Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Synergistic targeting of the regulatory and catalytic subunits of PI3Kδ in mature B cell malignancies

doi: 10.1158/1078-0432.CCR-17-2218

Figure Lengend Snippet: (A) Cell proliferation data for six independent DLBCL lines were accrued following 48h (HBL-1, WSU-NHL, OCI-Ly7,) or 72h (OCI-Ly3, OCI-Ly10, OCI-Ly18) exposure to the indicated compounds at progressively increasing doses. The combination of roflumilast and idelalisib synergistically suppressed cell proliferation relative to single-agent treatments as determined by the combination index (CI) analysis; CI <0.1 very strong synergism; CI=0.1–0.3 strong synergism; CI=0.3–0.85 synergism. (B) The combination of roflumilast and idelalisib significantly enhanced the suppression of PI3K activity relative to single agent treatments (*** p<0.001 ** p<0.01, ANOVA; Bonferroni’s multiple comparisons post-test, single agents relative to combination). Data shown are mean ± SD of experiments completed in triplicate. At least three biologic replicates were completed to all assays.

Article Snippet: Proliferation of DLBCL cell lines in response to increasing doses of the PDE4 inhibitor roflumilast (1.25 to 10μM) and the PI3Kδ inhibitor idelalisib (0.03 to 0.6μM), used as single agents or in combination, was measured using the CellTiter Proliferation assay (MTS; Promega, Madison, WI).

Techniques: Activity Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Synergistic targeting of the regulatory and catalytic subunits of PI3Kδ in mature B cell malignancies

doi: 10.1158/1078-0432.CCR-17-2218

Figure Lengend Snippet: (A) Tumor volume in mice inoculated with OCI-Ly7 and randomized into four treatment arms: 1) vehicle control, 2) roflumilast, 3) idelalisib 4) idelalisib and roflumilast. The cohort treated with the combination of idelalisib and roflumilast showed significantly reduced tumor volume relative to the single agent and vehicle groups at days 10 and 14 of dosing (*p<0.05, two tailed Student’s t-test). (B) All mice were sacrificed and tumors harvest on treatment day 14. PI3K activity was quantified in all tumors, and those from mice treated with the combination of PDE4 and PI3Kδ inhibitors showed significantly more pronounced suppression of PI3K activity relative to single-agent treatments (p<0.001, two-tailed Student’s t-test). Tumor volume data are mean ± SEM (6 mice/group), and PI3K activity data are mean ± SD of 24 tumors (6/group), each quantified in triplicate.

Article Snippet: Proliferation of DLBCL cell lines in response to increasing doses of the PDE4 inhibitor roflumilast (1.25 to 10μM) and the PI3Kδ inhibitor idelalisib (0.03 to 0.6μM), used as single agents or in combination, was measured using the CellTiter Proliferation assay (MTS; Promega, Madison, WI).

Techniques: Control, Two Tailed Test, Activity Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Synergistic targeting of the regulatory and catalytic subunits of PI3Kδ in mature B cell malignancies

doi: 10.1158/1078-0432.CCR-17-2218

Figure Lengend Snippet: (A) Annexin V data from 10 independent primary CLL samples was obtained by FACS following 96h of exposure to the indicated compounds either as single agents (10 μM roflumilast, 0.5μM idelalisib) or in combination. The combination of roflumilast and idelalisib significantly enhanced apoptosis relative to single-agent treatments (*** p<0.001, ** p<0.01, * P<0.05 – Bonferroni’s post-tests, single agents relative to combination; Two-way ANOVA P<0.0001). Data shown are mean ± SD of measurement performed in triplicate. (B) The combination of roflumilast and idelalisib is significantly more effective in suppression of PI3K activity in primary CLL samples than each agent used alone (p<0.05, two-sided Student’s t-test). Data are mean ± SD of primary CLL exposed the indicated agents, each sample quantified in triplicate.

Article Snippet: Proliferation of DLBCL cell lines in response to increasing doses of the PDE4 inhibitor roflumilast (1.25 to 10μM) and the PI3Kδ inhibitor idelalisib (0.03 to 0.6μM), used as single agents or in combination, was measured using the CellTiter Proliferation assay (MTS; Promega, Madison, WI).

