Journal: Journal of Virology

Article Title: Identification of Chicken CD74 as a Novel Cellular Attachment Receptor for Infectious Bursal Disease Virus in Bursa B Lymphocytes

doi: 10.1128/JVI.01712-19

Figure Lengend Snippet: Identification of CD74 as a putative receptor for IBDV. (A) Affinity purification with mAb against the IBDV major capsid protein VP2 in DT40 cells, incubated with VP2 (or the empty vector as a negative control), followed by mass spectrometry analysis. Many host proteins were associated with VP2, including chicken CD74. (B and C) Interaction between VP2 and the CD74 extracellular domain detected via a coimmunoprecipitation (co-IP) assay. (B) Western blot (WB) analysis using an antibody against the HA tag showing the bands corresponding to VP2 in the Flag co-IP assay. (C) Western blot analysis using an antibody against the HA tag showing the bands corresponding to the CD74 extracellular domain in the Flag co-IP assay. (D) To determine the distribution of CD74 in organs, the heart, liver, spleen, lung, kidney, thymus, and bursa of Fabricius of uninfected SPF chickens were collected to measure CD74 transcription levels. The most abundant CD74 expression was found in the bursa. (E) To confirm the involvement of chicken CD74 in the IBDV infection process, SPF chickens were challenged by vvIBDV or PBS. Bursas were collected for RT-qPCR analysis of CD74 transcription levels. CD74 mRNAs were significantly induced by vvIBDV at 12 h p.i. (*, P < 0.05; **, P < 0.01). The arithmetic means and standard deviations for at least three independent experiments performed in duplicate are shown.

Article Snippet: Total RNA was extracted from tissues or cells using the RNeasy minikit (Qiagen, Germany), and 1 μg RNA was reverse transcribed to cDNA using the ReverTra Ace qPCR RT master mix with a genomic DNA (gDNA) remover (Toyobo, Japan) in a 20-μl reaction mixture.

Techniques: Affinity Purification, Incubation, Plasmid Preparation, Negative Control, Mass Spectrometry, Co-Immunoprecipitation Assay, Western Blot, Expressing, Infection, Quantitative RT-PCR

Journal: Journal of Virology

Article Title: Identification of Chicken CD74 as a Novel Cellular Attachment Receptor for Infectious Bursal Disease Virus in Bursa B Lymphocytes

doi: 10.1128/JVI.01712-19

Figure Lengend Snippet: CD74 knockdown suppresses IBDV replication. The expression of the CD74 Ii-2 isoform was downregulated by siRNA interference or knockdown by shRNA in DT40 cells. The vvIBDV Gx strain at an MOI of 1 was added to the CD74 siRNA interference groups or the CD74 KD cell line. Infected cells were washed with PBS at 4 h p.i., and DT40 complete medium was then added. Cells and supernatants were collected at 24, 48, and 72 h p.i. for Western blotting and qPCR and at 72 h p.i. for ELD50 analysis. (A) CD74 expression levels in DT40 cells determined by Western blotting, showing that CD74 downregulation was effective. siSc., scrambled control siRNA. (B) Western blot assays showing IBDV VP2 expression declining at 24 to 48 h p.i. (C) qPCR analysis showing IBDV copy numbers dropping off at 48 h p.i. (6.34-fold decrease compared with the siRNA negative control; *, P < 0.05). (D) ELD50 assay showing the IBDV titer being downregulated at 48 h p.i. (14.0-fold decrease compared with the siRNA negative control; *, P < 0.05). (E) CD74 mRNA level in DT40 cells determined by qPCR, indicating that CD74 knockdown was effective. (F) IBDV VP2 protein expression was downregulated significantly at 24 to 72 h p.i. in the CD74 KD groups. (G) The IBDV copy number was downregulated at 24 to 72 h p.i. (103- to 104-fold decrease compared with the wild-type [WT] control; P < 0.05). (F) ELD50 assay showing the IBDV titer being downregulated at 72 h p.i. (4.42 × 104-fold decrease compared with wild-type cells; P < 0.05). The arithmetic means and standard deviations for at least three independent experiments performed in duplicate are shown.

Article Snippet: Total RNA was extracted from tissues or cells using the RNeasy minikit (Qiagen, Germany), and 1 μg RNA was reverse transcribed to cDNA using the ReverTra Ace qPCR RT master mix with a genomic DNA (gDNA) remover (Toyobo, Japan) in a 20-μl reaction mixture.

Techniques: Knockdown, Expressing, shRNA, Infection, Western Blot, Control, Negative Control

Journal: Journal of Virology

Article Title: Identification of Chicken CD74 as a Novel Cellular Attachment Receptor for Infectious Bursal Disease Virus in Bursa B Lymphocytes

doi: 10.1128/JVI.01712-19

Figure Lengend Snippet: CD74 overexpression promotes IBDV replication. CD74 Ii-2 isoform overexpression promotes IBDV infectivity. The expression of the CD74 Ii-2 isoform was upregulated by plasmid transfection in DT40 cells. Next, vvIBDV at an MOI of 1 was used to infected the cells as described in the text. Cells and supernatants were collected at 24, 48, and 72 h p.i. for Western blotting and at 48 h p.i. for qPCR analysis. (A) CD74 Ii-2 isoform overexpression remarkably promotes IBDV replication. The IBDV copy number was increased in the overexpression groups at 72 h p.i. compared with the nonoverexpression group (2.90-fold increase; *, P < 0.05). (B) IBDV VP2 protein expression is upregulated at 48 to 72 h p.i. in the overexpression group.

