Journal: International Journal of Biological Sciences

Article Title: CXCL8 Drives MMP1 Upregulation and Promotes Metastatic Progression in Oral Cancer Through CXCR1/2-Mediated JAK1/STAT3 Activation

doi: 10.7150/ijbs.115990

Figure Lengend Snippet: CXCL8 is overexpressed in OSCC and associated with poor prognosis. (A) Venn diagram of upregulated DEGs identified from three GEO datasets: GSE13601 (n=29 OSCC, 29 normal), GSE143905 (n=5 OSCC, 5 normal), and GSE31056 (n=23 OSCC, 24 normal). (B) Hub genes ranked by interaction degree within the protein-protein interaction network (Cytoscape). (C) Heatmap showing expression levels of the top 40 upregulated DEGs in OSCC tissues from GSE74530 (n=6 OSCC, 6 normal). (D) Volcano plot of significantly altered genes in GSE74530 ; red: upregulated, blue: downregulated. (E) Differential expression of CXCL8 between OSCC and normal tissues in GSE74530 . (F) Kaplan-Meier analysis showing overall survival in HNSCC patients with high and low CXCL8 expression (UCSC Xena).

Article Snippet: The human OSCC cell lines SCC9, SCC4 (from ATCC) and HSC3 (from Sigma-Aldrich) were used in this study.

Techniques: Expressing, Quantitative Proteomics

Journal: International Journal of Biological Sciences

Article Title: CXCL8 Drives MMP1 Upregulation and Promotes Metastatic Progression in Oral Cancer Through CXCR1/2-Mediated JAK1/STAT3 Activation

doi: 10.7150/ijbs.115990

Figure Lengend Snippet: CXCL8 expression positively correlates with OSCC cell motility. (A)-(B) Wound healing and Transwell migration assays comparing SCC9, SCC4, and HSC3 cell lines (n=4). (C) Western blot analysis of CXCL8 protein levels in the three cell lines (n=4). (D)-(G) The migration and wound closure were evaluated following CXCL8 (25-50 ng/ml) treatment in SCC4 and HSC3 cells (n=4). (H)-(I) Migration and wound healing ability of SCC4 subpopulations vs. parental SCC4 cells were assessed (n=4). (J)-(K) CXCL8 mRNA and protein expression in highly migratory SCC4 cells compared to parental cells were assessed (n=4). Results are expressed as the mean ± SD of four independent experiments. * p < 0.05 compared with the control group.

Article Snippet: The human OSCC cell lines SCC9, SCC4 (from ATCC) and HSC3 (from Sigma-Aldrich) were used in this study.

Techniques: Expressing, Migration, Western Blot, Control

Journal: International Journal of Biological Sciences

Article Title: CXCL8 Drives MMP1 Upregulation and Promotes Metastatic Progression in Oral Cancer Through CXCR1/2-Mediated JAK1/STAT3 Activation

doi: 10.7150/ijbs.115990

Figure Lengend Snippet: CXCL8 promotes OSCC cell migration by inducing MMP1 expression. (A) TIMER2.0 analysis of correlations between CXCL8 and MMPs in HNSC (n=522). (B)-(D) CXCL8-induced MMP1 expression was examined by qPCR and Western blot analysis in SCC4 and HSC3 cells (n=4). (E) Validation of MMP1 knockdown efficiency by Western blot (n=4). (F)-(I) Functional analysis confirmed the effect of MMP1 knockdown on CXCL8-induced cell migration and wound healing (n=4). (J)-(K) MMP1 mRNA and protein expression of highly migratory SCC4 subpopulations were analyzed by Western blotting and qPCR (n=4). Results are expressed as the mean ± SD of four independent experiments. * p < 0.05 compared with the control group; # p < 0.05 compared with the CXCL8-treated group.

Article Snippet: The human OSCC cell lines SCC9, SCC4 (from ATCC) and HSC3 (from Sigma-Aldrich) were used in this study.

Techniques: Migration, Expressing, Western Blot, Biomarker Discovery, Knockdown, Functional Assay, Control

Journal: Science advances

Article Title: The cohesin-associated protein Pds5A governs the meiotic spindle assembly via deubiquitination of Kif5B in oocytes.

doi: 10.1126/sciadv.adt6159

Figure Lengend Snippet: Fig. 1. Subcellular localization and protein expression patterns of Pds5A during mouse oocyte meiotic maturation. (A) Fluorescence images of Pds5A localization in oocytes. Mouse oocytes at different developmental stages were costained with Pds5A (Santa Cruz Biotechnology) and α-tubulin antibodies and counterstained with Hoechst 33342. Scale bar, 20 μm. (B) Fluorescence intensity profiles of Pds5A and α-tubulin along the yellow line in (A). (C) Immunoblotting analysis of Pds5A protein levels during oocyte meiosis corresponding to GV, GVBD, MI, and MII stages. The blots were probed with Pds5A (Proteintech) and β-actin antibodies, respectively. (D) Quantification of Pds5A protein levels during oocyte meiosis. The band intensity of Pds5A was normalized with that of β-actin. A.U., arbitrary unit.

