Figure 4.
Figure 4. FPN, hepcidin, and iron regulatory genes play a role in heme-mediated HIV-1 inhibition. (A) Hemin treatment induced ferroportin expression. THP-1 cells were treated with hemin for 24 hours. Cell lysates were resolved on 4% to 12% Bis-Tris gel and probed with antibodies against FPN. The β-actin was used as loading control. Results were quantified using Image Quant Software. Bars represent quantification from 4 SCD and control samples. The means ± SD are shown. P values were calculated using Student t test. (B) Expression of candidate genes in hemin-treated THP-1 cells. THP-1 cells were treated with hemin for 24 hours. RNA was extracted, reverse transcribed, and analyzed by RT-PCR using primers for IKBα, HO-1, p21, HIF-1α, and FPN. The18S rRNA was used as a reference for ΔΔCt analysis. The means ± SD are shown (n = 3 for each sample). P values were calculated using Student t test. (C) Restoration of hemin-inhibited HIV-1 replication in THP-1 cells and PBMCs. THP-1 cells and PBMCs were treated with hemin or with hemin and hepcidin and infected with HIV-1-LUC-G. Luciferase activity was measured 2 days PI. The means ± SD are shown (n = 3 for each sample). P values were calculated using Student t test. (D) Restoration of hemin-inhibited HIV-1 replication in MDMs. MDMs were treated with hemin or with hemin and hepcidin and infected with M-tropic HIV-1 (BAL) isolate. Expression of gag was measured by RT-PCR after 6 days PI. The means ± SD are shown (n = 3 for each sample). P values were calculated using Student t test. (E-I) Restoration of hemin-mediated HIV-1 replication by knockdowns of FPN, IKBα, HO-1, p21, and HIF-1α. THP-1 cells were stably transduced with lentiviruses expressing corresponding shRNA and infected HIV-1-LUC-G virus. Where indicated, the cells were also treated with 75 μM hemin. Luciferase activity was measured 2 days PI (left panels). The expression of the corresponding mRNAs was measured by RT-PCR with 18S RNA primers for internal control (right panels). Quantification from 3 independent experiments show the means ± SD and P values calculated using Student t test.

FPN, hepcidin, and iron regulatory genes play a role in heme-mediated HIV-1 inhibition. (A) Hemin treatment induced ferroportin expression. THP-1 cells were treated with hemin for 24 hours. Cell lysates were resolved on 4% to 12% Bis-Tris gel and probed with antibodies against FPN. The β-actin was used as loading control. Results were quantified using Image Quant Software. Bars represent quantification from 4 SCD and control samples. The means ± SD are shown. P values were calculated using Student t test. (B) Expression of candidate genes in hemin-treated THP-1 cells. THP-1 cells were treated with hemin for 24 hours. RNA was extracted, reverse transcribed, and analyzed by RT-PCR using primers for IKBα, HO-1, p21, HIF-1α, and FPN. The18S rRNA was used as a reference for ΔΔCt analysis. The means ± SD are shown (n = 3 for each sample). P values were calculated using Student t test. (C) Restoration of hemin-inhibited HIV-1 replication in THP-1 cells and PBMCs. THP-1 cells and PBMCs were treated with hemin or with hemin and hepcidin and infected with HIV-1-LUC-G. Luciferase activity was measured 2 days PI. The means ± SD are shown (n = 3 for each sample). P values were calculated using Student t test. (D) Restoration of hemin-inhibited HIV-1 replication in MDMs. MDMs were treated with hemin or with hemin and hepcidin and infected with M-tropic HIV-1 (BAL) isolate. Expression of gag was measured by RT-PCR after 6 days PI. The means ± SD are shown (n = 3 for each sample). P values were calculated using Student t test. (E-I) Restoration of hemin-mediated HIV-1 replication by knockdowns of FPN, IKBα, HO-1, p21, and HIF-1α. THP-1 cells were stably transduced with lentiviruses expressing corresponding shRNA and infected HIV-1-LUC-G virus. Where indicated, the cells were also treated with 75 μM hemin. Luciferase activity was measured 2 days PI (left panels). The expression of the corresponding mRNAs was measured by RT-PCR with 18S RNA primers for internal control (right panels). Quantification from 3 independent experiments show the means ± SD and P values calculated using Student t test.

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