EMBO/SourceData huggingface dataset:
Figure 5E MICAL2PV interacts with the C-terminal domain of Miro2 containing the transmembrane region. H1299 cells were co-transfected with plasmids encoding Flag-MICAL2PV and Myc-tagged Miro2 mutants as indicated. Following transfection, cells were lysed for immunoprecipitation using the anti-Flag antibody and Western blotting. Please note the Myc-Miro2 (410-618) protein band in lane 4 migrated as a faint band, slightly faster than the IgG light chain.F MICAL2PV inhibits the mitochondrial localization of Miro2 and mitochondrial infiltration. H1299 cells were co-transfected with Myc-tagged Miro2 and GFP-tagged MCAL2PV (GFP-M2) or control vector. Following transfection, cells were fixed and stained with anti-Tom20 (red) and anti-Myc antibodies (magenta). Confocal microscopic images were taken under the same objective and the same microscope settings. In cells co-expressing the GFP vector and Myc-Miro2 (upper panels), most of Miro2 signals (magenta) are co-colocalized with mitochondria (red Tom20 signals). However, a significant fraction of Miro2 signals is not colocalized with mitochondria in cells co-expressing GFP-M2 and Myc-Miro2 (marked by the yellow arrowheads). Mitochondria in cells expressing GFP-M2 show the perinuclear distribution pattern, whereas mitochondria in cells transfected by Myc-Miro2 without GFP-M2 (marked by the yellow stars) exhibit an infiltration pattern into the cortical cytoplasm. Scale bar: 20 μm.
Figure 6A Control and shM2 H1299 cells were transfected with control siRNA or siRNAs targeting Miro2. Following transfection, cells were stained with anti-Tom20 antibody (green) and phalloidin (red). Confocal microscopic images are shown. Scale bar: 20 μm. Data information: nuclei were stained with Hoechst 33342 (blue).F Down-regulating Miro2 inhibits mitochondrial transport in shM2 cells. Mito-GFP labelled control and shM2 H1299 cells were transfected with control siRNA or siRNAs targeting Miro2. These cells were then co-cultured with mCherry labelled recipient cells and analyzed by flow cytometry The percentage of mCherry recipient cells containing Mito-GFP was quantified. Twenty thousand events were analyzed for each condition Data information: Data were analyzed using two-way ANOVA with Bonferroni post-test and presented as mean ± SEM; * P < 0.05, *** P < 0.01, *** P < 0.001, ns, not significant. Three independent experiments were conducted.
Figure 7A Stable H1299 cells (shCtr or shM2) were treated by 5-FU (50 μM, 24 h) and then cultured alone or co-cultured with non-drug treated vehicle-treated cells stably expressing mito-mCherry in the absence or presence of Cyto B (500 nM, 24 h). The percentage of dead cells in each group was quantified. Three independent experiments were conducted. Data information: data were analyzed using two-way ANOVA with Bonferroni post-test and shown as mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001, ns, not significant.D Confocal microscopic images of TNTs in each group, white arrowheads and yellow triangle in enlarged views (zoom-in panels, zoom) marked the TNTs formed between non-drug treated (mCherry positive) and drug-treated cells. Scale bar: 25 μm.
Figure 8D MICAL2PV mutants containing the MO domain are active in depolymerizing F-actin. Following transfection of plasmids expressing different HA-MICAL2PV mutants (#1, #2, or #4) into shM2 cells, confocal microscopy was performed following staining with phalloidin (red) and anti-HA antibody (green). F-actin signals were reduced in cells expressing different mutants (HA-positive green cells marked by dashed lines). Scale bar: 20 μm. Data information: nuclei were stained with Hoechst 33342 (blue).G, H The MICAL2PV mutant containing an inactive MO domain does not inhibited TNT formation. Stable shM2 H1299 (G) or A549 (H) cells were transfected with plasmids expressing the MICAL2PV mutant in which the MO domain was inactivated by mutations in the FAD binding site (MOmut: amino acid residues 93-98 GxGxxG mutated to WxWxxW) or the control vector (Ctr). Following transfection, Western blotting and quantification of TNT formation were carried out as in panel E. 10 images were captured for quantification in each group. Three independent experiments were performed. Data information: Data were analyzed using a one-way ANOVA with Bonferroni post-test and shown as mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001, ns, not significant.
Figure 1(A) HEK293 cells were transfected with DAPK ΔCaM or with a control vector (pcDNA3-luciferase, LUC), together with shRNAs targeting beclin 1 or HcRed, and with GFP-LC3 plasmid. After 72 h, cells were counted and lysates were prepared. (A) The percentage of cells with punctate GFP-LC3 fluorescence per total GFP-LC3-positive cells was quantified. Data presented are the mean±s.d. from a triplicate of 100 transfected cells. The asterisks denote a significance level of P=0.001.(B) Western blot analysis was performed using the indicated antibodies.(C) Representative GFP-LC3 staining of cells transfected with the control shRNA (HcRed) together with pcDNA3‐luciferase or DAPKΔCaM. CaM, calmodulin; DAPK, death‐associated protein kinase; GFP, green fluorescent protein; HEK, human embryonic kidney; shRNA, short hairpin RNA.
