Journal: Autophagy

Article Title: The activation of KSHV lytic cycle blocks autophagy in PEL cells

doi: 10.1080/15548627.2015.1091911

Figure Lengend Snippet: The autophagic flux was blocked in BC3 cells undergoing KSHV lytic cycle activation. ( A ) Analysis of K-bZIP lytic antigen, RAB7 and LC3-I/-II expression in the presence or in the absence of Baf (used for the last 3 h at 20 nM) in BC3 cells induced to enter the lytic cycle by T/B treatment for 36 h or ( B ) in the KSHV-negative BJAB cells undergoing the same treatment. ( C ) Kinetic analysis of the expression of K-bZIP, RAB7 and LC3-II and effects of Baf on the expression of K-bZIP, RAB7 and LC3-II in BC3 cells induced to enter the lytic cycle by T/B at the indicated time points. ACTB was used as loading control and a representative experiment out of 3 is shown. The histograms represent the mean plus SD of the densitometric analysis of the ratio of specific proteins to ACTB of 3 different experiments. ( D ) BC3 cells were transfected with pEGFP-LC3 plasmid and induced to enter the lytic cycle by T/B treatment for 0, 18 and 36 h. Lysosomes were stained in red with LysoTracker while autophagosomes were indicated by GFP-LC3 puncta. Bar: 5 micron. ( E ) Gallery of BC3 cells transfected with pDest-mCherry-EGFP-LC3B plasmid and treated with T/B for 36 h or starved for 2 h. The yellow puncta indicate autophagosomes while red puncta indicate autolysosomes. A representative experiment out of 3 is shown. Bar: 5 micron.

Article Snippet: The knockdown of BECN1 (Santa Cruz Biotechnology, sc-29797) or RAB7 (Santa Cruz Biotechnology, sc-29460) was performed in PEL cell lines using specific small interfering RNA duplex.

Techniques: Activation Assay, Expressing, Transfection, Plasmid Preparation, Staining

Journal: Autophagy

Article Title: The activation of KSHV lytic cycle blocks autophagy in PEL cells

doi: 10.1080/15548627.2015.1091911

Figure Lengend Snippet: The autophagic flux was blocked in TRExBCBL1-Rta cells undergoing KSHV lytic cycle activation by doxycycline treatment. ( A ) Evaluation of the autophagic flux based on LC3-II accumulation in the presence or in the absence of Baf (used for the last 3 h at 20 nM) in TRExBCBL1-Rta cells induced to enter the lytic cycle by treatment with doxycycline for 48 h. TRExBCBL1-vector cells were used as control. ( B ) The expression of KSHV lytic antigen K8.1 and RAB7 was analyzed by western blot. TUBA1A was used as loading control and a representative experiment out of 3 is shown. The histograms represent the mean plus SD of the densitometric analysis of the ratio of LC3-II, K8.1 or RAB7 to TUBA1A of 3 different experiments. ( C ) Percentage of cells expressing K8.1 (red) lytic antigen analyzed by immunofluorescence. Nuclei were stained by DAPI (blue).

Article Snippet: The knockdown of BECN1 (Santa Cruz Biotechnology, sc-29797) or RAB7 (Santa Cruz Biotechnology, sc-29460) was performed in PEL cell lines using specific small interfering RNA duplex.

Techniques: Activation Assay, Plasmid Preparation, Expressing, Western Blot, Immunofluorescence, Staining

Journal: Autophagy

Article Title: The activation of KSHV lytic cycle blocks autophagy in PEL cells

doi: 10.1080/15548627.2015.1091911

Figure Lengend Snippet: RAB7 knockdown leads to an autophagic block in PEL cells. BCBL1 cells were knocked down for RAB7 or scramble (SC) treated and ( A ) RAB7 and ( B ) LC3-II expression level was evaluated in the presence or in the absence of Baf. TUBA1A was used as loading control and a representative experiment out of 3 is shown. The histograms represent the mean plus SD of the densitometric analysis of the ratio of LC3-II to TUBA1A of 3 different experiments.

Article Snippet: The knockdown of BECN1 (Santa Cruz Biotechnology, sc-29797) or RAB7 (Santa Cruz Biotechnology, sc-29460) was performed in PEL cell lines using specific small interfering RNA duplex.

Techniques: Blocking Assay, Expressing