Journal: Scientific Reports

Article Title: Hypoxia mimetics restore bone biomineralisation in hyperglycaemic environments

doi: 10.1038/s41598-022-18067-1

Figure Lengend Snippet: Hypoxia (1% O 2 ) and hyperglycaemia inhibited bone nodule formation. After 21 days, Alizarin Red stained (ARS) dense nodules were observed in ( a ) normal (5.5 mM) glucose, whilst ( b ) moderate (25 mM) and ( c ) high (50 mM) glucose conditions inhibited nodule formation. ( d ) Hypoxia normal glucose showed discrete biomineralisation that was not associated with collagen fibres. ( e ) Moderate and ( f ) high glucose inhibited biomineralisation. Transmission electron microscopy (TEM) micrographs of ( g ) normoxia normal glucose, ( h ) moderate glucose and ( i) high glucose showed a glucose concentration dependent inhibition of bone nodule formation. COL fibres were not observed in ( j ) hypoxia normal glucose, but ( k ) moderate and high glucose environments in hypoxia showed some collagen fibres. Scale bar for ( a – f ) is 200 µm and for ( g – i ) is 2 µm. (n = 5) (N: nodule, COL: collagen fibres, OB: osteoblast).

Article Snippet: Cells were seeded at a density of 6 × 10 4 cells/well into 12-well plates in Alpha modified minimum essential medium (α-MEM) supplemented with 10% foetal bovine serum (FBS; Thermo Fisher Scientific), 100 U/mL penicillin, 100 μg/mL streptomycin, 0.25 μg/mL amphotericin (Sigma-Aldrich) and 2 mM l -glutamine (Life Technologies) and kept at 37 °C in a humidified normoxia incubator under 20% O 2 and 5% CO 2 (Binder GmbH, Germany).

Techniques: Staining, Transmission Assay, Electron Microscopy, Concentration Assay, Inhibition

Journal: Scientific Reports

Article Title: Hypoxia mimetics restore bone biomineralisation in hyperglycaemic environments

doi: 10.1038/s41598-022-18067-1

Figure Lengend Snippet: Inhibition of bone nodule formation in hypoxia and hyperglycaemia quantified by ( a ) interferometry (area above 30 µm) and ( b ) image analysis of Alizarin Red staining (total area). Nodules cultured in normoxia normal (5.5 mM) glucose covered a substantially larger area ( a and b total area) than both normoxia moderate (25 mM) and high (50 mM) glucose. ( c ) ALP activity per unit protein revealed that all normoxic conditions had higher ALP production than hypoxic conditions. Error bars represent the SD from the mean values. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001 (n = 4).

Article Snippet: Cells were seeded at a density of 6 × 10 4 cells/well into 12-well plates in Alpha modified minimum essential medium (α-MEM) supplemented with 10% foetal bovine serum (FBS; Thermo Fisher Scientific), 100 U/mL penicillin, 100 μg/mL streptomycin, 0.25 μg/mL amphotericin (Sigma-Aldrich) and 2 mM l -glutamine (Life Technologies) and kept at 37 °C in a humidified normoxia incubator under 20% O 2 and 5% CO 2 (Binder GmbH, Germany).

Techniques: Inhibition, Staining, Cell Culture, Activity Assay

Journal: Scientific Reports

Article Title: Hypoxia mimetics restore bone biomineralisation in hyperglycaemic environments

doi: 10.1038/s41598-022-18067-1

Figure Lengend Snippet: The effects of HIF-1α mimetics and differing glucose environments on the ultrastructure of bone nodules. Transmission electron microscopy (TEM) of ( a – c ) moderate and high glucose levels showed reduced extracellular mineralised collagen fibres compared to normal glucose in normoxia. The HIF-1α mimetics ( d , j ) CoCl 2 and ( j , m ) DMOG, reduced extracellular mineralised collagen in normal (5 mM glucose conditions) but restored extracellular (bone-like) mineral in ( e , k , h ) moderate (25 mM) and ( f , i , l ) high (50 mM) glucose conditions compared to ( a – c ) untreated controls (with the exception of o ) 500 mM DMOG where no extracellular mineralised collagen fibres were observed. Scale bar for all images is 200 µm. (n = 5) (N: nodule, COL: collagen fibres, OB: osteoblast).

