Journal: FEBS letters

Article Title: Hypoxia inducible factor-1α suppresses Peroxiredoxin 3 expression to promote proliferation of CCRCC cells.

doi: 10.1016/j.febslet.2014.07.030

Figure Lengend Snippet: Fig. 2. The expression of Prx3 is regulated by VHL. (A–C) The mRNA and protein levels of the indicated genes were respectively detected by real-time quantitative RT-PCR and Western blot. RCC4/VHL and Caki-1 cells were infected with retroviral vectors harboring shRNAs against VHL (shVHL) or scrambled negative control (NC) (B and C). EV represents empty vector. The column represents mean with bar as S.D. of three independent experiments with triplicate samples (⁄P < 0.05; ⁄⁄P < 0.01).

Article Snippet: The proteins were probed by antibodies against human Prx3 (sc59661, Santa Cruz Biotech), HIF-1a (610958, BD Transduction Laboratories), VHL (NB100-1899, Novus biologicals), PDK1 (KAPPK112, Enzo life sciences) and Actin (JLA20, Merck).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Infection, Retroviral, Negative Control, Plasmid Preparation

Journal: FEBS letters

Article Title: Hypoxia inducible factor-1α suppresses Peroxiredoxin 3 expression to promote proliferation of CCRCC cells.

doi: 10.1016/j.febslet.2014.07.030

Figure Lengend Snippet: Fig. 3. Prx3 is negatively regulated by HIF-1a. The mRNA and protein levels of the indicated genes were respectively detected by real-time quantitative RT-PCR and Western blot. (A) RCC4/VHL cells were exposed to normal air or hypoxia (1% O2) for 12 h. (B) RCC4 cells were stably transfected with shRNAs against HIF-1a (a1and a2). (C) Caki-1 cells transfected with shRNAs against HIF-1a (a1 and a2) were incubated in normoxia or hypoxia for 12 h. Columns, means of three determinations; bars, S.D. (⁄P < 0.05; ⁄⁄P < 0.01).

Article Snippet: The proteins were probed by antibodies against human Prx3 (sc59661, Santa Cruz Biotech), HIF-1a (610958, BD Transduction Laboratories), VHL (NB100-1899, Novus biologicals), PDK1 (KAPPK112, Enzo life sciences) and Actin (JLA20, Merck).

Techniques: Quantitative RT-PCR, Western Blot, Stable Transfection, Transfection, Incubation

Journal: FEBS letters

Article Title: Hypoxia inducible factor-1α suppresses Peroxiredoxin 3 expression to promote proliferation of CCRCC cells.

doi: 10.1016/j.febslet.2014.07.030

Figure Lengend Snippet: Fig. 4. Two HREs in promoter of PRDX3 are essential for HIF-1a transactivity. (A) 293T cells were transfected with luciferase reporter plasmids driven by four putative HREs in PRDX3 promoter, which are shown on the top and middle panels, and were grown in the normal air or hypoxia for 12 h. In top diagram of putative HREs (black ovals), empty circles represent the transcriptional start point of PRDX3. (B) 293T cells were transfected with luciferase reporter plasmids driven by four putative HREs in PRDX3 promoter together with the indicated doses of HIF-1a and grown in the normal air for 36 h. Protein levels of HIF-1a were detected by western blot with Actin as a loading control (low panel). (C) Luciferase reporter plasmids driven by the HREs or CG/AA mutated HRE sequences as indicated were transfected together with HIF-1a expressing vector or empty vector into 293T cells for 36 h in normal air. All the relative luciferase activities of PRDX3 promoter were normalized by pSV40-Renilla and estimated as the relative folds against cells under normal air (A, lower panel) or empty vector-transfected cells (B and C). (D) RCC4/VHL cells were grown under normoxia and hypoxia for 12 h. Chromatin immunoprecipitation assay was performed as described in Section 2. The column represents mean with bar as S.D. of three independent experiments with triplicate samples (⁄P < 0.05; ⁄⁄P < 0.01).

