Journal:

Article Title: Transcriptional Regulation of Serine/Threonine Kinase-15 (STK15) Expression by Hypoxia and HIF-1

doi: 10.1091/mbc.E08-01-0042

Figure Lengend Snippet: Hypoxia increases STK15 mRNA and protein expression as well as STK15 promoter activity. (A) HepG2 cells were incubated under normoxia (16% O2) or hypoxia (3% O2) and then harvested at different time points. The STK15 mRNA levels were measured by Northern blot and HIF-1α levels were measured by Western blot analysis. The graphs represent STK15 mRNA and HIF-1α protein levels normalized against β-actin and indicate % induction compared with control levels. Values are means ± SEM of five independent culture experiments. (B) Representative Northern and Western blots. Total RNA (15 μg) was subjected to Northern blot analysis as outlined in Materials and Methods. For Western blots, protein samples were immunoblotted and sequentially probed with HIF-1α and β-actin antibodies as described in Materials and Methods. (C) HepG2 cells were pretreated with actinomycin D (Act D; 5 μg/ml) for 30 min, and then they were exposed to hypoxia (3% O2) for 4 h. The STK15 mRNA levels were measured by Northern blot, and STK15 protein levels were measured by Western blot analysis. The graphs represent STK15 mRNA and protein levels normalized against β-actin and indicate % induction compared with control levels. Values are means ± SEM of three independent culture experiments. (D) Representative Northern and Western blots. Total RNA (15 μg) was subjected to Northern blot analysis as outlined in Materials and Methods. For Western blots, protein samples (15 μg) were immunoblotted and sequentially probed with STK15 and β-actin antibodies as described in Materials and Methods. (E) A 1001-bp fragment of the human STK15 promoter 5′-flanking region (−986 to +15) was cloned into the luciferase vector pGL3-basic to give pGL3-STK15-WT. The STK15 promoter contains three potential HRE-sites at positions −336/−332 (HRE-1), −323/−319 (HRE-2), and −240/−236 (HRE-3). HepG2 cells were then transfected with the STK15 promoter construct (pGL3-STK15-WT) or with the pGL3-basic vector as a control as well as Renilla Luc vectors. Normalized Luc activity in the respective controls was set to 1. Values are means ± SEM of three independent experiments. Significant difference, 16% O2 versus 3% O2, *p ≤ 0.05.

Article Snippet: The primary mouse antibody against human HIF-1α (Becton Dickinson, Heidelberg, Germany) was used in a 1:2000 dilution.

Techniques: Expressing, Activity Assay, Incubation, Northern Blot, Western Blot, Control, Clone Assay, Luciferase, Plasmid Preparation, Transfection, Construct

Journal:

Article Title: Transcriptional Regulation of Serine/Threonine Kinase-15 (STK15) Expression by Hypoxia and HIF-1

doi: 10.1091/mbc.E08-01-0042

Figure Lengend Snippet: STK15 expression is induced by HIF-1α. (A) HepG2 cells were transfected with expression vectors for HIF-1α and the HIF-1α-PPN mutant (hHIF-1α P402A P564A N803A) and cultured under normoxia (16%) and mild hypoxia (8%) for 72h. The STK15 protein levels were measured by Western blot analysis and protein levels under normoxic conditions in the controls were set to 100%. Values are means ± SEM of three independent experiments. *Significant difference, 16% O2 versus 3% O2 and **significant difference, HIF-1α or HIF-1α PPN at 16% O2 and 3% O2 versus control at 16% O2, p ≤ 0.05. (B) Representative Western blot. Protein levels were analyzed by Western blot analysis with the STK15 402-, V5-, and β-actin antibodies. (C) HepG2 cells were cotransfected with the pGL3-STK15-WT construct and in controls with empty expression vector or with vectors for HIF-1α wild-type (HIF-1αV5) and the HIF-1α mutant (HIF-1αV5-PPN) as well as Renilla Luc vectors. The pGL3-Epo-HRE-Luc construct served as an independent control. Normalized Luc activity in the respective controls was set to 1. Values are means ± SEM of three independent experiments. Significant difference, HIF-1α or HIF-1α PPN versus control, *p ≤ 0.05.

