Journal: bioRxiv

Article Title: Direct observation of aggregate-triggered selective autophagy

doi: 10.1101/2021.04.21.440799

Figure Lengend Snippet: a) Coding strand of knock-in target site before exon 1 (black box) of the RAB7A locus. The sgRNA target site (orange) and PAM sequence (cyan) are indicated. Insert is EGFP followed by a linker sequence (grey box). DNA sequencing of the homozygous knock-in clone is aligned below. b) 1% agarose gel including PCR-amplification product of RAB7A locus of WT and EGFP-Rab7 KI lines.

Article Snippet: The HeLa GFP-RAB7 line was generated by transfecting HeLa cells (ATCC) with the donor plasmid pDONOR-GFP-RAB7 and pSpCas9(BB)-2A-Puro (PX459) V2.0 (Addgene, #62988) bearing the appropriate targeting sequence (5’-TAGTTTGAAGGATGACCTCT-3’; pX459v2-RAB7a sg3).

Techniques: Knock-In, Sequencing, DNA Sequencing, Agarose Gel Electrophoresis, Amplification

Journal: bioRxiv

Article Title: Direct observation of aggregate-triggered selective autophagy

doi: 10.1101/2021.04.21.440799

Figure Lengend Snippet: a) Endogenous GFP-RAB7 KI HeLa cell line transiently showing mKeima-PIM aggregates. b) Endogenous GFP-RAB7 signal showing larger RAB7 positive vesicles at the perinuclear area and smaller vesicles in the periphery. c) Zoom of the cell in (a) showing aggregates at neutral pH (green) and low pH (red) and the correlation with RAB7. d) Selected frames from a time-lapse movie showing the gradual recruitment of RAB7 and concomitant decrease of mKeima ratio. e) Analysis of RAB7 intensity (cyan) and mKeima ratio (black) of a 10 typical acidification events, including the one shown in (d). Data shown are mean (solid line) ± s.d. (shaded area) of 3-10 events. f-g) Time-lapse imaging of GFP-RAB7 KI HeLa cells showing putative kiss-and-run events with RAB7 positive vesicles (g) and full fusion event of RAB7 positive vesicle with the autolysosome (f). Scale bars: 10 µm (a) and 2µm (b,d-g)

Article Snippet: The HeLa GFP-RAB7 line was generated by transfecting HeLa cells (ATCC) with the donor plasmid pDONOR-GFP-RAB7 and pSpCas9(BB)-2A-Puro (PX459) V2.0 (Addgene, #62988) bearing the appropriate targeting sequence (5’-TAGTTTGAAGGATGACCTCT-3’; pX459v2-RAB7a sg3).

Techniques: Imaging

Journal: bioRxiv

Article Title: Direct observation of aggregate-triggered selective autophagy

doi: 10.1101/2021.04.21.440799

Figure Lengend Snippet: a) Monitoring a forming autolysosome in the perinuclear region (see schematic in b). Frames from a time-lapse movie depicting multiple rounds of transient associations with RAB7 positive vesicles (1.), subsequent fusion (2.) and full acidification (3.). Arrows and arrowheads indicate interactions and absence of interactions, respectively. b) Analysis of RAB7 intensity and mKeima ratio of (a). Scale bar: 2 µm

Article Snippet: The HeLa GFP-RAB7 line was generated by transfecting HeLa cells (ATCC) with the donor plasmid pDONOR-GFP-RAB7 and pSpCas9(BB)-2A-Puro (PX459) V2.0 (Addgene, #62988) bearing the appropriate targeting sequence (5’-TAGTTTGAAGGATGACCTCT-3’; pX459v2-RAB7a sg3).

Techniques:

Journal: bioRxiv

Article Title: Direct observation of aggregate-triggered selective autophagy

doi: 10.1101/2021.04.21.440799

Figure Lengend Snippet: First, DFCP1 is recruited to aggregates (omegasome formation). DFCP1 puncta are present only for a short time but the timing of DFCP1 puncta presence relative to acidification is variable (patterned area). Just before acidification of the cargo, STX17 is recruited for ∼7 min and STX17 leaves the autophagosome after start of cargo acidification. RAB7 is gradually recruited to the autophagolysosome while cargo acidification is also taking place.

Article Snippet: The HeLa GFP-RAB7 line was generated by transfecting HeLa cells (ATCC) with the donor plasmid pDONOR-GFP-RAB7 and pSpCas9(BB)-2A-Puro (PX459) V2.0 (Addgene, #62988) bearing the appropriate targeting sequence (5’-TAGTTTGAAGGATGACCTCT-3’; pX459v2-RAB7a sg3).

Techniques:

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