Journal: Frontiers in Cell and Developmental Biology

Article Title: SALL1 Modulates CBX4 Stability, Nuclear Bodies, and Regulation of Target Genes

doi: 10.3389/fcell.2021.715868

Figure Lengend Snippet: Plasmids used in the study.

Article Snippet: After that, membranes were incubated in blocking buffer for 1 h at RT or overnight at 4°C with the following primary antibodies: mouse monoclonal anti-HA (Sigma, 1:1000, #H3663), mouse monoclonal anti-β-Actin (Sigma, 1:1000, #A2228), mouse monoclonal anti-GFP (Roche, 1:1000, #11814460001), mouse monoclonal anti-SALL1 (R&D, 1:1000, #PP-K9814-00), rabbit polyclonal anti-CBX4 (Proteintech, 1:1000, #18544-1-AP), rabbit polyclonal anti-Avitag (GeneScript, 1:1000, #A00674), or rabbit monoclonal Vinculin (Cell Signaling, 1:1000, #13901S).

Techniques: Generated, Selection, Amplification, Modification, Plasmid Preparation

Journal: Frontiers in Cell and Developmental Biology

Article Title: SALL1 Modulates CBX4 Stability, Nuclear Bodies, and Regulation of Target Genes

doi: 10.3389/fcell.2021.715868

Figure Lengend Snippet: SALL1 and CBX4 do not colocalize in nuclear bodies. (A–F) Confocal images of U2OS cells showing expression of SALL1-YFP, SALL1ΔSUMO-YFP, or SALL1ΔSIM-YFP (green), and endogenous CBX4 (magenta in A–C ) or endogenous SUMO2/3 (magenta in D–F ). Nuclei were stained with DAPI. Black and white pictures show single green or magenta channels. Green arrowheads indicate SALL1 bodies, magenta arrowheads indicate Pc bodies (A–C) , or SUMO bodies (D–F) and white arrowheads indicate colocalization of SALL1 and SUMO2/3 (D–F) . Pictures were taken with a Leica DM IRE2 confocal microscope using a 63× objective. Scale bars indicate 5 μm.

Article Snippet: After that, membranes were incubated in blocking buffer for 1 h at RT or overnight at 4°C with the following primary antibodies: mouse monoclonal anti-HA (Sigma, 1:1000, #H3663), mouse monoclonal anti-β-Actin (Sigma, 1:1000, #A2228), mouse monoclonal anti-GFP (Roche, 1:1000, #11814460001), mouse monoclonal anti-SALL1 (R&D, 1:1000, #PP-K9814-00), rabbit polyclonal anti-CBX4 (Proteintech, 1:1000, #18544-1-AP), rabbit polyclonal anti-Avitag (GeneScript, 1:1000, #A00674), or rabbit monoclonal Vinculin (Cell Signaling, 1:1000, #13901S).

Techniques: Expressing, Staining, Microscopy

Journal: Frontiers in Cell and Developmental Biology

Article Title: SALL1 Modulates CBX4 Stability, Nuclear Bodies, and Regulation of Target Genes

doi: 10.3389/fcell.2021.715868

Figure Lengend Snippet: SALL1 interacts with CBX4 in a SUMOylation-independent manner. (A) Validation of the interaction between human SALL1 and human CBX4 or Drosophila melanogaster Pc proteins using BioID-based biotin pulldown in transfected HEK 293FT cells. In the Input panel, the relative expression of the HA-tagged SALL1 proteins (the full-length protein or a TBS-related truncation mutant) is shown. One asterisk indicates SALL1-HA, while two asterisks indicate SALL1 826 -HA. Negative controls (single expression of each individual protein) are shown in lanes 4–7. Anti-GAPDH was used as loading control. As shown in the Elution panel, CBX4-BirA* interact preferentially with full-length SALL1-HA (lane 1). Anti-biotin blot shows the efficiency of the different pulldowns. (B) Validation of the interaction between SALL1 and CBX4 using GFP-Trap. The Input panel shows the expression of epitope-tagged SALL1 and CBX4 proteins in transfected HEK 293FT cells. YFP alone and HA empty vector were used as controls. Lanes 1 and 2 of the Elution panel, show that CBX4 interacts with SALL1 full length and the truncated form. (C) SUMO-related SALL1 mutants interact with CBX4. Western blot analysis of proteins extracted from HEK 293FT cells transfected with the indicated plasmids. Pulldowns were performed using GFP-Trap. As shown in the Elution panel (lanes 4, 5, and 6), interaction between CBX4 and WT SALL1 or SALL1 mutants was readily detected in all blot images. (D) Graph showing that CBX4 levels increase when co-expressed with WT SALL1-YFP, SALL1ΔSUMO-YFP, or SALL1ΔSIM-YFP. The intensity of CBX4 bands in blots was quantified using ImageJ, normalized to b-Actin and reported as fold change relative to the YFP alone control. The mean plus SEM of three independent experiments is plotted. P -values were calculated using Mann–Whitney test. * P -value < 0.05. (A–C) Antibodies used are indicated to the left. Molecular weight markers are indicated to the right in KDa.

