Journal: Scientific Reports

Article Title: Bone secreted factors induce cellular quiescence in prostate cancer cells

doi: 10.1038/s41598-019-54566-4

Figure Lengend Snippet: Secretome of mouse calvariae.

Article Snippet: Antibodies for Ki67 (Dako #MIB-1), p27Kip1 (Cell Signaling #D69C12), phospho-p38MAPK (Thr180/Tyr182) (Cell Signaling #4511), DKK3 (Proteintech #10355-1-AP), BMP1 (abcam #ab205394), MIA (Santa Cruz #sc-37735), NGAL (Santa Cruz #sc-515876), and PEDF (R&D #AF-1177) were obtained from commercial sources.

Techniques: Sequencing, Migration, Protease Inhibitor

Journal: Scientific Reports

Article Title: Bone secreted factors induce cellular quiescence in prostate cancer cells

doi: 10.1038/s41598-019-54566-4

Figure Lengend Snippet: Bone-secreted factors induce cellular quiescence in PCa cells. ( A ) Dose response. C4-2B4 PCa cells were treated without or with various recombinant human bone-secreted factors at different concentrations and analyzed by live-cell imaging as in Fig. . About 100 cells were monitored for each factor at each concentration. Using this approach, we obtained an empirically-derived optimal concentration to be used for each factor (arrowheads): DKK3 (10 µg/mL), BMP1 (0.4 µg/mL), VASN (0.25 µg/mL), NEO1 (0.5 µg/mL), MIA (0.1 µg/mL), and NGAL (0.25 µg/mL). ( B ) (Left) Phase contrast brightfield images. Cells were plated in a Q4 glass-bottom dish, treated with and without various recombinant proteins, and analyzed by live-cell imaging. Representative images are shown for control cells and cells treated with recombinant human DKK3 (cell a), vasorin (cell b), neogenin (cell c), and BMP1 (cell d). Asterisks (*) follow a control cell through 3 cell divisions. Round cells are cells undergoing mitosis. (Right) Immunofluorescence images. At the end of live-cell imaging, cells were immediately fixed and co-immnostained for Ki67 (proliferation marker) and p27 (dormancy marker), and merged with phase contrast images. Cell outlines are traced for ease of view. All bars, 20 µm. ( C ) C4-2B4 cells were treated with various bone-secreted factors, using concentrations as determined in (A), and analyzed by live-cell imaging. PEDF was used at 0.25 µg/ml . The dormancy factor BMP7 (0.4 µg/ml) was used as a positive control. % quiescent cells that did not divide relative to total cells counted were quantified (mean ± s.e.m), except for PEDF and BMP7 (mean ± s.d.). n , number of cells monitored. N, number of independent experiments for control (N = 24), DKK3 (N = 10), BMP1 (N = 10), vasorin (N = 9), neogenin (N = 4), MIA (N = 4), NGAL (N = 6), PEDF (N = 2), BMP7 (N = 2). P values were by t test. ns, not significant. ( D ) C4-2b cells were treated with various bone-secreted factors as indicated, monitored by live-cell imaging, and analyzed as in ( C ). n , number of cells monitored. N, number of independent experiments for control (N = 3), DKK3 (N = 3), BMP1 (N = 3), vasorin (N = 2), neogenin (N = 2). P values were by t test.

Article Snippet: Antibodies for Ki67 (Dako #MIB-1), p27Kip1 (Cell Signaling #D69C12), phospho-p38MAPK (Thr180/Tyr182) (Cell Signaling #4511), DKK3 (Proteintech #10355-1-AP), BMP1 (abcam #ab205394), MIA (Santa Cruz #sc-37735), NGAL (Santa Cruz #sc-515876), and PEDF (R&D #AF-1177) were obtained from commercial sources.

