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Sino Biological rrm2 flag
Rrm2 Flag, supplied by Sino Biological, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rrm2+flag/10__1158_slash_0008___5472__can___25___3237-52-0-15?v=Sino+Biological
Average 94 stars, based on 1 article reviews

rrm2 flag - by Bioz Stars, 2026-07

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Journal: Journal of Virology

Article Title: Critical role for ribonucleoside-diphosphate reductase subunit M2 in ALV-J-induced activation of Wnt/β-catenin signaling via interaction with P27

doi: 10.1128/jvi.00267-23

Figure Lengend Snippet: Primer pairs used in this study

Article Snippet: Gallus RRM2 was cloned from DF-1 cells using the specific primers ( ). pRK5-flag- rrm2 , pCMV-myc- rrm2 , pEGFP- rrm2, and pEGFP- p27 were constructed by standard molecular biology techniques. pRK5-flag- p15 , pRK5-flag- p19 , pRK5-flag- p27 , pRK5-flag- p32 , pRK5-flag- p68 , pRK5-flag-g p37 , pRK5-flag-g p85 ,and pEGFP- p27 were kept in our laboratory.

Techniques: Sequencing

Infection of DF-1 cells with ALV-J enhances RRM2 expression. (A) DF-1 cells were infected with ALV-J SCAU1903 or DMEM as a control for 48or 72 hours, cell lysates were harvested and subjected to Western Blot analysis using anti-RRM2 antibodies. Endogenous GAPDH expression was examined as an internal control. (B) Relative levels of RRM2 in ALV-J-infected cells. The density of bands in A was quantitated by densitometry. The relative levels of RRM2 were calculated as follows: band density of RRM2/band density of GAPDH. The results of all experiments are reported as mean ± SD from three independent experiments. **P < 0.01.

Journal: Journal of Virology

Article Title: Critical role for ribonucleoside-diphosphate reductase subunit M2 in ALV-J-induced activation of Wnt/β-catenin signaling via interaction with P27

doi: 10.1128/jvi.00267-23

Figure Lengend Snippet: Infection of DF-1 cells with ALV-J enhances RRM2 expression. (A) DF-1 cells were infected with ALV-J SCAU1903 or DMEM as a control for 48or 72 hours, cell lysates were harvested and subjected to Western Blot analysis using anti-RRM2 antibodies. Endogenous GAPDH expression was examined as an internal control. (B) Relative levels of RRM2 in ALV-J-infected cells. The density of bands in A was quantitated by densitometry. The relative levels of RRM2 were calculated as follows: band density of RRM2/band density of GAPDH. The results of all experiments are reported as mean ± SD from three independent experiments. **P < 0.01.

Techniques: Infection, Expressing, Control, Western Blot

RRM2 facilitates ALV-J replication in DF-1 cells. DF-1 cells receiving RRM2-specific siRNA or control siRNA (A and B), or transfected with pCMV-myc-rrm2 or pCMV-myc as a control (C and D), or treated with 3-AP or DMSO as a control (E and F) were infected with ALV-J. Cell cultures were collected at different time points after infection and used to titrate viral titers using TCID50 assay, and cell lysates were examined by Western Blot analysis with indicated antibodies. (A) Effects of RRM2 RNAi on the expression of endogenous RRM2. (B) Effects of knockdown of RRM2 on ALV-J growth in DF-1 cells. (C) Determination of overexpression of RRM2 in DF-1 cells by Western Blot analysis. (D) Effects of overexpression of RRM2 on ALV-J growth in DF-1 cells. (E) Effects of 3-AP on the expression of viral protein gp85 in DF-1 cells. (F) Effects of 3-AP on ALV-J growth in DF-1 cells. Results are representative of three independent experiments. Error bars indicate mean ± SD from three independent experiments. **P < 0.01.

Journal: Journal of Virology

Article Title: Critical role for ribonucleoside-diphosphate reductase subunit M2 in ALV-J-induced activation of Wnt/β-catenin signaling via interaction with P27

doi: 10.1128/jvi.00267-23

Figure Lengend Snippet: RRM2 facilitates ALV-J replication in DF-1 cells. DF-1 cells receiving RRM2-specific siRNA or control siRNA (A and B), or transfected with pCMV-myc-rrm2 or pCMV-myc as a control (C and D), or treated with 3-AP or DMSO as a control (E and F) were infected with ALV-J. Cell cultures were collected at different time points after infection and used to titrate viral titers using TCID50 assay, and cell lysates were examined by Western Blot analysis with indicated antibodies. (A) Effects of RRM2 RNAi on the expression of endogenous RRM2. (B) Effects of knockdown of RRM2 on ALV-J growth in DF-1 cells. (C) Determination of overexpression of RRM2 in DF-1 cells by Western Blot analysis. (D) Effects of overexpression of RRM2 on ALV-J growth in DF-1 cells. (E) Effects of 3-AP on the expression of viral protein gp85 in DF-1 cells. (F) Effects of 3-AP on ALV-J growth in DF-1 cells. Results are representative of three independent experiments. Error bars indicate mean ± SD from three independent experiments. **P < 0.01.

