Journal: International Journal of Molecular Sciences

Article Title: Regulation and Role of Neuron-Derived Hemoglobin in the Mouse Hippocampus

doi: 10.3390/ijms23105360

Figure Lengend Snippet: Age-related decline of hemoglobin-α (Hb-α) with parallel increase in brain hypoxia in the intact male mouse brain. ( A ) Representative photomicrographs from male mouse hippocampal CA1, CA3 and cerebral cortex regions showing age-related decline in Hb-α staining and parallel increase in brain hypoxia as measured by pimonidazole (hypoxyprobe-1) staining. ( B ) Semi-quantitative intensity analysis of Hb-α and pimonidazole staining in hippocampal CA1 and CA3 regions and cerebral cortex of all male mice at different ages. ( C ) Correlation coefficient analysis between Hb-α and pimonidazole in hippocampal CA1, CA3 and cortex regions. *: p < 0.05 vs. 6 months; #: p < 0.05 vs. 12 months; n = 5. Scale bar: 20 μm.

Article Snippet: Primary antibodies used included Hemoglobin-α (rabbit, 1:5000, Proteintech, Rosemont, IL, USA, Cat# 14537-1-AP), and GAPDH (mouse, 1:2000, Santa Cruz Biotechnology, Dallas, TX, USA, Cat# sc-32233).

Techniques: Staining

Journal: International Journal of Molecular Sciences

Article Title: Regulation and Role of Neuron-Derived Hemoglobin in the Mouse Hippocampus

doi: 10.3390/ijms23105360

Figure Lengend Snippet: Age-related decline of hemoglobin-α (Hb-α) with parallel increase in brain hypoxia in the intact female mouse brain. ( A ) Representative photomicrographs from female mouse hippocampal CA1, CA3, and cerebral cortex regions showing age-related decline in Hb-α staining and parallel increase in brain hypoxia as measured by pimonidazole (hypoxyprobe-1) staining. ( B ) Semi-quantitative intensity analysis of Hb-α and pimonidazole staining in hippocampal CA1 and CA3 regions and cerebral cortex of all female mice at different ages. ( C ) Correlation coefficient analysis between Hb-α and pimonidazole in hippocampal CA1, CA3 and cortex regions. *: p < 0.05 vs. 6 months; #: p < 0.05 vs. 12 months; n = 5. Scale bar: 20 μm.

Article Snippet: Primary antibodies used included Hemoglobin-α (rabbit, 1:5000, Proteintech, Rosemont, IL, USA, Cat# 14537-1-AP), and GAPDH (mouse, 1:2000, Santa Cruz Biotechnology, Dallas, TX, USA, Cat# sc-32233).

Techniques: Staining

Journal: International Journal of Molecular Sciences

Article Title: Regulation and Role of Neuron-Derived Hemoglobin in the Mouse Hippocampus

doi: 10.3390/ijms23105360

Figure Lengend Snippet: Gender differences in neuronal hemoglobin-α (Hb-α) levels and brain hypoxia in the mouse brain in aging. Semi-quantitative intensity analysis of Hb-α ( A – C ) and pimonidazole staining ( D – F ) in hippocampal CA1 and CA3 regions and cerebral cortex of all female and male mice at different ages. *: p < 0.05 vs. female; n = 5.

Article Snippet: Primary antibodies used included Hemoglobin-α (rabbit, 1:5000, Proteintech, Rosemont, IL, USA, Cat# 14537-1-AP), and GAPDH (mouse, 1:2000, Santa Cruz Biotechnology, Dallas, TX, USA, Cat# sc-32233).

Techniques: Staining

Journal: International Journal of Molecular Sciences

Article Title: Regulation and Role of Neuron-Derived Hemoglobin in the Mouse Hippocampus

doi: 10.3390/ijms23105360

Figure Lengend Snippet: Effect of hemoglobin-α knockdown on HIF-1α expression in the male mouse hippocampal CA1 and CA3 regions following global cerebral ischemia (GCI). ( A )/( a ) and ( B )/( a ) Representative photomicrographs of HIF-1α immunostaining in 3-month-old male mouse hippocampal CA1 and CA3 regions from control (empty, upper row), scramble (lenti-CRISPRi-scramble, middle row) or Hb-α knockdown (lenti-CRISPRi-HBA1/2, lower row) groups. ( A )/( b ) and ( B )/( b ) Quantification of mean immunofluorescence (IF) demonstrates a mild increase in HIF-1α expression in sham after Hb-α knockdown (*: p < 0.05, n = 6). At 7 days after GCI reperfusion (R7d), Hb-α knockdown robustly increased HIF-1α expression in both hippocampal CA1 ( A ) and CA3 regions ( B ). (*: p < 0.05, **: p < 0.01, n = 6). Scale bar: 20 μm.

