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: Cell reports

Article Title: CCDC57 Cooperates with Microtubules and Microcephaly Protein CEP63 and Regulates Centriole Duplication and Mitotic Progression.

doi: 10.1016/j.celrep.2020.107630

Figure Lengend Snippet: Figure 6. CCDC57 Is Required for Efficient Ciliogenesis and Centriolar Satellite Distribution (A) Effect of CCDC57 loss on cilium formation. Control and CCDC57-depleted RPE1 cells were serum-starved for 48 h, and percentage of ciliated cells was determined by staining for acetylated-tubulin, glutamylated tubulin, PCM1, and DAPI. (B) Quantification of ciliogenesis experiments. Results shown are the mean of three independent experiments ± SEM (>300 cells per experiment; **p < 0.01). (C) Effect of CCDC57 depletion on cilium length. Cilium length was determined by staining for acetylated-tubulin and DAPI. Results shown are the mean of three independent experiments ± SEM (>100 cells per experiment; ****p < 0.0001). (D) mNeonGreen (mNG)-CCDC57 (1–502) stable expression rescues the ciliogenesis defect of CCDC57-depleted cells. U2OS cells stably expressing mNG, mNG-CCDC57 (1–502), or mNG-CCDC57 (503–916) were fixed 86 h after transfection with control siRNA or CCDC57 siRNA and 24 h serum starvation and were stained for cilium markers. Results shown are the mean of two independent experiments ± SEM (>100 cells per experiment; *p < 0.05; n.s, non-significant). (E) Effect of CCDC57 depletion on centriolar satellite distribution. U2OS cells were fixed 86 h after transfection with control siRNA or CCDC57 siRNA and stained for the indicated proteins. Images represent centrosomes in cells from the same coverslip taken with the same camera settings. (F) Quantification of (E). PCM1 and CEP131 fluorescence intensities at the centrosome were measured from SUM projections, and average means of the levels in control cells were normalized to 1. Data represent mean values from two experiments ± SEM (>50 cells per experiment; **p < 0.01; ****p < 0.0001). (G) Effects of PCM1 depletion on ciliogenesis efficiency of CCDC57-depleted cells. RPE1 cells were transfected with control siRNA, CCDC57 siRNA, or CCDC57/ PCM1 siRNAs for 86 h; serum-starved for the last 24 h; fixed; and stained for cilium markers. (H) Quantification of (G). Results shown are the mean of two independent experiments ± SEM (>100 cells per experiment; **p < 0.01; ***p < 0.001). Scale bars, 10 mm.

Article Snippet: Primary antibodies used for immunoblotting were rabbit anti PCM1 (Proteintech, 19856-1-AP) at 1:500, rabbit anti-beta-actin (Cell Signaling Technology) at 1:10000, mouse anti alpha-tubulin (Sigma, DM1A) at 1:5000, anti-CCDC57 (Sigma HPA023344) at 1:1000, anti-c-Myc (clone 9E10) at 1:500, rabbit anticapsase-3 (Proteintech) at 1:500 and mouse anti-acetylated tubulin (clone 6-11B, 32270, Thermo Fischer) at 1:5000. anti-PCM1 and anti-GFP antibodies were generated and used for immunobloting as previously described (Firat-Karalar et al., 2014).

Techniques: Control, Staining, Expressing, Stable Transfection, Transfection