Journal: bioRxiv

Article Title: Transcriptional control by Sall4 in blastocysts facilitates lineage commitment of inner cell mass cells

doi: 10.1101/194852

Figure Lengend Snippet: (A)Representative projection images of anti-Sall4 (white), anti-Oct4 (magenta) and DAPI (blue) staining in WT (n=3), Sall4 Δ/+ (Het, n=2) and Sall4 Δ/Δ (KO, n=3) 16 cell stage embryos. Scale bars = 50μM. (B)Representative projection images of DAPI (blue), anti Nanog (green), anti-Oct4 (magenta) and anti-Sall4 (white) staining in control (Het; N = 10) and Sall4 Δ/Δ (KO; N = 5) early blastocyst embryos (E3.5). Scale bars = 50μM. (C)Quantification of the number of cells per embryos from (B) (left), the percentage of Nanog positive cells per embryo (middle), and intensity values for the Nanog positive cells from (B) for control (Wt/Het) and KO embryos (right). Significance was determined using a Mann-Whitney test for all three comparisons (*** P≤ 0.001). (D)Representative projection images of anti-Nanog (green), anti-Gata4 (magenta), anti-Cdx2 (white) staining, plus DAPI (blue) in control (Het; n=7) and Sall4 Δ/Δ (KO, n=6) E3.75 embryos. Scale bar = 50μM. (E)Quantification of the number of cells per embryos from (D) (left), the percentage of Nanog positive cells per embryo (middle), and percentage of Gata4 positive cells per embryo (right) for control (Wt/Het) and KO embryos. Significance was determined using a Mann-Whitney test (** P≤ 0.01). (F)Single cell gene expression analysis from E3.25 ICM WT/Het and KO embryos at indicated stages, with Log 10 expression normalised to that of housekeeping genes (see Methods) on the y-axis. Each dot represents a single cell, and each plot shows data for the indicated gene. Statistical significance was calculated using a Kruskall-Wallis test (Supplemental Table 1), * indicates P ≤0.05, ** P ≤0.01, *** P ≤0.001, **** P <=0.0001. Published data from wild type 16-cell embryos are included for comparison . (G)Same as in (F), but for genes expressed in primitive endoderm cells.

Article Snippet: Primary antibodies were used at the following dilutions: anti-Oct4 (1/200, Santa Cruz Biotechnologies, sc-8628), anti-Nanog (1/200, Cosmo Bio, RCAB002P), anti-Cdx2 (1/200, Abcam, ab157524), anti-Gata6 (1/200, R&D Systems, AF1700), anti-Gata4 (1/200, Santa Cruz, sc-1237), anti-Sox17 (1/200, R&D Systems, AF1924), anti-Sall4 (1/200, Cosmo Bio, PPX-PP-PPZ0601-00), anticleaved caspase 3 (1/500, Cell Signaling Technologies, 9664S).

Techniques: Staining, MANN-WHITNEY, Expressing

Journal: bioRxiv

Article Title: Transcriptional control by Sall4 in blastocysts facilitates lineage commitment of inner cell mass cells

doi: 10.1101/194852

Figure Lengend Snippet: (A)Representative projection images of anti-Nanog (green), anti-Gata6 (magenta) staining, along with composite images of anti-Nanog and anti-Gata6 together, plus the inclusion of anti-Cdx2 (white) staining in control embryos (WT n=4) and Sall4 Δ/Δ (KO, n=5) E3.75 embryos. Scale bar = 50μM. (B)Representative projection images of anti-Nanog (green), anti-Gata4 (magenta), anti-Cdx2 (white) staining in control (WT n=9) and Sall4 Δ/Δ (KO, n=6) E4.5 late blastocysts. Scale bar = 50μM. (C)Quantification of the number of cells per embryo (left); the percentage of Nanog positive cells per embryo (middle); and the percentage of Gata4 positive cells per embryo at 4.5 dpc. *** P ≤0.001, **** P ≤0.0001 by Mann-Whitney test. (D)Representative confocal projection images of control (WT or Het) and Sall4 Δ/ Δ (KO) embryos flushed at 2.5dpc and cultured for 48hours in media only or with 0.5 μg/μl or 1 μg/μl Fgf2. Embryos were stained with anti-Gata4 (magenta), anti-Nanog (green) and anti-Cdx2 (white). Scale bar = 50μM. N=9 for WT or Het embryos cultured without Fgf2 and in 0.5μg/ml Fgf2, n=10 for those in 1μg/ml Fgf2. N=4 for KO embryos cultured without Fgf2 and in 1μg/ml and n=3 for those cultured in 0.5μg/ml Fgf2. (E)Percentage of Nanog positive (top) or Gata4 positive (middle) cells per embryo for control (WT and Het) or Sall4-null (KO) embryos after culture in indicated conditions for 48 hours. Each point represents one embryo. Statistical significance was tested using an unpaired Mann-Whitney test: * P ≤0.05, *** P ≤0.001.