Article Snippet: Total RNA was extracted from tissues or cells using the RNeasy minikit (Qiagen, Germany), and 1 μg RNA was reverse transcribed to cDNA using the ReverTra Ace qPCR RT master mix with a genomic DNA (gDNA) remover (Toyobo, Japan) in a 20-μl reaction mixture.

Techniques: Over Expression, Infection, Expressing, Plasmid Preparation, Transfection, Western Blot

Journal: Journal of Virology

Article Title: Identification of Chicken CD74 as a Novel Cellular Attachment Receptor for Infectious Bursal Disease Virus in Bursa B Lymphocytes

doi: 10.1128/JVI.01712-19

Figure Lengend Snippet: CD74 isoform Ii-2 confers to vvIBDV the ability to attach to a vvIBDV-nonpermissive cell line. (A) 293T cells (nonpermissive to vvIBDV) were transfected with a full-length chicken CD74 Ii-2 plasmid with an HA tag (or the empty vector as a negative control). Twenty-four hours after transfection, cells were incubated with vvIBDV at an MOI of 5 at 37°C for 36 h to investigate whether CD74 confers susceptibility to IBDV infection. (Top) Cells were processed for confocal analysis, using IBDV VP2 mAb and an HA tag antibody as the primary antibodies. No virus (green fluorescence) was detected in the empty vector control. (Bottom) In the CD74 Ii-2 overexpression group, CD74 (red fluorescence) accumulated on the cell membrane, colocalizing with the IBDV particles (green fluorescence). No virus was observed to enter CD74-overexpressing nonpermissive cells. CD74 could confer attachment ability but not susceptibility to IBDV infection. (B) 293T cells overexpressed HA-tagged chicken CD74 Ii-2 or the empty vector for 24 h and were then incubated with 200 μg SVPs at 4°C for 1 h. After washing with PBS 5 times, cells were processed for confocal analysis as described above. SVPs (green fluorescence) were bound to the membranes of CD74-overexpressing cells (red fluorescence) and colocalized with CD74, but no binding was observed for the empty vector group. (C and D) 293T and HeLa cells (both of which are nonpermissive to vvIBDV) were transfected with a eukaryotic expression plasmid of HA-tagged chicken CD74 Ii-2 (or the empty vector as a negative control) and maintained for 24 h under normal culture conditions. The cells were then incubated with vvIBDV at an MOI of 50 at 4°C for 1 h for the binding assay and washed with PBS 5 times to remove the unbound viruses. Cells were processed for confocal analysis using the same antibodies and procedure as the ones described above. No virus (green fluorescence) was found to bind or infect 293T (C) or HeLa (D) cells transfected with the empty vector. In contrast, IBDV (green fluorescence) colocalized with overexpressed chicken CD74 (red fluorescence) on the membrane of nonpermissive 293T (C) and HeLa (D) cells. (E and F) qPCR analysis indicating that CD74 overexpression promotes IBDV binding to 293T or HeLa cells compared with the empty vector transfection group (9.55-fold increase in 293T cells [E] and 5.07-fold increase in HeLa cells [F]; both P < 0.05). The arithmetic means and standard deviations for at least three independent experiments performed in duplicate are shown.

Article Snippet: Total RNA was extracted from tissues or cells using the RNeasy minikit (Qiagen, Germany), and 1 μg RNA was reverse transcribed to cDNA using the ReverTra Ace qPCR RT master mix with a genomic DNA (gDNA) remover (Toyobo, Japan) in a 20-μl reaction mixture.

Techniques: Transfection, Plasmid Preparation, Negative Control, Incubation, Infection, Virus, Fluorescence, Control, Over Expression, Membrane, Binding Assay, Expressing

Journal: Cells

Article Title: Cap-Specific m 6 Am Methyltransferase PCIF1/CAPAM Regulates mRNA Stability of RAB23 and CNOT6 through the m 6 A Methyltransferase Activity

doi: 10.3390/cells13201689

Figure Lengend Snippet: Exploration of PCIF1 target genes by genome-wide gene expression analysis. ( A ) RT-qPCR analysis of total RNAs isolated from HeLa cells treated with control siRNA (siNC) or two distinct PCIF1-targeted siRNAs (siPCIF1 #1 and #2). ( B ) Immunoblotting analysis of total protein extracts from HeLa cells treated with control siRNA (siNC) and two distinct PCIF1-targeted siRNAs (siPCIF1 #1 and #2) with anti-PCIF1 and anti-b-actin antibodies. ( C , D ) Venn diagrams showing the overlap between the two indicated siRNA-mediated downregulated ( C ) and upregulated ( D ) genes identified by the gene expression profile analyzed by DNA microarray using a Human Genome U133 Plus 2.0 Array (Affymetrix). The condition for selecting differentially expressed genes are as follows: (1) cut-off condition: expression > 100, (2) fold change: >2, (3) p -value < 0.05.

Article Snippet: An amount of 1.5 μg of anti-m 6 A antibody (Abcam Limited, Cambridge, UK, ab151230) or normal rabbit IgG (Medical & Biological Laboratories Co.) was incubated with 30 μL of Dynabeads Protein G (Thermo Fisher Scientific) at room temperature for 1 h. Total RNA (50 μg) was added to the antibody-Dynabead complexes and incubated at 4 °C for 4 h. After washing three times, the bound RNA was purified from the beads using SepasolTM-RNA I Super G. The first strand of cDNA was synthesized from m 6 A-RNA using ReverTra AceTM qPCR RT Master Mix with gDNA Remover (Toyobo Co., Osaka, Japan) with random hexamer primers according to the manufacturer’s instructions. cDNA was quantified using GeneAce SYBRTM qPCR Mix II (Nippon Gene Co., Tokyo, Japan) and a Mx3000P real-time PCR system (Agilent).