Techniques: Control, Transfection, Infection, TCID50 Assay, Western Blot, Expressing, Knockdown, Over Expression

RRM2 interacts with viral capsid protein P27. (A) Identification of viral proteins interacting with RRM2. DF-1 cells were transfected with the indicated expression plasmids. Twenty-four hours after transfection, cell lysates were prepared and immunoprecipitated (IP) with anti-FLAG antibody and immunoblotted with anti-GFP or anti-FLAG antibodies. (B) Interaction of exogenous RRM2 with ALV-J P27. DF-1 cells were transfected with indicated expression plasmids. Twenty-four hours after transfection, cell lysates were immunoprecipitated with anti-Flag antibody and immunoblotted with anti-Myc or anti-Flag antibodies. (C) Interaction of endogenous RRM2 with ALV-J P27. DF-1 cells were transfected with pRK5-flag-p27 or pRK5-flag as a control. Twenty-four hours after transfection, cell lysates were immunoprecipitated with anti-FLAG antibodies and immunoblotted with anti-FLAG or anti-RRM2 antibodies. (D) Interaction of endogenous RRM2 with ALV-J P27 under physiological conditions. DF-1 cells were infected with ALV-J. Forty-eight hours after infection, cell lysates were immunoprecipitated with anti-RRM2 antibody and immunoblotted with anti-RRM2 or anti-P27 antibodies.

Journal: Journal of Virology

Article Title: Critical role for ribonucleoside-diphosphate reductase subunit M2 in ALV-J-induced activation of Wnt/β-catenin signaling via interaction with P27

doi: 10.1128/jvi.00267-23

Figure Lengend Snippet: RRM2 interacts with viral capsid protein P27. (A) Identification of viral proteins interacting with RRM2. DF-1 cells were transfected with the indicated expression plasmids. Twenty-four hours after transfection, cell lysates were prepared and immunoprecipitated (IP) with anti-FLAG antibody and immunoblotted with anti-GFP or anti-FLAG antibodies. (B) Interaction of exogenous RRM2 with ALV-J P27. DF-1 cells were transfected with indicated expression plasmids. Twenty-four hours after transfection, cell lysates were immunoprecipitated with anti-Flag antibody and immunoblotted with anti-Myc or anti-Flag antibodies. (C) Interaction of endogenous RRM2 with ALV-J P27. DF-1 cells were transfected with pRK5-flag-p27 or pRK5-flag as a control. Twenty-four hours after transfection, cell lysates were immunoprecipitated with anti-FLAG antibodies and immunoblotted with anti-FLAG or anti-RRM2 antibodies. (D) Interaction of endogenous RRM2 with ALV-J P27 under physiological conditions. DF-1 cells were infected with ALV-J. Forty-eight hours after infection, cell lysates were immunoprecipitated with anti-RRM2 antibody and immunoblotted with anti-RRM2 or anti-P27 antibodies.

Techniques: Transfection, Expressing, Immunoprecipitation, Control, Infection

Colocalization of RRM2 with ALV-J P27. (A to C) Colocalization of exogenous RRM2 and ALV-J P27. HEK293T cells were cultured on confocal dish (Coverglass Bottom Dish) and transfected with pEGFP-p27 and pCMV-myc-rrm2. Twenty-four hours after transfection, cells were fixed with 4% paraformaldehyde and probed with mouse anti-Myc antibodies, followed by coraLite594-conjugated goat anti-mouse antibody. Cell nuclei were counterstained with DAPI (blue). The cell samples were observed with a laser confocal scanning microscope. (D to F) Colocalization of endogenous RRM2 and ALV-J P27. DF-1 cells were transfected with pEGFP-p27. Twenty-four hours after transfection, cells were fixed with 4% paraformaldehyde and probed with mouse anti-RRM2 antibodies, followed by coraLite594-conjugated goat anti-rabbit antibody. Cell nuclei were counterstained with DAPI (blue). The cell samples were observed with a laser confocal scanning microscope. (G to L) Colocalization of endogenous RRM2 and P27 during ALV-J infection. DF-1 cells were cultured on confocal dish (Coverglass Bottom Dish) and infected with ALV-J (J to L) or DMEM as s control (G to I). At 48 hours post infection, cells were fixed with 4% paraformaldehyde and probed with anti-P27 or anti-RRM2 antibodies, followed by coraLite488-conjugated goat anti-mouse antibody (green) and coraLite594-conjugated goat anti-rabbit antibody (red). Cell nuclei were counterstained with DAPI (blue). The cell samples were observed with a laser confocal scanning microscope.

