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Results: 6

1.
Fig 6

Fig 6. From: Heterogeneous Nuclear Ribonucleoprotein K Supports Vesicular Stomatitis Virus Replication by Regulating Cell Survival and Cellular Gene Expression.

hnRNP K regulates expression of cellular proteins required for VSV infection. (A and B) HeLa cells transfected with siRNA for 60 h were infected with VSV at an MOI of 0.01 for 12 h. The levels of the proteins were detected by WB using specific antibodies. (C) Quantification of ADAL and ARF1 protein levels (arbitrary units) from three independent experiments, with error bars showing the standard errors of means. The level of ADAL or ARF1 in the NT lane was arbitrarily set at 1. **, P < 0.01. (D) Involvement of hnRNP K in regulating expression of cellular proteins that affect VSV infection. hnRNP K inhibits TIA1a/b expression, which is known to inhibit VSV replication. Increased levels of antiapoptotic proteins such as Bcl-XL, Bag1, and Bcl-2 and/or reduction in the levels of apoptotic proteins Bcl-XS and Bik promotes cell survival for increased VSV replication, while maintenance of factors such as ADAL and ARF1 is required for VSV replication.

Phat X. Dinh, et al. J Virol. 2013 September;87(18):10059-10069.
2.
Fig 5

Fig 5. From: Heterogeneous Nuclear Ribonucleoprotein K Supports Vesicular Stomatitis Virus Replication by Regulating Cell Survival and Cellular Gene Expression.

VSV-mediated cell death in hnRNP K-depleted cells is associated with increased levels of proapoptotic proteins Bcl-XS and Bik. (A) HeLa or HEK293 cells transfected with siRNAs for 60 h were infected with VSV at an MOI of 1 for 6 h. The levels of proapoptotic proteins Bak, Bad, Noxa, and antiapoptotic factor FLIP were detected by WB using specific antibodies. (B and C) mRNA levels of proapoptotic proteins (B) or antiapoptotic proteins (C) in hnRNP K-depleted HeLa cells infected with VSV. siRNA treatment and virus infections were performed as described in panel A. Total RNA was converted to cDNA using oligo(dT) primers followed by PCR with specific primers. The mRNA level of cellular ribosomal protein L32 was used as an internal control. A DNA ladder (bp) is shown on the right. Values at the top or bottom of the lanes represent relative levels of mRNAs, using levels in the NT lane as 1. The Bcl-XS level is relative to the Bcl-XL level in the NT lane. The asterisk indicates the DNA product of an undocumented isoform of Bag1 or a nonspecific product. (D) mRNA levels of antiapoptotic or proapoptotic proteins in hnRNP K-depleted HEK293 cells infected with VSV. siRNA treatment, virus infection, and mRNA detection were as described above.

Phat X. Dinh, et al. J Virol. 2013 September;87(18):10059-10069.
3.
Fig 1

Fig 1. From: Heterogeneous Nuclear Ribonucleoprotein K Supports Vesicular Stomatitis Virus Replication by Regulating Cell Survival and Cellular Gene Expression.

hnRNP K is required for VSV infection. (A) Suppression of VSV replication in cells depleted of hnRNP K. HeLa cells were transfected with 10 nM siRNA for NT (lane 2) or 10 nM and 20 nM hnRNP K (siK) (lanes 3 and 4, respectively) for 60 h. Cells were then infected with VSV at an MOI of 0.01 for 12 h. Equal amounts of cell lysates were analyzed by WB with anti-hnRNP K and anti-M antibodies. Actin served as the loading control. (B) Quantification of M protein levels (arbitrary units) from three independent experiments as described in panel A with error bars representing the standard errors of means. The level of M expression in the NT siRNA-treated sample was set at 1. ***, P < 0.001. (C) Multicycle growth of VSV in cells depleted of hnRNP K. siRNA (10 nM) treatment and virus infections were performed as described in panel A; virus titers in supernatants collected at indicated time points were determined by plaque assay and are expressed as log10 PFU/ml. Statistical significance was determined for virus titers at 12, 18, and 24 hpi. ***, P < 0.001. (D) VSV infection is enhanced in hnRNP K-overexpressing cells. HeLa cells transfected with 1.5 μg of empty vector (EV) or an HA-hnRNP K (HA-K)-encoding plasmid for 48 h were infected with VSV at an MOI of 0.01 for 12 h, and cell lysates were analyzed by WB using anti-M or anti-HA antibodies. Actin served as the loading control. α, anti; siK, siRNA targeting hnRNP K.