Techniques: Genome Wide, Gene Expression, Quantitative RT-PCR, Isolation, Control, Western Blot, Microarray, Expressing

Journal: Cells

Article Title: Cap-Specific m 6 Am Methyltransferase PCIF1/CAPAM Regulates mRNA Stability of RAB23 and CNOT6 through the m 6 A Methyltransferase Activity

doi: 10.3390/cells13201689

Figure Lengend Snippet: Expression of RAB23 and CNOT6 is regulated by PCIF1 at both the mRNA and protein levels. ( A – C ) RT-qPCR analysis of total RNAs isolated from HeLa cells treated with control siRNA (siNC) and two distinct PCIF1-targeted siRNAs (siPCIF1 #1 and #3), using the specific primer set detecting PCIF1 ( A ), RAB23 ( B ), and CNOT6 ( C ) expression. ( D , E ) Immunoblotting analysis of total protein extracts from HeLa cells treated with control siRNA (siNC) and two distinct PCIF1-targeted siRNAs (siPCIF1 #1 and #3) with the indicated antibodies. Signal intensities obtained from immunoblotting were quantified using ImageJ software version 1.52. The y-axis represents the fold change relative to the levels in HeLa cells treated with control siRNA. Data are expressed as the mean ± standard deviation of three independent experiments. Asterisks represent statistically significant differences between the control siRNA treatment and the indicated PCIF1-targeted siRNA (Student’s t -test, * p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: An amount of 1.5 μg of anti-m 6 A antibody (Abcam Limited, Cambridge, UK, ab151230) or normal rabbit IgG (Medical & Biological Laboratories Co.) was incubated with 30 μL of Dynabeads Protein G (Thermo Fisher Scientific) at room temperature for 1 h. Total RNA (50 μg) was added to the antibody-Dynabead complexes and incubated at 4 °C for 4 h. After washing three times, the bound RNA was purified from the beads using SepasolTM-RNA I Super G. The first strand of cDNA was synthesized from m 6 A-RNA using ReverTra AceTM qPCR RT Master Mix with gDNA Remover (Toyobo Co., Osaka, Japan) with random hexamer primers according to the manufacturer’s instructions. cDNA was quantified using GeneAce SYBRTM qPCR Mix II (Nippon Gene Co., Tokyo, Japan) and a Mx3000P real-time PCR system (Agilent).

Techniques: Expressing, Quantitative RT-PCR, Isolation, Control, Western Blot, Software, Standard Deviation

Journal: Cells

Article Title: Cap-Specific m 6 Am Methyltransferase PCIF1/CAPAM Regulates mRNA Stability of RAB23 and CNOT6 through the m 6 A Methyltransferase Activity

doi: 10.3390/cells13201689

Figure Lengend Snippet: Ectopic expression of siRNA-resistant PCIF1 restores the normal levels of target mRNA expression. ( A , B ) RT-qPCR analysis of total RNAs isolated from HeLa cells transfected with a control empty vector (Vec) or a vector expressing siRNA-resistant PCIF1 (PCIF1siR) under treatment with control siRNA (siNC) or PCIF1-targeted siRNAs (siPCIF1 #3), using the specific primer set detecting RAB23 ( A ) and CNOT6 ( B ) expression. The y -axis represents the fold change relative to the levels in HeLa cells treated with control siRNA. Data are expressed as the mean ± standard deviation of three independent experiments. Asterisks represent statistically significant differences between indicated pairs (Student’s t -test, *** p < 0.001).

Article Snippet: An amount of 1.5 μg of anti-m 6 A antibody (Abcam Limited, Cambridge, UK, ab151230) or normal rabbit IgG (Medical & Biological Laboratories Co.) was incubated with 30 μL of Dynabeads Protein G (Thermo Fisher Scientific) at room temperature for 1 h. Total RNA (50 μg) was added to the antibody-Dynabead complexes and incubated at 4 °C for 4 h. After washing three times, the bound RNA was purified from the beads using SepasolTM-RNA I Super G. The first strand of cDNA was synthesized from m 6 A-RNA using ReverTra AceTM qPCR RT Master Mix with gDNA Remover (Toyobo Co., Osaka, Japan) with random hexamer primers according to the manufacturer’s instructions. cDNA was quantified using GeneAce SYBRTM qPCR Mix II (Nippon Gene Co., Tokyo, Japan) and a Mx3000P real-time PCR system (Agilent).

Techniques: Expressing, Quantitative RT-PCR, Isolation, Transfection, Control, Plasmid Preparation, Standard Deviation

Journal: Cells

Article Title: Cap-Specific m 6 Am Methyltransferase PCIF1/CAPAM Regulates mRNA Stability of RAB23 and CNOT6 through the m 6 A Methyltransferase Activity

doi: 10.3390/cells13201689

Figure Lengend Snippet: PCIF1 does not regulate expression of its target genes at the transcriptional level. ( A , E ) Schematic illustrations of the RAB23 ( A ) and CNOT6 ( E ) genes. The transcription start sites are indicated by arrows, the exons are shown as black boxes, and the polyadenylation signals are indicated by arrowheads. The positions of the PCR primer set for RT-qPCR amplification of the precursor and mature mRNAs are indicated by arrows. ( B – D , F – H ) RT-qPCR analysis of total RNAs isolated from HeLa cells treated with control siRNA (siNC) and two distinct PCIF1-targeted siRNAs (siPCIF1 #1 and #3) using the indicated primer sets. ( I , J ) ChIP analyses of the RAB23 ( I ) and CNOT6 ( J ) gene promoters (position 2 in ) using antibodies against Pol II in HeLa cells treated with control siRNA (siNC) and two distinct PCIF1 targeted siRNAs (siPCIF1 #1 and #3). Normal rabbit IgG was used as the negative control. Data are expressed as the mean ± standard deviation of three independent experiments. Asterisks represent statistically significant differences between the control siRNA treatment and the indicated PCIF1 targeted siRNA (Student’s t -test, n.s. p > 0.05, *** p < 0.001).

