Journal: bioRxiv

Article Title: Tropism of SARS-CoV-2 for Developing Human Cortical Astrocytes

doi: 10.1101/2021.01.17.427024

Figure Lengend Snippet: A) Experimental paradigm for viral infection of human cortical tissue. Primary cortical tissue was acutely sectioned and cultured at the air-liquid interface. The next day tissue was infected with SARS-CoV-2 at MOI 0.5 and cultured for 72 hours before being collected and processed. B) SARS-CoV-2 infects GFAP+AQP4+ astrocyte cells in the developing human cortex, which are predominantly located in the subventricular zone (SVZ), where >81% of cells assayed expressed markers of astrocytes. 100% of infected cells expressed both SARS-CoV-2+ nucleocapsid (N) antibody and double-stranded (ds)RNA antibody. White arrowheads indicate dsRNA+GFAP+ infected astrocytes (dsRNA+SARS-CoV-2 N+ n=31 cells, GFAP+AQP4+ n=74 cells across two biological samples and four technical replicates). C) Few other neural types were infected, as indicated by co-labeling of SARS-CoV-2 N or dsRNA, where <8% of cells assayed expressed NEUN, a marker of cortical neurons, and <17% expressed KI67, a marker of dividing cells (NEUN n=613 cells, KI67 n=186 cells across two biological samples and four technical replicates). D) Vascular cell types can also be infected where 7% of LAMININ+ blood vessels, 13% of CD31+ endothelial cells and 18% of PDGFR-β+ mural cells have infection. White arrowheads indicate infected vascular cells (Laminin n=269 cells, CD31 n=247 cells, PDGFRB=225 cells across two biological samples and four technical replicates).

Article Snippet: Primary Antibodies include: Mouse: dsRNA, clone rJ2 (Millipore, MABE1134, 1:100), Sox2 (Santa Cruz, sc-365823, 1:500), S100B (Sigma, S2532, 1:200), Ki67 (Abcam, ab156956, 1:500), CD31 (Agilent, GA61061–2, 1:100), Olig2 (Millipore, MABN50, 1:100), Rabbit: SARS-CoV-2 (Sino Biological, 40143-R001, 1:200), Pax6 (Biolegend, 901301, 1:500), Hopx (Proteintech, 11419–1-AP, 1:500), Cleaved Caspase-3 (Cell Signaling, 9661S, 1:250), Synm (Proteintech, 20735–1-AP, 1:100), Aqp4 (Proteintech, 16473–1-AP, 1:600), Egfr (Abcam, ab32077, 1:100), Dpp4 (Proteintech, 10940–1-AP, 1:50), CD147 (Invitrogen, 34–5600, 1:500), Arcn1 (Proteintech, 23843–1-AP, 1:50), Rat: Gfap (Thermofisher, 13–0300, 1:200), Laminin (Abcam, ab80580, 1:500), Nrp1 (Abcam, ab81321, 1:50), Chicken: Gfap (Abcam, ab4674, 1:500), Map2 (Abcam, ab5392, 1:200), Goat: Ace2 (R&D, AF933, 1:200), Ace2 (Thermofisher, MA5–32307, 1:200), Iba1 (Abcam, ab48004, 1:500), Pdgfrb (R&D, AF385, 1:100), Sheep: Eomes (R&D, AF6166, 1:200), Guinea pig: NeuN (Millipore, ABN90, 1:500), Sheep: CD34 (R&D, AF7227, 1:200).

Techniques: Infection, Cell Culture, Labeling, Marker

Journal: bioRxiv

Article Title: Tropism of SARS-CoV-2 for Developing Human Cortical Astrocytes

doi: 10.1101/2021.01.17.427024

Figure Lengend Snippet: A) Viral infection of cortical organoids. Human stem cells from several lines were aggregated and differentiated toward cortical identity in suspension. After 5, 10, 16 or 22 weeks of differentiation, organoids were exposed to SARS-CoV-2 for 2 hours, media was replaced and then collected 72 hours later. B) After 5, 10, or 16 weeks organoids only indicated rare infection (white arrowheads). At five and 10 weeks, SARS-CoV-2 N+ cells did not co-express SOX2, NEUN or GFAP indicating infected cells are not cortical progenitors, neurons or astrocytes and may instead be an off-target population. However, after 16 weeks, in one stem cell line a few GFAP+ astrocytes were infected. C) Although rare cells are infected at neurogenic stages, as indicated by coronavirus N antibody presence, there was no observed viral replication with dsRNA at these timepoints. D) At late gliogenic stages, by week 22 infection was readily observed in GFAP astrocytes but not NEUN+ neurons. E) 94% of infected cells stained positive for markers of astrocytes GFAP or AQP4, while only 4% are NEUN+ neurons. White arrowheads indicate SARS-CoV-2+ dsRNA+ GFAP+ AQP4+ astrocytes (GFAP+AQP4+ n=169 cells, NEUN n=143 cells).

