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Fig. 9. Effect of a thrombin-produced 15-kDa rcGH peptide on cellular viability as measured by MTT (A), and on caspase-3 activity (B). Cells were subjected to HLG (H) or maintained under normoxia conditions with no addition (N), or with 1nM (H + F1) or 10nM (H+F10) 15-kDa rcGH fragment for 1h, then re-oxygenated for 24 h prior to measuring cell viability and caspase-3 activity. Each bar represents the mean + SEM, n=5. Groups with different letters are significantly different from each other by one-way ANOVA and Tukey’s multiple-comparison post hoc tests, p<0.05.

Figure 9 Effect of a thrombin-produced 15-kDa rcGH peptide on cellular viability as measured by MTT (A), and on caspase-3 activity (B). Cells were subjected to HLG (H) or maintained under normoxia conditions with no addition (N), or with 1nM (H + F1) or 10nM (H+F10) 15-kDa rcGH fragment for 1h, then re-oxygenated for 24 h prior to measuring cell viability and caspase-3 activity. Each bar represents the mean + SEM, n=5. Groups with different letters are significantly different from each other by one-way ANOVA and Tukey’s multiple-comparison post hoc tests, p<0.05.

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Fig. 1. GH concentration measured by ELISA and expressed in ng of GH per mg of protein. (A) and (B) 15- and 18-day-old chicken embryos (ED15 and ED18), respectively, were subjected to hypoxia for 24 h followed by 24 h of re-oxygenation (H-24 h); after this time, embryos were immediately sacrificed, and whole brains were obtained for GH determination. (C) and (D) Chicken embryos (ED15 and ED18) were exposed to hypoxia for 24 h and then maintained under normoxic conditions until 1 day after hatching (H-1ph), when the animals were sacrificed and the GH concentrations in whole brains were obtained. (E) Embryonic chicks (ED15) were subjected to hypoxia for 24h followed by 24 h of re-oxygenation; after this time, embryos were immediately sacrificed and the cerebellum was collected for the GH measurements. (F) Primary cerebellar cell cultures from ED15 were subjected to hypoxia for 1 h followed by 24 h of re-oxygenation, and then the cells were harvested and maintained in an EDTA-free, protease- inhibitor cocktail (Mini-Complete, Roche) containing 1 mM PMSF, pH 9.0, and analyzed for GH concentration. Each bar represent mean + SEM for: [(A) and (B) panels] n= 7 (N) and 5 (H-24 h) independent embryos (3 three replicates for each); [(C) and (D) panels] n = 9 (N) and 5 (H-1ph) independent embryos (3 replicates for each); and [(E) and (D) panels] n=5 independent embryos (3 replicates for each) in both conditions.
Fig. 1. GH concentration measured by ELISA and expressed in ng of GH per mg of protein. (A) and (B) 15- and 18-day-old chicken embryos (ED15 and ED18), respectively, were subjected to hypoxia for 24 h followed by 24 h of re-oxygenation (H-24 h); after this time, embryos were immediately sacrificed, and whole brains were obtained for GH determination. (C) and (D) Chicken embryos (ED15 and ED18) were exposed to hypoxia for 24 h and then maintained under normoxic conditions until 1 day after hatching (H-1ph), when the animals were sacrificed and the GH concentrations in whole brains were obtained. (E) Embryonic chicks (ED15) were subjected to hypoxia for 24h followed by 24 h of re-oxygenation; after this time, embryos were immediately sacrificed and the cerebellum was collected for the GH measurements. (F) Primary cerebellar cell cultures from ED15 were subjected to hypoxia for 1 h followed by 24 h of re-oxygenation, and then the cells were harvested and maintained in an EDTA-free, protease- inhibitor cocktail (Mini-Complete, Roche) containing 1 mM PMSF, pH 9.0, and analyzed for GH concentration. Each bar represent mean + SEM for: [(A) and (B) panels] n= 7 (N) and 5 (H-24 h) independent embryos (3 three replicates for each); [(C) and (D) panels] n = 9 (N) and 5 (H-1ph) independent embryos (3 replicates for each); and [(E) and (D) panels] n=5 independent embryos (3 replicates for each) in both conditions.
