Journal: Science advances

Article Title: The emergence of human primordial germ cell-like cells in stem cell-derived gastruloids.

doi: 10.1126/sciadv.ado1350

Figure Lengend Snippet: Fig. 4. ISL1 KO hGs do not display PGCLC formation. (A) Confocal micrographs showing the lack of amnion markers ISL1 and GATA3 in the ISL1 KO hGs. Scale bars, 50 μm (n = 10 samples from three experiments). Red arrowheads indicate AMLCs. (B) Live microscopic images showing the detection of the fluorescence reporter NANOS3-tdTomato in WT but absent in the ISL1 KO hGs. NANOS3-tdTomato is rescued in gastruloids treated with exogenous BMP supplementation. Scale bars, 250 μm (WT) and 100 μm (KO and KO + BMP) (n = 300 samples from five experiments). (C) Confocal micrographs showing the maximum intensity projection of PGCLC markers, including SOX17 and TFAP2C in WT, KO, and KO + BMP–supplemented hGs. Scale bars, 50 μm (WT and KO) and 25 μm (KO + BMP) (n = 30 samples from three experiments). Red arrowheads indicate hPGCLCs. (D) Flow cytometry analysis showing the quantification of PGCLCs and AMLCs in the WT, ISL1 KO, and KO + BMP–supplemented hGs (n = 3 samples from three experiments). One-way ANOVA with Tukey’s multiple comparisons test was performed for statistical analysis. P < 0.05 was considered signifi- cant. ****P < 0.0001, ***P < 0.0005, and **P < 0.005. (E) Confocal micrographs with maximum intensity projection of SOX17, POU5F1 (OCT4), and ISL1 showing the distribution of PGCLCs and AMLCs in hGs. PGCLCs are indicated by red arrowheads. AMLCs are shown in white circles. Scale bars, 50 μm (n = 15 samples from three ex- periments). (F) Live microscopic images showing the detection of the fluorescence reporter SMAD1-RFP in hGs from D1 to D4. Scale bars, 100 μm (n = 300 samples from five experiments). (G) Confocal micrographs showing the expression of amniotic ectoderm markers, TFAP2A and GATA3, and pSMAD1/5/8, an intracellular target of BMP4 signaling in D1 hGs. Scale bars, 50 μm (WT). Red arrowheads indicate coexpression of pSMAD1 with either TFAP2A or GATA3 (n = 30 samples from three experiments).

Article Snippet: Quantitative flow cytometry was performed on a SONY SH800 cell sorter.

Techniques: Fluorescence, Flow Cytometry, Expressing

Journal: iScience

Article Title: The canonical single-stranded DNA-binding protein is not an essential replication factor but an RNA chaperon in Saccharolobus islandicus

doi: 10.1016/j.isci.2023.108389

Figure Lengend Snippet: Growth and flow cytometry analysis of the knockout strains of Sisssb and Sisdbp (A) PCR verification of the knockout strains Δ ssb , Δ dbp , and Δ dbp Δ ssb . Genomic DNA and two primer pairs, flanking F/R and gene specific F/R, were used for the analysis. (B) Western blotting analysis of the knockout strains using whole-cell lysate. Cells were collected at OD 600 = 0.5–0.8, disrupted by sonication. Primary antibody of SisDBP was added alone, while those of SisTBP and SisSSB were incubated simultaneously. Anti-SisTBP was used as the loading control. (C) Growth curves under normal conditions. Cells were cultured with shaking at 110 rpm and 75°C in STVU medium with an initial OD 600 0.05. The values were calculated based on three biological repeats. (D) Flow cytometry analysis of the knockout strains. Acetic acid (6 mM）was added into the culture medium when the OD 600 reached 0.15–0.2. Samples were taken at different time (0, 1, 2, 3, 4, 5, 6, 7, and 8 h) and analyzed as described in the materials and methods. “1C” and “2C” indicate one and two copies of chromosomes. (E) Growth curves of cells treated with 4-NQO. Cells were cultured as in (C) except that 4-NQO (3 μM）was added into the culture medium when the OD 600 reached 0.2 (at 12 h). The values were calculated based on three biological repeats. See also Figure S2 .