Article Snippet: The primary mouse antibody against human HIF-1α (Becton Dickinson, Heidelberg, Germany) was used in a 1:2000 dilution.

Techniques: Expressing, Transfection, Mutagenesis, Cell Culture, Western Blot, Control, Construct, Plasmid Preparation, Activity Assay

Journal:

Article Title: Transcriptional Regulation of Serine/Threonine Kinase-15 (STK15) Expression by Hypoxia and HIF-1

doi: 10.1091/mbc.E08-01-0042

Figure Lengend Snippet: Expression of PHD-2 reduces the induction of the STK15 promoter under hypoxia. (A) HepG2 cells were cotransfected with an expression vector for PHD-2 or catalytically inactive PHD-2-H374A and either the pGL3-STK15-WT or EPO-HRE-Luc construct. Luc values represent the fold induction under hypoxia in comparison to normoxia. Values are means ± SEM of three independent experiments. Significant difference, control versus PHD2, *p ≤ 0.05. (B) Representative Western blot. Proteins were detected by Western blot analysis with the STK15 402-, HIF-1α, V5-, and β-actin antibodies.

Article Snippet: The primary mouse antibody against human HIF-1α (Becton Dickinson, Heidelberg, Germany) was used in a 1:2000 dilution.

Techniques: Expressing, Plasmid Preparation, Construct, Comparison, Control, Western Blot

Journal:

Article Title: Transcriptional Regulation of Serine/Threonine Kinase-15 (STK15) Expression by Hypoxia and HIF-1

doi: 10.1091/mbc.E08-01-0042

Figure Lengend Snippet: Expression of HIF-1α siRNA decreases hypoxia-dependent STK15 induction. (A) HepG2 cells were transfected with an expression vector for HIF-1α shRNA (siSTRIKE U6-HIF-1α) and cultured under normoxia and hypoxia. Proteins were detected by Western blot analysis with the STK15 402- and HIF-1α antibodies. The STK15 protein levels in control cells at 16% were set to 100%. Values are means ± SEM of three independent experiments. *Significant difference, 16% O2 versus 3% O2, **significant difference, HIF-1α shRNA at 3% O2 versus control at 3% O2, p ≤ 0.05. (B) Representative Western blot. STK15 levels were analyzed with the STK15 402 antibodies. Analysis with anti-HIF-1α was performed to show transfection efficiency and knockdown of HIF-1α expression via shRNA. (C) HepG2 cells were cotransfected with the pGL3-STK15-WT Luc construct and siSTRIKE U6-HIF-1α as well as Renilla Luc vectors. Normalized Luc activity in the respective controls was set to 1. Values are means ± SEM of three independent experiments. Significant difference, 16% O2 versus 3% O2, *p ≤ 0.05.

Article Snippet: The primary mouse antibody against human HIF-1α (Becton Dickinson, Heidelberg, Germany) was used in a 1:2000 dilution.

Techniques: Expressing, Transfection, Plasmid Preparation, shRNA, Cell Culture, Western Blot, Control, Knockdown, Construct, Activity Assay

Journal:

Article Title: Transcriptional Regulation of Serine/Threonine Kinase-15 (STK15) Expression by Hypoxia and HIF-1