Techniques: Recombinant, Live Cell Imaging, Concentration Assay, Derivative Assay, Immunofluorescence, Marker, Positive Control

Journal: Cell Death & Disease

Article Title: Pigment epithelium-derived factor mediates retinal ganglion cell neuroprotection by suppression of caspase-2

doi: 10.1038/s41419-019-1379-6

Figure Lengend Snippet: a Fold change in CASP2 mRNA relative to β-actin reference gene in retinal cells freshly isolated and after 3d exposure to NBA, siCNL, 50 nM siCASP2 and PEDF/PEDF-34/PEDF-44. b % of surviving βIII-tubulin + RGC after 3d in culture in NBA, and after treatment with siCNL, 50 nM siCASP2 and PEDF/PEDF-34/PEDF-44. c Fold change in CASP2 mRNA relative to β-actin reference gene in retinal cells freshly isolated and after 3d exposure to suboptimal doses of siCASP2 (5–20 nM) with and without PEDF/PEDF-34/PEDF-44. d % of surviving βIII-tubulin + RGC after 3d in culture in with suboptimal concentrations of siCASP2 (5–20 nM) with and without PEDF/PEDF-34/PEDF-44 ( n = 9 wells/treatment)

Article Snippet: In a preliminary dose response experiment, the optimal concentration of goat polyclonal anti-rat PEDF antibody (Cat No. AF1177; R&D Systems Europe) to antagonise the effects of pre-optimised PEDF and PEDF-34 on RGC survival in vitro was determined , .

Techniques: Isolation

Journal: Cell Death & Disease

Article Title: Pigment epithelium-derived factor mediates retinal ganglion cell neuroprotection by suppression of caspase-2

doi: 10.1038/s41419-019-1379-6

Figure Lengend Snippet: a Fold change in CASP2 mRNA at 7d after ONC + PBS, ONC + siCNL and ONC + siCASP2 and ONC + PEDF treatment. b, c Western blot and subsequent densitometry to show that ONC-induced C-CASP2 (p12 fragment) was significantly suppressed by siCASP2 and PEDF treatment ( n = 18 retinae/treatment)

Article Snippet: In a preliminary dose response experiment, the optimal concentration of goat polyclonal anti-rat PEDF antibody (Cat No. AF1177; R&D Systems Europe) to antagonise the effects of pre-optimised PEDF and PEDF-34 on RGC survival in vitro was determined , .

Techniques: Western Blot

Journal: Cell Death & Disease

Article Title: Pigment epithelium-derived factor mediates retinal ganglion cell neuroprotection by suppression of caspase-2

doi: 10.1038/s41419-019-1379-6

Figure Lengend Snippet: a Fold change in CASP2 mRNA after ONC + saline and ONC + PEDF-34 daily eye-drops over 21d. b , c Western blot and subsequent densitometry to show that ONC-induced rise in C-CASP2 (p12 fragment) is significantly suppressed at 1d and 3d after injury and treatment, without changes in C-CASP3 or C-CASP6 levels. d Immunohistochemistry to show that ONC-induced localisation of C-CASP2 (red) in βIII-tubulin + (green) RGC (arrowheads) after ONC + saline treatment is suppressed by 1d and 3d after ONC + PEDF-34 eye-drops. Scale bar = 50 µm. GCL = ganglion cell layer. (n = 18 retinae/treatment)

Article Snippet: In a preliminary dose response experiment, the optimal concentration of goat polyclonal anti-rat PEDF antibody (Cat No. AF1177; R&D Systems Europe) to antagonise the effects of pre-optimised PEDF and PEDF-34 on RGC survival in vitro was determined , .

Techniques: Saline, Eye Drops, Western Blot, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: Pigment epithelium-derived factor mediates retinal ganglion cell neuroprotection by suppression of caspase-2

doi: 10.1038/s41419-019-1379-6

Figure Lengend Snippet: a Fold change in CASP2 mRNA in 5-FDU-treated cultures showing no change in CASP2 mRNA after siCASP2 or PEDF treatment compared to cultures with glia. b % of surviving βIII-tubulin + RGC in 5-FDU treated cultures in siCASP2 treated cells was unaffected but survival was reduced in the absence of glia. c Fold change in CASP2 mRNA in purified RGC cultures showing no change in CASP2 mRNA after siCASP2 or PEDF treatment. d % of surviving βIII-tubulin + RGC in purified RGC cultures in siCASP2 treated cells was unaffected but survival was reduced in the absence of glia ( n = 9 wells/treatment)

Article Snippet: In a preliminary dose response experiment, the optimal concentration of goat polyclonal anti-rat PEDF antibody (Cat No. AF1177; R&D Systems Europe) to antagonise the effects of pre-optimised PEDF and PEDF-34 on RGC survival in vitro was determined , .