Article Snippet: An amount of 1.5 μg of anti-m 6 A antibody (Abcam Limited, Cambridge, UK, ab151230) or normal rabbit IgG (Medical & Biological Laboratories Co.) was incubated with 30 μL of Dynabeads Protein G (Thermo Fisher Scientific) at room temperature for 1 h. Total RNA (50 μg) was added to the antibody-Dynabead complexes and incubated at 4 °C for 4 h. After washing three times, the bound RNA was purified from the beads using SepasolTM-RNA I Super G. The first strand of cDNA was synthesized from m 6 A-RNA using ReverTra AceTM qPCR RT Master Mix with gDNA Remover (Toyobo Co., Osaka, Japan) with random hexamer primers according to the manufacturer’s instructions. cDNA was quantified using GeneAce SYBRTM qPCR Mix II (Nippon Gene Co., Tokyo, Japan) and a Mx3000P real-time PCR system (Agilent).

Techniques: Expressing, Quantitative RT-PCR, Amplification, Isolation, Control, Negative Control, Standard Deviation

Journal: Cells

Article Title: Cap-Specific m 6 Am Methyltransferase PCIF1/CAPAM Regulates mRNA Stability of RAB23 and CNOT6 through the m 6 A Methyltransferase Activity

doi: 10.3390/cells13201689

Figure Lengend Snippet: PCIF1 regulates the stability of target gene mRNAs in opposite ways. After HeLa cells were treated with a negative control siRNA (siNC) and two PCIF1-targeted siRNAs (siPCIF1 #1 and #3) for 72 h, actinomycin D was added to inhibit transcription. Cells were harvested at 0, 2, 4, 8, and 12 h after treatment, and total RNA was isolated. The amount of residual mRNA was analyzed by RT-qPCR at each time point, using the specific primer set detecting PCIF1 ( A ), CRAB23 ( B ), and CNOT6 ( C ) mRNAs. The relative value was calculated using the expression level of β-actin mRNA (ACTB) as a normalizer. The relative values at each time point were calculated relative to time zero. Asterisks represent statistically significant differences between the control siRNA treatment and the indicated PCIF1-targeted siRNA (Student’s t -test, * p < 0.05, *** p < 0.001).

Article Snippet: An amount of 1.5 μg of anti-m 6 A antibody (Abcam Limited, Cambridge, UK, ab151230) or normal rabbit IgG (Medical & Biological Laboratories Co.) was incubated with 30 μL of Dynabeads Protein G (Thermo Fisher Scientific) at room temperature for 1 h. Total RNA (50 μg) was added to the antibody-Dynabead complexes and incubated at 4 °C for 4 h. After washing three times, the bound RNA was purified from the beads using SepasolTM-RNA I Super G. The first strand of cDNA was synthesized from m 6 A-RNA using ReverTra AceTM qPCR RT Master Mix with gDNA Remover (Toyobo Co., Osaka, Japan) with random hexamer primers according to the manufacturer’s instructions. cDNA was quantified using GeneAce SYBRTM qPCR Mix II (Nippon Gene Co., Tokyo, Japan) and a Mx3000P real-time PCR system (Agilent).

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

Journal: Cells

Article Title: Cap-Specific m 6 Am Methyltransferase PCIF1/CAPAM Regulates mRNA Stability of RAB23 and CNOT6 through the m 6 A Methyltransferase Activity

doi: 10.3390/cells13201689

Figure Lengend Snippet: Ectopic expression of siRNA-resistant wild-type but not methyltransferase-deficient mutant PCIF1 restored normal levels of target mRNA expression. ( A ) Immunoblotting analysis of total protein extracts from HeLa cells transfected with a control empty vector (Vec), a vector expressing siRNA-resistant wild-type PCIF1 (siR_wt), or methyltransferase-deficient mutant PCIF1 (siR_mut) under treatment with control siRNA (siNC) or PCIF1-targeted siRNAs (siPCIF1: siPCIF1 #3 was used for PCIF1 suppression) with the indicated antibodies. ( B , C ) RT-qPCR analysis of total RNAs isolated from HeLa cells treated as in ( A ), using the specific primer set detecting RAB23 ( B ) and CNOT6 ( C ) expression. Data are expressed as the mean ± standard deviation of three independent experiments. Asterisks represent statistically significant differences between the indicated pairs (Student’s t -test, n.s. p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: An amount of 1.5 μg of anti-m 6 A antibody (Abcam Limited, Cambridge, UK, ab151230) or normal rabbit IgG (Medical & Biological Laboratories Co.) was incubated with 30 μL of Dynabeads Protein G (Thermo Fisher Scientific) at room temperature for 1 h. Total RNA (50 μg) was added to the antibody-Dynabead complexes and incubated at 4 °C for 4 h. After washing three times, the bound RNA was purified from the beads using SepasolTM-RNA I Super G. The first strand of cDNA was synthesized from m 6 A-RNA using ReverTra AceTM qPCR RT Master Mix with gDNA Remover (Toyobo Co., Osaka, Japan) with random hexamer primers according to the manufacturer’s instructions. cDNA was quantified using GeneAce SYBRTM qPCR Mix II (Nippon Gene Co., Tokyo, Japan) and a Mx3000P real-time PCR system (Agilent).