doi: 10.1091/mbc.E08-01-0042

Figure Lengend Snippet: The HRE-2 mediates the HIF-1–dependent induction of the STK15 promoter. (A) HepG2 cells were transfected with either pGL3-STK15-WT or the respective STK15 promoter mutant constructs (pGL3-STK15-HRE1m-Luc, pGL3-STK15-HRE2m-Luc, pGL3-STK15-HRE3m-Luc, and STK15-HRE1m3m-Luc) as well as Renilla Luc vectors and cultured under normoxia and hypoxia. Normalized Luc values in the controls were set to 100%. Values are means ± SEM of three independent experiments. Significant difference, pGL3-STK15-WT versus mutants, *p ≤ 0.05. (B) Cells were incubated for 1 h under normoxia (N) and hypoxia (H) and ChIP was performed by using either no antibody (No), unspecific preimmune serum (pre) or the HIF-1α antibody (HIF). Quantitative PCR was performed with primers for the STK15 promoter fragment containing the three HREs and normalized to β-actin. (C) To detect binding of HIF-1 to the STK-15 promoter, a ChIP assay was performed with the STK15 promoter fragment containing the three HREs (STK-15-H) and a promoter fragment containing no HREs as a control (STK-15). I-16, input 16% O2; I-3, input 3% O2. (D) To detect binding of HIF-1 to HRE2, a ChIP assay was performed with a transfected STK15 promoter construct containing mutations in HRE-1 and HRE-3 (STK-15-HRE1mHRE3m), respectively. (E) EMSA with oligonucleotides spanning the HRE2 of the STK15 promoter. The respective 32P-labeled oligonucleotides with the wild-type HRE2 and mutant HRE2 (HREm) were incubated with 10 μg nuclear extracts. In supershift experiments the nuclear extracts were preincubated on ice for 45 min with preimmune serum or HIF-1α antibody before adding the labeled probes. The DNA–protein complexes were separated by electrophoresis on 5% native polyacrylamide gels and visualized by phosphoimaging.

Article Snippet: The primary mouse antibody against human HIF-1α (Becton Dickinson, Heidelberg, Germany) was used in a 1:2000 dilution.

Techniques: Transfection, Mutagenesis, Construct, Cell Culture, Incubation, Real-time Polymerase Chain Reaction, Binding Assay, Control, Labeling, Electrophoresis

Journal: EBioMedicine

Article Title: Bone marrow mesenchymal stem cells combined with ultra-purified alginate gel as a regenerative therapeutic strategy after discectomy for degenerated intervertebral discs

doi: 10.1016/j.ebiom.2020.102698

Figure Lengend Snippet: Cell sorting data for separation of caboxyfluorescein diactetate succinimidyl ester (CFDA-SE)-labeled bone-derived mesenchymal stem cells (BMSCs) and unlabeled nucleus pulposus cells (NPCs) following 7-day co-culture using UPAL gel. (a): Two-dimensional (2D) dot plot for co-cultured cells. The P1 gate was placed around single live cells. (b): 2D dot plot showing unlabeled NPCs in the P2 gate and CFDA-SE-labeled BMSCs in the P3 gate that were sorted. FSC-A: Forward scatter-area; FSC-W: Forward scatter-width; SSC-A: Side scatter-area; CFDA-SE-A: CFDA-SE-area. Gene expression for each cell was normalized to the housekeeping gene GAPDH and plotted on a log scale (y-axis). Data were averaged from four different rabbit NPC lines. (c): HIF-1α, (d): GLUT-1, (e): Brachyury, (f): CDMP-1, (g): TGF-β, (h): IGF-1, (i): type II collagen, and (j): aggrecan. Data are represented as the means ± SE. p -values were determined by one-way ANOVA with a post hoc Tukey–Kramer test.

Article Snippet: For HIF-1α, GLUT-1, and Brachyury staining, DyLight 550-conjugated rabbit polyclonal antibodies against HIF-1α (Novus Biologicals, Centennial, CO, USA; NB100-479R, RRID: AB_1642267), PE-conjugated rabbit polyclonal antibody against GLUT-1 (LS Bio, Seattle, WA, USA; LS-A109342-100), and unconjugated rabbit polyclonal antibodies against Brachyury (LS Bio; LS-C31179-100, RRID: AB_911118) were applied.