Article Snippet: The DNA content of the cells was analyzed using the ImageStreamX MarkII Quantitative imaging analysis for flow cytometry system (Merck Millipore, Germany).

Techniques: Flow Cytometry, Knock-Out, Western Blot, Sonication, Incubation, Control, Cell Culture

Journal: iScience

Article Title: The canonical single-stranded DNA-binding protein is not an essential replication factor but an RNA chaperon in Saccharolobus islandicus

doi: 10.1016/j.isci.2023.108389

Figure Lengend Snippet: SSB-deficient cells exhibited slow growth at lower temperature (A) Growth curves of SSB-deficient strains at 55°C. Cells were cultured in liquid STVU at 75°C to OD 600 = 0.6–0.8 and then used as inoculates with an initial OD 600 of 0.05. The cultivation was continued in STVU medium or in the arabinose-containing medium ATVU (indicated with “A”) at 55°C with shaking. The optical density was monitored and the values were calculated based on measurements of three biological replicates. (B) Cytometry profiles of the strains grown at 55°C. Samples were taken at 72 h and analyzed. Cells of E233S cultivated to middle logarithmic phase (OD 600 ∼0.4) at 75°C was used as a control. See also Figure S3 .

Article Snippet: The DNA content of the cells was analyzed using the ImageStreamX MarkII Quantitative imaging analysis for flow cytometry system (Merck Millipore, Germany).

Techniques: Cell Culture, Cytometry, Control

Journal: iScience

Article Title: The canonical single-stranded DNA-binding protein is not an essential replication factor but an RNA chaperon in Saccharolobus islandicus

doi: 10.1016/j.isci.2023.108389

Figure Lengend Snippet: Overexpression of SisSSB has global effect on cell cycle progression and growth (A) Assay of the expression levels in the promoter replacement strain Para::ssb under non-inducible and inducible conditions by western blotting. Cells were cultured in liquid STVU or ATVU medium and collected at OD 600 0.5–0.8. The cells were then disrupted by sonication and the cell lysates were subjected to SDS-PAGE and western blotting analysis with antibodies against SisSSB and SisTBP at the same time. (B) Quantitative analysis of the results in (A). The values were calculated based on three replicates. Error bars indicated the standard deviation. (C) Growth curves of the promoter substitution strain in comparison with the wild-type E233S. The cells were cultured in liquid ATVU medium with an initial OD 600 0.05 with shaking (110 rpm) at 75°C. OD 600 was monitored at 6 or 12 h interval. The values were based on measurements of three biological replicates. (D) Comparison of the cytometry profiles of the synchronized E233S and the promoter substitution stain. The cells were cultured in TSVU medium until the OD 600 reached 0.15–0.2 when acetic acid (6 mM) was added into the culture. After cultured for 3 h, 0.2% D-arabinose was added to induce the expression of SisSSB. After incubation for further 6 h, samples were taken at different time points (0, 1, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 8, 9, 10, 11, and 12 h) and subjected to the flow cytometry analysis. “1C” and “2C” indicate the cells containing one and two copies of chromosomes, respectively. (E) Comparison of the cell cycle periods of E233S and the SisSSB overexpression strain according to (D). G2→M→G1 (green), from the cell cycle release to 1C appearance; G1→S→G2 (gray): 1C appearance to 2C peak. See also Figure S1 .

Article Snippet: The DNA content of the cells was analyzed using the ImageStreamX MarkII Quantitative imaging analysis for flow cytometry system (Merck Millipore, Germany).

Techniques: Over Expression, Expressing, Western Blot, Cell Culture, Sonication, SDS Page, Standard Deviation, Comparison, Cytometry, Staining, Incubation, Flow Cytometry