Article Snippet: Rabbit polyclonal anti- Pds5A antibodies were purchased from Proteintech (Rosemont, IL, USA; catalog no.: 17485- 1- AP) and Bethyl Laboratories (Montgomery, TX, USA; catalog no.: A300- 089A- T); mouse monoclonal anti- Pds5A antibody was purchased from Santa Cruz Biotechnology (Dallas, Texas, USA; catalog no.: sc- 515263); mouse monoclonal anti–α- tubulin–fluorescein isothiocyanate (FITC) antibody was purchased from Sigma- Aldrich (St. Louis, MO, USA; catalog no.: F2168); sheep polyclonal anti- BubR1 antibody was purchased from Abcam (Cambridge, MA, USA; catalog no.: ab28193); rabbit polyclonal anti- Mad2L1 antibody was purchased from Proteintech (catalog no.: 10337- 1- AP); human anti- centromere antibody was purchased from Antibodies Incorporated (Davis, CA, USA; catalog no.: CA95617); rabbit polyclonal anti- Kif5B antibody was purchased from ABclonal Technology (Wuhan, China; catalog no.: A15284); mouse monoclonal anti- Kif5B antibody was purchased from Santa Cruz Biotechnology (catalog no.: sc- 133184); mouse monoclonal anti–β- actin antibody was purchased from Proteintech (catalog no.: 60008- 1- Ig); mouse monoclonal anti- Usp14 antibody was purchased from Santa Cruz Biotechnology (catalog no.: sc- 398009); rabbit polyclonal anti- Usp14 antibody was purchased from Proteintech (catalog no.: 14517- 1- AP); rabbit monoclonal anti- Rec8 antibody was purchased from Abcam (catalog no.: ab192241); mouse monoclonal anti- Rad21 antibody was purchased from Sigma- Aldrich (catalog no.: 05- 908); rabbit polyclonal anti- HA antibody was purchased from Proteintech (catalog no.: 51064- 2- AP); rabbit monoclonal anti- Myc antibody was purchased from ABclonal Technology (catalog no.: AE070).

Techniques: Expressing, Fluorescence, Western Blot

Journal: Science advances

Article Title: The cohesin-associated protein Pds5A governs the meiotic spindle assembly via deubiquitination of Kif5B in oocytes.

doi: 10.1126/sciadv.adt6159

Figure Lengend Snippet: Fig. 2. Effects of Pds5A depletion on the mouse oocyte meiotic progression. (A) Representative images of GVBD oocytes observed at 4 hours following release from 3-isobutyl-1-metyl-xanthine in control and Pds5A-MO groups. Scale bar, 80 μm. (B) The rate of GVBD in control (n = 169) and Pds5A-MO (n = 185) oocytes. (C) Representa- tive images of MII oocytes observed at 10 hours post-GVBD in control, Pds5A-MO, and Pds5A-rescue (Pds5A-MO + Pds5A-cRNA) groups. Scale bar, 80 μm. (D) The rate of PBE in control (n = 119), Pds5A-MO (n = 119), and Pds5A-rescue (n = 113) oocytes. (E) Representative images of chromosome morphology in Pds5A-MO oocytes without PB1 after maturation. Scale bar, 5 μm. (F) The percentage of oocytes arresting at different developmental stages in Pds5A-MO (n = 76) group. (G) Fluorescence images of BubR1 present on the chromosomes in control and Pds5A-MO oocytes at Pro-MI and MI stages. Scale bar, 5 μm. (H) The proportion of BubR1 presence on the chromo- somes in control (n = 73) and Pds5A-MO (n = 69) oocytes at MI stage. (I) Fluorescence images of Mad2 present on the chromosomes in control and Pds5A-MO oocytes at Pro-MI and MI stages. Scale bar, 5 μm. (J) The proportion of Mad2 presence on the chromosomes in control (n = 71) and Pds5A-MO (n = 86) oocytes at MI stage. Data in (B), (D), (F), (H), and (J) were designated as mean percentage (means ± SEM) of at least three independent experiments. **P < 0.01; ns, no significance.

Article Snippet: Rabbit polyclonal anti- Pds5A antibodies were purchased from Proteintech (Rosemont, IL, USA; catalog no.: 17485- 1- AP) and Bethyl Laboratories (Montgomery, TX, USA; catalog no.: A300- 089A- T); mouse monoclonal anti- Pds5A antibody was purchased from Santa Cruz Biotechnology (Dallas, Texas, USA; catalog no.: sc- 515263); mouse monoclonal anti–α- tubulin–fluorescein isothiocyanate (FITC) antibody was purchased from Sigma- Aldrich (St. Louis, MO, USA; catalog no.: F2168); sheep polyclonal anti- BubR1 antibody was purchased from Abcam (Cambridge, MA, USA; catalog no.: ab28193); rabbit polyclonal anti- Mad2L1 antibody was purchased from Proteintech (catalog no.: 10337- 1- AP); human anti- centromere antibody was purchased from Antibodies Incorporated (Davis, CA, USA; catalog no.: CA95617); rabbit polyclonal anti- Kif5B antibody was purchased from ABclonal Technology (Wuhan, China; catalog no.: A15284); mouse monoclonal anti- Kif5B antibody was purchased from Santa Cruz Biotechnology (catalog no.: sc- 133184); mouse monoclonal anti–β- actin antibody was purchased from Proteintech (catalog no.: 60008- 1- Ig); mouse monoclonal anti- Usp14 antibody was purchased from Santa Cruz Biotechnology (catalog no.: sc- 398009); rabbit polyclonal anti- Usp14 antibody was purchased from Proteintech (catalog no.: 14517- 1- AP); rabbit monoclonal anti- Rec8 antibody was purchased from Abcam (catalog no.: ab192241); mouse monoclonal anti- Rad21 antibody was purchased from Sigma- Aldrich (catalog no.: 05- 908); rabbit polyclonal anti- HA antibody was purchased from Proteintech (catalog no.: 51064- 2- AP); rabbit monoclonal anti- Myc antibody was purchased from ABclonal Technology (catalog no.: AE070).

