dynamics scanner, personal densitometer si  (Molecular Dynamics Inc)

Journal: Journal of neurochemistry

Article Title: Vital role of protein kinase C-related kinase (PRK1) in the formation and stability of neurites during hypoxia

doi: 10.1111/j.1471-4159.2010.06624.x

Figure Lengend Snippet: Hypoxia induces morphological changes and PRK1 activation in PC12 cells. PC12 cells were incubated under normoxic (21% O2; N-co) or hypoxic (1% O2; H-co) conditions. (a) After 6-8h incubation, cells were stained with Hoechst/PI for photomicrographs of viable (blue) and dead (pink) cells. (b) Quantification of the percentage of cell death [pink cells × 100/total cell number]. (c) F-actin–associated cytoskeletal reorganization over 0-24h normoxia/hypoxia in phalloidin-stained cells (green). Dashed lines in panels c(i-v) indicate the area selected for higher magnification in panels c(vi-x). (d) Neurite formation of PC12 cells after incubation under normoxic and hypoxic conditions for 24h. Three to five independent microscopic fields were studied and the percentage of neurite formation was calculated [neurite-bearing cells × 100/total cell number]. Extensions longer than twice the cell diameter were considered neurites. (e) Hypoxic treatment for 5 min increases PRK1 activation compared to normoxic cells. Cell lysates were analyzed by Western blot with anti-phospho-PRK1 and anti-pan-PRK1 antibodies. The intensity was scanned by a Personal Densitometer SI scanner (Molecular Dynamics) and the ratio (anti-phospho/anti-pan–PRK1) was calculated. Pictures and blots are representative of 3 to 5 independent experiments. Values represent the means ± S.E.M., n=3-5. Differences were analyzed using unpaired one-tailed t-test: (b) ++P<0.01, (d) ++P<0.01 and (e) +++P<0.001.

Article Snippet: For quantification, developed films were scanned with a special densitometer (Molecular-Dynamics-Personal-Densitometer-SI-scanner, Austria).

Techniques: Activation Assay, Incubation, Staining, Western Blot, One-tailed Test

Journal: Journal of neurochemistry

Article Title: Vital role of protein kinase C-related kinase (PRK1) in the formation and stability of neurites during hypoxia

doi: 10.1111/j.1471-4159.2010.06624.x

Figure Lengend Snippet: Guanosine and NGF mediate neuroprotective effects and increase PRK1 activation in PC12 cells. Hypoxic cells (H-co) were treated with 500μM guanosine (Guo), 5ng/mL NGF (NGF), or a combination of guanosine and NGF (Guo+NGF). (a) Cell death was quantified with Hoechst/PI after 6-8h treatment as described in experimental procedures. The white line indicates cell death in normoxic control. (b) F-actin–associated cytoskeleton was studied in phalloidin-stained cells (green) 24h after stimulation. Nuclei were labeled with Hoechst 33342 dye (blue). (c) Neurite formation was calculated in hypoxic PC12 cells (H-co), as well as in cells treated with 500μM guanosine (Guo), 5ng/mL NGF (NGF), or a combination of guanosine and NGF (Guo+NGF) for 24h. White lines indicate neurite formation under normoxic condition. (d) PRK1 activation was studied in cell lysates stimulated under normoxic/ hypoxic conditions and with 500μM guanosine (Guo), 5ng/mL NGF (NGF), or guanosine and NGF together (Guo+NGF) for 5 min by immunoprobing the blot with anti-phospho-PRK1 and anti-pan-PRK1 antibodies. White lines indicate PRK1 activation under normoxic condition. The intensity was scanned by a Personal Densitometer SI scanner (Molecular Dynamics) and the anti-phospho/anti-pan–PRK1 ratio was calculated. White lines indicate PRK1 activation under normoxic condition. Pictures and blots are representative of 4 to 11 independent experiments. Values represent the means ± S.E.M., n=3-11. Differences were analyzed using one-way ANOVA followed by Dunnett’s multiple comparison test: (a) *P<0.05, **P<0.01 (c) *P<0.05, ***P<0.001 and (d) *P<0.05, ***P<0.001.

Article Snippet: For quantification, developed films were scanned with a special densitometer (Molecular-Dynamics-Personal-Densitometer-SI-scanner, Austria).

Techniques: Activation Assay, Control, Staining, Labeling, Comparison

Journal: Journal of neurochemistry

Article Title: Vital role of protein kinase C-related kinase (PRK1) in the formation and stability of neurites during hypoxia

doi: 10.1111/j.1471-4159.2010.06624.x

Figure Lengend Snippet: siRNA-mediated PRK1 knockdown is specific and suppresses viability of hypoxic PC12 cells. PC12 cells were transfected with synthetic siRNA for PRK1 or scrambled non-targeting siRNA duplexes. (a) Cell extracts were analyzed 24h to 72h after transfection by immunoblotting for anti-pan-PRK1, anti-pan-PRK2 (specificity control) and anti-pan-AKT (loading control). (b) The intensity of protein bands was scanned by a Personal Densitometer SI scanner (Molecular Dynamics) and the anti-pan-PRK1/anti-pan-AKT ratio was calculated. (c) The effect of siRNA-mediated knockdown on cell viability was studied after additional stimulation of hypoxic PC12 cells (H-co) with 500μM guanosine (Guo) for 6-8h and staining for Hoechst/PI. (d) The percentage of hypoxic cell death in PRK1 knockdown cells was calculated. Pictures and blots are representative of 3 to 5 independent experiments and values represent the means ± S.E.M., n=3-5. Differences were analyzed using unpaired one-tailed t-test: (b) ++P<0.01, +++P<0.001 and (d) ++P<0.01 (control siRNA vs. PRK1 siRNA).