Journal: Journal of Virology

Article Title: Critical role for ribonucleoside-diphosphate reductase subunit M2 in ALV-J-induced activation of Wnt/β-catenin signaling via interaction with P27

doi: 10.1128/jvi.00267-23

Figure Lengend Snippet: Colocalization of RRM2 with ALV-J P27. (A to C) Colocalization of exogenous RRM2 and ALV-J P27. HEK293T cells were cultured on confocal dish (Coverglass Bottom Dish) and transfected with pEGFP-p27 and pCMV-myc-rrm2. Twenty-four hours after transfection, cells were fixed with 4% paraformaldehyde and probed with mouse anti-Myc antibodies, followed by coraLite594-conjugated goat anti-mouse antibody. Cell nuclei were counterstained with DAPI (blue). The cell samples were observed with a laser confocal scanning microscope. (D to F) Colocalization of endogenous RRM2 and ALV-J P27. DF-1 cells were transfected with pEGFP-p27. Twenty-four hours after transfection, cells were fixed with 4% paraformaldehyde and probed with mouse anti-RRM2 antibodies, followed by coraLite594-conjugated goat anti-rabbit antibody. Cell nuclei were counterstained with DAPI (blue). The cell samples were observed with a laser confocal scanning microscope. (G to L) Colocalization of endogenous RRM2 and P27 during ALV-J infection. DF-1 cells were cultured on confocal dish (Coverglass Bottom Dish) and infected with ALV-J (J to L) or DMEM as s control (G to I). At 48 hours post infection, cells were fixed with 4% paraformaldehyde and probed with anti-P27 or anti-RRM2 antibodies, followed by coraLite488-conjugated goat anti-mouse antibody (green) and coraLite594-conjugated goat anti-rabbit antibody (red). Cell nuclei were counterstained with DAPI (blue). The cell samples were observed with a laser confocal scanning microscope.

Techniques: Cell Culture, Transfection, Microscopy, Infection, Control

Inhibition of Wnt/β-catenin signaling suppresses ALV-J replication. (A and B) β-catenin involves ALV-J growth. CEF cells were transfected with β-catenin-specific siRNA or control siRNA, followed by infection with ALV-J or DMEM as a control. Forty-eight hours post infection, cell lysates were examined by Western Blot analysis with indicated antibodies, and cell cultures were collected at different time points after infection and used to titrate viral titers using TCID50 assay. (C) SKL2001 (agonist of Wnt/β-catenin pathway) enhances the expression of RRM2 and β-catenin in host cells. CEF cells were treated with SKL2001 or DMSO as a control, followed by infection with ALV-J. Forty-eight hours post infection, cell lysates were examined by Western Blot analysis with indicated antibodies. (D to F) Relative levels of β-catenin, Env, and RRM2 in SKL2001-treated cells. The density of bands in C was quantitated by densitometry. The relative levels of target proteins were calculated as follows: band density of RRM2 (β-catenin or Env)/band density of GAPDH. (G) PRI-724 (inhibitor of Wnt/β-catenin pathway) suppresses the expression of RRM2 and β-catenin. CEF cells were treated with PRI-724 or DMSO as a control, followed by infection with ALV-J. Forty-eight hours post infection, cell lysates were examined by Western Blot analysis with indicated antibodies. (H to J) Relative levels of β-catenin, Env, and RRM2 in PRI-724-treated cells. The density of bands in G was quantitated by densitometry. The relative levels of target proteins were calculated as follows: band density of RRM2 (β-catenin or Env)/band density of GAPDH. (K and L) The status of Wnt/β-catenin signaling involves ALV-J growth. CEF cells were treated with SKL2001 or PRI-724 or DMSO as a control, followed by infection with ALV-J. Cell cultures were collected at different time points after infection and used to titrate viral titers using TCID50 assay. Error bars indicate mean ± SD from three independent experiments. **P < 0.01.

Journal: Journal of Virology

Article Title: Critical role for ribonucleoside-diphosphate reductase subunit M2 in ALV-J-induced activation of Wnt/β-catenin signaling via interaction with P27