Article Snippet: Primary antibodies used included Hemoglobin-α (rabbit, 1:5000, Proteintech, Rosemont, IL, USA, Cat# 14537-1-AP), and GAPDH (mouse, 1:2000, Santa Cruz Biotechnology, Dallas, TX, USA, Cat# sc-32233).

Techniques: Knockdown, Expressing, Immunostaining, Control, Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: Regulation and Role of Neuron-Derived Hemoglobin in the Mouse Hippocampus

doi: 10.3390/ijms23105360

Figure Lengend Snippet: Effect of hemoglobin-α knockdown on expression of the HIF-1α-regulated pro-apoptotic gene, PUMA in the male mouse hippocampal CA1 and CA3 regions following global cerebral ischemia (GCI). ( A , C ) Representative photomicrographs of PUMA immunostaining in 3-month-old male mouse hippocampal CA1 ( A ) and CA3 ( C ) regions from control (empty, upper row), scramble (lenti-CRISPRi-scramble, middle row) or Hb-α knockdown (lenti-CRISPRi-HBA1/2, lower row) groups. Staining in red represents NeuN, a neuronal marker, and staining in green represents PUMA. ( B , D ) Quantification of mean immunofluorescence (IF) demonstrates a mild increase in PUMA intensity in sham after Hb-α knockdown. At 7 days after GCI reperfusion (R7d), Hb-α knockdown robustly increased PUMA intensity in the hippocampal CA1 and CA3 regions (**: p < 0.01, n = 6). Scale bar: 20 μm.

Article Snippet: Primary antibodies used included Hemoglobin-α (rabbit, 1:5000, Proteintech, Rosemont, IL, USA, Cat# 14537-1-AP), and GAPDH (mouse, 1:2000, Santa Cruz Biotechnology, Dallas, TX, USA, Cat# sc-32233).

Techniques: Knockdown, Expressing, Immunostaining, Control, Staining, Marker, Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: Regulation and Role of Neuron-Derived Hemoglobin in the Mouse Hippocampus

doi: 10.3390/ijms23105360

Figure Lengend Snippet: Effect of hemoglobin-α knockdown on expression of the HIF-1α-regulated pro-apoptotic gene, NOXA in the male mouse hippocampal CA1 and CA3 regions following global cerebral ischemia (GCI). ( A , C ) Representative photomicrographs of NOXA immunostaining in 3-month-old male mouse hippocampal CA1 and CA3 regions from control (empty, upper row), scramble (lenti-CRISPRi-scramble, middle row) or Hb-α knockdown (lenti-CRISPRi-HBA1/2, lower row) groups. ( B , D ). Quantification of mean immuno-fluorescence (IF) demonstrates a mild increase in NOXA expression in sham after Hb-α knockdown (**: p < 0.01, n = 6). At 7 days after GCI reperfusion (R7d), Hb-α knockdown robustly increased NOXA expression in both CA1 ( A ) and CA3 regions ( C ). (**: p < 0.01, n = 6). Scale bar: 20 μm.

Article Snippet: Primary antibodies used included Hemoglobin-α (rabbit, 1:5000, Proteintech, Rosemont, IL, USA, Cat# 14537-1-AP), and GAPDH (mouse, 1:2000, Santa Cruz Biotechnology, Dallas, TX, USA, Cat# sc-32233).

Techniques: Knockdown, Expressing, Immunostaining, Control, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Regulation and Role of Neuron-Derived Hemoglobin in the Mouse Hippocampus

doi: 10.3390/ijms23105360

Figure Lengend Snippet: Effect of hemoglobin-α knockdown on neurodegeneration in the male mouse hippocampal CA1 region following global cerebral ischemia (GCI). ( A ) Representative photomicrographs from male mouse hippocampal CA1 region showing staining with the neuronal marker NeuN or with the neurodegeneration marker Fluoro Jade C (FJC). ( B ) NeuN-positive cell number in hippocampal CA1 region of various groups. ( C ) Intensity analysis of FJC staining in hippocampal CA1 region of various groups. **: p < 0.01. Scale bar: 20 μm.

Article Snippet: Primary antibodies used included Hemoglobin-α (rabbit, 1:5000, Proteintech, Rosemont, IL, USA, Cat# 14537-1-AP), and GAPDH (mouse, 1:2000, Santa Cruz Biotechnology, Dallas, TX, USA, Cat# sc-32233).