Techniques: Control, Fluorescence

Journal: Science advances

Article Title: The cohesin-associated protein Pds5A governs the meiotic spindle assembly via deubiquitination of Kif5B in oocytes.

doi: 10.1126/sciadv.adt6159

Figure Lengend Snippet: Fig. 3. Effects of Pds5A depletion on the spindle organization and chromosome alignment in mouse oocytes. (A) Fluorescence images of spindle morphology and chromosome alignment in control, Pds5A-MO, and Pds5A-rescue oocytes at MI stage. The yellow arrow points to the misaligned chromosomes. Scale bar, 20 μm. (B) The rate of abnormal spindles in control (n = 72), Pds5A-MO (n = 73), and Pds5A-rescue (n = 80) oocytes at MI stage. (C) Representative images showing different types of spindle defects observed in Pds5A-MO oocytes at MI stage. Scale bar, 10 μm. (D) The number of different types of spindle defects in Pds5A-MO (n = 73) oocytes at MI stage. (E) Schematic picture showing how the spindle length, width, and pole width were determined. (F) The ratio of spindle length to width in control (n = 23), Pds5A-MO (n = 21), and Pds5A-rescue (n = 22) oocytes at MI stage. (G) The rate of misaligned bivalents in control (n = 72), Pds5A-MO (n = 73), and Pds5A-rescue (n = 80) oocytes at MI stage. (H) Quantification of MI plate width in control (n = 24), Pds5A-MO (n = 24), and Pds5A-rescue (n = 22) oocytes at MI stage. Data in (B) and (G) were designated as mean percentage (means ± SEM), and (F) and (H) were designated as mean value (means ± SD) of at least three independent experiments. **P < 0.01; ***P < 0.001.

Article Snippet: Rabbit polyclonal anti- Pds5A antibodies were purchased from Proteintech (Rosemont, IL, USA; catalog no.: 17485- 1- AP) and Bethyl Laboratories (Montgomery, TX, USA; catalog no.: A300- 089A- T); mouse monoclonal anti- Pds5A antibody was purchased from Santa Cruz Biotechnology (Dallas, Texas, USA; catalog no.: sc- 515263); mouse monoclonal anti–α- tubulin–fluorescein isothiocyanate (FITC) antibody was purchased from Sigma- Aldrich (St. Louis, MO, USA; catalog no.: F2168); sheep polyclonal anti- BubR1 antibody was purchased from Abcam (Cambridge, MA, USA; catalog no.: ab28193); rabbit polyclonal anti- Mad2L1 antibody was purchased from Proteintech (catalog no.: 10337- 1- AP); human anti- centromere antibody was purchased from Antibodies Incorporated (Davis, CA, USA; catalog no.: CA95617); rabbit polyclonal anti- Kif5B antibody was purchased from ABclonal Technology (Wuhan, China; catalog no.: A15284); mouse monoclonal anti- Kif5B antibody was purchased from Santa Cruz Biotechnology (catalog no.: sc- 133184); mouse monoclonal anti–β- actin antibody was purchased from Proteintech (catalog no.: 60008- 1- Ig); mouse monoclonal anti- Usp14 antibody was purchased from Santa Cruz Biotechnology (catalog no.: sc- 398009); rabbit polyclonal anti- Usp14 antibody was purchased from Proteintech (catalog no.: 14517- 1- AP); rabbit monoclonal anti- Rec8 antibody was purchased from Abcam (catalog no.: ab192241); mouse monoclonal anti- Rad21 antibody was purchased from Sigma- Aldrich (catalog no.: 05- 908); rabbit polyclonal anti- HA antibody was purchased from Proteintech (catalog no.: 51064- 2- AP); rabbit monoclonal anti- Myc antibody was purchased from ABclonal Technology (catalog no.: AE070).

Techniques: Fluorescence, Control

Journal: Science advances

Article Title: The cohesin-associated protein Pds5A governs the meiotic spindle assembly via deubiquitination of Kif5B in oocytes.

doi: 10.1126/sciadv.adt6159

Figure Lengend Snippet: Fig. 4. Effects of Pds5A depletion on the K-M attachment and chromosome ploidy in mouse oocytes. (A) Fluorescence images kinetochores and microtubule fibers in control, Pds5A-MO, and Pds5A-rescue oocytes at MI stage. White arrows indicate unattached kinetochores. Scale bars, 5 and 2.5 μm. (B) The percentage of unattached kinetochores in control (n = 90), Pds5A-MO, (n = 125), and Pds5A- rescue (n = 103) oocytes at MI stage. (C) Representative images of chromosome spreads in control, Pds5A-MO, and Pds5A-rescue oocytes at MII stage. Scale bar, 8 μm. (D) The percentage of aneuploid oocytes at MII stage in control (n = 76), Pds5A-MO (n = 78), and Pds5A-rescue (n = 76) groups. Data in (B) and (D) were designated as mean percentage (means ± SEM) of at least three independent ex- periments. **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: Rabbit polyclonal anti- Pds5A antibodies were purchased from Proteintech (Rosemont, IL, USA; catalog no.: 17485- 1- AP) and Bethyl Laboratories (Montgomery, TX, USA; catalog no.: A300- 089A- T); mouse monoclonal anti- Pds5A antibody was purchased from Santa Cruz Biotechnology (Dallas, Texas, USA; catalog no.: sc- 515263); mouse monoclonal anti–α- tubulin–fluorescein isothiocyanate (FITC) antibody was purchased from Sigma- Aldrich (St. Louis, MO, USA; catalog no.: F2168); sheep polyclonal anti- BubR1 antibody was purchased from Abcam (Cambridge, MA, USA; catalog no.: ab28193); rabbit polyclonal anti- Mad2L1 antibody was purchased from Proteintech (catalog no.: 10337- 1- AP); human anti- centromere antibody was purchased from Antibodies Incorporated (Davis, CA, USA; catalog no.: CA95617); rabbit polyclonal anti- Kif5B antibody was purchased from ABclonal Technology (Wuhan, China; catalog no.: A15284); mouse monoclonal anti- Kif5B antibody was purchased from Santa Cruz Biotechnology (catalog no.: sc- 133184); mouse monoclonal anti–β- actin antibody was purchased from Proteintech (catalog no.: 60008- 1- Ig); mouse monoclonal anti- Usp14 antibody was purchased from Santa Cruz Biotechnology (catalog no.: sc- 398009); rabbit polyclonal anti- Usp14 antibody was purchased from Proteintech (catalog no.: 14517- 1- AP); rabbit monoclonal anti- Rec8 antibody was purchased from Abcam (catalog no.: ab192241); mouse monoclonal anti- Rad21 antibody was purchased from Sigma- Aldrich (catalog no.: 05- 908); rabbit polyclonal anti- HA antibody was purchased from Proteintech (catalog no.: 51064- 2- AP); rabbit monoclonal anti- Myc antibody was purchased from ABclonal Technology (catalog no.: AE070).