doi: 10.1128/jvi.00267-23

Figure Lengend Snippet: Inhibition of Wnt/β-catenin signaling suppresses ALV-J replication. (A and B) β-catenin involves ALV-J growth. CEF cells were transfected with β-catenin-specific siRNA or control siRNA, followed by infection with ALV-J or DMEM as a control. Forty-eight hours post infection, cell lysates were examined by Western Blot analysis with indicated antibodies, and cell cultures were collected at different time points after infection and used to titrate viral titers using TCID50 assay. (C) SKL2001 (agonist of Wnt/β-catenin pathway) enhances the expression of RRM2 and β-catenin in host cells. CEF cells were treated with SKL2001 or DMSO as a control, followed by infection with ALV-J. Forty-eight hours post infection, cell lysates were examined by Western Blot analysis with indicated antibodies. (D to F) Relative levels of β-catenin, Env, and RRM2 in SKL2001-treated cells. The density of bands in C was quantitated by densitometry. The relative levels of target proteins were calculated as follows: band density of RRM2 (β-catenin or Env)/band density of GAPDH. (G) PRI-724 (inhibitor of Wnt/β-catenin pathway) suppresses the expression of RRM2 and β-catenin. CEF cells were treated with PRI-724 or DMSO as a control, followed by infection with ALV-J. Forty-eight hours post infection, cell lysates were examined by Western Blot analysis with indicated antibodies. (H to J) Relative levels of β-catenin, Env, and RRM2 in PRI-724-treated cells. The density of bands in G was quantitated by densitometry. The relative levels of target proteins were calculated as follows: band density of RRM2 (β-catenin or Env)/band density of GAPDH. (K and L) The status of Wnt/β-catenin signaling involves ALV-J growth. CEF cells were treated with SKL2001 or PRI-724 or DMSO as a control, followed by infection with ALV-J. Cell cultures were collected at different time points after infection and used to titrate viral titers using TCID50 assay. Error bars indicate mean ± SD from three independent experiments. **P < 0.01.

Techniques: Inhibition, Transfection, Control, Infection, Western Blot, TCID50 Assay, Expressing

RRM2 enhances the expression and translocation of β-catenin in birds. (A) Overexpression of RRM2 enhances the expression of β-catenin and promotes the translocation of β-catenin into nuclear in DF-1 cells. DF-1 cells were transfected with pEGFP-rrm2 or pEGFP-N1 as a control. Twenty-four hours after transfection, cells were harvested with trypsin-EDTA for cytoplasmic and nuclear protein extraction to detect the expression of β-catenin by Western Blot. (B) Relative levels of β-catenin in RRM2-expressing cells. The density of bands in A was quantitated by densitometry. (C) The knockdown of RRM2 inhibits the expression of β-catenin and blocks the translocation of β-catenin into nuclear in DF-1 cells. DF-1 cells were transfected with RRM2-specific siRNA or control siRNA. Twenty-four hours after transfection, cells were harvested with trypsin-EDTA for cytoplasmic and nuclear protein extraction to detect the expression of β-catenin by Western Blot. (D and E) Relative levels of RRM2 and β-catenin in lower RRM2-expressing DF-1 cells. (F) Overexpression of RRM2 enhances the expression of β-catenin and promotes the translocation of β-catenin into nuclear in CEF cells. CEF cells were transfected with pEGFP-rrm2 or pEGFP-N1 as a control. Twenty-four hours after transfection, cells were harvested with trypsin-EDTA for cytoplasmic and nuclear protein extraction to detect the expression of β-catenin by Western Blot. (G) Relative levels of β-catenin in RRM2-expressing CEF cells. The density of bands in F was quantitated by densitometry. (H) The knockdown of RRM2 inhibits the expression of β-catenin and blocks the translocation of β-catenin into nuclear in CEF cells. CEF cells were transfected with RRM2-specific siRNA or control siRNA. Twenty-four hours after transfection, cells were harvested with trypsin-EDTA for cytoplasmic and nuclear protein extraction to detect the expression of β-catenin by Western Blot. (I and J) Relative levels of RRM2 and β-catenin in lower RRM2-expressing CEF cells. The density of bands was quantitated by densitometry. The relative levels of target proteins in cytoplasm were calculated as follows: band density of target proteins/band density of GAPDH. The relative levels of target proteins in nucleus were calculated as follows: band density of target protein/band density of PCNA. Error bars indicate mean ± SD from three independent experiments. **P < 0.01.

Journal: Journal of Virology

Article Title: Critical role for ribonucleoside-diphosphate reductase subunit M2 in ALV-J-induced activation of Wnt/β-catenin signaling via interaction with P27