Fig. 2. GH-IR and NeuN-IR are present in chicken cerebellar cell cultures after 72 h of incubation with Neurobasal medium supplemented with B27. Confocal images at 40> magnification of double-stained ED15 cell cultures were analyzed to identify neurons containing NeuN (b, green, 1st Ab: monoclonal anti-NeuN antibody, 1:200; 2nd Ab: goa anti-mouse IgG-FITC 1:200) and GH (a, red, 1st Ab: anti-cGH 1:200; 2nd Ab: goat anti-rabbit IgG-TRITC 1:200). Panel c represents the overlap of GH and Neul immunoreactivity, indicating co-expression (yellow, arrows). Scale bars = 10 ttm. Higher magnification (63 x) is shown in d (cGH), e (NeuN), and f (merged). Immunoreactiv signal for NeuN is observed mainly in the nucleus (arrows), and it co-localizes with the GH signal. In panel g, GH immunoreactivity is also observed in the perikarya and i some cell processes; panel h clearly shows neural processes immunoreactive for f-tubulin III, and this signal also co-localized with GH, as shown in panel i. Magnificatio1 10x, bar: 50 tum (a-c); magnification 63 x, bar: 10 jum (d-f); magnification 40x, bar: 10 tim (g and h). (For interpretation of the references to colour in this figure legend, th reader is referred to the web version of this article.)
Fig. 2. GH-IR and NeuN-IR are present in chicken cerebellar cell cultures after 72 h of incubation with Neurobasal medium supplemented with B27. Confocal images at 40> magnification of double-stained ED15 cell cultures were analyzed to identify neurons containing NeuN (b, green, 1st Ab: monoclonal anti-NeuN antibody, 1:200; 2nd Ab: goa anti-mouse IgG-FITC 1:200) and GH (a, red, 1st Ab: anti-cGH 1:200; 2nd Ab: goat anti-rabbit IgG-TRITC 1:200). Panel c represents the overlap of GH and Neul immunoreactivity, indicating co-expression (yellow, arrows). Scale bars = 10 ttm. Higher magnification (63 x) is shown in d (cGH), e (NeuN), and f (merged). Immunoreactiv signal for NeuN is observed mainly in the nucleus (arrows), and it co-localizes with the GH signal. In panel g, GH immunoreactivity is also observed in the perikarya and i some cell processes; panel h clearly shows neural processes immunoreactive for f-tubulin III, and this signal also co-localized with GH, as shown in panel i. Magnificatio1 10x, bar: 50 tum (a-c); magnification 63 x, bar: 10 jum (d-f); magnification 40x, bar: 10 tim (g and h). (For interpretation of the references to colour in this figure legend, th reader is referred to the web version of this article.)
Fig. 3. (A) Hypoxia [0.5% O2] and Low Glucose [1 g/L] (HLG) incubation conditions induced cell death in chicken cerebellar cell cultures causing a reduction in the number o cells. GH-immunoreactive cells were observed in normoxia (a) and hypoxia (f) conditions in the nucleus and perikarya (arrows). Immunoreactive signal for NeuN wa. observed within the nucleus in normoxia (b) and hypoxia (g). The two signals co-localized when the confocal images were merged (c for normoxia, h for hypoxia). Panels « ind i show the cell nuclei stained with DAPI in order to quantify cells present in the culture. Scale bar: 50 ttm. Higher magnification (63x) showed marked difference. yetween the two conditions (e for normoxia and j for hypoxia). Scale bar: 20 ym. (B). Total number of cell nuclei stained with DAPI (in a surface of 19,400 1m?) unde conditions of normoxia (a), hypoxia and low glucose condition (HLG, b), and HLG with addition of 1 nM rcGH (HLG + GH, c). These cell nuclei were quantified (d) on 8-1( elds per culture dish and fluoresce-DAPI marked cells are reported as a percent of total cells present in each field; significant differences were observed between treatments Normoxia had the highest number of cells, and during HLG the number of cells dramatically decreased. With 1 nM rcGH a significant increase in the number of cells wa »bserved. Each bar represents the mean + SEM, n=5, *p < 0.05; “p< 0.01.