Article Snippet: Primary antibodies were used at the following dilutions: anti-Oct4 (1/200, Santa Cruz Biotechnologies, sc-8628), anti-Nanog (1/200, Cosmo Bio, RCAB002P), anti-Cdx2 (1/200, Abcam, ab157524), anti-Gata6 (1/200, R&D Systems, AF1700), anti-Gata4 (1/200, Santa Cruz, sc-1237), anti-Sox17 (1/200, R&D Systems, AF1924), anti-Sall4 (1/200, Cosmo Bio, PPX-PP-PPZ0601-00), anticleaved caspase 3 (1/500, Cell Signaling Technologies, 9664S).

Techniques: Staining, MANN-WHITNEY, Cell Culture

Journal: bioRxiv

Article Title: Transcriptional control by Sall4 in blastocysts facilitates lineage commitment of inner cell mass cells

doi: 10.1101/194852

Figure Lengend Snippet: (A)Diffusion plot of single cell gene expression data for Sall4-null (KO, left) and wild type (WT, right) ICM cells. Points represent individual cells of the indicated stages and genotypes. 8- and 16-cell data is taken from . (B)Heat map constructed from single ICM cell expression data based on hierarchical clustering. Normalised Ct values refer to –ΔCt values normalised to housekeeping genes ( Ppia, Gapdh and Hprt ). Each row is one cell with the genotype and the time isolated indicated by the annotation panel on the left and key on the right (dpc = days post coitum). Each column shows expression of one gene. The cells cluster into 5 groups which have been labelled according to their expression profiles: Early unspecified (EU), Making a decision (MD), Epiblast (Epi), Primitive Endoderm (PrE) and the outliers. (C)The percentage of cells in each cluster from (B) at each time point (E3.25 or E3.75) for KO and control (WT, including cells from Het embryos) ICM cells. (D)Absolute average correlation (see Figure S3) between cells for each developmental stage is plotted for control (WT, black) and Sall4-null (KO, blue) cells. CV for 8- and 16-cell stages were calculated using data from O’Shaugnessy-Kirwan et al 2015. Error bars represent SEM, and significance was determined using one-way ANOVA followed by Sidak’s multiple comparison test. *** P≤0.001, **** P≤0.0001.

Article Snippet: Primary antibodies were used at the following dilutions: anti-Oct4 (1/200, Santa Cruz Biotechnologies, sc-8628), anti-Nanog (1/200, Cosmo Bio, RCAB002P), anti-Cdx2 (1/200, Abcam, ab157524), anti-Gata6 (1/200, R&D Systems, AF1700), anti-Gata4 (1/200, Santa Cruz, sc-1237), anti-Sox17 (1/200, R&D Systems, AF1924), anti-Sall4 (1/200, Cosmo Bio, PPX-PP-PPZ0601-00), anticleaved caspase 3 (1/500, Cell Signaling Technologies, 9664S).

Techniques: Diffusion-based Assay, Expressing, Construct, Isolation

Journal: bioRxiv

Article Title: Transcriptional control by Sall4 in blastocysts facilitates lineage commitment of inner cell mass cells

doi: 10.1101/194852

Figure Lengend Snippet: (A)Single cell expression data for genes characteristic of Epiblast and Primitive Endoderm split into clusters as specified in for control (WT, red) and Sall4-null (KO, Blue) cells. Each dot represents a single cell. Statistical significance was calculated using a Kruskall-Wallis test (Supplemental Table 1): * P ≤0.05, ** P ≤0.01, *** P ≤0.001, **** P <=0.0001. (B)Model of how Sall4 facilitates lineage commitment. In early wild type ICMs (top left) most cells (purple circles) exist in an attractor state of a differentiation landscape associated with an unspecified identity (EU), though some cells occupy the MD state, which is essentially a ‘staging post’ en route towards differentiation. The probability of achieving a differentiated state (PrE or Epi) is very low. As the embryo matures (top right) the depth of the attractor state lessens and cells in the MD state are able to commit to either the PrE or Epi state. In the absence of Sall4 (bottom panels) the depth of the EU attractor state is more pronounced, and cells within this state show increased cell-cell variability. As the embryo matures the depth of the attractor is lessened, but remains deeper than in wild type cells so most Sall4-null cells still occupy the MD state and few have committed to either PrE or Epi. Black horizontal lines represent the threshold limits of the EU and MD states.