doi: 10.1128/jvi.00267-23

Figure Lengend Snippet: RRM2 enhances the expression and translocation of β-catenin in birds. (A) Overexpression of RRM2 enhances the expression of β-catenin and promotes the translocation of β-catenin into nuclear in DF-1 cells. DF-1 cells were transfected with pEGFP-rrm2 or pEGFP-N1 as a control. Twenty-four hours after transfection, cells were harvested with trypsin-EDTA for cytoplasmic and nuclear protein extraction to detect the expression of β-catenin by Western Blot. (B) Relative levels of β-catenin in RRM2-expressing cells. The density of bands in A was quantitated by densitometry. (C) The knockdown of RRM2 inhibits the expression of β-catenin and blocks the translocation of β-catenin into nuclear in DF-1 cells. DF-1 cells were transfected with RRM2-specific siRNA or control siRNA. Twenty-four hours after transfection, cells were harvested with trypsin-EDTA for cytoplasmic and nuclear protein extraction to detect the expression of β-catenin by Western Blot. (D and E) Relative levels of RRM2 and β-catenin in lower RRM2-expressing DF-1 cells. (F) Overexpression of RRM2 enhances the expression of β-catenin and promotes the translocation of β-catenin into nuclear in CEF cells. CEF cells were transfected with pEGFP-rrm2 or pEGFP-N1 as a control. Twenty-four hours after transfection, cells were harvested with trypsin-EDTA for cytoplasmic and nuclear protein extraction to detect the expression of β-catenin by Western Blot. (G) Relative levels of β-catenin in RRM2-expressing CEF cells. The density of bands in F was quantitated by densitometry. (H) The knockdown of RRM2 inhibits the expression of β-catenin and blocks the translocation of β-catenin into nuclear in CEF cells. CEF cells were transfected with RRM2-specific siRNA or control siRNA. Twenty-four hours after transfection, cells were harvested with trypsin-EDTA for cytoplasmic and nuclear protein extraction to detect the expression of β-catenin by Western Blot. (I and J) Relative levels of RRM2 and β-catenin in lower RRM2-expressing CEF cells. The density of bands was quantitated by densitometry. The relative levels of target proteins in cytoplasm were calculated as follows: band density of target proteins/band density of GAPDH. The relative levels of target proteins in nucleus were calculated as follows: band density of target protein/band density of PCNA. Error bars indicate mean ± SD from three independent experiments. **P < 0.01.

Techniques: Expressing, Translocation Assay, Over Expression, Transfection, Control, Protein Extraction, Western Blot, Knockdown

ALV-J P27 regulates Wnt/β-catenin signaling by binding to RRM2. (A to H) The knockdown of the endogenous RRM2 blocks P27-induced Wnt/β-catenin signaling. CEF cells were transfected with RRM2-specific siRNA or control siRNA, followed by transfection with pRK5-flag-p27. Twenty-four hours after transfection, cells were harvested to detect the expression of β-catenin (A) and the transcriptional level of lef1, tcf4, axin2, c-myc, and cyclin D1 (D to H). The density of bands in A was quantitated by densitometry (B and C). (I to O) Inhibition of RRM2 by 3-AP blocks P27-induced Wnt/β-catenin signaling. CEF cells were treated with 3-AP or DMSO as controls, followed by transfection with pRK5-flag-p27. Twenty-four hours after transfection, cells were harvested to detect the expression of β-catenin (I) and the transcriptional level of lef1, tcf4, axin2, c-myc, and cyclin D1 (K to O). The density of bands in I was quantitated by densitometry (J). The relative levels of target proteins were calculated as follows: band density of RRM2 (or β-catenin)/band density of GAPDH. Error bars indicate mean ± SD from three independent experiments. **P < 0.01.

Journal: Journal of Virology

Article Title: Critical role for ribonucleoside-diphosphate reductase subunit M2 in ALV-J-induced activation of Wnt/β-catenin signaling via interaction with P27

doi: 10.1128/jvi.00267-23

Figure Lengend Snippet: ALV-J P27 regulates Wnt/β-catenin signaling by binding to RRM2. (A to H) The knockdown of the endogenous RRM2 blocks P27-induced Wnt/β-catenin signaling. CEF cells were transfected with RRM2-specific siRNA or control siRNA, followed by transfection with pRK5-flag-p27. Twenty-four hours after transfection, cells were harvested to detect the expression of β-catenin (A) and the transcriptional level of lef1, tcf4, axin2, c-myc, and cyclin D1 (D to H). The density of bands in A was quantitated by densitometry (B and C). (I to O) Inhibition of RRM2 by 3-AP blocks P27-induced Wnt/β-catenin signaling. CEF cells were treated with 3-AP or DMSO as controls, followed by transfection with pRK5-flag-p27. Twenty-four hours after transfection, cells were harvested to detect the expression of β-catenin (I) and the transcriptional level of lef1, tcf4, axin2, c-myc, and cyclin D1 (K to O). The density of bands in I was quantitated by densitometry (J). The relative levels of target proteins were calculated as follows: band density of RRM2 (or β-catenin)/band density of GAPDH. Error bars indicate mean ± SD from three independent experiments. **P < 0.01.

Techniques: Binding Assay, Knockdown, Transfection, Control, Expressing, Inhibition