Fig. 3. (A) Hypoxia [0.5% O2] and Low Glucose [1 g/L] (HLG) incubation conditions induced cell death in chicken cerebellar cell cultures causing a reduction in the number o cells. GH-immunoreactive cells were observed in normoxia (a) and hypoxia (f) conditions in the nucleus and perikarya (arrows). Immunoreactive signal for NeuN wa. observed within the nucleus in normoxia (b) and hypoxia (g). The two signals co-localized when the confocal images were merged (c for normoxia, h for hypoxia). Panels « ind i show the cell nuclei stained with DAPI in order to quantify cells present in the culture. Scale bar: 50 ttm. Higher magnification (63x) showed marked difference. yetween the two conditions (e for normoxia and j for hypoxia). Scale bar: 20 ym. (B). Total number of cell nuclei stained with DAPI (in a surface of 19,400 1m?) unde conditions of normoxia (a), hypoxia and low glucose condition (HLG, b), and HLG with addition of 1 nM rcGH (HLG + GH, c). These cell nuclei were quantified (d) on 8-1( elds per culture dish and fluoresce-DAPI marked cells are reported as a percent of total cells present in each field; significant differences were observed between treatments Normoxia had the highest number of cells, and during HLG the number of cells dramatically decreased. With 1 nM rcGH a significant increase in the number of cells wa »bserved. Each bar represents the mean + SEM, n=5, *p < 0.05; “p< 0.01.
Fig. 4. Effect of recombinant growth hormone (rcGH) in embryonic ED15 primary cerebellar cultures treated during 1h with hypoxia and low glucose (HLG) anc followed by 24h of reoxygenation. Cell viability was evaluated with the trypan- blue exclusion assay (A) and the MTT assay (B). The graph shows viable cells as ¢ percent of total cells. Cells were subjected to HLG in the presence of 1nM GH (H + GH1), 10 nM GH (H + GH10), and 100 nM GH (H + GH100). Controls (N) were maintained under normoxic conditions (37 °C, 95% air, 5% CO). Each bar represents the mean+SEM, n=5 independent experiments with three replicates for each sample. Groups with different letters are significantly different from each other by ANOVA, p < 0.05.
Fig. 4. Effect of recombinant growth hormone (rcGH) in embryonic ED15 primary cerebellar cultures treated during 1h with hypoxia and low glucose (HLG) anc followed by 24h of reoxygenation. Cell viability was evaluated with the trypan- blue exclusion assay (A) and the MTT assay (B). The graph shows viable cells as ¢ percent of total cells. Cells were subjected to HLG in the presence of 1nM GH (H + GH1), 10 nM GH (H + GH10), and 100 nM GH (H + GH100). Controls (N) were maintained under normoxic conditions (37 °C, 95% air, 5% CO). Each bar represents the mean+SEM, n=5 independent experiments with three replicates for each sample. Groups with different letters are significantly different from each other by ANOVA, p < 0.05.