Techniques: Fluorescence, Control

Journal: Science advances

Article Title: The cohesin-associated protein Pds5A governs the meiotic spindle assembly via deubiquitination of Kif5B in oocytes.

doi: 10.1126/sciadv.adt6159

Figure Lengend Snippet: Fig. 5. The oocyte maturation and female fertility in Pds5A+/− mice. (A) Representative images of pups from WT and Pds5A+/− female mice. Scale bar, 1 cm. (B) The average number of pups per litter from WT (n = 9) and Pds5A+/− (n = 8) female mice. (C) Representative images of superovulated oocytes from WT and Pds5A+/− female mice. Scale bar, 80 μm. (D to F) The number, PBE rate, and death rate of superovulated oocytes in WT (n = 248) and Pds5A+/− (n = 173) groups. (G) Fluorescence images of spindles and chromosomes in WT and Pds5A+/− superovulated oocytes. Scale bars, 20 and 10 μm. (H) The rate of abnormal spindles in WT (n = 85) and Pds5A+/− (n = 84) superovulated oocytes. (I) The number of different defective spindles in Pds5A+/− (n = 40) superovulated oocytes. (J) The rate of misaligned chromosomes in WT (n = 85) and Pds5A+/− (n = 84) superovulated oocytes. (K) Representative images of chromosome spreads in WT and Pds5A+/− superovulated oocytes. Scale bar, 10 μm. (L) The percentage of aneuploid oocytes in WT (n = 84) and Pds5A+/− (n = 57) groups. (M) Fluorescence images of spindles and chromosomes in WT and Pds5A+/− MI oocytes. Scale bar, 20 μm. (N) The rate of abnormal spindles in WT (n = 100) and Pds5A+/− (n = 118) MI oocytes. (O) The number of different defective spindles in Pds5A+/− (n = 36) MI oocytes. (P) The rate of misaligned bivalents in WT (n = 100) and Pds5A+/− (n = 118) MI oocytes. Data in (B) and (D) were designated as means ± SD, and (E), (F), (H), (J), (L), (N), and (P) were designated as means ± SEM of at least three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: Rabbit polyclonal anti- Pds5A antibodies were purchased from Proteintech (Rosemont, IL, USA; catalog no.: 17485- 1- AP) and Bethyl Laboratories (Montgomery, TX, USA; catalog no.: A300- 089A- T); mouse monoclonal anti- Pds5A antibody was purchased from Santa Cruz Biotechnology (Dallas, Texas, USA; catalog no.: sc- 515263); mouse monoclonal anti–α- tubulin–fluorescein isothiocyanate (FITC) antibody was purchased from Sigma- Aldrich (St. Louis, MO, USA; catalog no.: F2168); sheep polyclonal anti- BubR1 antibody was purchased from Abcam (Cambridge, MA, USA; catalog no.: ab28193); rabbit polyclonal anti- Mad2L1 antibody was purchased from Proteintech (catalog no.: 10337- 1- AP); human anti- centromere antibody was purchased from Antibodies Incorporated (Davis, CA, USA; catalog no.: CA95617); rabbit polyclonal anti- Kif5B antibody was purchased from ABclonal Technology (Wuhan, China; catalog no.: A15284); mouse monoclonal anti- Kif5B antibody was purchased from Santa Cruz Biotechnology (catalog no.: sc- 133184); mouse monoclonal anti–β- actin antibody was purchased from Proteintech (catalog no.: 60008- 1- Ig); mouse monoclonal anti- Usp14 antibody was purchased from Santa Cruz Biotechnology (catalog no.: sc- 398009); rabbit polyclonal anti- Usp14 antibody was purchased from Proteintech (catalog no.: 14517- 1- AP); rabbit monoclonal anti- Rec8 antibody was purchased from Abcam (catalog no.: ab192241); mouse monoclonal anti- Rad21 antibody was purchased from Sigma- Aldrich (catalog no.: 05- 908); rabbit polyclonal anti- HA antibody was purchased from Proteintech (catalog no.: 51064- 2- AP); rabbit monoclonal anti- Myc antibody was purchased from ABclonal Technology (catalog no.: AE070).