Article Snippet: After that, membranes were incubated in blocking buffer for 1 h at RT or overnight at 4°C with the following primary antibodies: mouse monoclonal anti-HA (Sigma, 1:1000, #H3663), mouse monoclonal anti-β-Actin (Sigma, 1:1000, #A2228), mouse monoclonal anti-GFP (Roche, 1:1000, #11814460001), mouse monoclonal anti-SALL1 (R&D, 1:1000, #PP-K9814-00), rabbit polyclonal anti-CBX4 (Proteintech, 1:1000, #18544-1-AP), rabbit polyclonal anti-Avitag (GeneScript, 1:1000, #A00674), or rabbit monoclonal Vinculin (Cell Signaling, 1:1000, #13901S).

Techniques: Transfection, Expressing, Mutagenesis, Plasmid Preparation, Western Blot, MANN-WHITNEY, Molecular Weight

Journal: Frontiers in Cell and Developmental Biology

Article Title: SALL1 Modulates CBX4 Stability, Nuclear Bodies, and Regulation of Target Genes

doi: 10.3389/fcell.2021.715868

Figure Lengend Snippet: SALL1 and CBX4 interact in the nucleoplasm. (A–D) Confocal pictures of a proximity ligation assay (PLA) showing in situ interaction of SALL1 and CBX4 in the nucleus of U2OS cells, visualized as magenta spots. Cells were transfected with SALL1-HA or with the empty pcDNA3 vector as negative control. Antibodies used in the assay are indicated in magenta. Panel A shows SALL1 and CBX4 interaction, while panels B–D are negative controls. (E) Quantification of PLA signals per cell as in A–D . Bars represent mean plus SEM of three independent experiments. P -values were calculated using one-way ANOVA test. *** P -value < 0.001.

Article Snippet: After that, membranes were incubated in blocking buffer for 1 h at RT or overnight at 4°C with the following primary antibodies: mouse monoclonal anti-HA (Sigma, 1:1000, #H3663), mouse monoclonal anti-β-Actin (Sigma, 1:1000, #A2228), mouse monoclonal anti-GFP (Roche, 1:1000, #11814460001), mouse monoclonal anti-SALL1 (R&D, 1:1000, #PP-K9814-00), rabbit polyclonal anti-CBX4 (Proteintech, 1:1000, #18544-1-AP), rabbit polyclonal anti-Avitag (GeneScript, 1:1000, #A00674), or rabbit monoclonal Vinculin (Cell Signaling, 1:1000, #13901S).

Techniques: Proximity Ligation Assay, In Situ, Transfection, Plasmid Preparation, Negative Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: SALL1 Modulates CBX4 Stability, Nuclear Bodies, and Regulation of Target Genes

doi: 10.3389/fcell.2021.715868

Figure Lengend Snippet: SALL1 influences the levels of CBX4. (A) Western blot showing protein levels of CBX4-HA when co-expressed with SALL1-YFP or YFP alone in HEK 293FT cells. Actin expression was used as loading control. (B) Western blot showing expression levels of endogenous CBX4 protein in parental HEK 293FT cells (lane 1) or in HEK 293FT cells stably expressing GFS-SALL1 (lane 2). (C) Confocal microscopy images showing inducible expression SALL1-2xHA in HEK 293FT_TripZ-SALL1-2xHA cells. Cells were treated with different concentrations of doxycycline (Dox) to induce SALL1 expression as indicated. SALL1-2xHA was detected using anti-SALL1 primary antibody (green). Cell nuclei were stained with DAPI (blue). (D) Western blot analysis showing expression levels of endogenous CBX4 in HEK 293FT_TripZ-SALL1-2xHA cells treated with increasing concentrations of Dox. (E) Quantification of the expression levels of endogenous CBX4 in HEK 293FT_TripZ-SALL1-2xHA cells treated with increasing concentrations of Dox. Three independent experiments as the one shown in D were performed. The intensity of CBX4 bands was quantified using ImageJ, and the values were normalized to the levels of Actin. P -value was calculated using one-way ANOVA test. * P -value < 0.05. (F) RT-qPCR analysis of SALL1 and CBX4 mRNA expression in HEK 293FT_TripZ-SALL1-2xHA cells treated with increasing concentrations of Dox. SALL1 and CBX4 expression were normalized using GAPDH expression and shown as fold change relative to untreated control. (A,B,D) Molecular weight markers are shown to the right in KDa. Antibodies were used as indicated to the left. (E,F) The mean plus SEM of at least three independent experiments is shown.