RRM2 affects ALV-J replication by regulating Wnt/β-catenin pathway. (A to H) Overexpression of RRM2 enhance ALV-J growth by activating Wnt/β-catenin signaling. CEF cells were transfected with pEGFP-rrm2 or pEGFP-N1 as a control, followed by infection with ALV-J. Cells were harvested to detect the expression of viral protein and β-catenin using Western Blot (A to C), and the transcriptional level of lef1, tcf4, axin2, c-myc, and Cyclin D1 using qRT-PCR (D to H). The density of bands in A was quantitated by densitometry (B and C). (I to Q) Knockdown of RRM2 inhibits ALV-J growth by suppressing Wnt/β-catenin signaling. CEF cells receiving RRM2-specific siRNA or control siRNA were infected ALV-J or DMEM as a control. Cells were harvested to detect the expression of viral protein and the expression of β-catenin in cytoplasm and nucleus using Western Blot (I to L) and the transcriptional level of lef1, tcf4, axin2, c-myc, and Cyclin D1 using qRT-PCR (M to Q). The density of bands in I was quantitated by densitometry (J to L). (R to Y) Inhibition of RRM2 by 3-AP suppresses ALV-J growth by inhibiting Wnt/β-catenin signaling. CEF cells treated with 3-AP or DMSO as a control were infected ALV-J or DMEM as a control. Cells were harvested to detect the expression of viral protein and the expression of β-catenin in cytoplasm and nucleus using Western Blot (R to T) and the transcriptional level of lef1, tcf4, axin2, c-myc, and Cyclin D1 using qRT-PCR (U to Y). The density of bands in H was quantitated by densitometry (S and T). The relative levels of target proteins in cytoplasm were calculated as follows: band density of target proteins/band density of GAPDH. The relative levels of target proteins in nucleus were calculated as follows: band density of target protein/band density of PCNA. Error bars indicate mean ± SD from three independent experiments. *P < 0.05, **P < 0.01.

Journal: Journal of Virology

Article Title: Critical role for ribonucleoside-diphosphate reductase subunit M2 in ALV-J-induced activation of Wnt/β-catenin signaling via interaction with P27

doi: 10.1128/jvi.00267-23

Figure Lengend Snippet: RRM2 affects ALV-J replication by regulating Wnt/β-catenin pathway. (A to H) Overexpression of RRM2 enhance ALV-J growth by activating Wnt/β-catenin signaling. CEF cells were transfected with pEGFP-rrm2 or pEGFP-N1 as a control, followed by infection with ALV-J. Cells were harvested to detect the expression of viral protein and β-catenin using Western Blot (A to C), and the transcriptional level of lef1, tcf4, axin2, c-myc, and Cyclin D1 using qRT-PCR (D to H). The density of bands in A was quantitated by densitometry (B and C). (I to Q) Knockdown of RRM2 inhibits ALV-J growth by suppressing Wnt/β-catenin signaling. CEF cells receiving RRM2-specific siRNA or control siRNA were infected ALV-J or DMEM as a control. Cells were harvested to detect the expression of viral protein and the expression of β-catenin in cytoplasm and nucleus using Western Blot (I to L) and the transcriptional level of lef1, tcf4, axin2, c-myc, and Cyclin D1 using qRT-PCR (M to Q). The density of bands in I was quantitated by densitometry (J to L). (R to Y) Inhibition of RRM2 by 3-AP suppresses ALV-J growth by inhibiting Wnt/β-catenin signaling. CEF cells treated with 3-AP or DMSO as a control were infected ALV-J or DMEM as a control. Cells were harvested to detect the expression of viral protein and the expression of β-catenin in cytoplasm and nucleus using Western Blot (R to T) and the transcriptional level of lef1, tcf4, axin2, c-myc, and Cyclin D1 using qRT-PCR (U to Y). The density of bands in H was quantitated by densitometry (S and T). The relative levels of target proteins in cytoplasm were calculated as follows: band density of target proteins/band density of GAPDH. The relative levels of target proteins in nucleus were calculated as follows: band density of target protein/band density of PCNA. Error bars indicate mean ± SD from three independent experiments. *P < 0.05, **P < 0.01.

Techniques: Over Expression, Transfection, Control, Infection, Expressing, Western Blot, Quantitative RT-PCR, Knockdown, Inhibition

Phosphorylation of RRM2 at sites Ser18 is pivotal for activating Wnt/β-catenin signaling and enhancing ALV-J replication. (A) ALV-J enhances the phosphorylation of RRM2 in host cells. CEF cells were infected with ALV-J or DMEM as a control. At 48 hpi, cell lysates were immunoprecipitated with anti-RRM2 antibody, and immunoblotted with anti-phosphoserine or anti-RRM2 antibodies. (B) Identification of phosphorylated site of RRM2. DF-1 cells were transfected with pEGFP-rrm2 for 24 hours, cell lysates were immunoprecipitated with anti-GFP antibody, and subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. (C) Phosphorylation of RRM2 at sites Ser18 involves the expression and translocation of β-catenin in host cells. CEF cells were transfected with pEGFP-rrm2 and pEGFP-rrm2mut or pEGFP-N1 as a control. Twenty-four hours after transfection, the expression and distribution of β-catenin was detected using Western Blot. (D) Relative levels of β-catenin in RRM2-expressing CEF cells. The density of bands in C was quantitated by densitometry. (E to G) Phosphorylation of RRM2 at sites Ser18 involves ALV-J growth. CEF cells were transfected with pEGFP-rrm2 and pEGFP-rrm2mut or pEGFP-N1 as a control, followed by infection with ALV-J or DMEM as a control. The expression of viral protein Env and RRM2 were evaluated by Western Blot (E), and viral titers were titrated using TCID50 assay (G). The density of bands in E was quantitated by densitometry (F). (H to K) Phosphorylation of RRM2 at sites Ser18 is critical for ALV-J growth. CEF cells receiving RRM2-specific siRNA or control siRNA were transfected with pEGFP-rrm2 and pEGFP-rrm2mut or pEGFP-N1 as a control, followed by infection with ALV-J or DMEM as a control. Cell lysates and cultures were harvested to examine the expression of viral protein (H) and viral titers (K), respectively. The density of bands in E was quantitated by densitometry (I and J), the relative levels of RRM2 and Env were calculated as follows: band density of RRM2 (or Env)/band density of GAPDH. Error bars indicate mean ± SD from three independent experiments. **P < 0.01.