Techniques: FACS, Labeling, Derivative Assay, Co-Culture Assay, Cell Culture, Gene Expression

Journal: EBioMedicine

Article Title: Bone marrow mesenchymal stem cells combined with ultra-purified alginate gel as a regenerative therapeutic strategy after discectomy for degenerated intervertebral discs

doi: 10.1016/j.ebiom.2020.102698

Figure Lengend Snippet: Nucleus pulposus (NP) marker-positive cells in rabbit NPs. (a-c): Horizontal sections of rabbit intervertebral discs (IVDs) stained for HIF-1α, GLUT-1, and Brachyury at days 1, 7, and 28. Images are representative of four replicates (Intact control, Discectomy, and BMSCs + Gel, n = 4; days 1, 7, and 28). Scale bar = 50 μm. (d–f): Percentages of NP marker-positive cells relative to total cells. (g–i): Percentages of NP marker-positive cells relative to CFDA-SE-positive cells (representative of implanted BMSCs). Data are the means ± SE. p -values were determined using the paired t -test.

Article Snippet: For HIF-1α, GLUT-1, and Brachyury staining, DyLight 550-conjugated rabbit polyclonal antibodies against HIF-1α (Novus Biologicals, Centennial, CO, USA; NB100-479R, RRID: AB_1642267), PE-conjugated rabbit polyclonal antibody against GLUT-1 (LS Bio, Seattle, WA, USA; LS-A109342-100), and unconjugated rabbit polyclonal antibodies against Brachyury (LS Bio; LS-C31179-100, RRID: AB_911118) were applied.

Techniques: Marker, Staining, Control

Journal: bioRxiv

Article Title: HIF-1α Contributes to the Progression of Chronic Obstructive Pulmonary Disease

doi: 10.1101/2022.01.09.472256

Figure Lengend Snippet: To investigate the role of HIF-1α on CS-induced COPD, mice exposed to CS for 6 months were intragastrically treated with digoxin (0.02 and 0.1 mg/kg). (A) TLC, (B) FRC, (C) FVC, (D) Resistance, (E) FEV 50 /FVC and (F) Hematocrit were evaluated. All data were shown as the mean ± SD; n= 8-10 per group. Statistical significance was assessed by one-way ANOVA. * p< 0.05 and ** p< 0.01 versus control group; # p< 0.05 and ## p< 0.01 versus CS group.

Article Snippet: The primary antibodies used in our study were as follows: mouse-anti-β-actin antibody (sc-47778, Santa Cruz Biotechnology, Dallas, TX), rabbit-anti-HIF-1α antibody (NB100479, Novus Biologicals, Littleton, Colorado, USA), mouse anti-Ecadherin antibody (Cell Signaling Technology, Danvers, CA, United States), rabbit anti-Vimentin antibody (Cell Signaling Technology, Danvers, CA, United States), rabbit anti-ZO-1antibody (RA231621, Cell Signaling Technology, Danvers, CA, United States), rabbit anti-phospho-Smad3 antibody (9523S, Cell Signaling Technology, Danvers, CA, United States), and rabbit-anti-Smad3 (9520T, Cell Signaling Technology, Danvers, CA, United States). β-actin was used as the internal control.

Techniques: Control

Journal: bioRxiv

Article Title: HIF-1α Contributes to the Progression of Chronic Obstructive Pulmonary Disease

doi: 10.1101/2022.01.09.472256

Figure Lengend Snippet: (A-B) The number of total inflammatory cells and macrophages in BALF were measured by cell counting. The mRNA levels of TNF-α, IL-6, and IL-1β in mouse lung tissues were measured by real-time PCR (C-E). The protein levels of TNF-α, IL-6 and MCP-1 in BALF (F-H) were measured by ELISA. To further explore the effect of HIF-1α on CSE-induced inflammatory responses in vitro , A549 cells were treated with digoxin for 1 h before 48-h CSE exposure. IL-6 and IL-8 levels in cell supernatant (I-J) were measure by ELISA. All data were shown as the mean ± SD; n= 8-10 per group in mice, n=5 per group in A549 cells. Statistical significance was assessed by one-way ANOVA. * p< 0.05 and ** p< 0.01 versus control group; # p< 0.05 and ## p< 0.01 versus CS group.