Article Snippet: For quantification, developed films were scanned with a special densitometer (Molecular-Dynamics-Personal-Densitometer-SI-scanner, Austria).

Techniques: Knockdown, Transfection, Western Blot, Control, Staining, One-tailed Test

Journal: Journal of neurochemistry

Article Title: Vital role of protein kinase C-related kinase (PRK1) in the formation and stability of neurites during hypoxia

doi: 10.1111/j.1471-4159.2010.06624.x

Figure Lengend Snippet: siRNA-mediated PRK1 knockdown inhibits guanosine- and NGF-mediated cytoskeletal reorganization, GAP-43 expression, and neurite formation in hypoxic PC12 cells. PC12 cells were transfected with siRNA for PRK1 or control siRNA duplexes. (a) siRNA-transfected hypoxic PC12 cells (H-co) were treated with 500μM guanosine (Guo), 5ng/mL NGF (NGF), or both guanosine and NGF (Guo+NGF) for 24h and then stained with FITC-phalloidin (green); nuclei were labeled with Hoechst 33342 (blue) [panels a(i-iv and ix-xii)]. F-actin accumulation at neurite growth cones and branching points was observed (arrowheads). In parallel, hypoxic neurons (H-co) were immunostained for anti-pan-GAP-43 (red) [panels a(v-viii and xiii-xvi), the inserts in panel a(v-viii) represent normoxic cells] after stimulation with 500μM guanosine (Guo), 5ng/mL NGF (NGF), or guanosine plus NGF (Guo+NGF) for 24h. Condensed levels of GAP-43 in growth cones (asterisk) and in perinuclei (arrows) are shown. Photomicrographs are representative of 3 to 5 independent experiments, each with 4 to 6 independent microscopic fields. (b) GAP-43 expression was investigated by analyzing cell lysates from transfected hypoxic PC12 cells (H-co) and hypoxic cells treated with 500μM guanosine (Guo), 50ng/mL NGF (NGF), or a combination of both (Guo+NGF) for 3 days. Intensity was scanned by a Personal Densitometer SI scanner (Molecular Dynamics) and the anti-pan-GAP-43/anti-pan-AKT ratio was calculated. (c) The effect of PRK1 knockdown on guanosine- and NGF-mediated neurite formation by stimulation of siRNA-transfected cells with 500μM guanosine (Guo), 5ng/mL NGF (NGF), or guanosine plus NGF (Guo+NGF) under hypoxic conditions for 3 days was calculated as described in experimental procedures. (d) Transfected PC12 cells treated with guanosine (Guo), 5ng/mL NGF (NGF), or guanosine plus NGF (Guo+NGF) under hypoxic conditions for 24h were stained for gamma tubulin (green) and anti-pan-GAP-43 (red) [panels d(i-viii)]. Pictures are representative of 3 independent experiments. Values represent the means ± S.E.M., n=3-9. Differences were analyzed using unpaired one-tailed t-test: (b) +P<0.05, ++P<0.01 and (c) +P<0.05, ++P<0.01 (control siRNA vs. PRK1 siRNA).

Article Snippet: For quantification, developed films were scanned with a special densitometer (Molecular-Dynamics-Personal-Densitometer-SI-scanner, Austria).

Techniques: Knockdown, Expressing, Transfection, Control, Staining, Labeling, One-tailed Test

Journal: Journal of neurochemistry

Article Title: Vital role of protein kinase C-related kinase (PRK1) in the formation and stability of neurites during hypoxia

doi: 10.1111/j.1471-4159.2010.06624.x

Figure Lengend Snippet: PRK1 is essential for neuroprotection and GAP-43 expression in hypoxic cerebellar granule neurons. Freshly isolated primary cerebellar granule neurons were transfected with control or specific siRNA for PRK1. (a) PRK1 protein expression was studied 48h post-transfection by immunoblotting. The intensity was scanned by a Personal Densitometer SI scanner (Molecular Dynamics) and the anti-pan-PRK1/anti-pan-AKT ratio was calculated. (b) Transfected cerebellar granule neurons were exposed to hypoxic conditions (H-co) [panels b(i and ii)], treated with 500μM guanosine (Guo) [panels b(iii and iv)] for 6-8h, and stained with Hoechst/PI. (c) Quantification of the effect of PRK1 knockdown on cell death. (d) GAP-43 protein fluorescence intensity was studied by staining normoxic (N-co) [panel d(i)] and hypoxic cerebellar granule neurons (H-co) [panels d(ii and iii)]. Hypoxic cells were also treated with 500μM guanosine (Guo) [panels d(iv and v)]. Pictures and blots are representative of 3 to 4 independent experiments. Values represent the means ± S.E.M., n=4. Differences were analyzed using unpaired one-tailed t-test: (a) +P<0.05 and (c) ++P<0.01.

Article Snippet: For quantification, developed films were scanned with a special densitometer (Molecular-Dynamics-Personal-Densitometer-SI-scanner, Austria).

Techniques: Expressing, Isolation, Transfection, Control, Western Blot, Staining, Knockdown, Fluorescence, One-tailed Test