Phat X. Dinh, et al. J Virol. 2013 September;87(18):10059-10069.
4.
Fig 4

Fig 4. From: Heterogeneous Nuclear Ribonucleoprotein K Supports Vesicular Stomatitis Virus Replication by Regulating Cell Survival and Cellular Gene Expression.

hnRNP K is required for survival of VSV-infected cells. (A) Knockdown of hnRNP K reduces cell survival during VSV infection. HeLa cells were transfected with NT or hnRNP K siRNA at concentration of 10 nM for 60 h. Cells were mock infected or infected with VSV at an MOI of 0.01. At 6, 12, and 18 hpi, cell survival was determined by a CellTiter-Glo Kit. The percentage of cell viability in NT siRNA-transfected and mock-infected culture at the beginning of infection was set at 100%. Data from three independent experiments are shown with error bars representing the standard errors of means. **, P < 0.01; NS, nonsignificant. (B) Caspase 3 activation is more pronounced in infected cells lacking hnRNP K. The experiment was conducted as described in panel A. Infected cells were lysed and analyzed at the indicated time points to detect procaspase 3, activated caspase 3 (p17), hnRNP K, and VSV M protein using specific antibodies. Actin served as a loading control. (C) Quantification of p17 protein levels (arbitrary units) from three independent experiments as described in panel B. The level of activated caspase 3 (p17) in cells treated with siRNA targeting hnRNP K at 18 hpi was set arbitrarily at 1, and the relative levels of p17 in other samples were then determined. Error bars represent the standard errors of means from three independent experiments. ***, < 0.001; **, P < 0.01. (D) wt MEF or TIA1−/− MEF cells were transfected with NT or hnRNP K siRNA at a concentration of 20 nM for 60 h. Cells were mock infected (mock) or infected with VSV at an MOI of 1. At 4 and 8 hpi, cells were lysed and subjected to WB with the indicated antibodies. (E) Quantification of p17 levels (arbitrary units) from three independent experiments, with error bars representing the standard errors of means. The level of p17 in wt MEFs treated with siRNA targeting hnRNP K was set arbitrarily at 1, and the relative levels of p17 in other samples were then determined. ***, P < 0.001.

Phat X. Dinh, et al. J Virol. 2013 September;87(18):10059-10069.
5.
Fig 3

Fig 3. From: Heterogeneous Nuclear Ribonucleoprotein K Supports Vesicular Stomatitis Virus Replication by Regulating Cell Survival and Cellular Gene Expression.