Article Snippet: Cells were seeded at a density of 6 × 10 4 cells/well into 12-well plates in Alpha modified minimum essential medium (α-MEM) supplemented with 10% foetal bovine serum (FBS; Thermo Fisher Scientific), 100 U/mL penicillin, 100 μg/mL streptomycin, 0.25 μg/mL amphotericin (Sigma-Aldrich) and 2 mM l -glutamine (Life Technologies) and kept at 37 °C in a humidified normoxia incubator under 20% O 2 and 5% CO 2 (Binder GmbH, Germany).

Techniques: Transmission Assay, Electron Microscopy

Journal: Scientific Reports

Article Title: Hypoxia mimetics restore bone biomineralisation in hyperglycaemic environments

doi: 10.1038/s41598-022-18067-1

Figure Lengend Snippet: Raman spectra of rat calvarial native bone and rat calvarial osteoblasts in normoxia, hypoxia and 12.5 µM CoCl 2 all in normal glucose conditions (5.5 mM). ( a ) Average Raman peaks associated with proteins (Amide III, CH 2 , Amide I) are much reduced in hypoxia compared to normoxia, ( b ) with a much higher mineral to matrix ratio. The hypoxia mimetic CoCl 2 had a different effect than hypoxia and had a biochemical composition more similar to normal culture conditions and bone. **P ≤ 0.01; ***P ≤ 0.001*; ****P ≤ 0.0001 (the minimum number of bone nodules per treatment = 10).

Article Snippet: Cells were seeded at a density of 6 × 10 4 cells/well into 12-well plates in Alpha modified minimum essential medium (α-MEM) supplemented with 10% foetal bovine serum (FBS; Thermo Fisher Scientific), 100 U/mL penicillin, 100 μg/mL streptomycin, 0.25 μg/mL amphotericin (Sigma-Aldrich) and 2 mM l -glutamine (Life Technologies) and kept at 37 °C in a humidified normoxia incubator under 20% O 2 and 5% CO 2 (Binder GmbH, Germany).

Techniques:

Journal: iScience

Article Title: Predicting tuberculosis drug efficacy in preclinical and clinical models from in vitro data

doi: 10.1016/j.isci.2025.111932

Figure Lengend Snippet: In vitro assays compiled and their sources

Article Snippet: THP-1 Hypoxia , Evotec , S. Souriant et al. 2023 , Macrophage/slow-growing.

Techniques: In Vitro, Binding Assay, Ex Vivo, Alamar Blue Assay

Journal: Open Biology

Article Title: Differential sub-nuclear distribution of hypoxia-inducible factors (HIF)-1 and -2 alpha impacts on their stability and mobility

doi: 10.1098/rsob.160195

Figure Lengend Snippet: Sub-nuclear localization of HIF-1α and HIF-2α. ( a ) HeLa cells ectopically expressing HIF-1α and HIF-2α EGFP fusions compared with endogenous HIF-1α and HIF-2α labelled using immunostaining. Images of HIF-1α were taken following DMOG treatment (6 h; 0.5 mM). Scale bar, 5 μm. ( b ) HeLa cells transiently transfected with plasmids encoding (i) clover-HIF-2α (green, pseudocolour), (ii) dsRED-HIF-2α (red, pseudocolour), (iii) HIF-2α-venus (yellow pseudocolour) and (iv) Halotag-HIF-2α (green, pseudocolour). The cells expressing Halotag-HIF-2α were labelled with the fluorescent Oregon Green Halotag ligand (HL-OregonGreen; Promega, WI, USA) to visualize the fusion protein. ( c ) Confocal images of C2C12 (mouse myoblast; top) and HEK293T (Human embryonic kidney cells; bottom) cells ectopically expressing EGFP-HIF-2α. Scale bar, 5 μm. ( d ) HeLa cells transiently transfected with EGFP-HIF-2α were imaged with a CCD camera. One thousand frames were acquired per cell in normoxia, hypoxia (1% v/v O 2 , 16 h) or following treatment with DMOG (0.5 mM, 6 h). The average (±s.d.) number of speckles per nucleus in each condition was 64 ± 49 ( n = 25), 44 ± 24 ( n = 24) and 96 ± 33 ( n = 22), respectively. Mean of the sample data represented by the red dashed line. ( e ) Using the images from ( d ) the average speckle area per nucleus over the 1000 frames. The mean values (±s.d.) for each condition were 0.24 ± 0.09 µm ( n = 25), 0.21 ± 0.07 µm ( n = 24) and 0.27 ± 0.09 µm ( n = 22), respectively. The mean values for hypoxia and DMOG were compared with the normoxic values (independent t -test, significance value set at 5%). Mean of the sample data represented by the red dashed line.