Journal: Journal of Virology

Article Title: Critical role for ribonucleoside-diphosphate reductase subunit M2 in ALV-J-induced activation of Wnt/β-catenin signaling via interaction with P27

doi: 10.1128/jvi.00267-23

Figure Lengend Snippet: Phosphorylation of RRM2 at sites Ser18 is pivotal for activating Wnt/β-catenin signaling and enhancing ALV-J replication. (A) ALV-J enhances the phosphorylation of RRM2 in host cells. CEF cells were infected with ALV-J or DMEM as a control. At 48 hpi, cell lysates were immunoprecipitated with anti-RRM2 antibody, and immunoblotted with anti-phosphoserine or anti-RRM2 antibodies. (B) Identification of phosphorylated site of RRM2. DF-1 cells were transfected with pEGFP-rrm2 for 24 hours, cell lysates were immunoprecipitated with anti-GFP antibody, and subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. (C) Phosphorylation of RRM2 at sites Ser18 involves the expression and translocation of β-catenin in host cells. CEF cells were transfected with pEGFP-rrm2 and pEGFP-rrm2mut or pEGFP-N1 as a control. Twenty-four hours after transfection, the expression and distribution of β-catenin was detected using Western Blot. (D) Relative levels of β-catenin in RRM2-expressing CEF cells. The density of bands in C was quantitated by densitometry. (E to G) Phosphorylation of RRM2 at sites Ser18 involves ALV-J growth. CEF cells were transfected with pEGFP-rrm2 and pEGFP-rrm2mut or pEGFP-N1 as a control, followed by infection with ALV-J or DMEM as a control. The expression of viral protein Env and RRM2 were evaluated by Western Blot (E), and viral titers were titrated using TCID50 assay (G). The density of bands in E was quantitated by densitometry (F). (H to K) Phosphorylation of RRM2 at sites Ser18 is critical for ALV-J growth. CEF cells receiving RRM2-specific siRNA or control siRNA were transfected with pEGFP-rrm2 and pEGFP-rrm2mut or pEGFP-N1 as a control, followed by infection with ALV-J or DMEM as a control. Cell lysates and cultures were harvested to examine the expression of viral protein (H) and viral titers (K), respectively. The density of bands in E was quantitated by densitometry (I and J), the relative levels of RRM2 and Env were calculated as follows: band density of RRM2 (or Env)/band density of GAPDH. Error bars indicate mean ± SD from three independent experiments. **P < 0.01.

Techniques: Phospho-proteomics, Infection, Control, Immunoprecipitation, Transfection, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Expressing, Translocation Assay, Western Blot, TCID50 Assay

Graphic model for the critical role of RRM2 during ALV-J infection. RRM2 infection activates Wnt/β-catenin signaling by enhancing its phosphorylation at Ser18, facilitating ALV-J replication via interaction with viral capsid protein P27.

Journal: Journal of Virology

Article Title: Critical role for ribonucleoside-diphosphate reductase subunit M2 in ALV-J-induced activation of Wnt/β-catenin signaling via interaction with P27

doi: 10.1128/jvi.00267-23

Figure Lengend Snippet: Graphic model for the critical role of RRM2 during ALV-J infection. RRM2 infection activates Wnt/β-catenin signaling by enhancing its phosphorylation at Ser18, facilitating ALV-J replication via interaction with viral capsid protein P27.