Techniques: Activity Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Synergistic targeting of the regulatory and catalytic subunits of PI3Kδ in mature B cell malignancies

doi: 10.1158/1078-0432.CCR-17-2218

Figure Lengend Snippet: (A) Western blot analysis shows that elevation of intracellular cyclic-AMP levels with forskolin is associated with a decrease in phosphorylation of the P85/P55 subunit of PI3K, with a consequent reduction in phospholipid PI(3,4,5)P3 production by PI3K in the PDE4-null/low DLBCL cell lines SU-DHL4, SU-DHL6, and SU-DHL10; left and right panels, respectively (*** p<0.001 ** p<0.01, two-tailed Student’s t-test). (B) Western blots show that roflumilast suppresses P85 phosphorylation, and consequently PI3K activity, in the PDE4-high cell lines HBL-1, OCI-Ly3, and OCI-Ly10; left and right panels, respectively (***p<0.001, two tailed Student’s t-test). (C) Western blot analysis shows that expression of wild-type (WT) PDE4B but not of a phosphodiesterase-inactive (PI) mutant enzyme abrogates cyclic-AMP inhibitory effects on P85 phosphorylation and PI3K activity, left and right panels, respectively (*** p<0.001, two-tailed Student’s t-test). (D) Western blot analysis shows that stable expression of a constitutively active (CA) SYK variant blunts the cyclic-AMP inhibitory effects on P85 phosphorylation and PI3K activity, left and right panels, respectively. (**p<0.01, two- tailed Student’s t-test). Cell expressing PDE4B-WT and –PI are included as controls. (E) Western blot analysis shows that roflumilast but not idelalisib suppresses P85 phosphorylation in PDE4B-high DLBCL cell lines. All data shown are mean ± SD of assays performed in triplicate. A minimum of three biologic replicates were completed for each assay. Densitometric quantification of pP85/P55 suppression is shown at the bottom of the western blots.

Article Snippet: Proliferation of DLBCL cell lines in response to increasing doses of the PDE4 inhibitor roflumilast (1.25 to 10μM) and the PI3Kδ inhibitor idelalisib (0.03 to 0.6μM), used as single agents or in combination, was measured using the CellTiter Proliferation assay (MTS; Promega, Madison, WI).

Techniques: Western Blot, Phospho-proteomics, Two Tailed Test, Activity Assay, Expressing, Mutagenesis, Variant Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Synergistic targeting of the regulatory and catalytic subunits of PI3Kδ in mature B cell malignancies

doi: 10.1158/1078-0432.CCR-17-2218

Figure Lengend Snippet: (A) Western blot analysis shows that roflumilast suppresses BTK phosphorylation in multiple PDE4-high DLBCL cell lines (B) Western blots show that elevation of intracellular cyclic-AMP levels with forskolin suppresses BTK phosphorylation in multiple PDE4-null/low DLBCL cell lines (C) Western blot analysis shows that idelalisib does not suppress BTK phosphorylation at Y223. (D) Western blot shows that expression of PDE4B-WT or SYK-CA, but not PDE4B-PI, blunts the suppressive cyclic-AMP effects towards BTK phosphorylation. At least three biologic replicates were completed for each assay. Densitometry with quantification of pBTK suppression is shown at the bottom of the western blots.

Article Snippet: Proliferation of DLBCL cell lines in response to increasing doses of the PDE4 inhibitor roflumilast (1.25 to 10μM) and the PI3Kδ inhibitor idelalisib (0.03 to 0.6μM), used as single agents or in combination, was measured using the CellTiter Proliferation assay (MTS; Promega, Madison, WI).