Techniques: Expressing, Mutagenesis, Western Blot, Transfection, Control, Plasmid Preparation, Quantitative RT-PCR, Isolation, Standard Deviation

Journal: Cells

Article Title: Cap-Specific m 6 Am Methyltransferase PCIF1/CAPAM Regulates mRNA Stability of RAB23 and CNOT6 through the m 6 A Methyltransferase Activity

doi: 10.3390/cells13201689

Figure Lengend Snippet: PCIF1 suppression resulted in a significant decrease in m 6 A levels of both RAB23 and CNOT6 mRNAs. MeRIP-qPCR analysis was performed using HeLa cells treated with control siRNA (siNC) or PCIF1-targeted siRNA (siPCIF1: siPCIF1 #3 was used for PCIF1 suppression). RT-qPCR analysis of RNAs purified from the immunoprecipitates of HeLa cell extracts using the anti-m 6 A antibody, using the specific primer set detecting RAB23 ( A ), CNOT6 ( B ), and ACTB ( C ) mRNAs. The y -axis represents the fold change relative to the RNA levels in the immunoprecipitate by anti-m 6 A antibody from HeLa cells treated with control siRNA (siNC). Normal rabbit IgG (IgG) was used as a negative control. Data are expressed as the mean ± standard deviation of three independent experiments. Asterisks represent statistically significant differences between the indicated pairs (Student’s t -test, n.s. p > 0.05, *** p < 0.001).

Article Snippet: An amount of 1.5 μg of anti-m 6 A antibody (Abcam Limited, Cambridge, UK, ab151230) or normal rabbit IgG (Medical & Biological Laboratories Co.) was incubated with 30 μL of Dynabeads Protein G (Thermo Fisher Scientific) at room temperature for 1 h. Total RNA (50 μg) was added to the antibody-Dynabead complexes and incubated at 4 °C for 4 h. After washing three times, the bound RNA was purified from the beads using SepasolTM-RNA I Super G. The first strand of cDNA was synthesized from m 6 A-RNA using ReverTra AceTM qPCR RT Master Mix with gDNA Remover (Toyobo Co., Osaka, Japan) with random hexamer primers according to the manufacturer’s instructions. cDNA was quantified using GeneAce SYBRTM qPCR Mix II (Nippon Gene Co., Tokyo, Japan) and a Mx3000P real-time PCR system (Agilent).

Techniques: Control, Quantitative RT-PCR, Purification, Negative Control, Standard Deviation

Journal: Scientific Reports

Article Title: Concomitant attenuation of HMGCR expression and activity enhances the growth inhibitory effect of atorvastatin on TGF-β-treated epithelial cancer cells

doi: 10.1038/s41598-021-91928-3

Figure Lengend Snippet: Expression of EMT markers in NCI-H322M cells treated with TGF-β1. ( A ) Overview of experimental procedures. ( B–D ) Quantitative PCR analysis of E-cadherin ( B ), N-cadherin ( C ), and vimentin ( D ) mRNA expression in cells treated with TGF-β1. Data are normalized to 18S rRNA level in each sample and are expressed as values relative to that of the internal control. RNA isolated from cells cultured without TGF-β1 is used as control. The measurement values for each group are compared using the Bonferroni-Dunn post-hoc test. Mean ± SD, n = 3, * p < 0.05, ** p < 0.01; Comparison with TGF-β1 (−) control (0 ng/mL). ( E ) Protein levels of E-cadherin, N-cadherin, and vimentin are determined by western blot analyses. GAPDH expression is used as a loading control. Representative images from three independent experiments are shown. ( F ) Immunofluorescence staining images of EMT markers. E-cadherin (green), N-cadherin (red), and cell nuclei (blue, Hoechst staining) are shown. N-cadherin expression is only observed in TGF-β1 induced cells.

Article Snippet: The cells were incubated with a mixture of two primary antibodies: anti-E-cadherin rabbit monoclonal antibody (1:200 dilution, 24E10; Cell Signaling Technology) and anti-N-cadherin mouse monoclonal antibody (1:200 dilution, 610920; BD Biosciences) for 1 h at RT.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Isolation, Cell Culture, Comparison, Western Blot, Immunofluorescence, Staining

Journal: Scientific Reports

Article Title: Concomitant attenuation of HMGCR expression and activity enhances the growth inhibitory effect of atorvastatin on TGF-β-treated epithelial cancer cells

doi: 10.1038/s41598-021-91928-3

Figure Lengend Snippet: mRNA and protein expression levels of EMT-related molecules, regulator of cell proliferation, and target of statins in TGF-β (−) and TGF-β (+) groups. Real-time PCR analyses of expression of ( A ) E-cadherin, ( B ) N-cadherin, ( C ) vimentin, ( D ) c-Myc, and ( E ) HMGCR genes after the start of TGF-β1 incubation. Cells incubated with PBS are designated as TGF-β1 (−). Data are normalized to 18S rRNA levels in each sample and expressed as values relative to those of the internal control. The measurement values for each group are compared using the Bonferroni-Dunn post-hoc test. Mean ± SD, n = 3, * p < 0.05, ** p < 0.01, n.s. not significant. ( F ) Western blot analysis of the expression of E-cadherin, N-cadherin, vimentin, c-Myc, and HMGCR after induction with TGF-β1. GAPDH expression is used as the loading control. Representative images from three independent experiments are shown.