Techniques: Fluorescence

Journal: Science advances

Article Title: The cohesin-associated protein Pds5A governs the meiotic spindle assembly via deubiquitination of Kif5B in oocytes.

doi: 10.1126/sciadv.adt6159

Figure Lengend Snippet: Fig. 6. Identification of binding partners of Pds5A. (A) Representative bind- ing candidates of Pds5A as shown in MS data. (B) Costaining of Pds5A and Kif5B in oocytes at MI stage. Scale bars, 20 and 5 μm. (C) Fluorescence intensity pro- files of Pds5A and Kif5B along the white line. (D) Co-IP of Pds5A and Kif5B as precipitated with Pds5A antibody. The blots of precipitates were probed with Kif5B and Pds5A antibodies, respectively. (E) Co-IP of Pds5A and Kif5B as pre- cipitated with Kif5B antibody. The blots of precipitates were probed with Pds5A and Kif5B antibodies, respectively.

Article Snippet: Rabbit polyclonal anti- Pds5A antibodies were purchased from Proteintech (Rosemont, IL, USA; catalog no.: 17485- 1- AP) and Bethyl Laboratories (Montgomery, TX, USA; catalog no.: A300- 089A- T); mouse monoclonal anti- Pds5A antibody was purchased from Santa Cruz Biotechnology (Dallas, Texas, USA; catalog no.: sc- 515263); mouse monoclonal anti–α- tubulin–fluorescein isothiocyanate (FITC) antibody was purchased from Sigma- Aldrich (St. Louis, MO, USA; catalog no.: F2168); sheep polyclonal anti- BubR1 antibody was purchased from Abcam (Cambridge, MA, USA; catalog no.: ab28193); rabbit polyclonal anti- Mad2L1 antibody was purchased from Proteintech (catalog no.: 10337- 1- AP); human anti- centromere antibody was purchased from Antibodies Incorporated (Davis, CA, USA; catalog no.: CA95617); rabbit polyclonal anti- Kif5B antibody was purchased from ABclonal Technology (Wuhan, China; catalog no.: A15284); mouse monoclonal anti- Kif5B antibody was purchased from Santa Cruz Biotechnology (catalog no.: sc- 133184); mouse monoclonal anti–β- actin antibody was purchased from Proteintech (catalog no.: 60008- 1- Ig); mouse monoclonal anti- Usp14 antibody was purchased from Santa Cruz Biotechnology (catalog no.: sc- 398009); rabbit polyclonal anti- Usp14 antibody was purchased from Proteintech (catalog no.: 14517- 1- AP); rabbit monoclonal anti- Rec8 antibody was purchased from Abcam (catalog no.: ab192241); mouse monoclonal anti- Rad21 antibody was purchased from Sigma- Aldrich (catalog no.: 05- 908); rabbit polyclonal anti- HA antibody was purchased from Proteintech (catalog no.: 51064- 2- AP); rabbit monoclonal anti- Myc antibody was purchased from ABclonal Technology (catalog no.: AE070).

Techniques: Binding Assay, Fluorescence, Co-Immunoprecipitation Assay

Journal: Science advances

Article Title: The cohesin-associated protein Pds5A governs the meiotic spindle assembly via deubiquitination of Kif5B in oocytes.

doi: 10.1126/sciadv.adt6159

Figure Lengend Snippet: Fig. 7. Recruitment of Usp14 by Pds5A stabilizes Kif5B in mouse oocytes. (A) Immunoblotting analysis showing the protein levels of Kif5B in control, Pds5A-MO, and Pds5A-rescue oocytes. (B) Fluorescence images of Kif5B in control, Pds5A-MO, and Pds5A-rescue oocytes at MI stage. Scale bars, 20 and 5 μm. (C) The fluorescence inten- sity of Kif5B signals in control (n = 19), Pds5A-MO (n = 20), and Pds5A-rescue (n = 19) oocytes at MI stage. (D) qRT-PCR analysis showing the mRNA levels of Kif5B in control (n = 30) and Pds5A-MO (n = 30) oocytes. (E) Immunoblotting analysis showing the protein levels of Kif5B in control, Pds5A-MO, and Pds5A-MO+MG132 (10 μM) oocytes. (F) Representative binding DUBs of Pds5A as shown in MS data. (G to K) Immunoblotting analysis showing the protein levels of Kif5B in control, PR-619-treated, WP1130- treated, IU1-treated, USP5-IN-1-treated, and FT671-treated oocytes, respectively. PR-619 (10 μM), 10 μM WP1130, 25 μM IU1, 10 μM USP5-IN-1, and 10 μM FT671 were used to treat GV oocytes for 22 hours, respectively. (L) Co-IP of Pds5A and Usp14 as precipitated with Pds5A antibody. (M) Costaining of Pds5A and Usp14 in oocytes at MI stage. Scale bars, 20 and 5 μm. (N) Fluorescence intensity profiles of Pds5A and Usp14 along the white line in (M). (O) Immunoblotting analysis showing the protein levels of Usp14 in control and Pds5A-MO oocytes. (P) Fluorescence images of Usp14 in control and Pds5A-MO oocytes at MI stage. Scale bar, 5 μm. (Q) The fluorescence intensity of Usp14 signals in control (n = 26) and Pds5A-MO (n = 35) oocytes at MI stage. Data in (D) were designated as mean percentage (means ± SEM), and (C) and (Q) were designated as mean value (means ± SD) of at least three independent experiments. ***P < 0.001; ****P < 0.0001; ns, no significance.

