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1.
Fig 12

Fig 12. From: Kaposi's Sarcoma-Associated Herpesvirus Suppression of DUSP1 Facilitates Cellular Pathogenesis following De Novo Infection.

Proposed schematic representation of DUSP1 regulation by KSHV.

Zhiqiang Qin, et al. J Virol. 2013 Jan;87(1):621-635.
2.
Fig 7

Fig 7. From: Kaposi's Sarcoma-Associated Herpesvirus Suppression of DUSP1 Facilitates Cellular Pathogenesis following De Novo Infection.

miR-K12-11 repression of DUSP1 induces the secretion of promigratory factors and endothelial cell invasiveness. (A) HUVEC were transfected with either a control vector or pc-mi11 and, after 24 h, were transduced with either a control AdV or AdV-DUSP1. After 48 h, ELISAs were used to quantify the levels of IL-6, VEGF, and IL-8 secreted in culture supernatants. (B) In parallel experiments, aliquots either were treated as described for panel A or were transfected with constructs encoding xCT (pc-xCT) for 24 h; then they were transduced with either a control AdV or AdV-DUSP1. After an additional 24 h, transwell assays were used to quantify invasiveness as described in Materials and Methods. Error bars represent the standard errors of the means for three independent experiments. **, P < 0.01.

Zhiqiang Qin, et al. J Virol. 2013 Jan;87(1):621-635.
3.
Fig 11

Fig 11. From: Kaposi's Sarcoma-Associated Herpesvirus Suppression of DUSP1 Facilitates Cellular Pathogenesis following De Novo Infection.

Pharmacologic derepression of DUSP1 suppresses KSHV-induced endothelial cell pathogenesis. (A) HUVEC were incubated first with either a vehicle or 1 μM dexamethasone (DEX) for 24 h and then with purified KSHV for 2 h. After an additional 24 h, immunoblotting was performed to identify the protein expression of DUSP1, activated MAPKs, and β-actin (loading control). (B to D) Cells were treated as described for panel A, and supernatants were collected for the quantification of IL-6, VEGF, and IL-8 secretion by ELISA. (E) Transwell assays were used to quantify invasiveness for cells treated as described for panel A. Relative invasiveness was determined as described in Materials and Methods. Error bars represent the standard errors of the means for three independent experiments. **, P < 0.01.

Zhiqiang Qin, et al. J Virol. 2013 Jan;87(1):621-635.
4.
Fig 9

Fig 9. From: Kaposi's Sarcoma-Associated Herpesvirus Suppression of DUSP1 Facilitates Cellular Pathogenesis following De Novo Infection.

Targeting xCT or 14-3-3β results in derepression of DUSP1 during KSHV infection. (A and B) HUVEC were transfected with control (n-siRNA) or xCT-specific siRNA, and after 24 h, the cells either were transfected with a control or a pc-mi11 vector (A) or were incubated with or without KSHV for 2 h (B). Twenty-four hours later, protein expression was determined by immunoblotting. (C to G) HUVEC were transfected with control (n-siRNA) or 14-3-3β-specific siRNA, and after 24 h, cells either were transfected with a control or a pc-mi11 vector or were incubated with or without KSHV for 2 h. Assays were performed 24 h after viral incubation. DUSP1 protein and transcript expression was determined by immunoblotting (C and D) and qRT-PCR (E and F), respectively. (G) Transwell assays were used to quantify invasiveness as described in Materials and Methods. Error bars represent the standard errors of the means for three independent experiments. **, P < 0.01.

Zhiqiang Qin, et al. J Virol. 2013 Jan;87(1):621-635.
5.
Fig 5

Fig 5. From: Kaposi's Sarcoma-Associated Herpesvirus Suppression of DUSP1 Facilitates Cellular Pathogenesis following De Novo Infection.

Ectopic expression of DUSP1 suppresses cell invasiveness induced by de novo KSHV infection. (A) HUVEC (top row) or HFF (bottom row) were transduced as described above for DUSP1 overexpression prior to their incubation with KSHV for 2 h. Cells were then incubated for 24 h prior to the assessment of invasiveness. Relative invasiveness was calculated as described in Materials and Methods. (B) HUVEC were transduced with either a control AdV or AdV-DUSP1 for 48 h, transfected for an additional 24 h with constructs expressing ERK (pcERK) or a control vector (pc) as described above, and then incubated with KSHV for an additional 2 h. Transwell assays were used to quantify invasiveness 24 h following viral incubation. Error bars represent the standard errors of the means for three independent experiments. **, P < 0.01.