hnRNP K regulates the expression of the two isoforms of TIA1 but not PCBP1/2. (A) Depletion of hnRNP K does not affect the expression of PCBP1/2. HeLa cells were transfected with NT or hnRNP K siRNA at a concentration of 10 nM, and at 60 hpt, cells were infected with VSV at an MOI of 0.01 for 12 h. Cell lysates were used for WB to detect the indicated proteins. (B to D) hnRNP K depletion results in increased levels of total TIA1 and the TIA1b isoform. HeLa cells were mock transfected or transfected with NT or hnRNP K siRNA as a pool of four duplexes (pooled) or as individual duplexes (si DX1 to si DX4) at a concentration of 10 nM, and at 60 hpt, the cells were infected with VSV at an MOI of 0.01 for 12 h. Cell lysates were used for WB to detect the indicated proteins (B). Quantification of total TIA1 protein levels (arbitrary units) from three independent experiments is shown in panel C while the levels of TIA1a and TIA1b isoforms are shown in panel D. The level of TIA1a expression in the NT siRNA-treated sample was set at 1. ***, P < 0.001; **, P < 0.01. (E) Switching of TIA1 isoform expression is regulated by hnRNP K protein at the mRNA level. The experiment was done as described in panel B. Total RNA was subjected to semiquantitative RT-PCR to detect the two isoforms of TIA1. Ribosomal protein (RP) L32 mRNA served as an internal control. (F) Overexpression of TIA1a and TIA1b inhibits VSV gene expression. HeLa cells transfected with 1.5 μg of empty vector (EV) or HA-TIA1a- or HA-TIA1b-encoding plasmids for 48 h were infected with VSV at an MOI of 0.1 for 12 h (lanes 1 to 3) or with VSVΔG at an MOI of 0.5 for 8 h (lanes 4 to 6) or supertransfected for 6 h with viral NC prepared from VSV (lanes 7 to 9), and cell lysates were analyzed by WB using anti-M or anti-HA antibodies. Actin served as the loading control. siPCBP1, siRNA targeting PCBP1; siPCBP2, siRNA targeting PCBP2; siTIA1, siRNA targeting TIA1.

Phat X. Dinh, et al. J Virol. 2013 September;87(18):10059-10069.
6.
Fig 2

Fig 2. From: Heterogeneous Nuclear Ribonucleoprotein K Supports Vesicular Stomatitis Virus Replication by Regulating Cell Survival and Cellular Gene Expression.

hnRNP K is required for VSV propagation. (A and B) VSV mRNA and antigenomic RNA levels in cells depleted of hnRNP K and infected with the virus at a low (0.01) or high (1.0) MOI (panels A and B, respectively) were determined by qRT-PCR. Experimental conditions were as described in the legend of Fig. 1A. Error bars represent the standard errors of means from three independent experiments. ***, P < 0.001; *, P < 0.05. (C) hnRNP K is not required for early stages of the VSV life cycle. HeLa cells were transfected with 10 nM siRNA for NT or targeting hnRNP K for 60 h. Cells were then treated as follows: infected with VSV at an MOI of 0.01 for 12 h (lanes 1 and 2), supertransfected for 6 h with viral NC prepared from VSV (lanes 3 and 4), infected with VSVΔG at an MOI of 0.5 for 8 h (lanes 5 and 6), or infected with VSV at an MOI of 1 for 4 h (lane 7 and 8). Cell lysates corresponding to equal amounts of total proteins were analyzed by WB with anti-hnRNP K and anti-M antibodies. Actin served as the loading control. (D) HeLa cells were transfected with siRNA as described in the legend of Fig. 1A. At 60 hpt, cells were infected with VSV-PLuc at an MOI of 300. After 1 h of virus adsorption on ice, cells were washed with PBS, and cell extracts at various times postincubation were analyzed for luciferase activity. The luciferase activity in NT siRNA-treated cells at various times postinfection was set at 1. Data show relative luciferase activity and are expressed as the averages of three independent experiments, with error bars representing the standard errors of means. NS, nonsignificant. (E) HeLa cells were transfected with siRNA as described in the legend of Fig. 1A. At 60 hpt, cells were infected with VSV-PeGFP at an MOI of 0.05 or VSVΔG at an MOI of 0.5 for 12 h. The percentages of infected cells were determined by counting the number of cells expressing green fluorescence from PeGFP. Data show relative percent infection after normalizing the value of cells treated with siRNA targeting hnRNP K to that of NT siRNA-treated cells, which is set at 100, and are expressed as average of three independent experiments with error bars representing the standards error of means. ***, P < 0.001; NS, nonsignificant.

Phat X. Dinh, et al. J Virol. 2013 September;87(18):10059-10069.

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