Journal: Nucleic Acids Research

Article Title: A novel transcriptional cascade is involved in Fzr-mediated endoreplication

doi: 10.1093/nar/gkaa158

Figure Lengend Snippet: Specific knockdown of Fzr expression in Drosophila salivary gland blocks endoreplication and results in the initiation of CycB transcription. ( A ) RNAi-mediated Fzr knockdown driven by salivary gland-specific Sg -Gal4 resulted in a significant reduction in the gland size. At 120 h AEL, salivary glands from control and Fzr knockdown Drosophila were dissected and stained with DAPI. Two Fzr RNAi lines targeting different sequences were used in this experiment. Fzr -i #1 : UAS-Fzr RNAi line from VDRC (#V25550); Fzr -i #2 : UAS-Fzr RNAi line from TsingHua Fly Center (#TH2015000745.S). Scale bar, 180 μm. ( B ) C-value was quantified by DAPI fluorescence. The salivary glands from wandering Drosophila larvae at 120 h AEL were fixed and stained with DAPI. The integrated DAPI intensity was used to measure the DNA content. ( C ) Quantification of the DNA content in the salivary glands of Drosophila larvae at 120 h AEL. DNA was extracted from the indicated salivary gland cells and quantified via absorbance analysis. ( D , E ) EdU staining of DNA replication. In the salivary glands of larvae at 96 h AEL, the nuclei of most endocycling cells in the salivary glands as control could be strongly stained with EdU, indicating that DNA synthesis is ongoing. However, no replication signals were detected in salivary gland cells with Fzr knockdown. Scale bar, 30 μm. ( F–J ) Fzr knockdown in the salivary glands causes an accumulation of the CycB protein (F, G) and an appearance of the CycB mRNA (H–J) at 96 h AEL. The mRNA level was measured by fluorescence in situ hybridization (H, I) and RT-qPCR(J). Scale bar, 30 μm. (K) Fzr overexpression in Drosophila S2 cells significantly downregulated the transcription of the CycB gene. Data are presented as mean ± SE (error bars). For the significance test: *** P < 0.001 versus control. OE, overexpression. AEL, after egg laying.

Article Snippet: The antibodies and dilutions used in the study were as follows: goat anti- Drosophila Fzr (1:1000; Santa Cruz), rabbit anti- Drosophila Myc (1:1000; Santa Cruz), goat anti-human Fzr (1:1000; Santa Cruz), mouse anti-human Myc (1:1000; Santa Cruz), mouse anti- Drosophila CycB (1:5000; DSHB), rabbit anti-human CycB (1:5000; Cell Signaling), mouse anti-H2B (1:10 000; Beyotime), rabbit anti-H2Bub (1:20 000; Cell Signaling), mouse anti-V5 (1:5000; Abcam), rabbit anti-Flag (1:5000; Sigma), mouse anti-Myc tag (1:5000; Sigma), rabbit anti-MCM6 (1:5000; Zoonbio Biotechnology), and mouse anti-tubulin (1:10 000; Beyotime).

Techniques: Knockdown, Expressing, Control, Staining, Fluorescence, DNA Synthesis, In Situ Hybridization, Quantitative RT-PCR, Over Expression

Journal: Nucleic Acids Research

Article Title: A novel transcriptional cascade is involved in Fzr-mediated endoreplication

doi: 10.1093/nar/gkaa158

Figure Lengend Snippet: Fzr expression changes affect the transcription of transcription factor gene Myc and Myc is required for inhibition of CycB transcription in Drosophila salivary gland. ( A ) Transcriptome analysis of expression changes of several transcription factor genes following Fzr overexpression in Drosophila S2 cells. Normalized FPKM value was used for measuring relative expression level. Myc was upregulated following Fzr overexpression. Data are presented as mean + SE (error bars). For the significance test: * P < 0.05, ** P < 0.01, *** P < 0.001 versus control. ( B ) Fzr overexpression in S2 cells promoted mRNA transcription and protein expression of the Myc gene. ( C ) Fzr knockdown in the salivary glands reduced mRNA transcription and protein expression of the Myc gene at 96 h AEL. ( D–H ) Myc knockdown in the salivary glands causes an accumulation of the CycB mRNA (D, E and H) and the CycB protein (F, G) at 96 h AEL. ( I ) Myc overexpression in Drosophila S2 cells significantly downregulated the transcription of the CycB gene. Data are presented as mean ± SE (error bars). For the significance test: * P < 0.05, ** P < 0.01, *** P < 0.001 versus control. OE, overexpression. AEL, after egg laying. Scale bar, 30 μm.