Techniques: Infection, Phospho-proteomics

The functional assays of biomarker knockdown in A549 lung cancer cells using shRNAs against ADAM9, MTHFD2 , SLC2A1, and RRM2 individually or simultaneously (4G KD). (A) Western blots of control (shVOID), individual gene knockdown (shADAM9, shMTHFD2, shSLC2A1, and shRRM2), and 4G KD in A549 cells. (B) Colony formation of individual single gene knockdown and 4G KD A549 cells. Colonies were detected with crystal violet staining of the cell culture after 8 days (left) and quantified (right). (C) Cell migration ability of control, individual single gene knockdown, and 4G KD A549 cells. Cell migration was detected by time-lapse video microscopy (left) and quantified (right). (D) Western blots of control, individual gene knockdown, and 4G KD KD CL1-0 lung cancer cells. (E) Colony formation assay of control, individual gene knockdown, and 4G KD CL1-0 cells on day 11. (F) Cell migration ability of control, individual gene knockdown, and 4G KD CL1-0 cells. GAPDH served as the internal control. Error bars represent the mean ± SD of triplicate experiments. Statistical differences were analyzed using the unpaired t test ( * P < 0.05, ** P < 0.01).

Journal: Theranostics

Article Title: Identification of theranostic factors for patients developing metastasis after surgery for early-stage lung adenocarcinoma

doi: 10.7150/thno.53176

Figure Lengend Snippet: The functional assays of biomarker knockdown in A549 lung cancer cells using shRNAs against ADAM9, MTHFD2 , SLC2A1, and RRM2 individually or simultaneously (4G KD). (A) Western blots of control (shVOID), individual gene knockdown (shADAM9, shMTHFD2, shSLC2A1, and shRRM2), and 4G KD in A549 cells. (B) Colony formation of individual single gene knockdown and 4G KD A549 cells. Colonies were detected with crystal violet staining of the cell culture after 8 days (left) and quantified (right). (C) Cell migration ability of control, individual single gene knockdown, and 4G KD A549 cells. Cell migration was detected by time-lapse video microscopy (left) and quantified (right). (D) Western blots of control, individual gene knockdown, and 4G KD KD CL1-0 lung cancer cells. (E) Colony formation assay of control, individual gene knockdown, and 4G KD CL1-0 cells on day 11. (F) Cell migration ability of control, individual gene knockdown, and 4G KD CL1-0 cells. GAPDH served as the internal control. Error bars represent the mean ± SD of triplicate experiments. Statistical differences were analyzed using the unpaired t test ( * P < 0.05, ** P < 0.01).

Article Snippet: Expression plasmids of pCMV3-SLC2A1-His (HG12102-CH), pCMV3-MTHFD2-Myc (HG16324-CM), and pCMV3-RRM2-Flag (HG18283-CF) were purchased from Sino Biological.

Techniques: Functional Assay, Biomarker Assay, Knockdown, Western Blot, Control, Staining, Cell Culture, Migration, Microscopy, Colony Assay

The functional assays of biomarker overexpression in A549 and CL1-0 cells using transiently transfection of plasmids expressing ADAM9, SLC2A1, MTHFD2, and RRM2 proteins individually or combined plasmids (4G OE). (A) Western blots of vector control, individual gene overexpression, and 4G OE in A549 cells. (B) Colony formation of control, individual single gene, and 4G OE A549 cells on day 8. (C) Cell migration ability of control, individual single gene, and 4G OE A549 cells. (D) Western blots of control, individual single gene, and 4G OE CL1-0 cells. (E) Colony formation assay of control, individual single gene, and 4G OE CL1-0 cells on day 8. (F) Cell migration ability of control, individual single gene, and 4G OE CL1-0 cells. Tubulin served as the internal control. Error bars represent the mean ± SD of triplicate experiments. Statistical differences were analyzed using the unpaired t test ( * P < 0.05, ** P < 0.01).

Journal: Theranostics

Article Title: Identification of theranostic factors for patients developing metastasis after surgery for early-stage lung adenocarcinoma

doi: 10.7150/thno.53176

Figure Lengend Snippet: The functional assays of biomarker overexpression in A549 and CL1-0 cells using transiently transfection of plasmids expressing ADAM9, SLC2A1, MTHFD2, and RRM2 proteins individually or combined plasmids (4G OE). (A) Western blots of vector control, individual gene overexpression, and 4G OE in A549 cells. (B) Colony formation of control, individual single gene, and 4G OE A549 cells on day 8. (C) Cell migration ability of control, individual single gene, and 4G OE A549 cells. (D) Western blots of control, individual single gene, and 4G OE CL1-0 cells. (E) Colony formation assay of control, individual single gene, and 4G OE CL1-0 cells on day 8. (F) Cell migration ability of control, individual single gene, and 4G OE CL1-0 cells. Tubulin served as the internal control. Error bars represent the mean ± SD of triplicate experiments. Statistical differences were analyzed using the unpaired t test ( * P < 0.05, ** P < 0.01).

Article Snippet: Expression plasmids of pCMV3-SLC2A1-His (HG12102-CH), pCMV3-MTHFD2-Myc (HG16324-CM), and pCMV3-RRM2-Flag (HG18283-CF) were purchased from Sino Biological.

Techniques: Functional Assay, Biomarker Assay, Over Expression, Transfection, Expressing, Western Blot, Plasmid Preparation, Control, Migration, Colony Assay

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