Techniques: Purification

Journal: Cell Death & Disease

Article Title: Pigment epithelium-derived factor mediates retinal ganglion cell neuroprotection by suppression of caspase-2

doi: 10.1038/s41419-019-1379-6

Figure Lengend Snippet: a ELISA to determine levels of BDNF, CNTF, GDNF, NGF and NT-3 released into the culture medium. Treatment of retinal cultures with ELISA isolated levels of BDNF, GDNF and NGF or CNTF either alone or in combination partially suppresses b CASP2 mRNA and C-CASP2 protein c , d by western blot and subsequent densitometry. e Representative images to demonstrate morphology of βIII-tubulin + RGC (note: neurite outgrowth occurs in surviving βIII-tubulin + RGC in wells containing PEDF, BDNF/GDNF/NGF and CNTF). (n = 9 wells/treatment). Scale bar in f = 20 µm

Article Snippet: In a preliminary dose response experiment, the optimal concentration of goat polyclonal anti-rat PEDF antibody (Cat No. AF1177; R&D Systems Europe) to antagonise the effects of pre-optimised PEDF and PEDF-34 on RGC survival in vitro was determined , .

Techniques: Enzyme-linked Immunosorbent Assay, Isolation, Western Blot

Journal: Cell Death & Disease

Article Title: Pigment epithelium-derived factor mediates retinal ganglion cell neuroprotection by suppression of caspase-2

doi: 10.1038/s41419-019-1379-6

Figure Lengend Snippet: a – c Pre-treatment of PEDF and PEDF-34 with a polyclonal antibody to PEDF fails to reduce CASP2 mRNA ( a ) and protein levels ( b ) whilst PEDF is suppressed in the absence of PEDF polyclonal antibody treatment, as confirmed by densitometry ( c ). d Pre-treatment with PEDF polyclonal antibody also suppresses the RGC neuroprotective effects of PEDF and PEDF-34. (n = 9 wells/treatment). e Representative images of the treatments in d to show morphology of βIII-tubulin + RGC. Scale bar in e = 20 µm

Article Snippet: In a preliminary dose response experiment, the optimal concentration of goat polyclonal anti-rat PEDF antibody (Cat No. AF1177; R&D Systems Europe) to antagonise the effects of pre-optimised PEDF and PEDF-34 on RGC survival in vitro was determined , .

Techniques:

Journal: Cell Death & Disease

Article Title: Pigment epithelium-derived factor mediates retinal ganglion cell neuroprotection by suppression of caspase-2

doi: 10.1038/s41419-019-1379-6

Figure Lengend Snippet: a CASP2-dependent apoptosis requires the formation of the PIDDosome (Adapted from ). b PEDF treatment suppresses CASP2, RAIDD but not PIDD and downstream Bim mRNA. c , d PEDF treatment suppresses C-CASP2, RAIDD, phopho-c-Jun and downstream Bim as detected by western blot and densitometry. e , f Immunohistochemistry to show changes in Bim protein colocalised to caspase-2 in the ganglion cell layer (GCL), inner plexiform layer (IPL), occasional cells of the inner nuclear layer (INL) whilst high power views of the GCL showed that Bim and caspase-2 were colocalised to RGC. Scale bars in e and ( f ) = 50 µm ( n = 18 retinae/treatment)

Article Snippet: In a preliminary dose response experiment, the optimal concentration of goat polyclonal anti-rat PEDF antibody (Cat No. AF1177; R&D Systems Europe) to antagonise the effects of pre-optimised PEDF and PEDF-34 on RGC survival in vitro was determined , .