Techniques: Western Blot, Phospho-proteomics, Expressing

Journal: bioRxiv

Article Title: Image Correlation Spectroscopy is a Robust Tool to Quantify Cellular DNA Damage Response

doi: 10.1101/2024.08.05.606697

Figure Lengend Snippet: ( A ) RPA1 DA as a function of olaparib concentration for HT1080 and HCC1937 cells, shown are average with SEM, n >= 504 cells, 3 biological repeats, and linear fit. ( B ) RPA1 DA in cells treated with 1 μM PARPi overnight, shown are average, normalized to untreated control, with SEM, n >= 350 cells, 2 biological repeats. ( C ) RPA1 DA as a function of cell line IC50 for each cell line and PARPi shown in . Shown are average, normalized to untreated control, with SEM, n >= 350 cells, 2 biological repeats and linear fit (prism) for each cell line. ( D ) The linear fit slope of RPA DA vs. cell line IC50 as a function of average PARPi z-score for each cell line. Shown are slope fit with SEM and average z-score over three PARPi with st. dev. ( E ) Western blot of RPA1 expression levels for each cell line, with GAPDH loading control. ( F ) Normalized protein expression level in each cell line as a function of PARPi z score. Shown are average z-score over three PARPi with st. dev.

Article Snippet: Using ICS DA, we were able to detect the dose dependent response to two PARP inhibitors, matching or exceeding dose-dependent sensitivity of foci counting via Imaris Spots.

Techniques: Concentration Assay, Control, Western Blot, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Three-dimensional NMR Structure of Hen Egg Gallin (Chicken Ovodefensin) Reveals a New Variation of the β-Defensin Fold *

doi: 10.1074/jbc.M113.507046

Figure Lengend Snippet: Antimicrobial activity of gallin against E. coli ATCC 25922. Left, dose response of gallin and its inhibitory effect on E. coli ATCC 25922, reflected by the diameter of the clear zone (inhibition zone is expressed in units = the diameter in mm of the clear zone on the plate minus the diameter in mm of the well). Each point represents the means ± S.E. (error bars) of three separate experiments. Right, the best fit straight line was determined by linear regression. MIC of gallin versus E. coli ATCC 25922 was calculated by finding the intersection of the line with the x axis, indicating the lowest peptide concentration at which no clear zone was detected.

Article Snippet: Left , dose response of gallin and its inhibitory effect on E. coli ATCC 25922, reflected by the diameter of the clear zone (inhibition zone is expressed in units = the diameter in mm of the clear zone on the plate minus the diameter in mm of the well).

Techniques: Activity Assay, Inhibition, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Three-dimensional NMR Structure of Hen Egg Gallin (Chicken Ovodefensin) Reveals a New Variation of the β-Defensin Fold *

doi: 10.1074/jbc.M113.507046

Figure Lengend Snippet: MIC of synthesized gallin

Article Snippet: Left , dose response of gallin and its inhibitory effect on E. coli ATCC 25922, reflected by the diameter of the clear zone (inhibition zone is expressed in units = the diameter in mm of the clear zone on the plate minus the diameter in mm of the well).

Techniques: Synthesized

Journal: The Journal of Biological Chemistry

Article Title: TBK1 interacts with tau and enhances neurodegeneration in tauopathy

doi: 10.1016/j.jbc.2021.100760

Figure Lengend Snippet: IP-MS analysis of human postmortem brains identifies novel tau-interacting kinases. A , Gene ontology (GO) analysis of AD tau-interacting proteins identified terms associated with “kinase binding,” “phosphotransferase,” “phosphoprotein binding,” and “kinase activity” (highlighted in red ). B , identification and quantification of 21 tau-interacting kinases. Tau-interacting kinases were selected from our previously published tau interactome MS data generated from four control and four AD samples . A graph depicts increased log 2 intensities of tau-interacting kinases in AD (n = 4) compared with controls (n = 4) following co-IP MS as previously reported . The intensity for each kinase is divided by the group (AD or control) geomean and log 2 fold changes are plotted. All kinases had significant log 2 -fold increase in AD (Student’s t -test, p ≤ 0.05). Novel tau-interacting kinases (TBK1, DAPK3, and TNIK) are highlighted in bold . C , Western blot analysis of AD (n = 3) and control (n = 3) postmortem brain inputs using tau antibodies (Tau5) shows high-molecular-weight (HMW) tau species in AD, whereas DAPK3 (~52 kDa) and TBK1 (~84 kDa) western blots show equal levels of both kinases in AD and control samples. GAPDH was used a loading control in inputs. D , IP of tau followed by immunoblotting for DAPK3 and TBK1 in AD brain compared with controls showed interaction of AD tau with both DAPK3 and TBK1. Lysates (pooled AD or control) incubated with isotype matched IgG were used as controls. Asterisk indicates nonspecific heavy IgG chains in negative control samples.