Fig. 5. Effect of added recombinant growth hormone upon apoptosis of embryonic chicken cerebellar primary cell cultures after 1 h of HLG treatment followed by a 24-h reoxygenation. (A) Caspase-3 activity was measured with a colorimetric assay kit, and the activity is reported as percent of activity after normalizing the activity obtained under HLG value as 100%. Addition of 1 nM rcGH during HLG decreased the caspase-3 activity. (B) Apoptotic bodies (arrows) were revealed by the TUNEL assay. Control cell cultures under normoxic conditions (N) displayed very low TUNEL reactivity (i-iii); cells subjected only to HLG without GH treatment showed the highest TUNEL signal (iv- vi), and with GH (HLG + GH) the TUNEL signal decreased significantly (vii-ix). (C) Quantization was performed on 8-10 fields per culture dish, and fluoresce-marked cells (green, ii, v, vii) are reported as a percent of total cells present in each field, observed with DAPI using a confocal microscope (i, iv, vii). Each bar represents the mean + SEM, n=5. Groups with different asterisks are significantly different from each other by one-way ANOVA and Tukey’s multiple-comparison post hoc tests, p < 0.05.
Fig. 5. Effect of added recombinant growth hormone upon apoptosis of embryonic chicken cerebellar primary cell cultures after 1 h of HLG treatment followed by a 24-h reoxygenation. (A) Caspase-3 activity was measured with a colorimetric assay kit, and the activity is reported as percent of activity after normalizing the activity obtained under HLG value as 100%. Addition of 1 nM rcGH during HLG decreased the caspase-3 activity. (B) Apoptotic bodies (arrows) were revealed by the TUNEL assay. Control cell cultures under normoxic conditions (N) displayed very low TUNEL reactivity (i-iii); cells subjected only to HLG without GH treatment showed the highest TUNEL signal (iv- vi), and with GH (HLG + GH) the TUNEL signal decreased significantly (vii-ix). (C) Quantization was performed on 8-10 fields per culture dish, and fluoresce-marked cells (green, ii, v, vii) are reported as a percent of total cells present in each field, observed with DAPI using a confocal microscope (i, iv, vii). Each bar represents the mean + SEM, n=5. Groups with different asterisks are significantly different from each other by one-way ANOVA and Tukey’s multiple-comparison post hoc tests, p < 0.05.
Fig. 6. Effect of recombinant growth hormone (rcGH) and wortmannin upon cell viability and apoptosis in embryonic ED15 primary cerebellar cultures treated during 1h with hypoxia and low glucose (HLG) and followed by 24 h of re-oxygenation. (A) Cell viability was evaluated with the trypan-blue exclusion assay under normoxia condition (N), hypoxia and low glucose (HLG) or in presence of 1nM GH (N+ GH), (HLG + GH) respectively. Both groups were also incubated in presence of the PI3K/Akt pathway inhibitor (100 nM) wortmannin, N + W or HLG + W and the combination of N + W + GH or HLG + W + GH. (B). The proportion of apoptotic cells was evaluated by TUNEL assay. Treatment groups were similar to the above mentioned, with the inclusion of and additional control (N+ GH). Each bar represents the mean + SEM, n= 3 independent experiments with 5 replicates each. Groups with different letters are significantly different from each other by one-way ANOVA and Tukey’s multiple- comparison post hoc tests, p < 0.001.
Fig. 6. Effect of recombinant growth hormone (rcGH) and wortmannin upon cell viability and apoptosis in embryonic ED15 primary cerebellar cultures treated during 1h with hypoxia and low glucose (HLG) and followed by 24 h of re-oxygenation. (A) Cell viability was evaluated with the trypan-blue exclusion assay under normoxia condition (N), hypoxia and low glucose (HLG) or in presence of 1nM GH (N+ GH), (HLG + GH) respectively. Both groups were also incubated in presence of the PI3K/Akt pathway inhibitor (100 nM) wortmannin, N + W or HLG + W and the combination of N + W + GH or HLG + W + GH. (B). The proportion of apoptotic cells was evaluated by TUNEL assay. Treatment groups were similar to the above mentioned, with the inclusion of and additional control (N+ GH). Each bar represents the mean + SEM, n= 3 independent experiments with 5 replicates each. Groups with different letters are significantly different from each other by one-way ANOVA and Tukey’s multiple- comparison post hoc tests, p < 0.001.
Fig. 7. GH activation of the PI3K/Akt signaling pathway. (A and B) Cell cultures were maintained either under normoxic or 1 h HLG conditions, and exposed to the following treatments: without (N and H, respectively) or with addition of 1 nM rcGH (lane GH); purified IgGs against GH (lane A); purified IgGs against GH + 1 nM rcGH (lane A+ GH), 100 nM wortmannin (lane W); or 100 nM wortmannin + 1 nM rcGH (lane W + GH). After treatment, cell homogenates were prepared and analyzed by Western Blot for Akt, p- Akt, and actin. Activation of the PI3K/Akt pathway was indicated by the phosphorylation of Akt (60 kDa). (C and D) Densitometric analysis: each bar represents the mean of the p-Akt/Akt ratio + SEM, expressed in%; n = 5 independent experiments with three replicates for each sample. Groups with different letters are significantly different from each other by one-way ANOVA and Tukey’s multiple- comparison post hoc tests, p < 0.05.
Fig. 7. GH activation of the PI3K/Akt signaling pathway. (A and B) Cell cultures were maintained either under normoxic or 1 h HLG conditions, and exposed to the following treatments: without (N and H, respectively) or with addition of 1 nM rcGH (lane GH); purified IgGs against GH (lane A); purified IgGs against GH + 1 nM rcGH (lane A+ GH), 100 nM wortmannin (lane W); or 100 nM wortmannin + 1 nM rcGH (lane W + GH). After treatment, cell homogenates were prepared and analyzed by Western Blot for Akt, p- Akt, and actin. Activation of the PI3K/Akt pathway was indicated by the phosphorylation of Akt (60 kDa). (C and D) Densitometric analysis: each bar represents the mean of the p-Akt/Akt ratio + SEM, expressed in%; n = 5 independent experiments with three replicates for each sample. Groups with different letters are significantly different from each other by one-way ANOVA and Tukey’s multiple- comparison post hoc tests, p < 0.05.
Fig. 8. GH effects on Bcl-2 levels. (A and B) Cell cultures were maintained either under normoxic conditions (N) or 1h HLG (H), end exposed to the following treatments: without or in the presence of 1 nM rcGH (lane GH); purified IgGs against GH (lane A); purified IgGs against GH + 1 nM rcGH (lane A+ GH); 100 nM wortmannin (lane W); or 100 nM wortmannin + 1 nM rcGH (lane W + GH). After 24-h re-oxygenation, cell homogenates were analyzed by Western Blot for Bcl-2 levels. Detection of the Bcl-2 band (26 kDa) indicated activation of the Bcl-2 pathway. (C and D) Densitometric analysis: each bar represents the mean of the Bcl-2/Actin ratio + SEM, n=5 independent experiments with three replicates for each sample. Groups with different letters are significantly different from each other by one-way ANOVA and Tukey’s multiple- comparison post hoc tests, p < 0.05.
Fig. 8. GH effects on Bcl-2 levels. (A and B) Cell cultures were maintained either under normoxic conditions (N) or 1h HLG (H), end exposed to the following treatments: without or in the presence of 1 nM rcGH (lane GH); purified IgGs against GH (lane A); purified IgGs against GH + 1 nM rcGH (lane A+ GH); 100 nM wortmannin (lane W); or 100 nM wortmannin + 1 nM rcGH (lane W + GH). After 24-h re-oxygenation, cell homogenates were analyzed by Western Blot for Bcl-2 levels. Detection of the Bcl-2 band (26 kDa) indicated activation of the Bcl-2 pathway. (C and D) Densitometric analysis: each bar represents the mean of the Bcl-2/Actin ratio + SEM, n=5 independent experiments with three replicates for each sample. Groups with different letters are significantly different from each other by one-way ANOVA and Tukey’s multiple- comparison post hoc tests, p < 0.05.
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