Article Snippet: Primary Antibodies include: Mouse: dsRNA, clone rJ2 (Millipore, MABE1134, 1:100), Sox2 (Santa Cruz, sc-365823, 1:500), S100B (Sigma, S2532, 1:200), Ki67 (Abcam, ab156956, 1:500), CD31 (Agilent, GA61061–2, 1:100), Olig2 (Millipore, MABN50, 1:100), Rabbit: SARS-CoV-2 (Sino Biological, 40143-R001, 1:200), Pax6 (Biolegend, 901301, 1:500), Hopx (Proteintech, 11419–1-AP, 1:500), Cleaved Caspase-3 (Cell Signaling, 9661S, 1:250), Synm (Proteintech, 20735–1-AP, 1:100), Aqp4 (Proteintech, 16473–1-AP, 1:600), Egfr (Abcam, ab32077, 1:100), Dpp4 (Proteintech, 10940–1-AP, 1:50), CD147 (Invitrogen, 34–5600, 1:500), Arcn1 (Proteintech, 23843–1-AP, 1:50), Rat: Gfap (Thermofisher, 13–0300, 1:200), Laminin (Abcam, ab80580, 1:500), Nrp1 (Abcam, ab81321, 1:50), Chicken: Gfap (Abcam, ab4674, 1:500), Map2 (Abcam, ab5392, 1:200), Goat: Ace2 (R&D, AF933, 1:200), Ace2 (Thermofisher, MA5–32307, 1:200), Iba1 (Abcam, ab48004, 1:500), Pdgfrb (R&D, AF385, 1:100), Sheep: Eomes (R&D, AF6166, 1:200), Guinea pig: NeuN (Millipore, ABN90, 1:500), Sheep: CD34 (R&D, AF7227, 1:200).

Techniques: Infection, Staining

Journal: Scientific Reports

Article Title: Identification of proteins responsible for adriamycin resistance in breast cancer cells using proteomics analysis

doi: 10.1038/srep09301

Figure Lengend Snippet: Proteins indicated in red were significantly enriched within the GSH metabolism pathway. Red stars represent DEPs. 2.3.2.4.: GGCT; 1.1.1.42: IDH1 and IDH2; 1.1.1.49: G6PD; 2.5.1.18: GSTP1.

Article Snippet: The membranes were blocked with 5% nonfat dry milk for 2 h at room temperature and subsequently probed with the primary antibodies: rabbit-anti-G6PD polyclonal antibody (diluted 1:500 ABclonal, China), rabbit- anti-GGCT polyclonal antibody (diluted 1:400 Proteintech, China), rabbit-anti-IDH1 polyclonal antibody (diluted 1:500 ABclonal, China), rabbit-anti-IDH2 polyclonal antibody (diluted 1:500 ABclonal, China), rabbit-anti-GSTP1polyclonal antibody (diluted 1:500 ABclonal, China).

Techniques:

Journal: Scientific Reports

Article Title: Identification of proteins responsible for adriamycin resistance in breast cancer cells using proteomics analysis

doi: 10.1038/srep09301

Figure Lengend Snippet: Fourteen proteins were significantly enriched within the GSH metabolism pathway

Article Snippet: The membranes were blocked with 5% nonfat dry milk for 2 h at room temperature and subsequently probed with the primary antibodies: rabbit-anti-G6PD polyclonal antibody (diluted 1:500 ABclonal, China), rabbit- anti-GGCT polyclonal antibody (diluted 1:400 Proteintech, China), rabbit-anti-IDH1 polyclonal antibody (diluted 1:500 ABclonal, China), rabbit-anti-IDH2 polyclonal antibody (diluted 1:500 ABclonal, China), rabbit-anti-GSTP1polyclonal antibody (diluted 1:500 ABclonal, China).

Techniques:

Journal: Scientific Reports

Article Title: Identification of proteins responsible for adriamycin resistance in breast cancer cells using proteomics analysis

doi: 10.1038/srep09301

Figure Lengend Snippet: Some DEPs were identified in MCF-7/ADR compared to MCF-7

Article Snippet: The membranes were blocked with 5% nonfat dry milk for 2 h at room temperature and subsequently probed with the primary antibodies: rabbit-anti-G6PD polyclonal antibody (diluted 1:500 ABclonal, China), rabbit- anti-GGCT polyclonal antibody (diluted 1:400 Proteintech, China), rabbit-anti-IDH1 polyclonal antibody (diluted 1:500 ABclonal, China), rabbit-anti-IDH2 polyclonal antibody (diluted 1:500 ABclonal, China), rabbit-anti-GSTP1polyclonal antibody (diluted 1:500 ABclonal, China).