Article Snippet: For both time course in vitro kinase assay and TBK1 inhibitor dose–response reactions, 400 ng of purified tau and 200 ng of active TBK1 were used and reactions were quenched by boiling at 98 °C in Laemmli sample buffer (BioRad, 161-0737) for 5 min prior to western blot analysis.

Techniques: Protein-Protein interactions, Binding Assay, Activity Assay, Generated, Control, Co-Immunoprecipitation Assay, Western Blot, High Molecular Weight, Incubation, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: TBK1 interacts with tau and enhances neurodegeneration in tauopathy

doi: 10.1016/j.jbc.2021.100760

Figure Lengend Snippet: Increased activation and interaction of TBK1 in AD and related tauopathies. A , Western blot analysis of postmortem brain tissues from control and AD cases for total TBK1 and phospho-TBK1 (pS172-TBK1) in AD compared with controls. B , quantification of the pS172-TBK1 levels in AD and control samples shows a significant increase in AD. Each group is plotted as the mean ± standard deviation (Student’s t -test, n = 3, ∗∗∗ = p < 0.001). C , Western blot analysis of control ( n = 2), AD ( n = 2) and an FTDP-17 (R406W) (n = 1) postmortem brain inputs using total TBK1 and phospho-TBK1 (pS172-TBK1) antibodies showed increased pS172-TBK1 in AD and FTDP-17 (R406W). D , IP of tau (Tau5) followed by immunoblotting for TBK1 shows interaction of TBK1 with tau from AD (positive control) and FTDP-17 brain. Cell lysate incubated with beads alone was used as a negative control (nc). E , Western blot analysis of inputs from control (lane 1), AD (lane 2), and additional tauopathy cases: including FTDP-17 (R406W, lane 3), P301L (lanes 4 and 5), and CBD (n = 3) brain samples using Tau5 and TBK1 showed increased HMW tau species in AD and FTDP-17 and CBD cases (lanes 6, 7, and 8), while tau and TBK1 levels remained the same. F , Western blot analysis of tau IPs using TBK1 antibody showed interaction of tau and TBK1 in AD and other tauopathies. GAPDH was used as a loading control. Pooled (AD) lysate incubated with beads alone was used as a negative control (lane 9).

Article Snippet: For both time course in vitro kinase assay and TBK1 inhibitor dose–response reactions, 400 ng of purified tau and 200 ng of active TBK1 were used and reactions were quenched by boiling at 98 °C in Laemmli sample buffer (BioRad, 161-0737) for 5 min prior to western blot analysis.

Techniques: Activation Assay, Western Blot, Control, Standard Deviation, Positive Control, Incubation, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: TBK1 interacts with tau and enhances neurodegeneration in tauopathy

doi: 10.1016/j.jbc.2021.100760

Figure Lengend Snippet: TBK1 directly phosphorylates tau. A , MS analysis of in vitro TBK1 kinase assay using recombinant full-length tau (1 μg) and active TBK1 (0.5 μg) was conducted in the presence of 100 μM ATP. After 30 min incubation the assay was quenched, and proteins were digested with trypsin for subsequent MS analysis. A schematic diagram depicts nine putative TBK1-mediated phosphorylation sites identified by MS across tau domains and the phosphopeptide sequences identified. N-terminus (N-term), proline-rich-domain (PRD), microtubule-binding repeats (MTBR) and C-terminus (C-term). TBK1-targeted Ser/Thr residues are indicated in red with corresponding phosphopeptide intensities measured in the mass spectrometer. B , tandem MS/MS spectrum of a tau phosphopeptide with S214 phosphorylation site (residues 212–224) shows b- and y-ions and the diagnostic neutral loss of phosphoric acid (–98 Da) on the precursor peptide. C , Western blotting of in vitro kinase reactions using site-specific phospho-tau antibodies (pS214, pS324, and pS356) following 0, 5, 10, 20, and 40 min (m) incubation shows gradual increase in tau phosphorylation. D , inhibition of TBK1 in vitro tau phosphorylation with increasing concentration (5, 10, 20, or 40 μM) of a small-molecule TBK1 inhibitor (BX795). Assays were quenched after 30 min. Western blot analysis of the in vitro kinase assay using pS214-Tau shows a dose-dependent decrease in S214 phosphorylation with increasing BX795 concentration. Western blots for active p172-TBK1 and total tau serve as controls.