Article Snippet: The cells were incubated with a mixture of two primary antibodies: anti-E-cadherin rabbit monoclonal antibody (1:200 dilution, 24E10; Cell Signaling Technology) and anti-N-cadherin mouse monoclonal antibody (1:200 dilution, 610920; BD Biosciences) for 1 h at RT.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Incubation, Control, Western Blot

Journal: Journal for Immunotherapy of Cancer

Article Title: SPI1+CD68+ macrophages as a biomarker for gastric cancer metastasis: a rationale for combined antiangiogenic and immunotherapy strategies

doi: 10.1136/jitc-2024-009983

Figure Lengend Snippet: SPI1 was highly expressed in macrophages within the metastatic lesions of gastric cancer. ( A ) UMAP representation colored according to different cell types. ( B ) Density plot of SPI1 expression distribution in scRNA sequencing data. ( C ) UMAP indicated SPI1 staining of macrophages in primary and metastasis gastric cancer (GC). ( D ) Differential analysis of SPI1 expression in the primary and metastasis tumor of GC in scRNA sequencing data. ( E ) Infiltration of SPI1 + CD68 + TAMs in normal tissue, primary tumor, and metastasis sites of GC. ( F ) Differential analysis of SPI1 + CD68 + TAMs infiltration between primary and metastasis tumor according to double immunohistochemical staining. ( G ) SPI1 + CD68 + TAMs were screened by flow cytometry in primary and metastasis tumor of patients with GC. ( H ) Quantitative analysis of SPI1 + CD68 + TAMs infiltration based on flow cytometry. scRNA, single-cell RNA; SPI1, Spi-1 proto-oncogene; TAMs, tumor-associated macrophages; UMAP, Uniform Manifold Approximation and Projection.

Article Snippet: Subsequently, a mixture of anti-SPI1 antibody (mouse, Proteintech, 66618–2-Ig, diluted 1:250) and anti-CD68 antibody (rabbit, Proteintech, 25747–1-AP, diluted 1:500) was incubated on the slides overnight at 4°C.

Techniques: Expressing, Sequencing, Staining, Immunohistochemical staining, Flow Cytometry

Journal: Journal for Immunotherapy of Cancer

Article Title: SPI1+CD68+ macrophages as a biomarker for gastric cancer metastasis: a rationale for combined antiangiogenic and immunotherapy strategies

doi: 10.1136/jitc-2024-009983

Figure Lengend Snippet: SPI1 + CD68 + TAMs was an independent prognostic factor in patients with metastatic gastric cancer. ( A ) Overall survival (OS) and disease-free survival (DFS) of patients with gastric cancer (GC) in different SPI1 + CD68 + TAMs groups in the training cohort. ( B ) OS and DFS of patients with GC in different SPI1 + CD68 + TAMs groups in the external validation cohort. ( C ) OS and DFS of patients with GC in different SPI1 − CD68 + TAMs groups in the training cohort. ( D ) OS and DFS of patients with GC in different SPI1 − CD68 + TAMs groups in the external validation cohort. ( E ) Univariate cox regression analysis of patients with GC in the training cohort. ( F ) SPI1 + CD68 + TAMs was an independent prognostic factor of patients with GC in the training cohort. ( G ) Univariate cox regression analysis of patients with GC in the external validation cohort. ( H ) SPI1 + CD68 + TAMs was an independent prognostic factor of patients with GC in the external validation cohort. SPI1, Spi-1 proto-oncogene; TAMs, tumor-associated macrophages.

Article Snippet: Subsequently, a mixture of anti-SPI1 antibody (mouse, Proteintech, 66618–2-Ig, diluted 1:250) and anti-CD68 antibody (rabbit, Proteintech, 25747–1-AP, diluted 1:500) was incubated on the slides overnight at 4°C.

Techniques: Biomarker Discovery

Journal: Journal for Immunotherapy of Cancer

Article Title: SPI1+CD68+ macrophages as a biomarker for gastric cancer metastasis: a rationale for combined antiangiogenic and immunotherapy strategies

doi: 10.1136/jitc-2024-009983

Figure Lengend Snippet: SPI1 was associated with M2 polarization of macrophages. ( A ) Screening M1/M2 macrophages used flow cytometry with CD45, CD68, CD80, CD206 and SPI1 in patients with gastric cancer (GC). ( B ) Quantitative analysis of SPI1 + CD68 + TAMs infiltration in M1 and M2 type based on flow cytometry of patients with GC. ( C ) The markers of induced M1 and M2-type macrophages were detected by qRT-PCR. ( D ) Detecting the macrophage-related markers with qRT-PCR after SPI1 knockdown. ( E ) qRT-PCR was used to detect the macrophage-associated markers after SPI1 overexpression. ( F ) Western blot was used to detect the macrophage-related markers after SPI1 knockdown. ( G ) Macrophage-related markers were detected with western blot after SPI1 overexpression. ( H ) Flow cytometry was used to detect the macrophage-related markers after SPI1 knockdown. ( I ) Quantitative analysis of flow cytometry in SPI1 knockdown macrophages. ( J ) Flow cytometry was used to detect the macrophage-related markers after SPI1 overexpression. ( K ) Quantitative analysis of flow cytometry in SPI1 overexpression macrophages. IL-10, interleukin-10; mRNA, messenger RNA; qRT-PCR, quantitative Reverse Transcription Polymerase Chain Reaction; SPI1, Spi-1 proto-oncogene; TAMs, tumor-associated macrophages.