Techniques: Western Blot, Immunohistochemistry

Journal: EBioMedicine

Article Title: HtrA1 Mediated Intracellular Effects on Tubulin Using a Polarized RPE Disease Model

doi: 10.1016/j.ebiom.2017.12.011

Figure Lengend Snippet: HtrA1 overexpression leads to apical impairment. (A) IF confocal images of RPE at day 35 of culture. β-tubulin (red) and DAPI (blue). Left image, S328A overexpressing cells; right image, HtrA1 overexpressing cells. Scale bar, 50 μm. Close up images show ciliated cells. (A′) % of ciliated cells in the RPE population. Data represent the mean ± SD of three independent experiments, *** p < 0.001. See also Fig. S3. (B) Life cell imaging of RPE cells at day 35 of culture. Left, S328A overexpressing RPE; right, HtrA1 overexpressing RPE. The apical membrane faces the top. Scale bar, 10 μm. (C) Transmission Electron micrographs of S328A (above) and Htra1 (below) overexpressing cells. Scale bar, 5 μm. (C′) Microvilli per μm of apical membrane. Data represent the mean ± SD of one experiment, *** p < 0.001.

Article Snippet: The proteins were transferred to a Trans-Blot® TurboTM (Bio—Rad) membrane using the Trans-Blot® TurboTM Transfer System (Bio—Rad) for 7 min. Membranes were incubated with 5% Blotting Grade Blocker Non-fat Dry Milk (Bio—Rad) in Tris-buffered saline (TBS) (Sigma) + 0.05% Tween-20 (Sigma) for 1 h at RT prior to incubation with primary antibodies specific to Tubulin (1:250, MAB3408; Millipore, RRID: AB_94650 ), HtrA1 (1:1000, , Serpin F1/PEDF (1:250, AF1177, R&D Systems, RRID: AB_2187173 ), E -Cadherin (1:500, 610,182, BD Transduction LaboratoriesTM, RRID: AB_39758 ), C1QTNF5 (1:250, MAB3167, R&D Systems, RRID: AB_2065810 ) with TBP (1:250, ab51841, Abcam, RRID: AB_945758 ) as the loading control, overnight at 4 °C.

Techniques: Over Expression, Imaging, Membrane, Transmission Assay

Journal: EBioMedicine

Article Title: HtrA1 Mediated Intracellular Effects on Tubulin Using a Polarized RPE Disease Model

doi: 10.1016/j.ebiom.2017.12.011

Figure Lengend Snippet: HtrA1 overexpression associates with tubulin loss and localization at the adherens junctions. (A) IF representative confocal image of S328A (left) and HtrA1 (right) overexpressing RPE at day 35 of culture. β-tubulin (red), HtrA1 (cyan) and DAPI (blue); scale bar, 10 μm. (A’) β-tubulin intensity quantification in HtrA1 positive cells at day 35 of culture. Data represent the mean ± SD of three independent experiments, *** p < 0.001. (B) IF high-resolution confocal images ( xy plane) of tubulin, claudin-19 and E-cadherin (red) and HrA1 (cyan), DAPI (blue). The apical membrane faces the top. Yellow arrows indicate cells containing less tubulin. White arrows indicate localization of HtrA1 under claudin-19 and co-localization of HtrA1 and E-cadherin. Scale bar, 5 μm. (C) Transmission electron micrographs from immune-gold detection of HtrA1 in S328A, left panel, and HtrA1 overexpressing cells, right panel. Scale bars, 500 nm and 200 nm. Red arrows indicate gold localization. Dashed line indicates the limit between cells.