Article Snippet: After that, membranes were incubated in blocking buffer for 1 h at RT or overnight at 4°C with the following primary antibodies: mouse monoclonal anti-HA (Sigma, 1:1000, #H3663), mouse monoclonal anti-β-Actin (Sigma, 1:1000, #A2228), mouse monoclonal anti-GFP (Roche, 1:1000, #11814460001), mouse monoclonal anti-SALL1 (R&D, 1:1000, #PP-K9814-00), rabbit polyclonal anti-CBX4 (Proteintech, 1:1000, #18544-1-AP), rabbit polyclonal anti-Avitag (GeneScript, 1:1000, #A00674), or rabbit monoclonal Vinculin (Cell Signaling, 1:1000, #13901S).

Techniques: Western Blot, Expressing, Stable Transfection, Confocal Microscopy, Staining, Quantitative RT-PCR, Molecular Weight

Journal: Frontiers in Cell and Developmental Biology

Article Title: SALL1 Modulates CBX4 Stability, Nuclear Bodies, and Regulation of Target Genes

doi: 10.3389/fcell.2021.715868

Figure Lengend Snippet: SALL1 stabilizes CBX4 protein. (A,C) Western blot analysis of cycloheximide (CHX) chase experiments performed in HEK 293FT cells transfected with SALL1-YFP , SALL1ΔSUMO-YFP , or GFP-β-gal . Cells were treated with 50 μg/ml of CHX in the absence (A) or presence (C) of 10 μM of the proteasome inhibitor MG132. Cells were collected at different time points (0, 4, 8, and 16 h after initiation of treatment) and endogenous CBX4 levels were analyzed by Western blot. Vinculin was used as loading control. Molecular weight markers are shown to the right in KDa. Antibodies were used as indicated to the left. (B,D) CBX4 levels were quantified after CHX treatment alone (B) or in combination with MG132 (D) , normalized to Vinculin, and data from six different independent experiments were pooled together. Graphs show mean plus SEM. P -values were calculated using one-way ANOVA test. * P -value < 0.05; ** P -value < 0.01.

Article Snippet: After that, membranes were incubated in blocking buffer for 1 h at RT or overnight at 4°C with the following primary antibodies: mouse monoclonal anti-HA (Sigma, 1:1000, #H3663), mouse monoclonal anti-β-Actin (Sigma, 1:1000, #A2228), mouse monoclonal anti-GFP (Roche, 1:1000, #11814460001), mouse monoclonal anti-SALL1 (R&D, 1:1000, #PP-K9814-00), rabbit polyclonal anti-CBX4 (Proteintech, 1:1000, #18544-1-AP), rabbit polyclonal anti-Avitag (GeneScript, 1:1000, #A00674), or rabbit monoclonal Vinculin (Cell Signaling, 1:1000, #13901S).

Techniques: Western Blot, Transfection, Molecular Weight

Journal: Frontiers in Cell and Developmental Biology

Article Title: SALL1 Modulates CBX4 Stability, Nuclear Bodies, and Regulation of Target Genes

doi: 10.3389/fcell.2021.715868

Figure Lengend Snippet: CBX4 ubiquitination is reduced in presence of SALL1. (A) Western blot analysis of HEK 293FT cells transfected with CBX4-HA together with CMV-BirA-2A-bioUb or BirA as a negative control. Cells were treated with 50 μM of biotin in the presence or absence of 10 μM MG132. Protein lysates were subjected to pulldown with streptavidin beads and the results were analyzed by Western blot. Two asterisks indicate monoubiquitinated CBX4-HA protein and the vertical line indicates the polyubiquitination smear. (B) Western blot analysis of HEK 293FT_TripZ-SALL1-2xHA cells transiently transfected with CBX4-YFP together with BirA-2A-bioUb or BirA as control. The cells were treated or not with 1 μg/ml of doxycycline (Dox), in presence or absence of 10 μM of MG132. Protein lysates were incubated with streptavidin beads to isolate bioUb conjugated proteins and results were analyzed by Western blot. β-Actin was used as loading control. (C) The levels of ubiquitinated CBX4-YFP in Dox induced and not induced cells, in presence (right panel) or absence (left panel) of MG132, were quantified and normalized to the CBX4 levels in the input. (D) Western blot analysis of endogenous CBX4 in HEK 293FT cells transfected with CMV-SALL1-2xHA (lanes 2 and 4) or with pcDNA3 control plasmid (lanes 1 and 3), in presence (lanes 3 and 4) or absence (lanes 1 and 2) of 10 μM MG132. (E) Quantification of ubiquitinated CBX4 in the elution panel normalized to the CBX4 levels in the input, in cells expressing or not SALL1-HA, in presence (right panel) or absence (left panel) of MG132. (A,B,D) Molecular weight markers are shown to the right in KDa. Antibodies were used as indicated to the left. (C,E) Graphs represent mean plus SEM. P -values were calculated on n = 4 using Mann–Whitney test. * P -value < 0.05.

