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. Author manuscript; available in PMC: 2017 Oct 1.

Published in final edited form as: J Neurovirol. 2016 May 2;22(5):666–673. doi: 10.1007/s13365-016-0443-6

Fig. 3 — The effect of cystatin B modulation in STAT-1PY were examined in induced MDM. (A) Cystatin B inhibits the STAT-1PY nuclear translocation in IFN-B-induced MDM. Uninfected MDM induced by IFN-β were assayed with PLA in situ. Non-targeting siRNA and unstimulated MDM were used as controls. Both, unstimulated and IFN-β treated MDM showed a cytoplasmic localization of STAT1-PY. In IFN-β treated MDM, STAT-1PY is translocated to the nucleus after silencing of cystatin B. (B) Cystatin B inhibits STAT-1PY in HIV infected MDM. (1) In situ PLA was used to determine the STAT1PY in HIV infected MDM treated with cystatin B siRNA. siRNA treatment against cystatin B increased the STAT-1 tyrosine phosphorylation in HIV infected MDM. (2) An unpaired two-tailed Student’s t-test was used to compare the PLA signals of Tyrosine-phosphorylated-STAT1 in uninfected and HIV-infected MDM treated with or without anti-cystatin B siRNA. (C) HIVp24 antigen levels from MDM supernatants of two donors at 3,6 and 12 days post-infection shows a productive infection. (D) Experimental controls included: (1) Biological Positive control consisting of uninfected MDM stained for cystatin B/cathepsin B interacting proteins, (2) Biological Negative control consisting of HIV-infected MDM that lost cystatin B/cathepsin B interaction, (3) A Technical control included the absence of the primary antibody and (4) STAT-1PY in IFN-β-treated MDM. Images were obtained on a Nikon Eclipse E400 fluorescence microscope with a SPOT Insight QE camera and SPOT 5.1, 40x. These results are representative of two independent experiments using two different donors.

Fig. 3 — The effect of cystatin B modulation in STAT-1PY were examined in induced MDM. (A) Cystatin B inhibits the STAT-1PY nuclear translocation in IFN-B-induced MDM. Uninfected MDM induced by IFN-β were assayed with PLA in situ. Non-targeting siRNA and unstimulated MDM were used as controls. Both, unstimulated and IFN-β treated MDM showed a cytoplasmic localization of STAT1-PY. In IFN-β treated MDM, STAT-1PY is translocated to the nucleus after silencing of cystatin B. (B) Cystatin B inhibits STAT-1PY in HIV infected MDM. (1) In situ PLA was used to determine the STAT1PY in HIV infected MDM treated with cystatin B siRNA. siRNA treatment against cystatin B increased the STAT-1 tyrosine phosphorylation in HIV infected MDM. (2) An unpaired two-tailed Student’s t-test was used to compare the PLA signals of Tyrosine-phosphorylated-STAT1 in uninfected and HIV-infected MDM treated with or without anti-cystatin B siRNA. (C) HIVp24 antigen levels from MDM supernatants of two donors at 3,6 and 12 days post-infection shows a productive infection. (D) Experimental controls included: (1) Biological Positive control consisting of uninfected MDM stained for cystatin B/cathepsin B interacting proteins, (2) Biological Negative control consisting of HIV-infected MDM that lost cystatin B/cathepsin B interaction, (3) A Technical control included the absence of the primary antibody and (4) STAT-1PY in IFN-β-treated MDM. Images were obtained on a Nikon Eclipse E400 fluorescence microscope with a SPOT Insight QE camera and SPOT 5.1, 40x. These results are representative of two independent experiments using two different donors.