Article Snippet: The antibodies and dilutions used in the study were as follows: goat anti- Drosophila Fzr (1:1000; Santa Cruz), rabbit anti- Drosophila Myc (1:1000; Santa Cruz), goat anti-human Fzr (1:1000; Santa Cruz), mouse anti-human Myc (1:1000; Santa Cruz), mouse anti- Drosophila CycB (1:5000; DSHB), rabbit anti-human CycB (1:5000; Cell Signaling), mouse anti-H2B (1:10 000; Beyotime), rabbit anti-H2Bub (1:20 000; Cell Signaling), mouse anti-V5 (1:5000; Abcam), rabbit anti-Flag (1:5000; Sigma), mouse anti-Myc tag (1:5000; Sigma), rabbit anti-MCM6 (1:5000; Zoonbio Biotechnology), and mouse anti-tubulin (1:10 000; Beyotime).

Techniques: Expressing, Inhibition, Over Expression, Control, Knockdown

Journal: Nucleic Acids Research

Article Title: A novel transcriptional cascade is involved in Fzr-mediated endoreplication

doi: 10.1093/nar/gkaa158

Figure Lengend Snippet: Myc functions as a downstream effector of Fzr modulation during endoreplication in Drosophila salivary gland. ( A–H ) Epistasis analysis revealed that Myc overexpression in the salivary glands moderately rescued the effects of Fzr knockdown on gland size (A–C), C-value (D), and DNA content (E) at 120 h AEL as well as DNA replication (F–H) at 96 h AEL. ( I–K ) Fzr knockdown-induced CycB expression in the salivary glands was abrogated by salivary gland-specific Myc overexpression at 96 h AEL. Data are presented as mean ± SE (error bars). For the significance test: ** P < 0.01, *** P < 0.001 versus control. OE, overexpression. AEL, after egg laying. Scale bar, 30 μm.

Article Snippet: The antibodies and dilutions used in the study were as follows: goat anti- Drosophila Fzr (1:1000; Santa Cruz), rabbit anti- Drosophila Myc (1:1000; Santa Cruz), goat anti-human Fzr (1:1000; Santa Cruz), mouse anti-human Myc (1:1000; Santa Cruz), mouse anti- Drosophila CycB (1:5000; DSHB), rabbit anti-human CycB (1:5000; Cell Signaling), mouse anti-H2B (1:10 000; Beyotime), rabbit anti-H2Bub (1:20 000; Cell Signaling), mouse anti-V5 (1:5000; Abcam), rabbit anti-Flag (1:5000; Sigma), mouse anti-Myc tag (1:5000; Sigma), rabbit anti-MCM6 (1:5000; Zoonbio Biotechnology), and mouse anti-tubulin (1:10 000; Beyotime).

Techniques: Over Expression, Knockdown, Expressing, Control

Journal: Nucleic Acids Research

Article Title: A novel transcriptional cascade is involved in Fzr-mediated endoreplication

doi: 10.1093/nar/gkaa158

Figure Lengend Snippet: Myc regulates the transcription of the CycB and MCM6 genes by directly binding to specific motif within their promoters. ( A ) Schematic diagram of the Myc binding peaks and potential E-box motifs for Myc binding within the promoters of the CycB and MCM6 genes. One binding peak is located in the region from -921 to -461 within the promoter upstream of the translational start site of the CycB gene and one potential E-box for Myc binding is located within this region from -838 to -833. One binding peak exists within the region from –359 to +221 around the translational start site of the MCM6 gene and there is one potential E-box within this region from –83 to –78. ( B–C′ ) ChIP-PCR and ChIP-qPCR assays verified the direct binding of Myc to the promoters of the CycB and MCM6 genes in Drosophila salivary glands (B, B’) and S2 cells with Myc overexpression (C, C’). ( D, D′ ) Electrophoretic mobility shift assay (EMSA) confirmed that Myc can directly bind to specific E-box motifs for Myc binding within the promoters of the CycB (D) and MCM6 genes (D’). Anti-Myc antibody competitively impaired the binding of Myc to the probes targeting E-box motifs. ( E, E ′ ) Luciferase reporter analyses revealed that Myc inhibited and promoted activities of the CycB promoter (E) and the MCM6 promoter (E’), respectively. P1, complete promoters of the CycB and MCM6 genes containing potential E-box. P2, truncated promoters of the CycB and MCM6 genes without E-box. Data are presented as mean ± SE (error bars). For the significance test: ** P < 0.01 versus control. OE, overexpression.