Article Snippet: For both time course in vitro kinase assay and TBK1 inhibitor dose–response reactions, 400 ng of purified tau and 200 ng of active TBK1 were used and reactions were quenched by boiling at 98 °C in Laemmli sample buffer (BioRad, 161-0737) for 5 min prior to western blot analysis.

Techniques: In Vitro, Kinase Assay, Recombinant, Incubation, Phospho-proteomics, Binding Assay, Mass Spectrometry, Tandem Mass Spectroscopy, Diagnostic Assay, Western Blot, Inhibition, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: TBK1 interacts with tau and enhances neurodegeneration in tauopathy

doi: 10.1016/j.jbc.2021.100760

Figure Lengend Snippet: Increased phosphorylation of putative TBK1 tau phosphosites in AD brain. A , quantification of AD brain tau phosphosites from previously published brain IMAC phosphoproteome data . IMAC was used for phosphopeptide enrichment followed by LC-MS/MS analysis for phosphopeptide identification and quantification. A graph depicts the increased fold change of tau phosphosites in AD (n = 9) compared with controls (n = 10). The phosphopeptide intensity for each site is divided by the group (AD or control) geomean and log 2 fold changes are plotted. Putative TBK1 sites identified by in vitro kinase assay are highlighted in red boxes . B , Venn diagram shows an overlap in tau phosphosites identified from IMAC brain phosphoproteome , Tau5-IP , phospho-tau IP (PHF-Tau) , and TBK1 in vitro kinase assay.

Article Snippet: For both time course in vitro kinase assay and TBK1 inhibitor dose–response reactions, 400 ng of purified tau and 200 ng of active TBK1 were used and reactions were quenched by boiling at 98 °C in Laemmli sample buffer (BioRad, 161-0737) for 5 min prior to western blot analysis.

Techniques: Phospho-proteomics, Liquid Chromatography with Mass Spectroscopy, Control, In Vitro, Kinase Assay

Journal: The Journal of Biological Chemistry

Article Title: TBK1 interacts with tau and enhances neurodegeneration in tauopathy

doi: 10.1016/j.jbc.2021.100760

Figure Lengend Snippet: TBK1 interacts with and alters tau phosphorylation in HEK-293 cell lines. A , HEK-293 cells were transfected with Myc-TBK1 in the presence or absence of GFP-Tau. Western blot using pS172-TBK1 shows TBK1 activation independent of tau expression. B , Western blotting for Myc and GFP shows expression of both recombinant Myc-TBK1 and GFP-Tau, respectively, in cotransfected HEK-293 cell lysates (inputs). Tubulin was used as a loading control. C , IP of tau using a GFP antibody followed by western blotting for Myc and pS172-TBK1 antibodies shows a positive interaction of tau with total recombinant TBK1 and activated TBK1 (pS172-TBK1), respectively. Western blot for GFP served as a positive control and beads alone were used as a negative control. The bead control lane from the pS172-TBK1 blot was cropped from the same blot. D , IP for Myc followed by western blotting for GFP shows recombinant tau and TBK1 interaction in HEK-293 cells. Pull-downs from cells transfected with tau alone or with bead only served as negative controls. E , overexpression of TBK1 in HEK-293 cells results in increased tau phosphorylation. HEK-293 cells were transfected with recombinant Myc-tagged TBK1 (Myc-TBK1) and either recombinant GFP-tagged tau (GFP-Tau) or a mutant tau (GFP-Tau null ) with all nine TBK1 sites substituted with alanine. Cells were harvested 48 h posttransfection and subjected to western blot analysis using phospho-tau (S214, S324, S356, and T231) and GFP antibodies. Western blot analysis shows increased tau phosphorylation at TBK1 sites (S214, S324, and S356) but not at T231. Overexpression of TBK1 results in shift in GFP-Tau phosphorylated species (upper band, arrows ). In contrast, the mutant tau (Tau null ) showed loss of tau phosphorylation and decreased GFP-Tau phosphorylated species ( arrows ). F , a graph shows percent max phosphopeptide intensities for in vitro TBK1 tau sites. Phosphopeptide enrichment using IMAC and MS analysis of HEK-293 cell lysates described above showed increased phosphorylation of tau at putative TBK1 sites following TBK1 overexpression, whereas phosphorylation at other tau sites, not TBK1 in vitro sites, remained unchanged. (Student’s t -test, n = 3, ∗ ≤0.05, ∗∗ ≤0.01, ∗∗∗ ≤0.001). G , HEK-293 cells transfected with GFP-Tau were treated with 50 μM BX795 or DMSO for 6 h in triplicate. Western analysis using pS172-TBK1, phospho-Tau antibodies (S214, S324, and S356), and GFP antibodies shows decreased pS172-TBK1 and phospho-Tau signals, whereas TBK1 levels remained unchanged. H , quantification of protein intensities shows a significant decrease in pS172-TBK1 and phospho-tau (S214, S324, and S356) protein levels upon BX795 treatment. Protein intensities were normalized using GAPDH protein intensities (ANOVA, n = 3, ∗∗∗= p < 0.001, graphs represent mean ± SD).