Article Snippet: The proteins were probed by antibodies against human Prx3 (sc59661, Santa Cruz Biotech), HIF-1a (610958, BD Transduction Laboratories), VHL (NB100-1899, Novus biologicals), PDK1 (KAPPK112, Enzo life sciences) and Actin (JLA20, Merck).

Techniques: Transfection, Luciferase, Western Blot, Control, Expressing, Plasmid Preparation, Chromatin Immunoprecipitation

Journal: BMC Oral Health

Article Title: BACH1 regulates the proliferation and odontoblastic differentiation of human dental pulp stem cells

doi: 10.1186/s12903-022-02588-2

Figure Lengend Snippet: BACH1 was mainly located in the odontoblast layer of human dental pulp tissues. a Immunohistochemical staining was performed to show the BACH1 expression in healthy human dental pulp tissues; arrowheads indicate BACH1 expression. Scale bars = 50 µm. b BACH1 (red) and HO-1 (green) expression was detected by double immunofluorescence staining. Blue, DAPI-stained cells. c Positive cell rate analysis indicated that the expression of BACH1 and HO-1 increased in the odontoblast layer (red) compared with the cell rich zone (yellow). Scale bar = 20 µm. Data were presented as the mean ± standard error of the mean (n = 7). ** P < 0.01, *** P < 0.001

Article Snippet: Rabbit polyclonal antibodies against BACH1 (1:150; Proteintech) were used as the primary antibodies for 24 h at 4 °C.

Techniques: Immunohistochemical staining, Staining, Expressing, Double Immunofluorescence Staining

Journal: BMC Oral Health

Article Title: BACH1 regulates the proliferation and odontoblastic differentiation of human dental pulp stem cells

doi: 10.1186/s12903-022-02588-2

Figure Lengend Snippet: The expression of BACH1 was changed during the odontoblastic differentiation of hDPSCs. a Changes in the mRNA level of BACH1 , DMP1 and DSPP on OM days 0,1,3,7 and 14. GAPDH was used as an internal control. b Changes in the total protein level of BACH1, DMP1 and DSPP on OM days 0, 1, 3, 7 and 14. Quantification of the relative protein expression via Image Pro Plus software, in which GAPDH was served as the internal control. c BACH1 protein expression of nuclear fraction on OM days 0, 1, 3, 7 and 14 had been tested by Western blot analysis. Lamin B1 was used as loading control. d Representative immunofluorescent labelling images are displayed. BACH1 (red), DAPI (blue) and Phalloidin (green) were imaged to show the subcellular distribution of BACH1 on OM days 0, 1 and 14. Scale bar = 10 µm. Data were presented as the mean ± standard error of the mean (n = 3). Statistically significant compared to the day 0 group. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. The gels in b, c were cropped, full-length gels are presented in Additional file : Original Band of Western blot Analysis Figs. S1 and S2. In Fig. 3b, the bands of BACH1 and GAPDH were from the same gel, while the bands of DSPP and DMP1 were from different gels. In Fig. 3c, the bands of BACH1 and Lamin B1 were from the same gel

Article Snippet: Rabbit polyclonal antibodies against BACH1 (1:150; Proteintech) were used as the primary antibodies for 24 h at 4 °C.

Techniques: Expressing, Control, Software, Western Blot

Journal: BMC Oral Health

Article Title: BACH1 regulates the proliferation and odontoblastic differentiation of human dental pulp stem cells

doi: 10.1186/s12903-022-02588-2

Figure Lengend Snippet: The downregulation of BACH1 impeded the proliferation and induced cell cycle arrest of hDPSCs in vitro. a The effect of BACH1 silencing on the cell proliferation of hDPSCs was determined by CCK-8 assay. BACH1 silencing group (LV-shBACH1) and control group (LC-NC). b EdU assay for cell proliferation. Red, EdU-positive cells; Blue, Hoescht-stained cells. The percentage of EdU-positive cells in the LV-shBACH1 and LC-NC was recorded. Scale bar = 50 µm. c Percentage of cell cycle distribution tested by flow cytometry. Statistically significant compared to the LV-NC group. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Rabbit polyclonal antibodies against BACH1 (1:150; Proteintech) were used as the primary antibodies for 24 h at 4 °C.

Techniques: In Vitro, CCK-8 Assay, Control, EdU Assay, Staining, Flow Cytometry

Journal: BMC Oral Health

Article Title: BACH1 regulates the proliferation and odontoblastic differentiation of human dental pulp stem cells

doi: 10.1186/s12903-022-02588-2

Figure Lengend Snippet: BACH1 affected the odontoblastic differentiation of hDPSCs in vitro. a Transduced hDPSCs were cultured with OM for 7 days. ALP activity was evaluated by ALP staining and ALP activity assay in the LV-NC and LV-shBACH1 group. b hDPSCs were cultured with OM for 21 days. Calcium nodule deposition was evaluated by ARS staining assay. Scale bar = 200 µm. c mRNA level of BACH1 , HMOX-1 and odontogenic markers in LV-NC and LV-shBACH1 hDPSCs on OM 14 days. GAPDH was used as an internal control. d Total protein level of BACH1, HO-1 and odontogenic markers in LV-NC and LV-shBACH1 hDPSCs on OM 14 days. Quantification of the relative protein expression via Image Pro Plus software. GAPDH was used as an internal control. Data were presented as the mean ± standard error of the mean (n = 3). Statistically significant compared to the LV-NC group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. The gels in d were cropped, full-length gels are presented in Additional file Original Band of Western blot Analysis Fig. S3. The bands of BACH1, DSPP, HO-1 and GAPDH were from the same gel, while the bands of RUNX2 and DMP1 were from different gels

Article Snippet: Rabbit polyclonal antibodies against BACH1 (1:150; Proteintech) were used as the primary antibodies for 24 h at 4 °C.

Techniques: In Vitro, Cell Culture, Activity Assay, Staining, ALP Activity Assay, Control, Expressing, Software, Western Blot

Journal: BMC Oral Health

Article Title: BACH1 regulates the proliferation and odontoblastic differentiation of human dental pulp stem cells

doi: 10.1186/s12903-022-02588-2

Figure Lengend Snippet: The downregulation of BACH1 impaired the odontoblastic differentiation of hDPSCs was HO-1-independent. a Transduced hDPSCs were cultured for 7 days. ALP activity was evaluated by ALP staining and ALP activity assay in the LV-NC, LV-shBACH1 and LV-shBACH1 + SnPP group. b Total protein level of odontogenic markers in LV-NC, LV-shBACH1 and LV-shBACH1+ SnPP hDPSCs on 14 days. Quantification of the relative protein expression of odontogenic markers via Image Pro Plus software. GAPDH was used as an internal control. Statistically significant compared to the control group. c hDPSCs were cultured for 21 days. Calcium nodule deposition was evaluated by ARS staining assay. Scale bar = 1 mm. SnPP, Tin-protoporphyrin IX. hDPSCS were cultured in OM or OM + SnPP (10 µM). Data were presented as the mean ± standard error of the mean (n = 3). Statistically significant compared to every other group. * P < 0.05, ** P < 0.01, *** P < 0.001. The gels in b were cropped, full-length gels are presented in Additional file Original Band of Western blot Analysis Fig. S4. The bands of DSPP, RUNX2 and GAPDH were from the same gel, while the band of DMP1 was from other gel

Article Snippet: Rabbit polyclonal antibodies against BACH1 (1:150; Proteintech) were used as the primary antibodies for 24 h at 4 °C.

Techniques: Cell Culture, Activity Assay, Staining, ALP Activity Assay, Expressing, Software, Control, Western Blot