Article Snippet: The proteins were transferred to a Trans-Blot® TurboTM (Bio—Rad) membrane using the Trans-Blot® TurboTM Transfer System (Bio—Rad) for 7 min. Membranes were incubated with 5% Blotting Grade Blocker Non-fat Dry Milk (Bio—Rad) in Tris-buffered saline (TBS) (Sigma) + 0.05% Tween-20 (Sigma) for 1 h at RT prior to incubation with primary antibodies specific to Tubulin (1:250, MAB3408; Millipore, RRID: AB_94650 ), HtrA1 (1:1000, , Serpin F1/PEDF (1:250, AF1177, R&D Systems, RRID: AB_2187173 ), E -Cadherin (1:500, 610,182, BD Transduction LaboratoriesTM, RRID: AB_39758 ), C1QTNF5 (1:250, MAB3167, R&D Systems, RRID: AB_2065810 ) with TBP (1:250, ab51841, Abcam, RRID: AB_945758 ) as the loading control, overnight at 4 °C.

Techniques: Over Expression, Membrane, Transmission Assay

Journal: EBioMedicine

Article Title: HtrA1 Mediated Intracellular Effects on Tubulin Using a Polarized RPE Disease Model

doi: 10.1016/j.ebiom.2017.12.011

Figure Lengend Snippet: HtrA1 overexpression is accompanied by a decrease in the phagocytosis activity. (A) Representative confocal images of Zymosan A treated hfRPE from control cells, control cells treated with Cytochalasyn D, overexpressing S328A cells and HtrA1 overexpressing cells. Zymosan A particles (red) and Hoechst (blue). Scale bar, 10 μm (A′) Phagocytic activity (in % to control cells) from S328A overexpressing cells, HtrA1 overexpressing cells and control cells treated with Cytochalasyn D at day 35 of culture. Mean ± SD of three independent experiments, *** p < 0.001. (B) Life cell imaging of RPE cells while the process of phagocytosis. Left, S328A overexpressing cells; right, HtrA1 overexpressing cells. POS (red), GFP (green) and Hoechst (blue). Scale bar, 20 μm. (C) Close up image of S328A cells in the moment of particle uptake. POS (red), GFP (green) and Hoechst (blue). Scale bar, 10 μm (D) % of phagocyting GFP cells. Mean ± SD of two independent experiments, *** p < 0.001.

Article Snippet: The proteins were transferred to a Trans-Blot® TurboTM (Bio—Rad) membrane using the Trans-Blot® TurboTM Transfer System (Bio—Rad) for 7 min. Membranes were incubated with 5% Blotting Grade Blocker Non-fat Dry Milk (Bio—Rad) in Tris-buffered saline (TBS) (Sigma) + 0.05% Tween-20 (Sigma) for 1 h at RT prior to incubation with primary antibodies specific to Tubulin (1:250, MAB3408; Millipore, RRID: AB_94650 ), HtrA1 (1:1000, , Serpin F1/PEDF (1:250, AF1177, R&D Systems, RRID: AB_2187173 ), E -Cadherin (1:500, 610,182, BD Transduction LaboratoriesTM, RRID: AB_39758 ), C1QTNF5 (1:250, MAB3167, R&D Systems, RRID: AB_2065810 ) with TBP (1:250, ab51841, Abcam, RRID: AB_945758 ) as the loading control, overnight at 4 °C.

Techniques: Over Expression, Activity Assay, Control, Imaging

Journal: EBioMedicine

Article Title: HtrA1 Mediated Intracellular Effects on Tubulin Using a Polarized RPE Disease Model

doi: 10.1016/j.ebiom.2017.12.011

Figure Lengend Snippet: Polarized RPE cell model characterization. (A) Retinal histological sections immunostained against HtrA1 (pink) from a healthy subject (left) and an AMD patient (right). Nuclei stained with eosin. Yellow arrows indicate RPE cells. Green arrows indicate the BM (B) Differentiation protocol. (C) Panel of genes that by RNA sequencing experienced an increase along the differentiation time. (D) Representative images of several characteristic RPE markers: Na + K + ATPase, PEDF, E-cadherin, N-cadherin, claudin 19 and occludin (green); DAPI (blue); scale bar, 10 μm. (E) Transmission electron micrographs from hfRPE at day 35 of differentiation; scale bar, 5 μm. Asterisks indicate the nucleus (F) Phase contrast image of hfRPE cells at the end of the differentiation; scale bar, 50 μm. (G) Mechanical characterization along the differentiation at 4, 9, 14 and 35 days in culture. Mean values and ± SD for nuclei, cytoplasm and cell junctions from three independent experiments. (G′) Representative force-indentation maps at days 4, 9, 14 and 35 of culture; scale bar, 10 μm. See also Fig. S1.