Zhiqiang Qin, et al. J Virol. 2013 Jan;87(1):621-635.
6.
Fig 4

Fig 4. From: Kaposi's Sarcoma-Associated Herpesvirus Suppression of DUSP1 Facilitates Cellular Pathogenesis following De Novo Infection.

Ectopic expression of DUSP1 suppresses ERK-dependent secretion of promigratory factors during de novo KSHV infection. (A to D) HUVEC (A and B) and HFF (C and D) were transduced with AdV-DUSP1 (or a control AdV) for 48 h prior to their incubation with purified KSHV for 2 h. Culture supernatants were collected 24 h later for the quantification of VEGF and IL-8 secretion by ELISA. (E and F) In parallel, qRT-PCR was used to quantify VEGF (E) and IL-8 (F) receptor transcript expression within HUVEC. (G and H) HUVEC were transduced for ectopic DUSP1 expression and, after 48 h, were transfected with a recombinant ERK construct (pcERK) or a control vector (pc) for 24 h; then they were incubated with purified KSHV for an additional 2 h. Culture supernatants were collected 24 h later for the quantification of VEGF and IL-8 secretion by ELISA. Error bars represent the standard errors of the means for three independent experiments. *, P < 0.05; **, P < 0.01.

Zhiqiang Qin, et al. J Virol. 2013 Jan;87(1):621-635.
7.
Fig 3

Fig 3. From: Kaposi's Sarcoma-Associated Herpesvirus Suppression of DUSP1 Facilitates Cellular Pathogenesis following De Novo Infection.

ERK activation restores latent KSHV gene expression in the presence of DUSP1 overexpression. HUVEC were transduced with AdV-DUSP1 (or a control AdV) for 48 h and were then transfected with a recombinant ERK construct (pcERK) or a control vector (pc) for 24 h prior to their incubation with KSHV for 2 h. Cells transduced with the control AdV and cells incubated with UV-inactivated virus (UV-K) were used as controls. (A) Immunoblot analyses were performed 12 h after KSHV incubation to determine total and activated ERK expression. β-Actin was used as a loading control. (B and C) LANA expression was identified by IFA and was quantified relative to that in KSHV-infected/control-transduced cells as described above. Error bars represent the standard errors of the means for three independent experiments. **, P < 0.01.

Zhiqiang Qin, et al. J Virol. 2013 Jan;87(1):621-635.
8.
Fig 8

Fig 8. From: Kaposi's Sarcoma-Associated Herpesvirus Suppression of DUSP1 Facilitates Cellular Pathogenesis following De Novo Infection.

Targeting miR-K12-11 restores DUSP1 expression and reduces the secretion of promigratory factors and invasiveness for KSHV-infected endothelial cells. (A) HUVEC were incubated with purified KSHV for 2 h and were then transfected with 300 pmol of either a miR-K12-11-specific or a miR-K12-12-specific antagomir. Forty-eight hours later, immunoblotting was performed for the identification of DUSP1 protein expression. β-Actin was used as a loading control. Numbers in immunoblots indicate relative expression as determined using ImageJ software. Data are the results of one of three independent experiments. (B and C) Cells were treated as described for panel A, and culture supernatants were collected for the quantification of VEGF and IL-8 secretion by ELISA. (D) Cells were treated as described for panel A, and transwell assays were used to quantify endothelial cell invasiveness. Error bars represent the standard errors of the means for three independent experiments. *, P < 0.05; **, P < 0.01.

Zhiqiang Qin, et al. J Virol. 2013 Jan;87(1):621-635.
9.
Fig 10

Fig 10. From: Kaposi's Sarcoma-Associated Herpesvirus Suppression of DUSP1 Facilitates Cellular Pathogenesis following De Novo Infection.

miR-K12-1 represses DUSP1 expression. (A) HUVEC were transfected with constructs encoding miR-K12-1 (pc-mi1) or an empty control vector (pc). Twenty-four hours later, immunoblotting was performed to identify DUSP1 and MAPK protein expression. (B) Cells were incubated with or without (mock) purified KSHV for 2 h, and infected cells were subsequently transfected with or without 300 pmol 2′ OMe RNA antagomirs targeting miR-K12-1. Forty-eight hours later, immunoblotting was performed for the identification of DUSP1 protein expression. β-Actin was used as a loading control. Numbers in immunoblots indicate relative expression as determined using ImageJ software. Data in panels A and B represent one of three independent experiments. (C) Cells were cotransfected with the pGL3-DUSP1 3′ UTR and either a control vector or pc-mi1, and luciferase expression was quantified as described in Materials and Methods. Error bars represent the standard errors of the means for three independent experiments.