Article Snippet: Subsequently, a mixture of anti-SPI1 antibody (mouse, Proteintech, 66618–2-Ig, diluted 1:250) and anti-CD68 antibody (rabbit, Proteintech, 25747–1-AP, diluted 1:500) was incubated on the slides overnight at 4°C.

Techniques: Flow Cytometry, Quantitative RT-PCR, Knockdown, Over Expression, Western Blot, Reverse Transcription, Polymerase Chain Reaction

Journal: Journal for Immunotherapy of Cancer

Article Title: SPI1+CD68+ macrophages as a biomarker for gastric cancer metastasis: a rationale for combined antiangiogenic and immunotherapy strategies

doi: 10.1136/jitc-2024-009983

Figure Lengend Snippet: SPI1 + CD68 + TAMs promoted tumor angiogenesis through VEGF pathway. ( A ) GSVA analysis between SPI1-high and SPI1-low TAMs groups in scRNA sequencing data. ( B ) Incoming and outgoing interaction strength in different types of cells. ( C ) Cell–cell communication between different cell types in the VEGF signaling pathway. ( D ) Response to bevacizumab in patients with different TAMs infiltrates in the external validation cohort. ( E ) Immunofluorescence revealed the infiltration of SPI1 + CD68 + TAMs around the tumor blood vessels (CD31 marked). ( F ) Difference in the number of SPI1 + CD68 + TAMs and SPI1 − CD68 + TAMs around tumor vessels. ( G ) Difference in distance between blood vessels with SPI1 + CD68 + TAMs and SPI1 − CD68 + TAMs. DAPI, 4',6-Diamidino-2-Phenylindole; GSVA, gene set variation analysis; NK, Natural Killer cells; scRNA, single-cell RNA; SPI1, Spi-1 proto-oncogene; TAMs, tumor-associated macrophages; VEGFA, vascular endothelial growth factor A.

Article Snippet: Subsequently, a mixture of anti-SPI1 antibody (mouse, Proteintech, 66618–2-Ig, diluted 1:250) and anti-CD68 antibody (rabbit, Proteintech, 25747–1-AP, diluted 1:500) was incubated on the slides overnight at 4°C.

Techniques: Sequencing, Biomarker Discovery, Immunofluorescence

Journal: Journal for Immunotherapy of Cancer

Article Title: SPI1+CD68+ macrophages as a biomarker for gastric cancer metastasis: a rationale for combined antiangiogenic and immunotherapy strategies

doi: 10.1136/jitc-2024-009983

Figure Lengend Snippet: The role of SPI1 in GC cell growth and metastasis in vivo. ( A ) The representative images of the xenograft tumor. ( B ) Quantitative analysis of tumor growth curve. ( C ) Quantitative analysis of tumor weight in different SPI1 expression group. ( D ) Bioluminescence images of tumor-bearing mice individually treated with shNC, shSPI1, Vector, SPI1 transfected macrophages at day 5, 10, 15, 20, 25, 30, 40, and 50. ( E ) Representative photographs of peritoneum and mesentery metastasis lesions in different SPI1 expression groups. ( F ) The fluorescence intensity of tumors in various groups of mice. ( G ) Quantitative analysis of peritoneum nodules. ( H ) Survival curves of mice in different groups. GC, gastric cancer; SPI1, Spi-1 proto-oncogene.

Article Snippet: Subsequently, a mixture of anti-SPI1 antibody (mouse, Proteintech, 66618–2-Ig, diluted 1:250) and anti-CD68 antibody (rabbit, Proteintech, 25747–1-AP, diluted 1:500) was incubated on the slides overnight at 4°C.

Techniques: In Vivo, Expressing, Plasmid Preparation, Transfection, Fluorescence

Journal: Journal for Immunotherapy of Cancer

Article Title: SPI1+CD68+ macrophages as a biomarker for gastric cancer metastasis: a rationale for combined antiangiogenic and immunotherapy strategies

doi: 10.1136/jitc-2024-009983

Figure Lengend Snippet: SPI1 closely interacted with endothelial cells and regulated VEGFA transcription. ( A ) Wound healing assay was used to detect the migration ability of HUVEC cells cultured with conditioned medium of SPI1 knockdown macrophages. ( B ) Compared with the control group, the overexpression of SPI1 enhanced the migration ability of HUVEC cells. ( C ) Tube formation assay was used to detect the angiogenic ability of HUVEC cells after cultured with conditioned medium of SPI1 knockdown macrophages. ( D ) On overexpressing SPI1, the angiogenesis ability of HUVEC cells was augmented. ( E ) The mRNA level of VEGFA changed after knockdown or overexpression of SPI1. ( F ) The protein level of VEGFA changed after knockdown or overexpression of SPI1. ( G ) Motif sequence logo plot of SPI1 according to JASPAR database. ( H ) Putative SPI1 and VEGFA promoter binding sites. ( I ) ChIP analysis of the direct interaction between SPI1 and the promoter of VEGFA. ( J ) The binding site of SPI1 to the VEGFA promoter was assessed using ChIP-qPCR. ChIP, chromatin immunoprecipitation; HUVEC, Human Umbilical Vein Endothelial Cells; mRNA, messenger RNA; qPCR, quantitative PCR; SPI1, Spi-1 proto-oncogene; VEGFA, vascular endothelial growth factor A.