Article Snippet: The primary antibodies used in our study were as follows: mouse-anti-β-actin antibody (sc-47778, Santa Cruz Biotechnology, Dallas, TX), rabbit-anti-HIF-1α antibody (NB100479, Novus Biologicals, Littleton, Colorado, USA), mouse anti-Ecadherin antibody (Cell Signaling Technology, Danvers, CA, United States), rabbit anti-Vimentin antibody (Cell Signaling Technology, Danvers, CA, United States), rabbit anti-ZO-1antibody (RA231621, Cell Signaling Technology, Danvers, CA, United States), rabbit anti-phospho-Smad3 antibody (9523S, Cell Signaling Technology, Danvers, CA, United States), and rabbit-anti-Smad3 (9520T, Cell Signaling Technology, Danvers, CA, United States). β-actin was used as the internal control.

Techniques: Cell Counting, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, In Vitro, Control

Journal: bioRxiv

Article Title: HIF-1α Contributes to the Progression of Chronic Obstructive Pulmonary Disease

doi: 10.1101/2022.01.09.472256

Figure Lengend Snippet: (A) The expressions of Fibronectin, Ecadherin, HIF-1[, and vimentin in lung tissues were measured by Western blot. To further explore the effect of HIF-1α on CSE-induced EMT in vitro , A549 cells were treated with digoxin before 48-h CSE exposure. (B) The expressions of ZO-1, HIF-1[, and vimentin in A549 cells were measured by Western blot. All data are shown as the mean ± SD; n=8-10 per group in mice, n=5 per group in A549 cells. Statistical significance was assessed by one-way ANOVA. * p< 0.05 and ** p< 0.01 versus control group; # p< 0.05 and ## p< 0.01 versus CS group.

Article Snippet: The primary antibodies used in our study were as follows: mouse-anti-β-actin antibody (sc-47778, Santa Cruz Biotechnology, Dallas, TX), rabbit-anti-HIF-1α antibody (NB100479, Novus Biologicals, Littleton, Colorado, USA), mouse anti-Ecadherin antibody (Cell Signaling Technology, Danvers, CA, United States), rabbit anti-Vimentin antibody (Cell Signaling Technology, Danvers, CA, United States), rabbit anti-ZO-1antibody (RA231621, Cell Signaling Technology, Danvers, CA, United States), rabbit anti-phospho-Smad3 antibody (9523S, Cell Signaling Technology, Danvers, CA, United States), and rabbit-anti-Smad3 (9520T, Cell Signaling Technology, Danvers, CA, United States). β-actin was used as the internal control.

Techniques: Western Blot, In Vitro, Control

Journal: bioRxiv

Article Title: HIF-1α Contributes to the Progression of Chronic Obstructive Pulmonary Disease

doi: 10.1101/2022.01.09.472256

Figure Lengend Snippet: The mRNA levels of HIF-1α, TGF-β1 and Smad3 in lung tissues or A549 cells were measured by real-time PCR (A-F). To further determine the role of Smad3 in COPD-associated EMT, CSE-exposed A549 cells were pretreated with S7959 (a Smad3 inhibitor) for 1 h before 48-h CSE stimulation. The protein levels of p-Smad3 and E-cadherin in A549 cells were measured by Western Blot. All data are shown as the mean ± SD; n=8-10 per group in mice, n=5 per group in A549 cells. Statistical significance was assessed by one-way ANOVA. * p< 0.05 and ** p< 0.01 versus control group; # p< 0.05 and ## p< 0.01 versus CS group.