Article Snippet: Incubation conditions were as follows: normoxia (20% O 2 v/v, 5% CO 2 v/v, 37°C; Sanyo Electric Biomedical Co., Japan); hypoxia (1% O 2 v/v, 5% CO 2 v/v, 37°C; Don Whitley Scientific, UK).

Techniques: Expressing, Immunostaining, Transfection

Journal: Open Biology

Article Title: Differential sub-nuclear distribution of hypoxia-inducible factors (HIF)-1 and -2 alpha impacts on their stability and mobility

doi: 10.1098/rsob.160195

Figure Lengend Snippet: Analysis of the HIF-2α speckles trajectories. ( a ) (i) HIF-2α speckles were tracked over time. (ii) The speckles were identified in each frame and then linked frame to frame to create a trajectory map. (iii) Coordinates of these trajectories were exported to E xcel and plotted. ( b ) Speed at which HIF-2α speckles move. Average (±s.d.) for normoxia 0.47 ± 0.17 µm s −1 ( N = 25, n = 522), hypoxia 0.38 ± 0.13 µm s −1 ( N = 24, n = 760) and DMOG 0.36 ± 0.10 µm s −1 ( N = 25, n = 1402). Independent t -test, significance level 1%; normoxia compared with hypoxia: t 1280 = 11.238, p < 0.001; normoxia compared with DMOG: t 1922 = 17.954, p < 0.001. ( c ) SMSS values for individual speckles in normoxia, hypoxia and DMOG. Average (±s.d.) SMSS (indicated by red line) was 0.12 ± 0.07 ( N = 25, n = 522), 0.13 ± 0.07 ( N = 24, n = 760) and 0.12 ± 0.08 ( N = 22, n = 1402), respectively.

Article Snippet: Incubation conditions were as follows: normoxia (20% O 2 v/v, 5% CO 2 v/v, 37°C; Sanyo Electric Biomedical Co., Japan); hypoxia (1% O 2 v/v, 5% CO 2 v/v, 37°C; Don Whitley Scientific, UK).

Techniques:

Journal: Open Biology

Article Title: Differential sub-nuclear distribution of hypoxia-inducible factors (HIF)-1 and -2 alpha impacts on their stability and mobility

doi: 10.1098/rsob.160195

Figure Lengend Snippet: Fluorescence recovery after photo-bleaching (FRAP) of EGFP-HIF-2α. ( a ) A series of confocal images of HeLa cell ectopically expressing EGFP-HIF-2α that has been photo-bleached in half of the nucleus. Images at different time points showing gradual recovery of fluorescence into the bleached region (outlined in white). ( b ) The mobile fraction of EGFP-HIF-2α molecules per nucleus. The average (±s.d.) mobile fraction of EGFP-HIF-2α was 0.95 ± 0.15 ( n = 89) in normoxia, 0.99 ± 0.16 ( n = 115) in hypoxia and 0.94 ± 0.13 ( n = 43) following treatment with DMOG. For HIF-2α-DM-EGFP, the mobile fraction was 0.9 ± 0.15 ( n = 23). ( c ) The time taken for recovery to reach half the final intensity ( t half ) per nucleus in normoxia, hypoxia and DMOG. The average (±s.d.) half-time was 0.56 ± 0.46 ( n = 89) minutes, 0.68 ± 0.36 ( n = 115) minutes and 0.40 ± 0.18 ( n = 43) minutes, respectively. The half-time for HIF-2α-DM-EGFP was 0.44 ± 0.31 ( n = 23) minutes. An independent t -test ( α = 0.05) was used to compare the mean normoxic half-time to those from the hypoxic ( t 202 = 2.122, p = 0.35) and hypoxia mimic ( t 130 = 2.156, p = 0.033) conditions. The mean value is represented by a black line on graph.