Article Snippet: Rabbit polyclonal anti- Pds5A antibodies were purchased from Proteintech (Rosemont, IL, USA; catalog no.: 17485- 1- AP) and Bethyl Laboratories (Montgomery, TX, USA; catalog no.: A300- 089A- T); mouse monoclonal anti- Pds5A antibody was purchased from Santa Cruz Biotechnology (Dallas, Texas, USA; catalog no.: sc- 515263); mouse monoclonal anti–α- tubulin–fluorescein isothiocyanate (FITC) antibody was purchased from Sigma- Aldrich (St. Louis, MO, USA; catalog no.: F2168); sheep polyclonal anti- BubR1 antibody was purchased from Abcam (Cambridge, MA, USA; catalog no.: ab28193); rabbit polyclonal anti- Mad2L1 antibody was purchased from Proteintech (catalog no.: 10337- 1- AP); human anti- centromere antibody was purchased from Antibodies Incorporated (Davis, CA, USA; catalog no.: CA95617); rabbit polyclonal anti- Kif5B antibody was purchased from ABclonal Technology (Wuhan, China; catalog no.: A15284); mouse monoclonal anti- Kif5B antibody was purchased from Santa Cruz Biotechnology (catalog no.: sc- 133184); mouse monoclonal anti–β- actin antibody was purchased from Proteintech (catalog no.: 60008- 1- Ig); mouse monoclonal anti- Usp14 antibody was purchased from Santa Cruz Biotechnology (catalog no.: sc- 398009); rabbit polyclonal anti- Usp14 antibody was purchased from Proteintech (catalog no.: 14517- 1- AP); rabbit monoclonal anti- Rec8 antibody was purchased from Abcam (catalog no.: ab192241); mouse monoclonal anti- Rad21 antibody was purchased from Sigma- Aldrich (catalog no.: 05- 908); rabbit polyclonal anti- HA antibody was purchased from Proteintech (catalog no.: 51064- 2- AP); rabbit monoclonal anti- Myc antibody was purchased from ABclonal Technology (catalog no.: AE070).

Techniques: Western Blot, Control, Fluorescence, Quantitative RT-PCR, Binding Assay, Co-Immunoprecipitation Assay

Journal: Scientific Reports

Article Title: MicroRNA-205-5p inhibits skin cancer cell proliferation and increase drug sensitivity by targeting TNFAIP8

doi: 10.1038/s41598-021-85097-6

Figure Lengend Snippet: TNFAIP8 expression is upregulated in BCC, SCC, and melanoma patient skin tissues. ( A ) Relative immunohistochemical TNFAIP8 expression in indicated skin cancer tissues is presented. ( B ) Immunohistochemical expression levels of TNFAIP8 protein from normal skin (n = 6), BCC (n = 6), H-SCC (n = 4), M-SCC (n = 4), L-SCC (n = 4), nevus (n = 6), and melanoma (n = 6) tissues were quantified using ImageJ software ( https://imagej.nih.gov/ij/ ) and plotted. The data represent mean ± SEM from 4 to 6 skin cancer tissues. * P < 0.05 relative to normal skin tissues. ( C ) Expression of TNFAIP8 transcripts between cutaneous melanoma (n = 45) and melanoma precursor (n = 18) samples were analyzed and presented from the Oncomine dataset ( https://www.oncomine.org/resource/login.html accessed October 17, 2020). *** P < 0.001 relatives to cutaneous melanoma tissues. ( D , E ) Expression of wild-type B-RAF, mutant B-RAF V600E , and TNFAIP8 in HaCaT, SK-MEL-2, A431, A375, and A2058 cells were analyzed by western blotting. Fifty micrograms of lysates from normal and skin cancer cells were immunoblotted with B-RAF, TNFAIP8, GAPDH, and β-actin antibodies. Immunoreactive bands were visualized using ECL chemiluminescence detection reagents after exposing the blots on X-ray films ( D ) or by scanning the blots with an Odyssey CLx Imager ( E ). The immunoblot scans were converted into grayscale and presented. ( F ) Expression of TNFAIP8 mRNA from indicated normal skin cells and skin cancer cells were analyzed by RT/qPCR as described in the “ ” section.

Article Snippet: Anti-β-actin antibody from Sigma (St. Louis, MO) and anti-TNFAIP8 antibody from Proteintech Group (Rosemont, IL) was also purchased.

Techniques: Expressing, Immunohistochemical staining, Software, Mutagenesis, Western Blot, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: MicroRNA-205-5p inhibits skin cancer cell proliferation and increase drug sensitivity by targeting TNFAIP8

doi: 10.1038/s41598-021-85097-6

Figure Lengend Snippet: TNFα-induced TNFAIP8 expression in skin cancer cells ( A ) Schematic represents TNFAIP8 isoform-specific forward and reverse primer design. ( B ) The expression of different variants/isoforms of TNFAIP8 in normal HaCaT and skin cancer cells was analyzed by RT-PCR. NC–negative control (no cDNA). ( C ) HaCaT, A431, A375, and A2058 cells were treated with vehicle or TNFα (10–50 ng/ml) for 30 h, and cell lysates were immunoblotted with TNFAIP8 or β-actin antibodies. Immunoreactive bands were visualized using ECL chemiluminescence detection reagents and the blots were scanned using an Odyssey CLx imager. The immunoblot scans were converted into grayscale and presented. ( D ) Similarly, normal and skin cancer cells were treated with vehicle or TNFα (10-50 ng/ml) for 30 h, and TNFAIP8 mRNA expression was analyzed by RT/qPCR as described in the “ ” section. * P < 0.05, ** P < 0.01, *** P < 0.001 relative to control/vehicle-treated cells. ( E ) HaCaT, A431, A375 and, A2058 cells were treated with vehicle or TNFα (10–50 ng/ml) for 48 h and, relative cell survival was analyzed by MTT assay as described in the “ ” section. Results are representative of three independent experiments. * P < 0.05, *** P < 0.001 relative to control/vehicle-treated cells.