Techniques: Cell Differentiation, Membrane, Coagulation, Activity Assay, Protein Binding, Binding Assay

Journal: Scientific Reports

Article Title: Identification of proteins responsible for adriamycin resistance in breast cancer cells using proteomics analysis

doi: 10.1038/srep09301

Figure Lengend Snippet: DEPs information obtained from HPRD

Article Snippet: The membranes were blocked with 5% nonfat dry milk for 2 h at room temperature and subsequently probed with the primary antibodies: rabbit-anti-G6PD polyclonal antibody (diluted 1:500 ABclonal, China), rabbit- anti-GGCT polyclonal antibody (diluted 1:400 Proteintech, China), rabbit-anti-IDH1 polyclonal antibody (diluted 1:500 ABclonal, China), rabbit-anti-IDH2 polyclonal antibody (diluted 1:500 ABclonal, China), rabbit-anti-GSTP1polyclonal antibody (diluted 1:500 ABclonal, China).

Techniques:

Journal: Scientific Reports

Article Title: Identification of proteins responsible for adriamycin resistance in breast cancer cells using proteomics analysis

doi: 10.1038/srep09301

Figure Lengend Snippet: The expression of DEPs was focused on GSH metabolism pathway. G6PD, GGCT, IDH1 and IDH2 were found down–regulated in MCF-7/ADR cells compared with MCF-7 cells. Up-regulated expression was observed for GSTP1. GAPDH was used as the loading control.

Article Snippet: The membranes were blocked with 5% nonfat dry milk for 2 h at room temperature and subsequently probed with the primary antibodies: rabbit-anti-G6PD polyclonal antibody (diluted 1:500 ABclonal, China), rabbit- anti-GGCT polyclonal antibody (diluted 1:400 Proteintech, China), rabbit-anti-IDH1 polyclonal antibody (diluted 1:500 ABclonal, China), rabbit-anti-IDH2 polyclonal antibody (diluted 1:500 ABclonal, China), rabbit-anti-GSTP1polyclonal antibody (diluted 1:500 ABclonal, China).

Techniques: Expressing, Control

Journal: Molecular Medicine

Article Title: Histone acetyltransferase P300 deficiency promotes ferroptosis of vascular smooth muscle cells by activating the HIF-1α/HMOX1 axis

doi: 10.1186/s10020-023-00694-7

Figure Lengend Snippet: P300 and P53 competitively bind HIF-1α to regulate HMOX1 expression during ferroptosis of HASMCs. A-B. Western blot analysis and quantification results showing SLC7A11, GPX4, and FSP1 protein levels after CD (A), and IKE (B) stimulation in HASMCs infected with lenti-shRNA, lenti-shP300-1 and lenti-shP300-2 (A-B), β-actin served as a loading control (n = 4 per group). C-D . Western blot analysis and quantification results showing HMOX1 protein levels in HASMCs infected with lenti-shRNA, lenti-shP300-1 and lenti-shP300-2 after CD and IKE stimulation. β-actin served as a loading control (n = 4 per group). E . Co-immunoprecipitation results showed that endogenous P300 interacted with exogeneous HIF-1α in HASMCs after treatment with MG132. F . The interaction between endogenous HIF-1α and exogenous P300 in 293T cells after treatment with MG132 was verified by Co-immunoprecipitation. G . Co-immunoprecipitation showed the interaction between endogenous HIF-1α and endogenous P300 and P53 after treatment with CD and MG132 in nucleus of HASMCs. Values are means ± SD; *** p < 0.001, NS, no significant

Article Snippet: Primary antibodies used were: P300 (sc-48,343, Santa Cruz), HIF-1α (20960-1-AP, Proteintech Group), H3K18ac (ab40888, Abcam), H3K27ac (GTX128944, GeneTex), H2BK5ac (12799, Cell Signaling Technology), AIFM2/FSP1 (HPA042309, Atlas Antibodies; 20886-1-AP, Proteintech Group), GPX4 (ab125066, Abcam), β-actin (8457, Cell Signaling Technology), SLC7A11 (NB300-318, Novus Biologicals), P53 (A5804, ABclonal Technology), Lamin B1 (12987-1-AP, Proteintech Group) and GAPDH (AC033, ABclonal Technology).

Techniques: Expressing, Western Blot, Infection, shRNA, Immunoprecipitation