Article Snippet: The antibodies and dilutions used in the study were as follows: goat anti- Drosophila Fzr (1:1000; Santa Cruz), rabbit anti- Drosophila Myc (1:1000; Santa Cruz), goat anti-human Fzr (1:1000; Santa Cruz), mouse anti-human Myc (1:1000; Santa Cruz), mouse anti- Drosophila CycB (1:5000; DSHB), rabbit anti-human CycB (1:5000; Cell Signaling), mouse anti-H2B (1:10 000; Beyotime), rabbit anti-H2Bub (1:20 000; Cell Signaling), mouse anti-V5 (1:5000; Abcam), rabbit anti-Flag (1:5000; Sigma), mouse anti-Myc tag (1:5000; Sigma), rabbit anti-MCM6 (1:5000; Zoonbio Biotechnology), and mouse anti-tubulin (1:10 000; Beyotime).

Techniques: Binding Assay, ChIP-qPCR, Over Expression, Electrophoretic Mobility Shift Assay, Luciferase, Control

Journal: Cell reports

Article Title: HIV-1 Nef interacts with the cyclin K/CDK13 complex to antagonize SERINC5 for optimal viral infectivity

doi: 10.1016/j.celrep.2021.109514

Figure Lengend Snippet: (A) The indicated CD8-ICL4 fusion proteins were expressed with HIV-1 expressing Nef with a dileucine substitution (Nef LL/AA). Proteins were co-immunoprecipitated with a polyclonal anti-Nef antibody and detected by WB using the indicated antibodies. (B) CoIP samples 1, 2, and 3 in (A) were analyzed by MS after trypsin digestion. Nef, CD8, and ICL4 peptides detected from these samples are underlined. Three ICL4 peptides are numbered (1, 2, 3). (C) Levels of Nef, CD8, and ICL4 peptides detected by MS in (B) were quantified by label-free quantification (LFQ) and their intensity is presented as relative values, with the value of WT CD8-ICL4 set to 100%. Nef and CD8 were quantified by all detectable peptides and ICL4 was quantified by peptide-1 (SSSDALQGR). The error bars indicate SEM calculated from three independent experiments. Statistical analysis: *p < 0.05, **p < 0.01; ns, p > 0.05. (D) A proposed model of how S360 phosphorylation promotes SERINC5 downregulation.

Article Snippet: Horseradish peroxidase (HRP)-conjugated mouse monoclonal anti-FLAG, anti-HA antibodies, and anti-actin were purchased from Sigma; HRP-conjugated mouse monoclonal anti-GAPDH was purchased from Proteintech; rabbit polyclonal anti-CrkRS (CDK12) and anti-CDC2L5 (CDK13) were purchased from Novus; rabbit polyclonal anti-CycK was purchased from Abcam; HRP-conjugated anti-human, -rabbit, and -mouse immunoglobulin G secondary antibodies were purchased from Thermo Fisher.

Techniques: Expressing, Immunoprecipitation

Journal: Cell reports

Article Title: HIV-1 Nef interacts with the cyclin K/CDK13 complex to antagonize SERINC5 for optimal viral infectivity

doi: 10.1016/j.celrep.2021.109514

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Horseradish peroxidase (HRP)-conjugated mouse monoclonal anti-FLAG, anti-HA antibodies, and anti-actin were purchased from Sigma; HRP-conjugated mouse monoclonal anti-GAPDH was purchased from Proteintech; rabbit polyclonal anti-CrkRS (CDK12) and anti-CDC2L5 (CDK13) were purchased from Novus; rabbit polyclonal anti-CycK was purchased from Abcam; HRP-conjugated anti-human, -rabbit, and -mouse immunoglobulin G secondary antibodies were purchased from Thermo Fisher.

Techniques: Recombinant, Protease Inhibitor, Mutagenesis, Clone Assay, Luciferase, Staining, Knock-Out, Marker, Software