Article Snippet: The primary antibodies used in our study were as follows: mouse-anti-β-actin antibody (sc-47778, Santa Cruz Biotechnology, Dallas, TX), rabbit-anti-HIF-1α antibody (NB100479, Novus Biologicals, Littleton, Colorado, USA), mouse anti-Ecadherin antibody (Cell Signaling Technology, Danvers, CA, United States), rabbit anti-Vimentin antibody (Cell Signaling Technology, Danvers, CA, United States), rabbit anti-ZO-1antibody (RA231621, Cell Signaling Technology, Danvers, CA, United States), rabbit anti-phospho-Smad3 antibody (9523S, Cell Signaling Technology, Danvers, CA, United States), and rabbit-anti-Smad3 (9520T, Cell Signaling Technology, Danvers, CA, United States). β-actin was used as the internal control.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: Frontiers in cell and developmental biology

Article Title: Osteocytic HIF-1α Pathway Manipulates Bone Micro-structure and Remodeling via Regulating Osteocyte Terminal Differentiation.

doi: 10.3389/fcell.2021.721561

Figure Lengend Snippet: FIGURE 1 | Continuous activation of HIF-1α in osteocytes causes excessive bone formation and impairs bone material properties. (A) Representative images of immunohistochemical staining of HIF-1α in the femurs of 2-month-old Vhlf/f-Dmp-1-Cre+ and Vhlf/f-mp-1-Cre−/−male mice. Scale bars represent 100 μm. (B) Representative images showing hematoxylin and eosin (H&E) staining of femurs of 2-month-old Vhlf/f-Dmp-1-Cre+ and Vhlf/f-Dmp-1-Cre−/−male mice. Red and black scale bars represent 500 and 100 μm, respectively. (C) Representative micro-computed tomography (micro-CT) images of femurs and the corresponding trabecular bone from the femoral metaphysis of 4-, 8-, and 24-week-old Vhlf/f-Dmp-1-Cre+ and Vhlf/f-Dmp-1-Cre−/−male mice. (D–G) The trabecular bone volume (BV/ TV) (D), trabecular number (Tb.N) (E), trabecular separation (Tb.Sp) (F), and trabecular thickness (Tb.Th) (G) parameters of 4-, 8-, and 24-week-old Vhlf/f-Dmp-1-Cre+

Article Snippet: Subsequently, sections were incubated overnight with a primary antibody against HIF-1α (NB100-105, 1/50 dilution; Novus, Centennial, CO) or RANKL (AB45039, 1/100 dilution; Abcam, United Kingdom).

Techniques: Activation Assay, Immunohistochemical staining, Staining, Micro-CT

Journal: Frontiers in cell and developmental biology

Article Title: Osteocytic HIF-1α Pathway Manipulates Bone Micro-structure and Remodeling via Regulating Osteocyte Terminal Differentiation.

doi: 10.3389/fcell.2021.721561

Figure Lengend Snippet: FIGURE 2 | Constitutive activation of HIF-1α in osteocytes leads to abnormal mineralization and disrupted collagen fiber formation in bone. (A,B) Von Kossa staining and bar graph showing the proportional Von Kossa-positive surface area relative to total bone surface area (Von Kossa/BS) in the femurs of 2-month-old Vhlf/f- Dmp-1-Cre+ and Vhlf/f-Dmp-1-Cre−/−male mice. Red and white scale bars represent 500 and 100 μm, respectively. (C) Sirius Red staining of the femurs of 2-month-old Vhlf/f-Dmp-1-Cre+ and Vhlf/f-Dmp-1-Cre−/−male mice. Red and white scale bars represent 500 and 100 μm, respectively. (D–G) Representative images of double calcein/alizarin labeling of the femurs of 2-month-old Vhlf/f-Dmp-1-Cre+ and Vhlf/f-Dmp-1-Cre−/−male mice (D) used to calculate the proportional mineral surface area over total bone surface area (MS/BS) (E), mineral apposition rate (MAR) (F), and bone formation rate per unit bone surface (BFR/BS) (G). Scale bars represent 100 μm ***p < 0.001. p-values were determined using two-tailed t-tests.

Article Snippet: Subsequently, sections were incubated overnight with a primary antibody against HIF-1α (NB100-105, 1/50 dilution; Novus, Centennial, CO) or RANKL (AB45039, 1/100 dilution; Abcam, United Kingdom).

