rlv ef1 mcherry mito9 lentivirus  (TaKaRa)

Figure Legend Snippet: ( A ) MEIS/HOXA9 cells transduced with rLV.EF1.mCherry-Mito9 lentivirus (MEIS/HOXA9-luci) or MN1-GFP cells (1 × 10 6 ) were injected into busulfan-treated C57/BL6 mice. Animals were treated with either CNTs or CLs on day 19 after injection and sacrificed on day 23. ( B ) Representative flow plot and gating strategy for BMMs (CD45 + GR1 – CD115 lo/int F4/80 + ). FSC, forward scatter; SSC, side scatter. ( C ) Flow cytometric analysis of the number of BMMs ( n = 5 mice). ( D ) In vivo imaging analysis of animals engrafted with MEIS/HOXA9-luci on days 19 and 23, representing before and after CL and CNT treatment. Graphs show bioluminescence analysis of the CL and CNT treatment groups ( n = 5 mice). ( E ) Bioluminescence analysis of apoptotic and live MEIS/HOXA9-luci cells. ( F ) Representative flow plots for MN1-GFP engraftment in the CL and CNT treatment groups. BM was extracted and analyzed for ( G ) MN1-GFP engraftment and ( H ) annexin V staining (AnnV + AML) of MN1-GFP cells ( n = 5 mice). ( I ) MN1- or vehicle-treated (PBS) cells (1 × 10 6 ) were injected into busulfan-treated C57/BL6 mice, and BM was harvested 14 days later. BMM F4/80 + cells were isolated via magnetic separation and incubated with zymosan A bioparticles for 2 hours, followed by imaging via fluorescence microscopy. ( J and K ) The number of bioparticles (red) per macrophage was counted for control and MN1-associated BMMs via microscopy ( n = 25 BMMs). Scale bar: 10 μm. ( L and M ) The number of bioparticles (red) and LC3 (green) per MN1-associated BMM was counted and compared with the number of bioparticles without LC3 ( n = 25 BMMs). Scale bar: 10 μm. Data indicate the mean ± SD * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001, by 2-tailed Mann-Whitney U test.

Techniques Used: Transduction, Injection, In Vivo Imaging, Staining, Isolation, Incubation, Imaging, Fluorescence, Microscopy, MANN-WHITNEY

Figure Legend Snippet: ( A ) ABs were isolated from MN1 and nonmalignant LSK cells and cultured with BMMs from C57/BL6 mice for 24 hours, and RNA was extracted for qPCR analysis. ( B ) Relative gene expression of Gbp2 , Irf7 , and Ifit3 in BMMs cultured with MN1 and LSK ABs ( n = 5). ( C ) Representative images of nonmalignant LSK and MN1 cells stained with MitoTracker Green (MTG) or VybrantDil (VD). ABs were isolated and analyzed via image flow cytometry. Scale bar: 7 μm. ( D ) Percentage of ABs from LSK and MN1 cells that were positive for MitoTracker Green and VybrantDil ( n = 5). ( E ) Representative confocal microscopy images of human AML cells that were transduced with a GFP membrane virus and stained with MitoTracker Red (MTR) and Hoechst. Arrows indicate blebs containing mitochondria. Scale bar: 10 μm. ( F ) Nonmalignant CD34, MN1, and human AML cells were stained with MitoTracker Red before isolating the ABs and analyzing them via flow cytometry for the percentage of ABs containing MitoTracker Red (mito + ) ( n = 5). ( G ) Schematic diagram of the experimental design. Primary AML cells were transduced with rLV.EF1.mCherry-Mito9 lentivirus (mCh-AML) and injected into NSG mice and left for 35 days ( n = 3). ( H ) BM was extracted and BMMs were analyzed by flow cytometry for mCherry fluorescence (MFI). ( I ) mCh-AML cells were cocultured with BMSCs and BMMs and analyzed by microscopy for mitochondria uptake, as determined by mCherry MFI ( n = 25). Scale bar: 10 μm. Data indicate the mean ± SD. * P < 0.05 and ** P < 0.01, by Mann-Whitney U test ( B , D , and I ) and Kruskal-Wallis test ( F and H ).

Techniques Used: Isolation, Cell Culture, Expressing, Staining, Flow Cytometry, Confocal Microscopy, Transduction, Injection, Fluorescence, Microscopy, MANN-WHITNEY

Figure Legend Snippet: ( A ) Primary AML cells were transduced with rLV.EF1.mCherry-Mito9 lentivirus (mCh-AML), and the ABs were isolated (mCh-AB). mCh-ABs were cultured with BMMs from Atg16L1 E230+ and Atg16L1 E230– mice for 24 hours. ( B ) mCherry intensity between Atg16L1 E230+ and Atg16L1 E230– BMMs was analyzed at 4 and 24 hours by confocal microscopy ( n = 5). ( C ) Relative mtDNA levels of MN1 cells and ρ 0 -generated MN1 cells normalized to DNA levels using TaqMan PCR and Tert and ND3 probes ( n = 5). ( D ). Nonmalignant LSK cells and MN1 cells were stained for MitoTracker Red, and the ABs were isolated before being sorted on the basis of a positive MitoTracker Red signal. ρ 0 MN1 cell ABs were also isolated. The ABs were cultured for 24 hours with BMMs from C57/BL6 mice, and RNA was extracted for qPCR analysis. ( E ) Relative gene expression of Gbp2 , Irf7 , and Ifit3 in BMMs cultured with sorted mitochondria containing ABs from MN1, LSK, and ρ 0 MN1 cells AB ( n = 4). ( F ) Nonmalignant LSK and MN1 cells were stained for MitoTracker Red, and the ABs were isolated before sorting on the basis of a positive MitoTracker Red signal. The ABs were cultured for 24 hours with BMMs from Atg16L1 E230– and Atg16L1 E230+ mice, and RNA was extracted for qPCR analysis. ( G ) Relative gene expression of Gbp2 , Irf7 , and Ifit3 in Atg16L1 E230– and Atg16L1 E230+ BMMs cultured with sorted mitochondria containing ABs from MN1 and LSK cells ( n = 5). Data indicate the mean ± SD. * P < 0.05 and ** P < 0.01, by Mann-Whitney U test ( B and C ) and Kruskal-Wallis test ( E and G ).

Techniques Used: Transduction, Isolation, Cell Culture, Confocal Microscopy, Generated, Staining, Expressing, MANN-WHITNEY