Article Snippet: Anti-β-actin antibody from Sigma (St. Louis, MO) and anti-TNFAIP8 antibody from Proteintech Group (Rosemont, IL) was also purchased.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Negative Control, Western Blot, Quantitative RT-PCR, Control, MTT Assay

Journal: Scientific Reports

Article Title: MicroRNA-205-5p inhibits skin cancer cell proliferation and increase drug sensitivity by targeting TNFAIP8

doi: 10.1038/s41598-021-85097-6

Figure Lengend Snippet: TNFAIP8 regulates cell proliferation and migration in skin cancer cells. ( A ) Western blotting analysis of TNFAIP8-Myc tagged protein expression in HaCaT and skin cancer cells. Immunoreactive bands were developed using ECL chemiluminescence detection reagents and the blots were scanned using an Odyssey CLx imager. The immunoblot scans were converted into grayscale and presented. (B) Effect of TNFAIP8-Myc protein overexpression on HaCaT, A431, A375 and, A2058 on cell survival was measured by MTT assay. Results are representative of three independent experiments. * P < 0.05 relative to EV transfected cells. ( C ) The effect of overexpression of TNFAIP8-Myc on cell colony formation was analyzed and plotted (upper and lower panels) as described in the “ ” section. * P < 0.05 relative to EV transfected cells. ( D ) HaCaT, A431, A375 and, A2058 cells were transfected with control siRNA or TNFAIP8 siRNA (100 nM) for 30 h, and cell lysates were western blotted with anti-TNFAIP8 and β-actin antibodies. Western blots were developed using ECL chemiluminescence detection reagents and the blots were scanned using an Odyssey CLx imager. The immunoblot scans were converted into grayscale and presented. TNFAIP8 and β-actin protein levels were quantified using ImageJ software ( https://imagej.nih.gov/ij/ ) and presented below. ( E ) The effect of TNFAIP8 knockdown by siRNA on cell survival was analyzed by MTT assay. The experiments were independently performed three times. * P < 0.05 relative to control siRNA transfected cells. ( F ) Control siRNA and TNFAIP8 siRNA-transfected HaCaT, A431, A375 and A2058 cells (3000 cells/well) were re-plated in 6-well plates in triplicate for 7–10 days and skin cancer cell colony formation was analyzed and plotted (upper and lower panels) as described in the “ ” section. * P < 0.05 relative to control siRNA transfected cells. ( G ) Wound healing assay: HaCaT, A431, A375 and A2058 cells were transfected with control siRNA or TNFAIP8 siRNA for 48 h. The effect of the TNFAIP8 knockdown on cell migration was analyzed using the wound healing assay. Representative images of the wound healing assay (top) and the calculated scratch area (bottom) are shown. * P < 0.05 relative to control siRNA transfected cells.

Article Snippet: Anti-β-actin antibody from Sigma (St. Louis, MO) and anti-TNFAIP8 antibody from Proteintech Group (Rosemont, IL) was also purchased.

Techniques: Migration, Western Blot, Expressing, Over Expression, MTT Assay, Transfection, Control, Software, Knockdown, Wound Healing Assay

Journal: Scientific Reports

Article Title: MicroRNA-205-5p inhibits skin cancer cell proliferation and increase drug sensitivity by targeting TNFAIP8

doi: 10.1038/s41598-021-85097-6

Figure Lengend Snippet: miR-205-5p targets the 3′UTR of TNFAIP8 and inhibits TNFAIP8 expression. ( A ) The binding site of miR-205-5p in the 3′UTR of TNFAIP8 was analyzed by TargetScan ( http://www.targetscan.org/vert_72/ ) and presented. We also generated a wild type and mutated binding nucleotides of miR-205-5p to the 3′UTR of TNFAIP8. Wild-type miR-205-5p and mutant miR-205-5p mimics are shown. ( B ) Relative expression of miR-205-5p in HaCaT, A431, A375, A2058, and SK-MEL-2 cell lines was analyzed by RT/qPCR as described in the “ ” section. ( C ) Luciferase reporter assay: A431 and A2058 cells were transfected with TNFAIP8-3′UTR-Luciferase reporter construct (0.5 µg) for 18 h and then transfected with NC mimic or wild-type miR-205-5p mimic or mutant miR-205-5p mimic for an additional 24 h. Transfected cells were lysed, and luciferase activity was measured. Results are representative of two independent experiments. ** P < 0.01, *** P < 0.001 compared with NC or mutant-type miR-205-5p mimic. ( D , E ) Normal or skin cancer cells were transfected with NC or miR-205-5p mimic for 48 h and the overexpression of miR-205-5p mimic and TNFAIP8 transcripts were analyzed by RT/qPCR. *** P < 0.001 compared with NC-transfected cells. * P < 0.05 compared with NC-transfected cells. ns-no significance. ( F ) HaCaT, A431 and A2058 cell lines were transfected with NC mimic or miR-205-5p mimic for 40 h and expression of endogenous TNFAIP8 protein were analyzed by western blotting. Western blots were developed using ECL chemiluminescence detection reagents, the blots were exposed to X-Ray films, scans were converted into grayscale and presented. ( G ) HaCaT, 431 and A2058 cell lines were grown on a coverslip and transfected with Cy3-labeled NC mimic or Cy3-labeled miR-205-5p mimic and the effect of Cy3-labeled miR-205-5p mimic on endogenous TNFAIP8 expression (green) was visualized by immunofluorescence as described in the “ ” section.

Article Snippet: Anti-β-actin antibody from Sigma (St. Louis, MO) and anti-TNFAIP8 antibody from Proteintech Group (Rosemont, IL) was also purchased.