Techniques: Knockdown, Staining, Marker

Journal: International Journal of Molecular Sciences

Article Title: Regulation and Role of Neuron-Derived Hemoglobin in the Mouse Hippocampus

doi: 10.3390/ijms23105360

Figure Lengend Snippet: Effect of hemoglobin-α knockdown on neurodegeneration in the male mouse hippocampal CA3 region following global cerebral ischemia (GCI). ( A ) Representative photomicrographs from male mouse hippocampal CA3 region showing staining with the neuronal marker NeuN or with the neurodegeneration marker Fluoro Jade C (FJC). ( B ) NeuN-positive cell number in hippocampal CA3 region of various groups. ( C ) Intensity analysis of FJC staining in hippocampal CA3 region of various groups. *: p < 0.05, **: p < 0.01. Scale bar: 20 μm.

Article Snippet: Primary antibodies used included Hemoglobin-α (rabbit, 1:5000, Proteintech, Rosemont, IL, USA, Cat# 14537-1-AP), and GAPDH (mouse, 1:2000, Santa Cruz Biotechnology, Dallas, TX, USA, Cat# sc-32233).

Techniques: Knockdown, Staining, Marker

Journal: Molecular Medicine

Article Title: Further corroboration of distinct functional features in SCN2A variants causing intellectual disability or epileptic phenotypes

doi: 10.1186/s10020-019-0073-6

Figure Lengend Snippet: Schematic drawing of SCN2A indicating the location of studied pathogenic variants. The three patients with ID without seizures carried de novo heterozygous variants in SCN2A (shown in blue), two of which are nonsense variants (p.L611Vfs*35 and p.W1716*) and one is a missense variant in the pore-forming loop of domain 2 of the channel (p.R937C). The two patients with early onset EE harbored de novo heterozygous missense variants (shown in red) in the fifth transmembrane segment of the third channel domain (p.L1342P) or the C-terminal domain (p.E1803G), respectively. The heterozygous inherited missense variant previously reported by others in a family with BFNIE (Heron et al. ) (shown in green) is located in the second transmembrane segment of domain 4 (p.L1563V). Adapted from Meisler and Kearney

Article Snippet: Human SCN2A (‘adult’ isoform (Kasai et al. ), RefSeq NM_021007 (O’Leary et al. )), SCN1B (NM_001037) and SCN2B (NM_004588) cDNA in pCMV6-XL4/5 expression vectors were purchased from OriGene (Rockville, MD, USA).

Techniques: Variant Assay

Journal: Molecular Medicine

Article Title: Further corroboration of distinct functional features in SCN2A variants causing intellectual disability or epileptic phenotypes

doi: 10.1186/s10020-019-0073-6

Figure Lengend Snippet: Effect of amino acid exchanges on the SCN2A structure. a Top view on a model of the SCN2A structure. The four homologous repeats are shown in different colors (blue, cyan, orange, red) and the III-IV domain linker is shown in purple. The site of the pore is marked by a black diamond and the positions of the missense variants investigated in the present study are indicated. The extracellular sequence stretch spanning residues 275–359 has been omitted for clarity. b Side-view on the SCN2A structure. Color coding as in ( a ). c Model of the SCN2A pore region. The four residues that are critical for Na + selectivity are shown in stick presentation. d In the wild-type SCN2A (left panel) the sidechain orientation of E942, which is part of the selectivity filter, is stabilized by polar interactions with R937 (see encircled region). These interactions cannot be formed in the R937C variant (right panel) by the shorter and uncharged cysteine sidechain thus impeding fixation of the E942 sidechain (flexibility is indicated by the magenta arrow). e E1803 forms interactions with residues Q1510 and P1512 of the III-IV domain linker, which cannot be formed in the ( f ) E1803G variant. The lacking interactions are highlighted by a blue dotted circle. g L1342 forms sidechain interactions with W864 of the adjacent repeat. h In the L1342P variant, these interactions cannot be formed by the less-extended proline sidechain. i L1563 forms hydrophobic interactions within the fourth domain. k These interactions are partially lost in the L1563V variant by the shorter valine sidechain (indicated by a blue dotted circle)

Article Snippet: Human SCN2A (‘adult’ isoform (Kasai et al. ), RefSeq NM_021007 (O’Leary et al. )), SCN1B (NM_001037) and SCN2B (NM_004588) cDNA in pCMV6-XL4/5 expression vectors were purchased from OriGene (Rockville, MD, USA).

Techniques: Sequencing, Variant Assay