Techniques: Activation Assay, Staining, Labeling, Two Tailed Test

Journal: Frontiers in cell and developmental biology

Article Title: Osteocytic HIF-1α Pathway Manipulates Bone Micro-structure and Remodeling via Regulating Osteocyte Terminal Differentiation.

doi: 10.3389/fcell.2021.721561

Figure Lengend Snippet: FIGURE 3 | Constitutive activation of HIF-1α impairs osteocytes terminal differentiation in vivo. (A,B) Representative images of Safranin O and toluidine blue staining in the femurs of 2-month-old Vhlf/f-Dmp-1-Cre+ and Vhlf/f-Dmp-1-Cre−/−male mice. Red and white scale bars represent 500 and 100 μm, respectively. (C–E) Immunostaining of E11 (C), DMP-1 (D), and sclerostin (E) in femur sections of 2-month-old Vhlf/f-Dmp-1-Cre+ and Vhlf/f-Dmp-1-Cre−/−male mice. Scale bar represents 100 μm. (F–H) Bar charts showing the percentages of E11- (F), DMP-1- (G), and sclerostin-positive (H) cells shown in (C–E). (I) Representative transmission electron microscopy (TEM) images showing the ultrastructure of osteocytes. Red and white scale bars represent 2 and 1 μm, respectively. (J) Texas red-X-conjugated phalloidin and 4′,6-diamidino-2-phenylindole (DAPI) staining of decalcified femoral cortices of 2-month-old Vhlf/f-Dmp-1-Cre+ and Vhlf/f-Dmp-1-Cre−/−male mice. Scale bars represent 100 μm. (K,L) Bar graphs showing the dendrites length (K) and dendrites number (L) of phalloidin-positive osteocytes depicted in (J). (M) Representative scanning electron microscopy (SEM) images showing the ultrastructure of osteocytes. Scale bar represents 10 μm ***p < 0.001. p-values were determined using two- tailed t-tests.

Article Snippet: Subsequently, sections were incubated overnight with a primary antibody against HIF-1α (NB100-105, 1/50 dilution; Novus, Centennial, CO) or RANKL (AB45039, 1/100 dilution; Abcam, United Kingdom).

Techniques: Activation Assay, In Vivo, Staining, Immunostaining, Transmission Assay, Electron Microscopy, Two Tailed Test

Journal: Frontiers in cell and developmental biology

Article Title: Osteocytic HIF-1α Pathway Manipulates Bone Micro-structure and Remodeling via Regulating Osteocyte Terminal Differentiation.

doi: 10.3389/fcell.2021.721561

Figure Lengend Snippet: FIGURE 4 | HIF-1α activation disrupts the terminal differentiation of osteocytes in vitro. (A–D) Time courses of E11 (A), DMP-1 (B), sclerostin (SOST) (C), and RANKL (D) mRNA expression in IDG-SW3 cells after osteoblastic induction with or without Vhl-or Hif-1α-small interfering RNA (siRNA) transfection. (E) Representative images of Alizarin red staining in IDG-SW3 cells after 14 days of osteoblastic induction. Scale bar represents 5 mm. (F) Three-dimensional images of Texas red-X- conjugated phalloidin staining of IDG-SW3 cells transfected with or without Vhl or Hif-1α-siRNA. Scale bar represents 20 μm. (G) Bar graphs showing the dendrite length of phalloidin-positive osteocytes depicted in (F). ***p < 0.001. p-values were determined using two-way ANOVA in (A–D) and two-tailed t-tests in (G).

Article Snippet: Subsequently, sections were incubated overnight with a primary antibody against HIF-1α (NB100-105, 1/50 dilution; Novus, Centennial, CO) or RANKL (AB45039, 1/100 dilution; Abcam, United Kingdom).