Article Snippet: Primary antibodies were used at the following dilutions: anti-Oct4 (1/200, Santa Cruz Biotechnologies, sc-8628), anti-Nanog (1/200, Cosmo Bio, RCAB002P), anti-Cdx2 (1/200, Abcam, ab157524), anti-Gata6 (1/200, R&D Systems, AF1700), anti-Gata4 (1/200, Santa Cruz, sc-1237), anti-Sox17 (1/200, R&D Systems, AF1924), anti-Sall4 (1/200, Cosmo Bio, PPX-PP-PPZ0601-00), anticleaved caspase 3 (1/500, Cell Signaling Technologies, 9664S).

Techniques: Expressing

Journal: Cell Death & Disease

Article Title: SALL4 promotes cancer stem-like cell phenotype and radioresistance in oral squamous cell carcinomas via methyltransferase-like 3-mediated m6A modification

doi: 10.1038/s41419-024-06533-9

Figure Lengend Snippet: A , B SALL4 mRNA and protein expression in CD44(+) cells and CD44(-) cells. C Predicted binding sites for SALL4 mRNA with METTL3. D The subcellular locations of METTL3 and SALL4, as detected by Immunofluorescence assay. Scale bars: 10 μm. E , F qRT-PCR and western blot analysis showed the SALL4 expression in CD44( + )-OSCC cells transfected with METTL3 silencing or overexpression. G RIP-qPCR showed the interaction between METTL3 and SALL4 mRNA. H The interaction of METTL3 and SALL4 was confirmed by CO-IP assay in CD44( + )-OSCC cells. IgG served as a negative control. I Relative protein expression levels of CSCs-related genes. J Positive correlation of SOX2, Nanog, BMI1, and CD44 with SALL4 as per the GEO-OSCC platform. K ALDH1/CD44 percentage in the negative controls, and the flow cytometry of cells with SALL4 knockdown.

Article Snippet: Cells were processed by permeabilization using Triton-X100 (1%) for 30 min and blocking in BSA (1%) for 30 min after fixing in paraformaldehyde (4%) at room temperature for 20 min. Then, overnight incubation at 4 °C with the primary antibodies β-catenin (1:50, GTX101435, GeneTex), SALL4 (1:50, GTX109983, GeneTex), CD44 (1:200, Abcam, ab254530), METTL3 (1:100, Proteintech Group, 15073-1-AP, 67733-1-Ig).

Techniques: Expressing, Binding Assay, Immunofluorescence, Quantitative RT-PCR, Western Blot, Transfection, Over Expression, Co-Immunoprecipitation Assay, Negative Control, Flow Cytometry, Knockdown

Journal: Cell Death & Disease

Article Title: SALL4 promotes cancer stem-like cell phenotype and radioresistance in oral squamous cell carcinomas via methyltransferase-like 3-mediated m6A modification

doi: 10.1038/s41419-024-06533-9

Figure Lengend Snippet: A mRNA levels of SALL4 expression in CD44( + )-OSCC cells after irradiation. B Representative immunofluorescence images showing the CD44 protein expression level in radioresistant and parental cells. Scale bars: 50 μm. C Flow cytometry assessment of cellular cycles. SALL4 knockdown and overexpression diminished and increased cells in the G2/M phase, respectively. D SCC15-CD44(+) cells apoptosis was detected via flow cytometry. E Relative Caspase-3 activity of CD44( + )-OSCC cells, with knockdown or overexpression of SALL4, 24 h after irradiation at 10 Gy. F CD44( + )-OSCC cells were irradiated at 10 Gy and recultured under suitable circumstances for 1, 2, 4, 6, 12, and 24 h. Western blotting showing the levels of γ-H2AX. 0 h, irradiated cells without DNA repair time. G Expression of β-catenin in the cytoplasm and nucleus after SALL4 overexpression. H The nuclear localization and expression level of β-catenin in CD44( + )-OSCC cells from the indicated groups detected by immunofluorescence staining. (red fluorescence indicates β-catenin, and green fluorescence indicates zsgreen). Scale bars: 50 μm. I Western blotting analysis showing β-catenin, Wnt3a, TCF1, c-MYC, and MMP9 expression in CD44( + )-OSCC cells after SALL4 knockdown and overexpression, and J following treatment with iCRT3.

Article Snippet: Cells were processed by permeabilization using Triton-X100 (1%) for 30 min and blocking in BSA (1%) for 30 min after fixing in paraformaldehyde (4%) at room temperature for 20 min. Then, overnight incubation at 4 °C with the primary antibodies β-catenin (1:50, GTX101435, GeneTex), SALL4 (1:50, GTX109983, GeneTex), CD44 (1:200, Abcam, ab254530), METTL3 (1:100, Proteintech Group, 15073-1-AP, 67733-1-Ig).