Article Snippet: For both time course in vitro kinase assay and TBK1 inhibitor dose–response reactions, 400 ng of purified tau and 200 ng of active TBK1 were used and reactions were quenched by boiling at 98 °C in Laemmli sample buffer (BioRad, 161-0737) for 5 min prior to western blot analysis.

Techniques: Phospho-proteomics, Transfection, Western Blot, Activation Assay, Expressing, Recombinant, Control, Positive Control, Negative Control, Over Expression, Mutagenesis, In Vitro

Journal: The Journal of Biological Chemistry

Article Title: TBK1 interacts with tau and enhances neurodegeneration in tauopathy

doi: 10.1016/j.jbc.2021.100760

Figure Lengend Snippet: Altering TBK1 expression in vivo affects tau S214 phosphorylation and neurotoxicity in flies. A , transgenic flies expressing either Drosophila TBK1 (dTBK1) or human TBK1 ( hTBK1 ) in conjunction with human mutant Tau R406W in the adult brain ( Elav>Tau R406W + UAS hTBK1-cDNA or Elav>Tau R406W + UAS dTBK1-cDNA ) enhances Tau expression and S214 phosphorylation levels when compared with Elav>Tau R406W and quantified in ( B , n = 5). C , qPCR quantification ( n = 3) shows that RNAi-mediated knockdown of dTBK1 causes a 60% loss of mRNA transcript levels in flies when compared with controls. Flies not expressing human tau ( Elav alone) serve as negative control and show no human tau expression. D , RNAi knockdown of TBK1 suppresses tau expression and S214 phosphorylation in Elav>Tau R406W flies ( Elav>Tau R406W + UAS dTBK1-RNAi ) when compared with controls (Elav>Tau R406W ) and quantified in ( E , n = 5). Protein level quantification in panels B and E shows total-Tau (Tau) expression normalized to actin, and S214 phosphorylation (pTau-S214) is quantified as pS214 levels divided by normalized total-Tau. F , TUNEL staining in 10-day-old transgenic fly brains expressing either human TBK1 (hTBK1) or Drosophila TBK1 (dTBK1) in conjunction with human mutant Tau R406W ( Elav>Tau R406W + UAS hTBK1-cDNA or Elav>Tau R406W + UAS dTBK1-cDNA ) enhances neuronal death when compared with controls ( Elav>Tau R406W ). In contrast, RNAi knockdown of TBK1 in Elav>Tau R406W flies ( Elav>Tau R406W + UAS dTBK1-RNAi ) suppressed neuronal death when compared with controls ( Elav>Tau R406W , panels F and G ). G , quantification of TUNEL-positive nuclei based on analysis of at least ten flies for each genotype. Empty symbols mean no mutant Tau expression in these animals. H , assessment of retinal function using electroretinograms (ERGs). Representative traces showing light-induced depolarization (Left) in the following genotypes: (i) Control ( Rh1-Gal4; +/+ ), (ii) dTBK-RNAi ( Rh1-Gal4; UAS-dTBK-RNAi/+ ), (iii) Tau WT ( Rh1-Gal4; UAS-Tau WT /+ ), and (iv) dTBK-RNAi + Tau WT ( Rh1-Gal4; UAS-Tau WT /UAS-dTBK-RNAi ). I , RNAi knockdown of dTBK1 in transgenic flies expressing human wild-type Tau in the retina. Empty symbols mean no wild-type Tau expression in these animals. All error bars denote SEM. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; ns, not significant. Statistical tests performed were Student’s t -test ( C ), two-way ( B , E ), and one-way ( G , I ) ANOVA followed by Dunnett’s post hoc test for multiple comparisons.