Techniques: Expressing, Binding Assay, Generated, Mutagenesis, Quantitative RT-PCR, Luciferase, Reporter Assay, Transfection, Construct, Activity Assay, Over Expression, Western Blot, Labeling, Immunofluorescence

Journal: Scientific Reports

Article Title: MicroRNA-205-5p inhibits skin cancer cell proliferation and increase drug sensitivity by targeting TNFAIP8

doi: 10.1038/s41598-021-85097-6

Figure Lengend Snippet: miR-205-5p targets TNFAIP8 and reduces TNFAIP8-mediated cell proliferation and autophagy. ( A ) HaCaT, A431 and A2058 cells were transfected with NC mimic or miR-205-5p mimic for 48 h and relative cell survival was analyzed by MTT assay. The experiments were independently repeated three times. * P < 0.05 compared with NC mimic transfected cells. ( B ) NC mimic or miR-205-5p mimic transfected cells were re-plated in 6-well plates for 7–10 days and cell colony formation was analyzed and plotted (upper and lower panels) as described in the “ ” section. The experiment was repeated twice in triplicates. * P < 0.05 relative to NC mimic transfected cells. ( C ) HaCaT, A431 and A2058 cells were transfected EV or TNFAIP8-Myc plasmids for 30 h and lysates were western blotted with anti-Myc, anti-LC3β I/II, and anti-β-actin antibodies. Western blots were developed using ECL chemiluminescence detection reagents, the blots were exposed on X-ray films, converted into grayscale, and presented. ( D ) HaCaT, lanoma A431 and A2058 cells were transfected NC mimic or miR-205-5p mimic for 30 h and lysates were western blotted with anti-TNFAIP8, anti-LC3β I/II, anti-p62, and anti-β-actin antibodies. Western blots were developed using ECL chemiluminescence detection reagents, exposed on X-Ray films, converted into grayscale, and presented. ( E ) HaCaT, A431 and A2058 cells were grown on a coverslip transfected with Cy3-labeled NC mimic or Cy3-labeled miR-205-5p mimic and the effect of Cy3-labeled miR-205-5p mimic on endogenous LC3β I/II expression (green) and related puncta formation were visualized by immunofluorescence (upper panels) as described in the “ ” section (upper panel). LC3β I/II and related puncta were measured from individual cells (n = 10) and plotted (lower panels). ** P < 0.01, *** P < 0.001 relatives to Cy3 labeled-NC mimic transfected cells.

Article Snippet: Anti-β-actin antibody from Sigma (St. Louis, MO) and anti-TNFAIP8 antibody from Proteintech Group (Rosemont, IL) was also purchased.

Techniques: Transfection, MTT Assay, Western Blot, Labeling, Expressing, Immunofluorescence

Journal: Scientific Reports

Article Title: MicroRNA-205-5p inhibits skin cancer cell proliferation and increase drug sensitivity by targeting TNFAIP8

doi: 10.1038/s41598-021-85097-6

Figure Lengend Snippet: miR-205-5p targets TNFAIP8 in skin cancer cells and increase sensitivity to vemurafenib. ( A ) HaCaT cells, and skin cancer SK-MEL-2, A375 and A2058 cell lines were treated with increasing concentrations of vemurafenib for 48 h and relative cell survival/proliferation was measured by MTT assay. The experiments were independently repeated three times. ** P < 0.01, *** P < 0.001 compared to vehicle-treated cells. ( B ) HaCaT cells and skin cancer SK-MEL-2, A375 and A2058 cell lines were transfected with NC mimic or miR-205-5p mimic for 24 h and treated with vemurafenib or vehicle for an additional 48 h and relative cell survival were measured by MTT assay as described in the “ ” section. * P < 0.05, ** P < 0.01, *** P < 0.001 compared to NC mimic vehicle-treated cells or NC mimic and vemurafenib-treated cells. ns-no significance. ( C ) Melanoma A375 cells were transfected with NC mimic or miR-205-5p mimic for 18 h and then cells were treated with vemurafenib for an additional 24 h. Fifty micrograms of lysates were immunoblotted with anti-cleaved-PARP and anti-β-tubulin antibodies. Western blots were developed using ECL chemiluminescence detection reagents, the blots were scanned using Azure C-500 Biosystem, scans were converted into grayscale and presented. ( D ) Melanoma A375 cells were transfected with EV of TNFAIP8-Myc plasmid for 18 h first and then cells were treated with vemurafenib or paclitaxel for 24 h as indicated and fifty micrograms lysates were immunoblotted with anti-Myc, anti-cleaved-PARP, and anti-β-actin antibodies. Western blots were developed using ECL chemiluminescence detection reagents and the blots were scanned using an Odyssey CLx imager. The immunoblot scans were converted into grayscale and presented. ( E ) Fifty micrograms of lysates from A375 and A2058 cells or A375R and A2058R (vemurafenib resistant) cells were immunoblotted with anti-TNFAIP8, anti-LC3β I/II, and anti-β-actin antibodies. Western blots were developed using ECL chemiluminescence detection reagents, the blots were exposed on X-Ray films, scans were converted into grayscale and presented. ( F ) Relative cell proliferation/cell survival rate of A375 and A2058 cells, or A375R and A2058R (vemurafenib resistant) cells were measured by using WST-1 reagent (Cayman Chemical) (upper panel) or MTT assay reagent (lower panel) and plotted. ** P < 0.01, *** P < 0.001 compared to A375 and A2058 parent cells. Results are representative of three independent experiments. Vem. Vemurafenib.

Article Snippet: Anti-β-actin antibody from Sigma (St. Louis, MO) and anti-TNFAIP8 antibody from Proteintech Group (Rosemont, IL) was also purchased.

Techniques: MTT Assay, Transfection, Western Blot, Plasmid Preparation