Techniques: Expressing, Irradiation, Immunofluorescence, Flow Cytometry, Knockdown, Over Expression, Activity Assay, Western Blot, Staining, Fluorescence

Journal: Cell Death & Disease

Article Title: SALL4 promotes cancer stem-like cell phenotype and radioresistance in oral squamous cell carcinomas via methyltransferase-like 3-mediated m6A modification

doi: 10.1038/s41419-024-06533-9

Figure Lengend Snippet: A METTL3 knockdown inhibited sphere formation potential, which was partially restored by SALL4 overexpression ( n = 3 independent experiments). Scale bars: 200 μm. B Statistics of the tumor sphere formation assay. C ALDH1A1 percentages in the negative control, shMETTL3, shMETTL3+vector, and shMETTL3 + SALL4 groups as flow cytometry revealed. D ALDH1A1 proportion statistic data in each group. E Western blot analysis showed the protein levels of CSCs-related factors in the relevant groups. F Representative images of clonogenic assay. G Clonogenic survival fraction were performed using CAL27-CD44(+) cells in the relevant groups at radiation doses of 0, 4, and 8 Gy. H Relative Caspase-3 activity 24 h following irradiation (10 Gy) of CAL27-CD44(+) cells in the indicated groups. I Levels of γ-H2AX in CAL27-CD44(+) cells irradiated at 10 Gy and recultured under suitable circumstances for 0, 2, and 6 h.

Article Snippet: Cells were processed by permeabilization using Triton-X100 (1%) for 30 min and blocking in BSA (1%) for 30 min after fixing in paraformaldehyde (4%) at room temperature for 20 min. Then, overnight incubation at 4 °C with the primary antibodies β-catenin (1:50, GTX101435, GeneTex), SALL4 (1:50, GTX109983, GeneTex), CD44 (1:200, Abcam, ab254530), METTL3 (1:100, Proteintech Group, 15073-1-AP, 67733-1-Ig).

Techniques: Knockdown, Over Expression, Tube Formation Assay, Negative Control, Plasmid Preparation, Flow Cytometry, Western Blot, Clonogenic Assay, Activity Assay, Irradiation

Journal: Cell Death & Disease

Article Title: SALL4 promotes cancer stem-like cell phenotype and radioresistance in oral squamous cell carcinomas via methyltransferase-like 3-mediated m6A modification

doi: 10.1038/s41419-024-06533-9

Figure Lengend Snippet: A CAL27-CD44(+) cell line with a stable transfection subcutaneously injected into mice, followed by radiation therapy. B Representative tumor images from treated and untreated groups on the 15th day. C Growth curvatures of CAL27-CD44(+) cell xenografts. D Tumor weight of CAL27-CD44(+) cell xenografts. E Immunofluorescence analysis of treated tumors for CD44 (green) and SALL4 (red) expression. Scale bar = 20 µm.

Article Snippet: Cells were processed by permeabilization using Triton-X100 (1%) for 30 min and blocking in BSA (1%) for 30 min after fixing in paraformaldehyde (4%) at room temperature for 20 min. Then, overnight incubation at 4 °C with the primary antibodies β-catenin (1:50, GTX101435, GeneTex), SALL4 (1:50, GTX109983, GeneTex), CD44 (1:200, Abcam, ab254530), METTL3 (1:100, Proteintech Group, 15073-1-AP, 67733-1-Ig).

Techniques: Stable Transfection, Injection, Immunofluorescence, Expressing

Journal: Cell Death & Disease

Article Title: SALL4 promotes cancer stem-like cell phenotype and radioresistance in oral squamous cell carcinomas via methyltransferase-like 3-mediated m6A modification

doi: 10.1038/s41419-024-06533-9

Figure Lengend Snippet: Schematic model depicting that SALL4 promotes OSCC radioresistance through Wnt/β-catenin signaling pathway.

Article Snippet: Cells were processed by permeabilization using Triton-X100 (1%) for 30 min and blocking in BSA (1%) for 30 min after fixing in paraformaldehyde (4%) at room temperature for 20 min. Then, overnight incubation at 4 °C with the primary antibodies β-catenin (1:50, GTX101435, GeneTex), SALL4 (1:50, GTX109983, GeneTex), CD44 (1:200, Abcam, ab254530), METTL3 (1:100, Proteintech Group, 15073-1-AP, 67733-1-Ig).

Techniques:

Journal: Journal of the National Cancer Center

Article Title: New substituted molecular classifications of advanced gastric adenocarcinoma: characteristics and probable treatment strategies

doi: 10.1016/j.jncc.2021.11.004

Figure Lengend Snippet: Primary antibody list used in this study.

Article Snippet: SALL4 , 6E3 , Merck-Millipore , Nuclear.

Techniques: Membrane