Article Snippet: Incubation conditions were as follows: normoxia (20% O 2 v/v, 5% CO 2 v/v, 37°C; Sanyo Electric Biomedical Co., Japan); hypoxia (1% O 2 v/v, 5% CO 2 v/v, 37°C; Don Whitley Scientific, UK).

Techniques: Fluorescence, Expressing

Journal: Open Biology

Article Title: Differential sub-nuclear distribution of hypoxia-inducible factors (HIF)-1 and -2 alpha impacts on their stability and mobility

doi: 10.1098/rsob.160195

Figure Lengend Snippet: Molecular mobility of HIF-2α measured using FLIP. ( a ) Confocal images of a HeLa cell ectopically expressing EGFP-HIF-2α that has been continually bleached in one region (red box). Nucleus outlined in yellow. ( b ) The average trend of fluorescence loss in normoxia, hypoxia and DMOG. Y -error bars represent standard deviation. The data were grouped (‘binned’) based on time and the X -error bars represent the standard deviation of these bins. ( c ) Half-time values were extrapolated from the curves fitted (as in ( b )) for each cell in the three conditions. Average half-time (represented by black line on graph) ± s.d.: normoxia = 3.47 ± 1.98 min ( n = 53), hypoxia = 3.96 ± 1.61 min ( n = 36), DMOG = 2.88 ± 0.88 min ( n = 23).

Article Snippet: Incubation conditions were as follows: normoxia (20% O 2 v/v, 5% CO 2 v/v, 37°C; Sanyo Electric Biomedical Co., Japan); hypoxia (1% O 2 v/v, 5% CO 2 v/v, 37°C; Don Whitley Scientific, UK).

Techniques: Expressing, Fluorescence, Standard Deviation

Journal: Open Biology

Article Title: Differential sub-nuclear distribution of hypoxia-inducible factors (HIF)-1 and -2 alpha impacts on their stability and mobility

doi: 10.1098/rsob.160195

Figure Lengend Snippet: Comparing molecular availability and mobility of HIF-1α and HIF-2α. ( a ) The mobile fraction of EGFP-HIF-2α compared to HIF-1α-EGFP. The average (±s.d.) mobile fraction and half-time for EGFP-HIF-2α are the same as in figure 5 . Only hypoxia and DMOG conditions are compared as HIF-1α-EGFP cannot be observed in normoxia and so cannot be photo-bleached in this condition. The average (±s.d.) mobile fraction of HIF-1α-EGFP per nucleus was 0.92 ± 0.11 ( n = 31) hypoxia and 0.92 ± 0.12 ( n = 29) following treatment with DMOG (0.5 mM, 6 h). Independent t -test ( α = 0.05) revealed that the mobile fraction of HIF-1α-EGFP in hypoxia is significantly less than EGFP-HIF-2α ( t 144 = 2.412, p = 0.017). ( b ) The half-time recovery of EGFP-HIF-2α compared with that of HIF-1α-EGFP in hypoxia and DMOG. In both hypoxic (independent t -test: t 144 = 9.532, p < 0.001) and DMOG (independent t -test: t 70 = 10.278, p < 0.001) conditions the half-time of HIF-1α-EGFP was significantly less than EGFP-HIF-2α. Average values represented by black line on graph.

Article Snippet: Incubation conditions were as follows: normoxia (20% O 2 v/v, 5% CO 2 v/v, 37°C; Sanyo Electric Biomedical Co., Japan); hypoxia (1% O 2 v/v, 5% CO 2 v/v, 37°C; Don Whitley Scientific, UK).

Techniques: