Journal: Bone research

Article Title: Hypoxia-inducible factor-1α restricts the anabolic actions of parathyroid hormone

doi: 10.1038/boneres.2014.5

Figure Lengend Snippet: Parathyroid hormone increases the expression of Hif-1α both in vitro and in vivo . ( a and b ) Levels of Hif-1α, Hif-2α and Hif-1β protein were measured in wild-type osteoblasts at the indicated time points after treatment with 10 nmol·L −1 PTH ( a ) and after 6 h of treatment with 200 nmol·L −1 DFO ( b ). ( c ) Immunohistochemical analysis of Hif-1α expression in the trabecular bone of 10-week-old female wild-type mice treated with 40 μg·kg −1 PTH or vehicle for 4 h. Representative tissue sections show increased Hif-1α expression in the osteoblasts (arrows) of PTH-treated mice relative to untreated controls. ( d and e ) mRNA levels of Hif-1α and Hif-2α were measured in vitro ( d ) and in vivo ( e ) after treatment with PTH or vehicle. Spp1 (Osteopontin) and Ptgs2 (Cox2) were used as controls. * P ≤0.05. DFO, desferoxamine.

Article Snippet: Antibodies for Hif-1α (NB100–105), Hif-2α (NB100–122) and Hif-1β (NB100–124) were obtained from Novus Biological.

Techniques: Expressing, In Vitro, In Vivo, Immunohistochemical staining

Journal: Bone research

Article Title: Hypoxia-inducible factor-1α restricts the anabolic actions of parathyroid hormone

doi: 10.1038/boneres.2014.5

Figure Lengend Snippet: cAMP/PKA signaling induces Hif-1α expression. ( a ) Levels of Hif-1α protein were measured in wild-type osteoblasts following 4 h of treatment with 10 nmol·L −1 PTH or vehicle (NT), preceded by 30 min of treatment with 100 μg·mL −1 cycloheximide (Chx). ( b ) Levels of Akt (S473) and p70 S6 kinase protein phosphorylation were measured in wild-type osteoblasts following 1 h of treatment with 10 nmol·L −1 PTH. ( c ) Hif-1α protein expression was measured in control and mTOR-deficient osteoblasts following treatment with 10 nmol·L −1 PTH for 4 h. ( d ) Densitometric quantitation of Hif-1α protein as shown in a and c . ( e and g ) Akt and p70 S6 kinase phosphorylation ( e ) and Hif-1α protein expression ( g ) were measured in wild-type osteoblasts following 30 min ( e ) or 6 hours ( g ) of treatment with Forskolin at the indicated concentrations. ( f ) Quanitification of Akt phosphorylation shown in e in Forskolin-treated osteoblasts relative to untreated. ( h ) Relative expression of Hif-1α protein as shown in g . ( i and l ) Levels of Akt and p70 S6 kinase phosphorylation ( i ) and Hif-1α protein expression ( l ) were measured following 30 min ( i ) or 6 h ( l ) of treatment with 10 nmol·L −1 PTH, preceded by 30 min of treatment with 10 μmol·L −1 H-89. ( j and k ) Quantification of Akt ( j ) and p70 S6 kinase ( k ) phosphorylation shown in i in PTH- and/or H-89-treated osteoblasts relative to untreated. ( m ) Relative expression of Hif-1α protein as shown in l . * P ≤0.05.

Article Snippet: Antibodies for Hif-1α (NB100–105), Hif-2α (NB100–122) and Hif-1β (NB100–124) were obtained from Novus Biological.

Techniques: Expressing, Phospho-proteomics, Control, Quantitation Assay

Journal: Bone research

Article Title: Hypoxia-inducible factor-1α restricts the anabolic actions of parathyroid hormone

doi: 10.1038/boneres.2014.5

Figure Lengend Snippet: Bone formation after PTH administration is increased in mice lacking Hif-1α in osteoblasts and osteocytes. Ten-week-old female control and Hif-1α mutant (ΔHif-1α) mice were treated with vehicle or PTH for 6 weeks by daily s.c. injection. ( a ) Representative microCT images illustrate trabecular bone structure in the distal femur of control and ΔHif-1α mice after treatment with PTH at the indicated doses. ( b – d ) Bone volume per tissue volume (BV/TV) ( b ), trabecular number (Tb. N) ( c ) and trabecular thickness (Tb. Th) ( d ) were quantified by microCT. ( e – g ) The mineralizing surface per bone surface (MS/BS) ( e ), mineral apposition rate (MAR) ( f ) and bone formation rate per bone surface (BFR/BS) ( g ) were assessed by dynamic histomorphometry. ( h and i ) Levels of P1NP ( h ) and CTx ( i ) were measured in serum collected from control and ΔHif-1α mice. Results from e through i are presented as % change from baseline levels in saline-treated animals, which were equivalent in control and ΔHif-1α mice. * P ≤0.05 vs. control mice with matched treatment. P1NP, amino pro-peptide of type 1 collagen; CTx, C-terminal telopeptide; s.c., subcutaneous.

Article Snippet: Antibodies for Hif-1α (NB100–105), Hif-2α (NB100–122) and Hif-1β (NB100–124) were obtained from Novus Biological.

Techniques: Control, Mutagenesis, Injection, Saline

Journal: Bone research

Article Title: Hypoxia-inducible factor-1α restricts the anabolic actions of parathyroid hormone

doi: 10.1038/boneres.2014.5

Figure Lengend Snippet: Hif-1α inhibits β-catenin activity following PTH administration.( a ) Relative expression of Hif-1α mRNA in osteoblasts following adenoviral Cre-mediated deletion. ( b ) Levels of cAMP production were measured by immunoassay after control and Hif-1α-deficient (ΔHif-1α) osteoblasts were treated with 10 nmol·L −1 PTH for 15 min. ( c ) Phosphorylation levels of Creb and Erk in control and ΔHif-1α osteoblasts were measured following 0–30 min of treatment with 10 nmol·L −1 PTH. ( d and e ) Relative expression of Axin2 ( d ) and Nkd2 ( e ) mRNA in control and ΔHif-1α osteoblasts treated with 10 nmol·L −1 PTH for 8 h or left untreated (NT). ( f ) Immunoblot analysis of Hif-1α levels in control and Vhl-deficient (ΔVhl) osteoblasts 48 h after adenoviral Cre infection. ( g ) Relative expression of Axin2 mRNA in control and ΔVhl osteoblasts treated with 10 nmol·L −1 PTH for 4 h or left untreated (NT). ( h ) Protein levels of β-catenin in nuclear extracts isolated from control and ΔHif-1α osteoblasts treated with 10 nmol·L −1 PTH for 4 h or left untreated (NT). Tbp was used as a nuclear control. ( i ) IP analysis of Hif-1α and β-catenin interaction following 4 h of 10 nmol·L −1 PTH treatment in wild-type cells. ( j ) Quantitative chromatin IP analysis of β-catenin occupancy at the Axin2 promoter in control and ΔHif-1α osteoblasts after 8 h of treatment with 10 nmol·L −1 PTH. ( k ) Relative expression of Axin2 mRNA in samples collected from the femur of control and ΔHif-1α mice after treatment with saline (NT) or 40 µg·kg −1 PTH for 16 h. * P ≤0.05. IP, immunoprecipitation; Tbp, TATA-binding protein.

Article Snippet: Antibodies for Hif-1α (NB100–105), Hif-2α (NB100–122) and Hif-1β (NB100–124) were obtained from Novus Biological.

Techniques: Activity Assay, Expressing, Control, Phospho-proteomics, Western Blot, Infection, Isolation, Chromatin Immunoprecipitation, Saline, Immunoprecipitation, Binding Assay

Journal: PLoS ONE

Article Title: Activation of Hypoxia Inducible Factor 1 Is a General Phenomenon in Infections with Human Pathogens

doi: 10.1371/journal.pone.0011576

Figure Lengend Snippet: (A) In uninfected skin, rare and faint nuclear HIF-1α staining occurred in the epidermis whereas the dermis was negative for HIF-1α (A1). (B to L) In all cutaneous infections, a strong nuclear HIF-1α signal was detected in keratinocytes, predominantly of the spinal cell layer (examplified in L). Moreover, endothelial cells of dermal capillaries frequently stained positive (arrows in B1, C1, D1, E1, G, I1). Arrowheads: (B2) border zone of the positive abscess; (C2): positive neutrophils; (F): positive dermal lympho-histiocytic infiltrates; (F1): large positive nuclei of a multi-nuclear giant cell; (F2): skin lesion surrounded by a HIF-1 positive inflammatory infiltrate; (H): positive sub-corneal neutrophils (higher power in H1); (I): positive intra-epidermal infiltrate; (K): intradermal nodules containing leishmaniae. (A) Control: uninfected sample; (B–E): bioptic samples of patients suffering from infections with humanpathogenic bacteria [(B) S. aureus , (C) S. agalactiae and S. aureus , (D) A. baumanii and E. coli and (E) B. burgdorferi ], (F, G) with humanpathogenic Herpes-viruses [(F) Varicella zoster virus (VZV) and (G) Human Herpes Virus-8 (HHV 8)], (H–J) with humanpathogenic fungi [(H+I) T. rubrum and (J) C. albicans ] or (K, L) with the protozoic pathogen L. donovani . Magnifications: all 250×, except for A1, B1, B2, C1, D1, E1, F2, I1 (all 400×), and C2, F1, H1, L (all 1,000×).

Article Snippet: Peritoneal HIF-1α activation was analyzed by immunoblotting of the shock-frozen peritoneal specimens using the Nuclear Extract Kit (Active Motif; Rixensart, Belgium), a mouse monoclonal anti-HIF-1α (H1α67) antibody (Novus Biologicals, Littleton, USA) and horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG antibodies (Dako).

Techniques: Staining, Control, Bacteria, Virus

Journal: PLoS ONE

Article Title: Activation of Hypoxia Inducible Factor 1 Is a General Phenomenon in Infections with Human Pathogens

doi: 10.1371/journal.pone.0011576

Figure Lengend Snippet: Four to six hours upon infection, HIF-1 activation was determined by Western blotting (loading control: actin). pO 2 levels were quantified in the medium of control and infected cells (A: S. aureus ATCC 33592, MOI 20, infection time: six hours; B: S. epidermidis ATCC 12228, MOI 20, infection time: six hours; C: E. coli ATCC 25992, MOI 10, infection time: four hours). Negative control: uninfected cells, positive control: hypoxia or DFO (200 µmol/L).

Article Snippet: Peritoneal HIF-1α activation was analyzed by immunoblotting of the shock-frozen peritoneal specimens using the Nuclear Extract Kit (Active Motif; Rixensart, Belgium), a mouse monoclonal anti-HIF-1α (H1α67) antibody (Novus Biologicals, Littleton, USA) and horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG antibodies (Dako).

Techniques: Infection, Activation Assay, Western Blot, Control, Negative Control, Positive Control

Journal: PLoS ONE

Article Title: Activation of Hypoxia Inducible Factor 1 Is a General Phenomenon in Infections with Human Pathogens

doi: 10.1371/journal.pone.0011576

Figure Lengend Snippet: Four to six hours upon infection, HIF-1 activation was determined by Western blotting (loading control: actin). pO 2 levels were quantified in the medium of control and infected cells (D: P. aeruginosa ATCC 27853, MOI 10, infection time: four hours; E: S. agalactiae SK 43, MOI 200, infection time: five hours; F: C. albicans ATCC 90028, MOI 20, infection time: six hours). Negative control: uninfected cells, positive control: hypoxia or DFO (200 µmol/L).

Article Snippet: Peritoneal HIF-1α activation was analyzed by immunoblotting of the shock-frozen peritoneal specimens using the Nuclear Extract Kit (Active Motif; Rixensart, Belgium), a mouse monoclonal anti-HIF-1α (H1α67) antibody (Novus Biologicals, Littleton, USA) and horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG antibodies (Dako).

Techniques: Infection, Activation Assay, Western Blot, Control, Negative Control, Positive Control

Journal: PLoS ONE

Article Title: Activation of Hypoxia Inducible Factor 1 Is a General Phenomenon in Infections with Human Pathogens

doi: 10.1371/journal.pone.0011576

Figure Lengend Snippet: Six hours upon infection, HIF-1 activation was analyzed in (A) Western blots (loading control: actin) and by determining (B) the induction of the HIF-1-dependent luciferase activity. HeLa-229 cells were transfected with a 4xHRE-luc reporter construct 24 hours before infection (triplicate means given; for details see ). Induction was determined by chemiluminescence. Bacterial induced (C) VEGF and (D) HIF-1α transcription. Total mRNA was prepared six hours upon infection, transcribed into cDNA and quantified by real-time Light-Cycler-PCR (ratio: VEGF/actin or HIF-1α/actin transcripts; triplicate means given). Negative control: uninfected cells; S. aureus wt (8325-4); S. aureus Δ hemB ( S. aureus ermB Ω hemB 8325-4, SCV); S. aureus Δ hemB , hemB -complemented ( S. aureus pCX19Ω hemB 8325-4, complemented SCV mutant) ; positive control: DFO (200 µmol/L). Multiplicity of infection (MOI): 20. * significant difference to control cells (P<0.05).

Article Snippet: Peritoneal HIF-1α activation was analyzed by immunoblotting of the shock-frozen peritoneal specimens using the Nuclear Extract Kit (Active Motif; Rixensart, Belgium), a mouse monoclonal anti-HIF-1α (H1α67) antibody (Novus Biologicals, Littleton, USA) and horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG antibodies (Dako).

Techniques: Infection, Activation Assay, Western Blot, Control, Luciferase, Activity Assay, Transfection, Construct, Negative Control, Mutagenesis, Positive Control

Journal: PLoS ONE

Article Title: Activation of Hypoxia Inducible Factor 1 Is a General Phenomenon in Infections with Human Pathogens

doi: 10.1371/journal.pone.0011576

Figure Lengend Snippet: The HIF-1α protein was analyzed in Western blots six hours upon infection (loading control: actin) of HeLa 229 cells with the following bacterial strains: (A) S. aureus 8325-4 (wt), S. aureus ermB Ω hemB 8325-4 (SCV), S. aureus SA113 (wt), S. aureus SA113 Δ ypf::ermB (reduced in lipoteichoic acid synthesis), S. aureus SA113 Δ tagO (defective in producing wall teichoic acid); (B) S. aureus ATCC 25923 (wt), S. epidermidis ATCC 12228 (wt), S. aureus Newman (wt), S. aureus Newman mAH12 (defective in producing the extracellular adherence protein EAP). Negative control: uninfected cells, positive control: DFO (200 µM). Multiplicity of infection (MOI): 20.

Article Snippet: Peritoneal HIF-1α activation was analyzed by immunoblotting of the shock-frozen peritoneal specimens using the Nuclear Extract Kit (Active Motif; Rixensart, Belgium), a mouse monoclonal anti-HIF-1α (H1α67) antibody (Novus Biologicals, Littleton, USA) and horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG antibodies (Dako).

Techniques: Western Blot, Infection, Control, Negative Control, Positive Control

Journal: PLoS ONE

Article Title: Activation of Hypoxia Inducible Factor 1 Is a General Phenomenon in Infections with Human Pathogens

doi: 10.1371/journal.pone.0011576

Figure Lengend Snippet: The HIF-1α protein was analyzed in Western blots six hours upon infection (loading control: actin). The following patient isolates were used: S. aureus patient isolate 1 (wt; S. aureus A22223 I), S. aureus patient isolate 2 (clinical SCV; S. aureus A22223 II), S. aureus patient isolate 3 (wt; S. aureus OM 1a), S. aureus patient isolate 4 (clinical SCV; S. aureus OM 1b). Negative control: uninfected cells, positive control: hypoxia. Multiplicity of infection (MOI): 20.

Article Snippet: Peritoneal HIF-1α activation was analyzed by immunoblotting of the shock-frozen peritoneal specimens using the Nuclear Extract Kit (Active Motif; Rixensart, Belgium), a mouse monoclonal anti-HIF-1α (H1α67) antibody (Novus Biologicals, Littleton, USA) and horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG antibodies (Dako).

Techniques: Western Blot, Infection, Control, Negative Control, Positive Control

Journal: PLoS ONE

Article Title: Activation of Hypoxia Inducible Factor 1 Is a General Phenomenon in Infections with Human Pathogens

doi: 10.1371/journal.pone.0011576

Figure Lengend Snippet: (A) Control and infected HeLa-229 cells were incubated with the hypoxic marker pimonidazole hydrochloride which was visualized by using specific Cy3 labelled antibodies (for details see ). Scale bare: 40 µm. (B) pO 2 levels were quantified in the medium of control and infected cells (triplicate means given). (C) Hela-229 cells were incubated in gas permeable (gas-perm) and conventional (conv) culture dishes and HIF 1α protein was analyzed in Western blots (loading control: actin). (D) pO 2 levels in the medium of control or S. aureus infected HeLa-229 cells (means of doubles). Negative control: uninfected cells; S. aureus wt (8325-4); S. aureus Δ hemB ( S. aureus ermB Ω hemB 8325-4, SCV); S. aureus Δ hemB , hemB -complemented ( S. aureus pCX19Ω hemB 8325-4, complemented SCV mutant). Multiplicity of infection (MOI): 20. * significant difference to control cells (P<0.05).

Article Snippet: Peritoneal HIF-1α activation was analyzed by immunoblotting of the shock-frozen peritoneal specimens using the Nuclear Extract Kit (Active Motif; Rixensart, Belgium), a mouse monoclonal anti-HIF-1α (H1α67) antibody (Novus Biologicals, Littleton, USA) and horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG antibodies (Dako).

Techniques: Control, Infection, Incubation, Marker, Western Blot, Negative Control, Mutagenesis

Journal: PLoS ONE

Article Title: Activation of Hypoxia Inducible Factor 1 Is a General Phenomenon in Infections with Human Pathogens

doi: 10.1371/journal.pone.0011576

Figure Lengend Snippet: (A) HIF 1α immunohistochemistry and (B) hematoxilin-eosin staining of renal mouse abscesses induced by intravenous infection with S. aureus seven days upon infection (following sections). HIF 1α positive nuclei are most abundant within the abscesses borders, some interstitial staining also occurs within the renal parenchyma. In addition, renal tubules in the vicinity of the abscess stain also positively (halfed arrow in B). A: arcuate artery, G: glomerulus, V: arcuate vein; arrowheads: margin of abscesses. Magnifications: (A) 250×, (B) 100×.

Article Snippet: Peritoneal HIF-1α activation was analyzed by immunoblotting of the shock-frozen peritoneal specimens using the Nuclear Extract Kit (Active Motif; Rixensart, Belgium), a mouse monoclonal anti-HIF-1α (H1α67) antibody (Novus Biologicals, Littleton, USA) and horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG antibodies (Dako).

Techniques: Immunohistochemistry, Staining, Infection

Journal: PLoS ONE

Article Title: Activation of Hypoxia Inducible Factor 1 Is a General Phenomenon in Infections with Human Pathogens

doi: 10.1371/journal.pone.0011576

Figure Lengend Snippet: (A) Survival of NMRI mice after intraperitoneal infection with S. aureus (wt; S. aureus 8325-4; n = 8) or S. aureus Δ hemB (SCV, S. aureus erm Ω hemB 8325-4; n = 8). Note the higher susceptibility of mice infected with S. aureus 8325-4 compared to the mice infected with S. aureus Δ hemB . *significant difference: P<0.001. (B) Inhibition of DFO induced HIF-1 activation in HeLa-229 cells by 17-DMAG. Cells were incubated with 17 DMAG (10 µmol/L) for 16 hours following induction of HIF 1 activation by the iron chelator DFO (200 µmol/L) for six hours. HIF 1α protein was analyzed in Western blots (loading control: actin). Negative control: uninfected cells. (C) Survival rate of NMRI mice after intraperitoneal infection with S. aureus ( S. aureus 8325-4). One group of mice was treated 24 h and 16 h before infection with the HIF-1 inhibiting compound 17-DMAG (25 mg/g body weight). Note the higher survival rate of 17-DMAG-treated mice (n = 45) compared with control mice (n = 45). *significant difference: P = 0.005 (Kaplan-Meier analysis).

Article Snippet: Peritoneal HIF-1α activation was analyzed by immunoblotting of the shock-frozen peritoneal specimens using the Nuclear Extract Kit (Active Motif; Rixensart, Belgium), a mouse monoclonal anti-HIF-1α (H1α67) antibody (Novus Biologicals, Littleton, USA) and horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG antibodies (Dako).

Techniques: Infection, Inhibition, Activation Assay, Incubation, Western Blot, Control, Negative Control

Journal: eLife

Article Title: mTORC1-induced retinal progenitor cell overproliferation leads to accelerated mitotic aging and degeneration of descendent Müller glia

doi: 10.7554/elife.70079

Figure Lengend Snippet: Figure 6. Rescue of Tsc1fl/fl;Tyrp1-Cre mouse retinal phenotypes by concomitant deletion of hypoxia-induced factor 1-alpha (Hif1a). (A) Levels of senescence markers in P30 mouse retinas with indicated genotypes were analyzed by Western blot (WB). Relative amounts of proteins used in each sample were determined by WB detection of β-actin. (B) Distributions of mTORC1-active cells, which are positive to pS6, MG, which are positive to Sox9, and senescent cells, which are positive to β-gal and p53, were examined by immunostaining of the eye sections. Expression of those markers in the Cre-affected cells were determined by comparing the expression of the Cre reporter R26tdTom. (C) Numbers of Sox9-positive MG in the mouse retinas were counted and the relative numbers are shown in the graph. (D) R26tdTom-negative wild-type cells and R26tdTom-positive Cre-affected cells in the retinas were determined byFACS and shown in the graph. (E) The lengths of telomeres of the cells isolated from P30 mouse retinas with the indicated genotypes were compared with that of control sample, and their relative values are shown in the graph. Error bars denote SD. Numbers of samples analyzed are shown in the graphs. n.s., not significant; *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: 70079 18 of 27 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody Anti- BrdU(CldU) (Rat monoclonal) Novus Biologicals NB500- 169 IHC (1:200) Antibody Anti- BrdU(IdU) (Mouse monoclonal) EXBIO 11–286 C100 IHC (1:200) Antibody Anti- Brn3b (Rabbit polyclonal) Santa Cruz Biotechnology sc- 31989 IHC (1:200) Antibody Anti- Calbindin (Rabbit polyclonal) Swant Inc. CB- 38 IHC (1:200) Antibody Anti- Cdo (Goat polyclonal) R&D Systems AF2429 IHC (1:200) Antibody Anti- c- myc (Mouse monoclonal) Santa Cruz Biotechnology sc40 WB (1:1000) Antibody Anti- Cleaved caspase- 3 (Rabbit polyclonal) Cell Signaling Technology 9661 IHC (1:200) Antibody Anti- Ezrin (Mouse monoclonal) Invitrogen Biotechnology 35–7300 IHC (1:200) Antibody Anti- GFAP (Rabbit polyclonal) Abcam ab48050 IHC (1:200) Antibody Anti- GFP (Chicken polyclonal) Abcam ab13970 IHC (1:2000) Antibody Anti- Glutamine synthase (Rabbit polyclonal) Sigma- Aldrich G- 2781 IHC (1:200) Antibody Anti- Hif1a (Mouse monoclonal) R&D Systems MAB1536 WB (1:1000) Antibody Anti- Hif2α/Epas1 (Rabbit polyclonal) Novus Biologicals NB100- 122 WB (1:1000) Antibody Anti- Hk2 (Rabbit polyclonal) Cell Signaling Technology 2867 WB 1:1,000 IHC (1:200) Antibody Anti- Hydroxy- Hif1alpha (Rabbit polyclonal) Cell Signaling Technology 3434 WB (1:1000) Antibody Anti- M- opsin Merck Millipore AB5405 IHC (1:200) Antibody Anti- Otx2 (Rabbit polyclonal) Abcam ab25985 IHC (1:200) Antibody Anti- Otx2 (Rabbit polyclonal) Abcam ab183951 IHC (1:200) Antibody Anti- Otx2 (Goat polyclonal) R&D Systems AF1979- SP IHC (1:200) Antibody Anti- p21/Cip1 (Mouse monoclonal) Santa Cruz Biotechnology SC817 WB (1:1000) IHC (1:200) Antibody Anti- p53 (Mouse monoclonal) Merck Millipore OP03- 100 WB (1:1000) IHC (1:200) Antibody Anti- Pax6 (Rabbit polyclonal) Covance PRB- 278P IHC (1:200) Antibody Anti- Phospho Smad1/5(S463/465) (Rabbit polyclonal) Invitrogen Biotechnology 700047 IHC (1:200) Antibody Anti- phospho Smad2(ser465/467) (Rabbit polyclonal) - Cell Signaling Technology 18338 IHC (1:200) Antibody Anti- phospho- Histone H3(S10) (pH3; Rabbit polyclonal) Merck Millipore 04–1093 IHC (1:200) Antibody Anti- phospho- S6(S235/236) (pS6; Rabbit polyclonal) Cell Signaling Technology 2211 WB (1:1000) IHC (1:200) Continued Continued on next page Lim et al. eLife 2021;10:e70079.

Techniques: Western Blot, Immunostaining, Expressing, Isolation, Control

Journal: eLife

Article Title: mTORC1-induced retinal progenitor cell overproliferation leads to accelerated mitotic aging and degeneration of descendent Müller glia

doi: 10.7554/elife.70079

Figure Lengend Snippet: Figure 7. Hif1a supports mTORC1-induced retinal progenitor cell (RPC) proliferation through the expression of glycolytic enzymes. (A) The Cre-affected cells in P0 mouse retinas with the indicated genotypes were visualized by R26tdTom Cre reporter. Distribution of mTORC1-active cells in the boxed areas was also examined by immunostaining of pS6 and shown in the second row. Proliferation and cell cycle progression of RPCs in the mouse retinas were also examined by 5-ethynyl-2′-deoxyuridine (EdU)/5-chloro-2'-deoxyuridine (CldU) labeling and chasing experiments as described in Materials and methods. (B) R26tdTom-positive Cre-affected cell population in the mouse retinas were quantified by FACS and shown in the graph. Error bars, SD. Numbers of samples analyzed are shown in the graph (four independent litters). (C) RPCs that had progressed from S-phase to G2/M-phase for 3 hr (EdU;pH3-positive); PRs that had been born for 12 hr (EdU;Otx2-positive); and RPCs that had reentered cell cycle for 15 hr (CldU;IdU-positive) in the mouse retinas were counted and shown in the graph. Error bars denote SD (n = 5, five independent litters). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. (D) Relative mRNA levels of Hif1a, hexokinase 2 (Hk2), and pyruvate kinase M2 (Pkm2) in P0 mouse retinas with the indicated genotypes were examined by real-time quantitative polymerase chain reaction (RT-qPCR). The y-axis values are relative 2-ΔCt values against those of Tsc1fl/+;Tyrp1- Cre mouse retinas. Error bars, SD (n = 4, three independent litters). (E) Expressions of the indicated proteins in the mouse retinas were examined by Western blot (WB). Relative amounts of the proteins used in each sample were determined by WB detection of β-actin. (F) Relative WB band intensities of the proteins are determined by the ImageJ software and shown in the graph. Error bars, SD (n = 3, three independent litters). (G) Distributions of Pkm2, Tom20, and hypoxyprobe-labeled proteins in the mouse retinas were examined by immunostaining. (H) P0 Tsc1fl/fl;Tyrp1-Cre littermate mice were injected with a chemical inhibitor of glycolysis (2DG) or mitochondrial oxidative phosphorylation (Metformin), and the eye sections of the mice were

Article Snippet: 70079 18 of 27 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody Anti- BrdU(CldU) (Rat monoclonal) Novus Biologicals NB500- 169 IHC (1:200) Antibody Anti- BrdU(IdU) (Mouse monoclonal) EXBIO 11–286 C100 IHC (1:200) Antibody Anti- Brn3b (Rabbit polyclonal) Santa Cruz Biotechnology sc- 31989 IHC (1:200) Antibody Anti- Calbindin (Rabbit polyclonal) Swant Inc. CB- 38 IHC (1:200) Antibody Anti- Cdo (Goat polyclonal) R&D Systems AF2429 IHC (1:200) Antibody Anti- c- myc (Mouse monoclonal) Santa Cruz Biotechnology sc40 WB (1:1000) Antibody Anti- Cleaved caspase- 3 (Rabbit polyclonal) Cell Signaling Technology 9661 IHC (1:200) Antibody Anti- Ezrin (Mouse monoclonal) Invitrogen Biotechnology 35–7300 IHC (1:200) Antibody Anti- GFAP (Rabbit polyclonal) Abcam ab48050 IHC (1:200) Antibody Anti- GFP (Chicken polyclonal) Abcam ab13970 IHC (1:2000) Antibody Anti- Glutamine synthase (Rabbit polyclonal) Sigma- Aldrich G- 2781 IHC (1:200) Antibody Anti- Hif1a (Mouse monoclonal) R&D Systems MAB1536 WB (1:1000) Antibody Anti- Hif2α/Epas1 (Rabbit polyclonal) Novus Biologicals NB100- 122 WB (1:1000) Antibody Anti- Hk2 (Rabbit polyclonal) Cell Signaling Technology 2867 WB 1:1,000 IHC (1:200) Antibody Anti- Hydroxy- Hif1alpha (Rabbit polyclonal) Cell Signaling Technology 3434 WB (1:1000) Antibody Anti- M- opsin Merck Millipore AB5405 IHC (1:200) Antibody Anti- Otx2 (Rabbit polyclonal) Abcam ab25985 IHC (1:200) Antibody Anti- Otx2 (Rabbit polyclonal) Abcam ab183951 IHC (1:200) Antibody Anti- Otx2 (Goat polyclonal) R&D Systems AF1979- SP IHC (1:200) Antibody Anti- p21/Cip1 (Mouse monoclonal) Santa Cruz Biotechnology SC817 WB (1:1000) IHC (1:200) Antibody Anti- p53 (Mouse monoclonal) Merck Millipore OP03- 100 WB (1:1000) IHC (1:200) Antibody Anti- Pax6 (Rabbit polyclonal) Covance PRB- 278P IHC (1:200) Antibody Anti- Phospho Smad1/5(S463/465) (Rabbit polyclonal) Invitrogen Biotechnology 700047 IHC (1:200) Antibody Anti- phospho Smad2(ser465/467) (Rabbit polyclonal) - Cell Signaling Technology 18338 IHC (1:200) Antibody Anti- phospho- Histone H3(S10) (pH3; Rabbit polyclonal) Merck Millipore 04–1093 IHC (1:200) Antibody Anti- phospho- S6(S235/236) (pS6; Rabbit polyclonal) Cell Signaling Technology 2211 WB (1:1000) IHC (1:200) Continued Continued on next page Lim et al. eLife 2021;10:e70079.

Techniques: Expressing, Immunostaining, Labeling, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Software, Injection, Phospho-proteomics

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