Techniques: Activation Assay, In Vitro, Expressing, Small Interfering RNA, Transfection, Staining, Two Tailed Test

Journal: Frontiers in cell and developmental biology

Article Title: Osteocytic HIF-1α Pathway Manipulates Bone Micro-structure and Remodeling via Regulating Osteocyte Terminal Differentiation.

doi: 10.3389/fcell.2021.721561

Figure Lengend Snippet: FIGURE 5 | Constitutive activation of the HIF-1α in osteocytes drives osteoblastic differentiation of bone marrow mesenchymal stem cells (BMSCs). (A) Schematic diagram depicting the two-dimensional co-culture (2D co-culture) system of BMSCs and IDG-SW3 cells with or without VHL-siRNA transfection in Transwell plates. (B) Bar graph showing the sclerostin concentrations in the supernatants of osteocytes with or without VHL-siRNA transfection depicted in (A). (C–F) mRNA expression levels of Osterix (D), RUNX2 (E), ALP (F), and osteocalcin (OC) (G) in BMSCs cultured in the system depicted in (A). (G) Alkaline phosphatase (ALP) staining of BMSCs cultured in the system depicted in (A). Scale bar represents 4 mm. (H) Osterix immunohistochemical staining in femur sections of 2-month-old Vhlf/f-Dmp-1- Cre+ and Vhlf/f-Dmp-1-Cre−/−male mice. Scale bar represents 50 μm. (I) Bar graph represents the relative protein expression of Osterix in (H). (J) Bar graph showing the serum concentrations of procollagen type 1 N-terminal propeptide (P1NP) in Vhlf/f-Dmp-1-Cre+ and Vhlf/f-Dmp-1-Cre−/−mice. *p < 0.05, **p < 0.01, ***p < 0.001. p-values were determined using two-tailed t-tests.

Article Snippet: Subsequently, sections were incubated overnight with a primary antibody against HIF-1α (NB100-105, 1/50 dilution; Novus, Centennial, CO) or RANKL (AB45039, 1/100 dilution; Abcam, United Kingdom).

Techniques: Activation Assay, Co-Culture Assay, Transfection, Expressing, Cell Culture, Staining, Immunohistochemical staining, Two Tailed Test

Journal: Frontiers in cell and developmental biology

Article Title: Osteocytic HIF-1α Pathway Manipulates Bone Micro-structure and Remodeling via Regulating Osteocyte Terminal Differentiation.

doi: 10.3389/fcell.2021.721561

Figure Lengend Snippet: FIGURE 6 | Constitutive activation of the HIF-1α in osteocytes impairs osteoclastic differentiation of bone marrow monocytes (BMMs). (A) Schematic diagram depicting the two-dimensional system used for the co-culture of BMMs and IDG-SW3 cells with or without VHL-siRNA transfection in Transwell plates. (B) Western blot showing RANKL expression in IDG-SW3 cells cultures in the system depicted in (A). (C) Representative images of tartrate-resistant acid phosphatase (TRAP) staining of BMMs cultured in the system depicted in (a). Scale bar represents 50 μm. (D) Bar graph showing the percentage of TRAP-positive area calculated from the images shown in (C). (E) Bar graph showing the relative TRAP activity of BMMs cultured in the system depicted in (A). (F) Schematic diagram depicting the three-dimensional system used to co-culture BMMs and IDG-SW3 cells with or without VHL-siRNA transfection in Transwell plates. (G) Representative images of TRAP staining of BMMs cultured in the system depicted in (F). Red and black scale bar represents 2 mm and 50 μm, respectively. (H) Schematic diagram showing IDG-SW3 cells co-cultured with BMSCs (co-up) with or without (co-down) cell-to-cell contact. (I) Bar graph showing the RANKL concentrations in the supernatants of co-up and co-down cultures depicted in (H). (J) Bar graph showing the relative TRAP activity of BMMs cultured in the co-up and co-down systems depicted in (H). *p < 0.05, **p < 0.01, ***p < 0.001. p-values were determined using two-tailed t-tests.

Article Snippet: Subsequently, sections were incubated overnight with a primary antibody against HIF-1α (NB100-105, 1/50 dilution; Novus, Centennial, CO) or RANKL (AB45039, 1/100 dilution; Abcam, United Kingdom).

Techniques: Activation Assay, Co-Culture Assay, Transfection, Western Blot, Expressing, Staining, Cell Culture, Activity Assay, Two Tailed Test