Article Snippet: The proteins were transferred to a Trans-Blot® TurboTM (Bio—Rad) membrane using the Trans-Blot® TurboTM Transfer System (Bio—Rad) for 7 min. Membranes were incubated with 5% Blotting Grade Blocker Non-fat Dry Milk (Bio—Rad) in Tris-buffered saline (TBS) (Sigma) + 0.05% Tween-20 (Sigma) for 1 h at RT prior to incubation with primary antibodies specific to Tubulin (1:250, MAB3408; Millipore, RRID: AB_94650 ), HtrA1 (1:1000, , Serpin F1/PEDF (1:250, AF1177, R&D Systems, RRID: AB_2187173 ), E -Cadherin (1:500, 610,182, BD Transduction LaboratoriesTM, RRID: AB_39758 ), C1QTNF5 (1:250, MAB3167, R&D Systems, RRID: AB_2065810 ) with TBP (1:250, ab51841, Abcam, RRID: AB_945758 ) as the loading control, overnight at 4 °C.

Techniques: Staining, RNA Sequencing, Transmission Assay

Journal: EBioMedicine

Article Title: HtrA1 Mediated Intracellular Effects on Tubulin Using a Polarized RPE Disease Model

doi: 10.1016/j.ebiom.2017.12.011

Figure Lengend Snippet: HtrA1 interacts with tubulin. (A) Immunoprecipitation against β-tubulin and detection of HtrA1 and β-tubulin in lysates obtained from HtrA1 and S328A HtrA1 overexpressing RPE cells. Left, input material in the column. Right, bound fraction. See also Fig. S2. (B) Western blot gels for HtrA1 and β-tubulin, representative of a single experiment from cell lysates incubated in increasing concentration of anti-HtrA1 competing antibody. Left, lysate from HtrA1 overexpressing cells. Right, lysate from S328A overexpressing cells. (C) Mass Spectrometry identification of possible HtrA1 interactors by immunocompetition assays. Concentration of HtrA1 and other beta subunits decay as the antibody competition antibody increases. Orange, lysate from HtrA1 overexpressing cells. Blue, lysate from S328A overexpressing cells.

Article Snippet: The proteins were transferred to a Trans-Blot® TurboTM (Bio—Rad) membrane using the Trans-Blot® TurboTM Transfer System (Bio—Rad) for 7 min. Membranes were incubated with 5% Blotting Grade Blocker Non-fat Dry Milk (Bio—Rad) in Tris-buffered saline (TBS) (Sigma) + 0.05% Tween-20 (Sigma) for 1 h at RT prior to incubation with primary antibodies specific to Tubulin (1:250, MAB3408; Millipore, RRID: AB_94650 ), HtrA1 (1:1000, , Serpin F1/PEDF (1:250, AF1177, R&D Systems, RRID: AB_2187173 ), E -Cadherin (1:500, 610,182, BD Transduction LaboratoriesTM, RRID: AB_39758 ), C1QTNF5 (1:250, MAB3167, R&D Systems, RRID: AB_2065810 ) with TBP (1:250, ab51841, Abcam, RRID: AB_945758 ) as the loading control, overnight at 4 °C.