Article Snippet: After that, membranes were incubated in blocking buffer for 1 h at RT or overnight at 4°C with the following primary antibodies: mouse monoclonal anti-HA (Sigma, 1:1000, #H3663), mouse monoclonal anti-β-Actin (Sigma, 1:1000, #A2228), mouse monoclonal anti-GFP (Roche, 1:1000, #11814460001), mouse monoclonal anti-SALL1 (R&D, 1:1000, #PP-K9814-00), rabbit polyclonal anti-CBX4 (Proteintech, 1:1000, #18544-1-AP), rabbit polyclonal anti-Avitag (GeneScript, 1:1000, #A00674), or rabbit monoclonal Vinculin (Cell Signaling, 1:1000, #13901S).

Techniques: Western Blot, Transfection, Negative Control, Incubation, Plasmid Preparation, Expressing, Molecular Weight, MANN-WHITNEY

Journal: Frontiers in Cell and Developmental Biology

Article Title: SALL1 Modulates CBX4 Stability, Nuclear Bodies, and Regulation of Target Genes

doi: 10.3389/fcell.2021.715868

Figure Lengend Snippet: SALL1 expression increases the number and size of CBX4-containing Pc bodies and enhances downregulation of CBX4 targets. (A,B) Graphs represent the number of CBX4-containing Pc bodies (A) and their mean area in pixels quantified using Fiji software (B) in U2OS cells expressing SALL1-YFP, SALL1ΔSUMO-YFP, or GFP-β-gal as a negative control. (C) Graph showing the mRNA expression levels of several CBX4 target genes in HEK 293FT cells expressing SALL1-YFP, SALL1ΔSUMO-YFP, or GFP-β-gal as control. Data shown correspond to the mean plus SEM of at least five independent RT-qPCR experiments. Gene expression data were normalized to GAPDH and are shown as relative fold change over β-Gal expressing cells (magenta line). P -values were calculated using one-way ANOVA test. * P -value < 0.05; ** P -value < 0.01; *** P -value < 0.001.

Article Snippet: After that, membranes were incubated in blocking buffer for 1 h at RT or overnight at 4°C with the following primary antibodies: mouse monoclonal anti-HA (Sigma, 1:1000, #H3663), mouse monoclonal anti-β-Actin (Sigma, 1:1000, #A2228), mouse monoclonal anti-GFP (Roche, 1:1000, #11814460001), mouse monoclonal anti-SALL1 (R&D, 1:1000, #PP-K9814-00), rabbit polyclonal anti-CBX4 (Proteintech, 1:1000, #18544-1-AP), rabbit polyclonal anti-Avitag (GeneScript, 1:1000, #A00674), or rabbit monoclonal Vinculin (Cell Signaling, 1:1000, #13901S).

Techniques: Expressing, Software, Negative Control, Quantitative RT-PCR

Journal: Frontiers in Cell and Developmental Biology

Article Title: SALL1 Modulates CBX4 Stability, Nuclear Bodies, and Regulation of Target Genes

doi: 10.3389/fcell.2021.715868

Figure Lengend Snippet: SALL1 influences regulation of CBX4 target genes. Hypothetical model showing speculative scenarios whereby SALL1 could influence CBX4-mediated regulation of target genes. Binding to SALL1 (SUMOylated or non-SUMOylated) could stabilize CBX4 by interfering with its ubiquitination and its consequent degradation by the proteasome. CBX4 stabilization entails an increment of its protein levels and its accumulation in Pc bodies. Binding to SUMOylated SALL1 increases CBX4-mediated transcriptional repression of its target genes. At least two non-exclusive hypothetical mechanisms might underlie this effect. Under one hypothetical scenario (left side), it could be due to the concurrent recruitment of other essential cofactors. In another hypothetical scenario (right side), SUMOylated SALL1 could increase CBX4 transcriptional repression by facilitating its SUMOylation through recruitment of SUMOylation machinery components. Discontinuous arrows indicate speculative events that have not been proven experimentally.

Article Snippet: After that, membranes were incubated in blocking buffer for 1 h at RT or overnight at 4°C with the following primary antibodies: mouse monoclonal anti-HA (Sigma, 1:1000, #H3663), mouse monoclonal anti-β-Actin (Sigma, 1:1000, #A2228), mouse monoclonal anti-GFP (Roche, 1:1000, #11814460001), mouse monoclonal anti-SALL1 (R&D, 1:1000, #PP-K9814-00), rabbit polyclonal anti-CBX4 (Proteintech, 1:1000, #18544-1-AP), rabbit polyclonal anti-Avitag (GeneScript, 1:1000, #A00674), or rabbit monoclonal Vinculin (Cell Signaling, 1:1000, #13901S).

Techniques: Binding Assay