Journal: Physiological reports

Article Title: Hyperoxia causes senescence and increases glycolysis in cultured lung epithelial cells.

doi: 10.14814/phy2.14839

Figure Lengend Snippet: FIGURE 2 Hyperoxia causes DNA damage and p53 activation. Mouse lung epithelial (MLE-12) cells were exposed to 21% O2/5% CO2 (Air) or 95% O2/5% CO2 (O2) for 24 h. For immunofluorescence, Blue = DAPI, nuclei; Green = protein of interest, Cyan = overlapping blue and green signal. (a) Measured γH2AX levels using immunofluorescence. (b) Measured 53BP1 levels using immunofluorescence, quantified by macros script. (c) Transcriptional levels of p53, pRB, p16, and p21 measured by RT-qPCR. (d, e) p53 and pRB protein levels measured by western blot. Western blot normalized using calnexin (CNX) levels. (f) p53 nuclear and cytoplasmic levels measured by western blot from nuclear and cytoplasmic extracts. (g) Nuclear quantification of p53 using immunofluorescence. (h) Levels of phosphorylated p53 at Ser15 as measured by ELISA. Sample turns blue according to phosphorylated levels of p53 at Ser15, absorbance measured at 450 nm. *p < 0.05 versus air groups. RT- qPCR, quantitative real time PCR

Article Snippet: Primary antibodies included p53 (abcam 31333 at 1:200 dilution), phospho- histone H2A Ser139 (Millipore 05- 636 at 1:100 dilution) and 53BP1 (Novus Biologicals NB100- 304 at 1:1000 dilution).

Techniques: Activation Assay, Immunofluorescence, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

Journal: Physiological reports

Article Title: Hyperoxia causes senescence and increases glycolysis in cultured lung epithelial cells.

doi: 10.14814/phy2.14839

Figure Lengend Snippet: FIGURE 3 Hyperoxia-induced senescence is p53-dependent and pRB-independent. (a) Cas9 knock-in, p53KO, and pRBKO shown by Western blot, and calnexin (CNX) as a housekeeping control. (b) Senescence levels using C12FDG FACS of WT, pRBKO, and p53KO MLE-12 cells exposed to 21% O2/5% CO2 (Air, white) or 95% O2/5% CO2 (O2, black) for 24 h. (c) Emission spectrum of senescent cells is shown in blue for Air, and O2 is in red. (d) γH2AX and (e) 53BP1 levels were measured using immunofluorescence. Blue = DAPI, nuclei; Green = protein of interest, Cyan = overlapping blue and green signal. (f) γH2AX protein levels were measured by Western blot in p53KO and WT cells exposed to hyperoxia. (g) Transcriptional levels of p21, pRB, E2F1, p16, MMP3, MMP10 measured by RT-qPCR. *p < 0.05. MLE-12, mouse lung epithelial

Article Snippet: Primary antibodies included p53 (abcam 31333 at 1:200 dilution), phospho- histone H2A Ser139 (Millipore 05- 636 at 1:100 dilution) and 53BP1 (Novus Biologicals NB100- 304 at 1:1000 dilution).

Techniques: Knock-In, Western Blot, Control, Immunofluorescence, Quantitative RT-PCR

Journal: Hypertension

Article Title: Identification of Bona Fide Alternative Renin Transcripts Expressed Along Cortical Tubules and Potential Roles in Promoting Insulin Resistance In Vivo Without Significant Plasma Renin Activity Elevation

doi: 10.1161/hypertensionaha.114.03394

Figure Lengend Snippet: Figure 7. A, The results of quantitative reverse transcription polymerase chain reaction analyses of inflammatory molecules in heart, kidney, and liver of both ARen2TG and wild-type littermates are shown as relative abundance, using GAPDH expression as an internal control. *, **, #, ##Significantly elevated compared with the expressions of wild-type littermates. Repeated measure ANOVA and subsequent post hoc analyses were performed. B, Representative results of in situ histological examinations for livers of both transgenic mice and wild-type littermate using rabbit polyclonal anti-angiotensin II antibodies (NBP1-30027; Novus Biologicals, LLC, Littleton, CO) were shown. a–c, Enhanced immunohistochemical staining in livers of transgenic mice compared with those of wild-type littermates (Bd), suggesting enhanced angiotensin II production in the organs.

Article Snippet: We performed experiments to compare ARen2TG mice with wild-type littermates in terms of following phenotypes: measurement of blood pressure,16,17 measurement of plasma renin activity,16,18,19 the results of in vivo near-infrared imaging using the renin substrate analog ReninSense 680 FAST (Perkin Elmer, Waltham, MA),20 the results of insulin tolerance tests,21 fasting plasma glucose levels, the expressions of inflammatory molecules in various tissues on quantitative RT-PCR analyses,16,18,19 and in situ histological examinations for livers of both transgenic mice and wild-type littermate using rabbit polyclonal anti-angiotensin II antibodies (NBP1-30027; Novus Biologicals, LLC, Littleton, CO).22,23 All experiments were performed according to the protocols of manufacturer, the references, and our reports as specified above.

Techniques: Reverse Transcription, Polymerase Chain Reaction, Expressing, Control, In Situ, Transgenic Assay, Immunohistochemical staining, Staining