Journal: Open Biology

Article Title: Notch stimulates growth by direct regulation of genes involved in the control of glycolysis and the tricarboxylic acid cycle

doi: 10.1098/rsob.150155

Figure Lengend Snippet: Metabolic genes are Notch targets in vivo and in primary human cells and Notch activity can rescue growth in nutrient-deprivation conditions. ( a ) The expression pattern of HexA–Gal4 reporter in the wing discs (crossed to UAS-lacZ). The strength of the signal from HexA–Gal4 reporter was quantified in I mage J by calculating the integrated density of HexA immunostaining signal from the whole disc (sum of pixel values), after background subtraction, using Z -stacks of confocal pictures spanning the whole disc thickness. The integrated density was divided by area of the disc and plotted as ‘HexA intensity per area’. ( b ) The intensity of HexA–Gal4 reporter in wing discs after deactivation of Notch signalling in flies with thermosensitive allele N ts2 , relative to the expression of HexA–Gal4 reporter in wild-type control Oregon R flies. The y -axis represents intensity of HexA reporter per area of the disc, x- axis indicates hours after shifting flies from 18°C to the non-permissive temperature of 29°C. Significance is according to one-tailed Student's t -test, compared with values at time 0. ( c ) The change of mRNA expression of metabolic genes in wing discs with thermosensitive allele N ts2 . The ratio of mRNA levels at 29°C against 18°C in N ts2 was compared relative to mRNA levels in control wild-type Oregon R flies at the same temperatures. ( d ) The intensity of HexA–Gal4 reporter in wing discs after blocking Notch activation via the expression of dominant negative Mastermind. Control flies express UAS-GFP instead of Mastermind. The y -axis represents intensity of HexA reporter per area of the disc. ( e ) The intensity of HexA–Gal4 reporter in wing discs after Notch activation via the expression of Notch intracellular domain (Nicd MH3 ). Control flies express UAS-GFP instead of N icd . The y -axis represents intensity of HexA reporter per area of the disc. ( f ) The fold changes of mRNA in human microvascular endothelial cells (HMVECnd) after blocking γ -secretase with 10 mM DAPT for 6 h, in comparison with cells treated with DMSO. ( g ) Immunostaining of wing disc showing the anterior part (stained with Ci , magenta) and posterior part ( Ci negative, engrailed ). ( h ) The effect of inhibiting Notch pathway or metabolic genes on the size of en domain in wing disc. The thermosensitive form of Gal80 repressor was used with the en-Gal4 to drive expression of N RNAi , N DN , Mam DN and GFP constructs at 29°C for 96 h before dissections of L3 larval wing discs. Two copies of en-Gal4 driver were used to drive two copies of UAS-RNAi of metabolic genes or of RNAi against white gene. The ratio between en/Ci domains was plotted. Significance relative to values in control flies (grey). ( i ) The size of adult wings when larvae of indicated genotype were raised on nutrient rich (++) or nutrient poor (−−) food. The N 55e11 and H 2 mutants were crossed to yw before scoring the heterozygous progeny. ( j ) The intensity of HexA–Gal4 reporter in wild-type control Oregon R and in Hairless (H 2 ) mutant flies. The y -axis represents relative intensity per area. ( k ) Dry weight of yw and H 2 flies raised on nutrient high (++) and nutrient low (−−) diets. Bodies of 20 males with dissected wings were placed per tube, dried on a lyophilizer, and average weight per fly was calculated from three replicates. ( b–e,h–k ) Data from 15 to 30 wing discs or 40 wings; error bars show standard error of the mean or min and max values ( h ). Significance is according to one-tailed ( b ) or two-tailed ( d , e , h – k ) Student's t -test.

Article Snippet: Human microvascular endothelial cells from neonatal dermis cells (HMVECnd) were purchased from ThermoFisher Scientific and grown at 37°C in a humidified atmosphere containing 5% CO 2 , in Medium 131 containing penicillin/streptomycin and Microvascular growth supplement (all from ThermoFisher).

Techniques: In Vivo, Activity Assay, Expressing, Immunostaining, One-tailed Test, Blocking Assay, Activation Assay, Dominant Negative Mutation, Staining, Construct, Mutagenesis, Two Tailed Test

Journal: Open Biology

Article Title: Notch stimulates growth by direct regulation of genes involved in the control of glycolysis and the tricarboxylic acid cycle

doi: 10.1098/rsob.150155

Figure Lengend Snippet: Metabolic genes are Notch targets in vivo and in primary human cells and Notch activity can rescue growth in nutrient-deprivation conditions. ( a ) The expression pattern of HexA–Gal4 reporter in the wing discs (crossed to UAS-lacZ). The strength of the signal from HexA–Gal4 reporter was quantified in I mage J by calculating the integrated density of HexA immunostaining signal from the whole disc (sum of pixel values), after background subtraction, using Z -stacks of confocal pictures spanning the whole disc thickness. The integrated density was divided by area of the disc and plotted as ‘HexA intensity per area’. ( b ) The intensity of HexA–Gal4 reporter in wing discs after deactivation of Notch signalling in flies with thermosensitive allele N ts2 , relative to the expression of HexA–Gal4 reporter in wild-type control Oregon R flies. The y -axis represents intensity of HexA reporter per area of the disc, x- axis indicates hours after shifting flies from 18°C to the non-permissive temperature of 29°C. Significance is according to one-tailed Student's t -test, compared with values at time 0. ( c ) The change of mRNA expression of metabolic genes in wing discs with thermosensitive allele N ts2 . The ratio of mRNA levels at 29°C against 18°C in N ts2 was compared relative to mRNA levels in control wild-type Oregon R flies at the same temperatures. ( d ) The intensity of HexA–Gal4 reporter in wing discs after blocking Notch activation via the expression of dominant negative Mastermind. Control flies express UAS-GFP instead of Mastermind. The y -axis represents intensity of HexA reporter per area of the disc. ( e ) The intensity of HexA–Gal4 reporter in wing discs after Notch activation via the expression of Notch intracellular domain (Nicd MH3 ). Control flies express UAS-GFP instead of N icd . The y -axis represents intensity of HexA reporter per area of the disc. ( f ) The fold changes of mRNA in human microvascular endothelial cells (HMVECnd) after blocking γ -secretase with 10 mM DAPT for 6 h, in comparison with cells treated with DMSO. ( g ) Immunostaining of wing disc showing the anterior part (stained with Ci , magenta) and posterior part ( Ci negative, engrailed ). ( h ) The effect of inhibiting Notch pathway or metabolic genes on the size of en domain in wing disc. The thermosensitive form of Gal80 repressor was used with the en-Gal4 to drive expression of N RNAi , N DN , Mam DN and GFP constructs at 29°C for 96 h before dissections of L3 larval wing discs. Two copies of en-Gal4 driver were used to drive two copies of UAS-RNAi of metabolic genes or of RNAi against white gene. The ratio between en/Ci domains was plotted. Significance relative to values in control flies (grey). ( i ) The size of adult wings when larvae of indicated genotype were raised on nutrient rich (++) or nutrient poor (−−) food. The N 55e11 and H 2 mutants were crossed to yw before scoring the heterozygous progeny. ( j ) The intensity of HexA–Gal4 reporter in wild-type control Oregon R and in Hairless (H 2 ) mutant flies. The y -axis represents relative intensity per area. ( k ) Dry weight of yw and H 2 flies raised on nutrient high (++) and nutrient low (−−) diets. Bodies of 20 males with dissected wings were placed per tube, dried on a lyophilizer, and average weight per fly was calculated from three replicates. ( b–e,h–k ) Data from 15 to 30 wing discs or 40 wings; error bars show standard error of the mean or min and max values ( h ). Significance is according to one-tailed ( b ) or two-tailed ( d , e , h – k ) Student's t -test.

Article Snippet: Human microvascular endothelial cells from neonatal dermis cells (HMVECnd) were purchased from ThermoFisher Scientific and grown at 37°C in a humidified atmosphere containing 5% CO 2 , in Medium 131 containing penicillin/streptomycin and Microvascular growth supplement (all from ThermoFisher).

Techniques: In Vivo, Activity Assay, Expressing, Immunostaining, One-tailed Test, Blocking Assay, Activation Assay, Dominant Negative Mutation, Staining, Construct, Mutagenesis, Two Tailed Test