Article Snippet: For both time course in vitro kinase assay and TBK1 inhibitor dose–response reactions, 400 ng of purified tau and 200 ng of active TBK1 were used and reactions were quenched by boiling at 98 °C in Laemmli sample buffer (BioRad, 161-0737) for 5 min prior to western blot analysis.

Techniques: Expressing, In Vivo, Phospho-proteomics, Transgenic Assay, Mutagenesis, Knockdown, Negative Control, TUNEL Assay, Staining, Control

Journal: PLOS Pathogens

Article Title: DCLK1 isoform (DCLK1-S) as a critical player in promoting inflammation, tissue remodeling, and EMT in mouse models of colitis

doi: 10.1371/journal.ppat.1013360

Figure Lengend Snippet: A. Paraffin sections prepared from the colons of Control, CR or CR+DBZ group of Dclk1 ΔIEC mice were subjected to IMC. MMP13 (green) is overlayed with DCLK1 (red) and DNA (blue). Boxed areas in CR group indicates significant co-localization of MMP13 with DCLK1. Scale bars as indicated (100 μ m); 8-10 mice /group. B. Box plots of DCLK1 and MMP13 counts based on IMC data set in the Control, CR, CR+DBZ groups. C. DCLK1 and MMP13 staining from IMC in control and Dclk1-S OE group after DSS-induced colitis. Lane 1 DCLK1(red) staining is overlayed with DNA (blue), Lane 2 MMP13 (red) staining is overlayed with DNA (blue), Lane 3 DCLK1(red)/MMP13(green)/DNA(Blue) colocalization in the Dclk1-S OE mice. P values as indicated. D. MMP13 promoter-reporter activity (*, ** p <0.05; n = 3 independent experiments). Ei. Western blot data of HCT116 colon cancer cells treated with PMA and PMA+DBZ. Eii. In silico molecular docking studies to predict MMP13 and DCLK1 binding. CR: Citrobacter rodentium , CR+DBZ: Citrobacter rodentium + Dibenzazepine (DBZ), PMA: Phorbol 12-Myristate 13-Acetate.

Article Snippet: By utilizing RKO colon cancer cell line, we discovered that in response to PMA, a significant increase in MMP13 enzymatic activity was recorded with a dose-dependent decrease in response to MMP13 inhibitor WAY-170523 (Cat.# 2633, TOCRIS Biosciences; ).

Techniques: Control, Staining, Activity Assay, Western Blot, In Silico, Binding Assay

Journal: PLOS Pathogens

Article Title: DCLK1 isoform (DCLK1-S) as a critical player in promoting inflammation, tissue remodeling, and EMT in mouse models of colitis

doi: 10.1371/journal.ppat.1013360

Figure Lengend Snippet: RKO cells were treated with PMA or PMA plus selective and potent MMP13 inhibitor WAY 170523 at varying doses as indicated for 30 min followed by measurement of enzymatic activity. A. Reference curve showing relative fluorescence units (RFUs). B, C. Dose-dependent decrease in MMP13 enzymatic activity (n = 3 independent experiments; * p <0.05). D. Western blots showing relative protein abundance in three colon cancer cell lines. Boxed area represents levels of the indicated proteins in RKO cells. E. Subcellular compartmentalization of proteins from RKO cells. 1: Cytosolic Fraction, 2: Nuclear Fraction, 3: Membrane Fraction, 4: Cytoskeletal fraction (n = 3 independent experiments). F. Promoters for DCLK1-L and DCLK1-S isoforms were cloned and transfected in HEK293 cells and promoter-reporter activity assays were performed using Dual-Luciferase Reporter Assay System (E1910, Promega, Madison, WI). Luminescence was measured using a BioTek Synergy Neo luminometer. P values as indicated; n = 3 independent experiments.