Zhiqiang Qin, et al. J Virol. 2013 Jan;87(1):621-635.
10.
Fig 1

Fig 1. From: Kaposi's Sarcoma-Associated Herpesvirus Suppression of DUSP1 Facilitates Cellular Pathogenesis following De Novo Infection.

KSHV suppresses DUSP1 expression following de novo infection of endothelial cells and fibroblasts. (A) HUVEC were incubated with purified KSHV or UV-inactivated virus (UV-KSHV), and cell lysates were collected at the indicated time points for immunoblotting to identify DUSP1, as well as total and activated ERK (t-ERK and p-ERK, respectively). β-Actin was used as a loading control. Numbers indicate relative expression as determined using ImageJ software. (B) qRT-PCR was used to quantify DUSP1 transcripts 48 h after incubation of HUVEC with KSHV or medium alone (mock). Error bars represent the standard errors of the means for three independent experiments. **, P < 0.01. (C and D) HUVEC (C) or HFF (D) were transduced using a recombinant human adenovirus encoding DUSP1 (AdV-DUSP1), or a control adenovirus (AdV), for 48 h prior to their incubation with purified KSHV (K) for 2 h. After an additional 24 h, protein expression was quantified by immunoblotting. Each immunoblot shown represents one of three independent experiments.

Zhiqiang Qin, et al. J Virol. 2013 Jan;87(1):621-635.
11.
Fig 2

Fig 2. From: Kaposi's Sarcoma-Associated Herpesvirus Suppression of DUSP1 Facilitates Cellular Pathogenesis following De Novo Infection.

Ectopic expression of DUSP1 suppresses latent KSHV gene expression during de novo infection. HUVEC were transduced with AdV-DUSP1 (or a control AdV) for 48 h prior to their incubation with purified KSHV for 2 h. (A and B) IFA to identify LANA expression were performed 24 h after viral incubation using an anti-LANA monoclonal antibody and a secondary antibody conjugated to Texas Red, along with DAPI for nuclear colocalization (blue). LANA expression, signified by the typical punctate intranuclear staining pattern (red dots), was quantified relative to that in KSHV-infected/control-transduced cells as described in Materials and Methods. (C) qRT-PCR was used to quantify ORF73 (LANA) transcript expression 2 h after viral incubation. (D and E) qPCR was used to quantify intracellular KSHV DNA contents 2 h (D) and 24 h (E) after incubation of cells with the virus. Error bars represent the standard errors of the means for three independent experiments. **, P < 0.01.

Zhiqiang Qin, et al. J Virol. 2013 Jan;87(1):621-635.
12.
Fig 6

Fig 6. From: Kaposi's Sarcoma-Associated Herpesvirus Suppression of DUSP1 Facilitates Cellular Pathogenesis following De Novo Infection.

miR-K12-11 represses DUSP1 expression. (A and B) HUVEC were transfected with constructs encoding miR-K12-11 (pc-mi11), xCT (pc-xCT), or an empty control vector (pc). Twenty-four hours later, immunoblotting was performed to identify DUSP1, MAPK, xCT, and 14-3-3β protein expression (A), and qRT-PCR was performed to identify DUSP1 transcript expression (B). Error bars represent the standard errors of the means for three independent experiments. **, P < 0.01. (C) Cells were cotransfected with the pGL3-DUSP1 3′ UTR and either a control vector or pc-mi11, and luciferase expression was quantified as described in Materials and Methods. (D) HUVEC were cotransfected with either a control vector or pc-mi11, along with the luciferase reporter construct for miR-K12-11 (mi11-sensor). Aliquots were also incubated with 300 pmol 2′ OMe RNA antagomirs targeting either miR-K12-11 or miR-K12-12 (the latter was used as a negative control). Forty-eight hours later, luciferase expression was quantified as described in Materials and Methods. (E) Immunoblotting was performed 48 h after the transfection of cells with either a control vector or pc-mi11 in the presence of 300 pmol of either a control antagomir or a miR-K12-11-specific antagomir. (F) HUVEC were incubated with purified KSHV; then miR-K12-11 expression was quantified at the time points indicated using qRT-PCR. Expression at each time point was quantified relative to that in cells incubated with KSHV for 10 min. (G) HUVEC were transfected with luciferase reporter constructs for miR-K12-11 for 24 h and were then incubated with purified KSHV or medium (mock). Luciferase expression was quantified as described in Materials and Methods for cells collected at the indicated time points. Error bars represent the standard errors of the means for three independent experiments.

Zhiqiang Qin, et al. J Virol. 2013 Jan;87(1):621-635.

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