Techniques: Immunoprecipitation, Western Blot, Incubation, Concentration Assay, Mass Spectrometry

Journal: EBioMedicine

Article Title: HtrA1 Mediated Intracellular Effects on Tubulin Using a Polarized RPE Disease Model

doi: 10.1016/j.ebiom.2017.12.011

Figure Lengend Snippet: The nanomechanical fingerprint of RPE cells overexpressing HrA1 and S328A variant. (A/B, left panels) The cell viability and position of the AFM probe is monitored in the brightfield signal for each experiment. Live-Fluorescence imaging allowed for identifying the overexpressing cells (white insert). AFM topography and stiffness data belonging to the measurements (white insert). (A, right panel) Stiffness data, S328A variant cells exhibit similar properties when compared to the neighboring negative cells. (B, right panel) Stiffness data, HtrA1 overexpressing cells showed a 2-fold decrease for RPEs overexpressing HtrA1 (red line) when compared to cells having a low HtrA1 content (green line). (C) Quantitative stiffness analysis over multiple measurements. N = 9 maps ( n = 92 cells) for S328A and N = 17 maps ( n = 168 cells) for HtrA1, * p < 0.05. (D) Live cell imaging Halotag TMR ligand for HtrA1 (red), SiR-Tubulin (green) and Hoechst (blue). Scale bar, 20 μm.

Article Snippet: The proteins were transferred to a Trans-Blot® TurboTM (Bio—Rad) membrane using the Trans-Blot® TurboTM Transfer System (Bio—Rad) for 7 min. Membranes were incubated with 5% Blotting Grade Blocker Non-fat Dry Milk (Bio—Rad) in Tris-buffered saline (TBS) (Sigma) + 0.05% Tween-20 (Sigma) for 1 h at RT prior to incubation with primary antibodies specific to Tubulin (1:250, MAB3408; Millipore, RRID: AB_94650 ), HtrA1 (1:1000, , Serpin F1/PEDF (1:250, AF1177, R&D Systems, RRID: AB_2187173 ), E -Cadherin (1:500, 610,182, BD Transduction LaboratoriesTM, RRID: AB_39758 ), C1QTNF5 (1:250, MAB3167, R&D Systems, RRID: AB_2065810 ) with TBP (1:250, ab51841, Abcam, RRID: AB_945758 ) as the loading control, overnight at 4 °C.

Techniques: Variant Assay, Fluorescence, Imaging, Live Cell Imaging

Journal: EBioMedicine

Article Title: HtrA1 Mediated Intracellular Effects on Tubulin Using a Polarized RPE Disease Model

doi: 10.1016/j.ebiom.2017.12.011

Figure Lengend Snippet: HtrA1 degrades preformed MT and avoids polymerization in vitro. (A) MT polymerized in vitro and incubated with S328A HtrA1 (center) and HtrA1 (right). Images acquired at 5 min, 6 h and 24 h after adding the protease. Scale bar, 10 μm. (B) Quantification of the area occupied by the MT 5 min, 6 h and 24 h after adding the protease. (C) Microtubule polymerization reactions in the absence or presence of S328A HtrA1 and HtrA1. As a representative growth signal, the absorbance at 340 nm was monitored in vitro as a function of time for three independent replicates. (C′) The average growth signal for each variant was calculated and normalized relative to the overall maximum. Best fits to sigmoidal function are shown with solid lines. (D) Comparison of the growth amplitudes ( S rel ), (E) kinetic rate constants ( k F ), (F) nucleation time ( t lag ) of microtubule polymerization reaction in the absence or presence of S328A HtrA1 and HtrA1. Mean ± SD for three replicates are reported, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The proteins were transferred to a Trans-Blot® TurboTM (Bio—Rad) membrane using the Trans-Blot® TurboTM Transfer System (Bio—Rad) for 7 min. Membranes were incubated with 5% Blotting Grade Blocker Non-fat Dry Milk (Bio—Rad) in Tris-buffered saline (TBS) (Sigma) + 0.05% Tween-20 (Sigma) for 1 h at RT prior to incubation with primary antibodies specific to Tubulin (1:250, MAB3408; Millipore, RRID: AB_94650 ), HtrA1 (1:1000, , Serpin F1/PEDF (1:250, AF1177, R&D Systems, RRID: AB_2187173 ), E -Cadherin (1:500, 610,182, BD Transduction LaboratoriesTM, RRID: AB_39758 ), C1QTNF5 (1:250, MAB3167, R&D Systems, RRID: AB_2065810 ) with TBP (1:250, ab51841, Abcam, RRID: AB_945758 ) as the loading control, overnight at 4 °C.