Article Snippet: By utilizing RKO colon cancer cell line, we discovered that in response to PMA, a significant increase in MMP13 enzymatic activity was recorded with a dose-dependent decrease in response to MMP13 inhibitor WAY-170523 (Cat.# 2633, TOCRIS Biosciences; ).

Techniques: Activity Assay, Fluorescence, Western Blot, Quantitative Proteomics, Membrane, Clone Assay, Transfection, Luciferase, Reporter Assay

Journal: PLOS Pathogens

Article Title: DCLK1 isoform (DCLK1-S) as a critical player in promoting inflammation, tissue remodeling, and EMT in mouse models of colitis

doi: 10.1371/journal.ppat.1013360

Figure Lengend Snippet: A-D. Molecular docking study showing the interaction of proMMP13 (A) and mature MMP13 (C) with DCLK1-S. B. Ser 73 and Ser 114 highlighted in green in proMMP13, represent the phosphorylation sites for DCLK1-S. D. Arg207 and Gln211 are donor sites from MMP13 in mature protein close to Thr197 (circled). E. Both rhMMP13 and rhDCLK1 were incubated in kinase buffer in presence or absence of ATP at 30 °C for 1 hr. rhMMP13 didn’t show any autophosphorylation in the same condition. Phosphorylation was detected by pan phospho-Serine/Threonine antibody. F. rhMMP13 was incubated with rhDCLK1 in presence of ATP at 30 °C for 1 hr. Phosphorylated rhMMP13 was detected with immunoblotting using pan phospho-Serine/Threonine antibody. Representative immunoblots showing the levels of phosphorylation of rhMMP13. Right panel represents the Coomassie staining (n = 3 independent experiments). G, H. Protein band intensity ratios, pMMP13/pDCLK1 and pMMP13/Total MMP13, showing rhMMP13 phosphorylation by rhDCLK1.

Article Snippet: By utilizing RKO colon cancer cell line, we discovered that in response to PMA, a significant increase in MMP13 enzymatic activity was recorded with a dose-dependent decrease in response to MMP13 inhibitor WAY-170523 (Cat.# 2633, TOCRIS Biosciences; ).

Techniques: Phospho-proteomics, Incubation, Western Blot, Staining

Journal: PLOS Pathogens

Article Title: DCLK1 isoform (DCLK1-S) as a critical player in promoting inflammation, tissue remodeling, and EMT in mouse models of colitis

doi: 10.1371/journal.ppat.1013360

Figure Lengend Snippet: A. Tissue sections prepared from the colons of indicated groups of mice were subjected to immunostaining with antibodies against MMP13 (red), α -SMA (green), Collagen (green), E-cadherin (green) and Vimentin (green). Samples were analyzed using the Hyperion Imaging System (Standard BioTools). DAPI (blue) was used to label DNA. Scale bars = 100 μ m (n = 8-10mice/group). B. Box plots of MMP13 and Collagen counts based on IMC data set in the Control, CR and CR+DBZ groups. C. An overlay of Collagen (Red) and MMP13 (green) in tissue sections of the indicated groups. The boxed area represents a magnified image of collagen accumulation in the CR+DBZ group. Scale bars = 100 μ m (n = 8-10mice/group). D. t-SNE plots showing MMP13 intensity across different groups. E. Masson’s Trichrome staining of tissue sections prepared from the colons of Control, CR, and CR+DBZ mice. Please note increases in collagenous fibrous tissue (stained blue) in both CR and CR+DBZ groups. Scale bars = 200 μ m; n = 8-10mice/group. CR: Citrobacter rodentium , CR+DBZ: Citrobacter rodentium + Dibenzazepine (DBZ).

Article Snippet: By utilizing RKO colon cancer cell line, we discovered that in response to PMA, a significant increase in MMP13 enzymatic activity was recorded with a dose-dependent decrease in response to MMP13 inhibitor WAY-170523 (Cat.# 2633, TOCRIS Biosciences; ).

Techniques: Immunostaining, Imaging, Control, Staining