques: Membrane, Control