Techniques: In Vitro, Incubation, Variant Assay, Comparison

Journal: Investigative Ophthalmology & Visual Science

Article Title: Corneal Mesenchymal Stromal Cells Are Directly Antiangiogenic via PEDF and sFLT-1

doi: 10.1167/iovs.17-22680

Figure Lengend Snippet: Co-MSCs have an antiangiogenic profile by protein expression. (A) Human angiogenesis array was used to profile pro- and antiangiogenesis mediators in the Co-MSC secretome. It revealed it has high levels of antiangiogenic factors, whereas low levels of proangiogenic factors. (B) The secretome from Co-MSCs contains high levels of sFLT-1 (1875 ± 677 pg/mL); PEDF (4829 ± 2342 pg/mL); TSG-6 (643.3 ± 149.1 pg/mL); and low amount of VEGF-A (106.9 ±103.3 pg/mL; n = 5). The values shown are mean ± SD (error bars).

Article Snippet: Co-MSC secretome was incubated with 25 μg/mL anti-PEDF (Cat. No. AF1177); anti-sFlt1 (Cat. No. AF321); or normal Goat IgG (Cat. No. AB 108-C) antibody (all from R&D) at 4°C overnight.

Techniques: Expressing

Journal: Investigative Ophthalmology & Visual Science

Article Title: Corneal Mesenchymal Stromal Cells Are Directly Antiangiogenic via PEDF and sFLT-1

doi: 10.1167/iovs.17-22680

Figure Lengend Snippet: Direct antiangiogenic properties of Co-MSCs depend on PEDF and sFLT-1. (A) Removing sFLT-1 and PEDF with neutralizing antibody capturing and immunoprecipitation abrogated the antiangiogenic property of Co-MSC secretome in a HUVEC assay (n = 5 1-way ANOVA and then 2-sided t-test were used to compare the groups). *P < 0.001. (B) The efficiency of immunoprecipitation for PEDF and sFLT1 was more than 95% determined by ELISA (n = 5, 2-sided t-test: *P < 0.0001). Boxes show the interquartile (25%–75%) range; whiskers encompass the range (minimum–maximum); and horizontal lines represent the mean. Scale bars: 100 μm. IP, immunoprecipitation.

Article Snippet: Co-MSC secretome was incubated with 25 μg/mL anti-PEDF (Cat. No. AF1177); anti-sFlt1 (Cat. No. AF321); or normal Goat IgG (Cat. No. AB 108-C) antibody (all from R&D) at 4°C overnight.

Techniques: Immunoprecipitation, HUVEC Assay, Enzyme-linked Immunosorbent Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: Corneal Mesenchymal Stromal Cells Are Directly Antiangiogenic via PEDF and sFLT-1

doi: 10.1167/iovs.17-22680

Figure Lengend Snippet: Direct antiangiogenic properties of Co-MSCs are in part due to PEDF. Knocking down PEDF in Co-MSCs by siRNA obviates the antiangiogenic effects of their secretome and increases tubule formation in a HUVEC assay (n = 4, 1-way ANOVA, and then 2-sided t-test were used to compare the groups). *P < 0.001. (B) Knocking down PEDF by siRNA decreased PEDF protein by almost 90% compared to scrambled siRNA (n = 4, 2-sided t-test: *P < 0.0001). Boxes show the interquartile (25%–75%) range; whiskers encompass the range (minimum–maximum); and horizontal lines represent the mean. Scale bars: 100 μm. SC, Scrambled.

Article Snippet: Co-MSC secretome was incubated with 25 μg/mL anti-PEDF (Cat. No. AF1177); anti-sFlt1 (Cat. No. AF321); or normal Goat IgG (Cat. No. AB 108-C) antibody (all from R&D) at 4°C overnight.

Techniques: HUVEC Assay