Article Snippet: Subsequently, a mixture of anti-SPI1 antibody (mouse, Proteintech, 66618–2-Ig, diluted 1:250) and anti-CD68 antibody (rabbit, Proteintech, 25747–1-AP, diluted 1:500) was incubated on the slides overnight at 4°C.

Techniques: Wound Healing Assay, Migration, Cell Culture, Knockdown, Control, Over Expression, Tube Formation Assay, Sequencing, Binding Assay, ChIP-qPCR, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

Journal: Journal for Immunotherapy of Cancer

Article Title: SPI1+CD68+ macrophages as a biomarker for gastric cancer metastasis: a rationale for combined antiangiogenic and immunotherapy strategies

doi: 10.1136/jitc-2024-009983

Figure Lengend Snippet: In vivo response to anti-PD-1 immunotherapy and antiangiogenesis treatment. ( A ) Response to immunotherapy in patients with different CPS scores and SPI1 + CD68 + TAMs infiltration. ( B ) Difference analysis of immunotherapy response in patients with different SPI1 + CD68 + TAMs infiltration. ( C ) Correlation analysis of SPI1 and PD-L1. ( D–G ) ROC curve of CPS score, CD68 + cells, SPI1 + CD68 + TAMs, and CPS score plus SPI1 + CD68 + TAMs. ( H ) Multiple immunofluorescences staining of PD-1 + CD8 + T cells and SPI1 + CD68 + TAMs. ( I ) Spatial distribution of SPI1 + CD68 + TAMs, SPI1 − CD68 + TAMs and PD-1 + CD8 + T cells analyzed by HALO. ( J ) Average distance from PD-1 + CD8 + T cells to SPI1 + CD68 + TAMs and SPI1 − CD68 + TAMs (p<0.05). ( K ) The discrepancy in the quantity of SPI1 + CD68 + TAMs and SPI1 − CD68 + TAMs surrounding PD-1 + CD8 + T cells (p<0.05). ( L ) Bioluminescence images of NOD/SCID mice with different treatment in week 1, 3, and 6. ( M ) The fluorescence intensity of intraperitoneal tumors in various groups of mice. CPS, Combined Positive Score; DAPI, 4',6-Diamidino-2-Phenylindole; PD-1, programmed cell death protein-1; PD-L1, programmed death-ligand 1; ROC, receiver operating characteristic; SPI1, Spi-1 proto-oncogene; TAMs, tumor-associated macrophages.

Article Snippet: Subsequently, a mixture of anti-SPI1 antibody (mouse, Proteintech, 66618–2-Ig, diluted 1:250) and anti-CD68 antibody (rabbit, Proteintech, 25747–1-AP, diluted 1:500) was incubated on the slides overnight at 4°C.

Techniques: In Vivo, Staining, Fluorescence

Journal: Frontiers in Microbiology

Article Title: Characterization of winged helix domain fusion endonucleases as N6-methyladenine-dependent type IV restriction systems

doi: 10.3389/fmicb.2024.1286822

Figure Lengend Snippet: (A) Divalent cation requirement for HhiV4I restriction activity. Divalent cations were supplemented in a medium salt buffer (50 mM NaCl, 10 mM Tris–HCl, pH 7.5) at 10, 1, and 0.1 mM final concentration, respectively. HhiV4I (1 μg) was used in the digestion of pBR322 (Dam + , 1 μg) at 37°C for 1 h. Mn 2+ supports HhiV4I activity as the preferred cofactor. In 10 mM Mg 2+ buffer, the enzyme showed partial nicking activity. In Co 2+ and Ni 2+ buffers, the enzyme showed weak but detectable activity. (B) HhiV4I incubation with 5mC−, 5hmC−, or dZ− (2-aminoadenine, 2,6-aminopurine) modified PCR DNAs. DpnI, MspJI, and HhiV4I digests were carried out in 1× NEB buffer 2.1, CutSmart buffer, and B2.1 plus 1 mM Mn 2+ , respectively. Modified and unmodified DNA substrates: (1) dC regular PCR-unmodified (2.9 kb), (2) dZ PCR (2-aminoadenine modified, 4 kb), (3) a mixture of 5mC (2 kb) and 5hmC (2.9 kb) PCR products, and two minor PCR products (0.4–0.5 kb), (4) HindIII (H3)-prelinearized pUC19 (Dam + Dcm + , 2.7 kb), (5) HindIII (H3)-prelinearized pUC19 (Dam − Dcm − , 2.7 kb). DpnI and HhiV4I digested linear Dam + pUC19 DNA. MspJI digested 5mC/5hmC modified PCR DNA and Dcm + linear pUC19 (MspJI site 5(h)mCNNR, Dcm-methylated sites C 5mC WG G ).

Article Snippet: E. coli T7 expression strains C2566 (Dam + ) and its isogenic Dam-deficient strain ER2948 [constructed and provided by Dr. Lise Raleigh, New England Biolabs (NEB)], expression vector pTXB1, pBR322, phage λ DNA (Dam + or Dam − ), 2-log (1 kb plus) DNA ladder, chitin beads, restriction enzymes, EcoGII methylase (frequent adenine methylase), Q5 DNA polymerase PCR master mix and cloning kit (Hi-Fi DNA assembly enzyme mix), NEBExpress Ni-NTA magnetic beads, and dZTP (2-aminoadenine triphosphate is abbreviated as base Z in the literature, here we use dZ to denote the 2’-deoxynucleoside) were provided by Michael Kuska (NEB Organic Synthesis Division).

Techniques: Activity Assay, Concentration Assay, Incubation, Modification, Methylation