Figure 2(A) Flag‐tagged DAPK (100 ng) was incubated with GST (900 ng) or GST-beclin‐1 (750 ng) in the presence of Ca2+, calmodulin and [γ‐33P]ATP for 30 min or 60 min. Phosphorylated proteins were visualized by X‐ray film exposure, and GST/GST-beclin‐1 levels were visualized by Ponceau S staining. The autophosphorylation of DAPK indicates that its catalytic activity was intact in all samples.(B) Flag‐tagged DAPK (60 ng) was incubated with Flag‐tagged beclin 1 (250 ng), which was purified from HEK293T cells, and a kinase assay was performed for 60 min. Where indicated (+LiCl), beclin‐1‐bound beads were first washed stringently in 0.5 M LiCl and 0.5 M KCl. Phosphorylated proteins were visualized by X‐ray film exposure, and the levels of beclin 1 were visualized by Western blot analysis using beclin 1 antibodies. DAPK, death‐associated protein kinase; GST, glutathione S‐transferase; HEK, human embryonic kidney.
Figure 3(Aa) Protein extracts from COS7 cells, HEK293T cells, or no extracts were added to bacterially produced GST or GST-beclin‐1. The pulled‐down proteins, as well as the total cell extracts, were blotted with the indicated antibodies. (Ab) Ponceau S staining of GST and GST-beclin‐1 to which the extracts were added.(B) HEK293 cells were co‐transfected with Flag‐tagged beclin 1 or with Flag‐tagged beclin 1 lacking the Bcl‐2‐binding domain (ΔBD) together with Bcl‐XL and HA‐tagged DAPK. Beclin 1 was immunoprecipitated using Flag antibodies, and the co‐immunoprecipitated proteins, as well as the total cell extracts, were blotted with DAPK, Bcl‐XL and beclin 1 antibodies.(C) HEK293 cells were transfected with Flag-beclin‐1 or Flag-GFP, and the Flag‐tagged proteins were immunoprecipitated using Flag antibodies, and eluted with an excess of Flag peptides. The blot was reacted with DAPK antibodies or with Flag antibodies at different time exposures
Figure 4(A) A bacterially purified catalytic domain of DAPK was incubated for 15 min at 30°C with increasing concentrations (5-10 nmol) of a peptide corresponding to the BH3 domain of beclin 1 (aa 108-127) and with the same peptide in which Thr 119 was substituted by alanine. An in vitro kinase assay was performed, and the reactions were applied to Whatman filters. Total levels of TCA‐insoluble counts were measured and plotted against substrate concentration. Data are the mean±s.d. of three experiments.(C) Flag‐tagged DAPK (60 ng) was incubated with GST-WT beclin 1 or with GST-T119A beclin 1 (1000 ng) in the presence of Ca2+, calmodulin and ATP for 30 min. GST‐beclin 1 levels were visualized by Ponceau S staining, and phosphorylation on Thr 119 was detected by a phosphoThr 119 antibody (Western blot).(D) HEK293 cells were co‐transfected with Flag‐tagged beclin 1 with or without ΔCaM DAPK. After 24 h, beclin 1 was immunoprecipitated from cells, using Flag antibodies, and the immunoprecipitates were reacted with phosphoThr 119 antibodies. The Ponceau staining shows equal amounts of immunoprecipitated beclin 1. The cell extract blots were reacted with haemagglutinin antibodies or with beclin 1 antibodies. CaM, calmodulin; DAPK, death‐associated protein kinase; GST, glutathione S‐transferase; HEK, human embryonic kidney; TCA, trichloro‐acetic acid.
Figure 5(A) HEK293 cells were transfected with Flag‐tagged beclin 1 and Bcl‐XL with or without haemagglutinin‐tagged DAPK. Beclin 1 was immunoprecipitated using Flag antibodies, and the co‐immunoprecipitated proteins, as well as the total cell extracts, were blotted using the indicated antibodies.(B) HEK293 cells were co‐transfected with Flag‐tagged T119A or T119E beclin 1 mutants and Bcl‐XL. Beclin 1 was immunoprecipitated using Flag antibodies, and the co‐immunoprecipitated proteins, as well as the total cell extracts, were blotted using the indicated antibodies.(C) HEK293 cells were transfected with 5 or 10 μg T119A or T119E beclin 1 mutants together with GFP-LC3 plasmid. After 24 h, cells were counted and lysates were prepared. (a) Representative GFP-LC3 staining. (b) The percentage of cells with punctate GFP-LC3 fluorescence per total GFP-LC3‐positive cells was quantified. Data presented are the mean±s.d. from a triplicate experiment with 100 transfected cells. The asterisks denote significance level: *P=0.01; **P=0.005. (c) Western blot analysis was performed using the indicated antibodies.(D) HEK293 cells were transfected with Bcl‐XL, Flag‐tagged beclin 1 (WT) or Flag‐tagged T119A beclin 1 mutant with or without haemagglutinin‐tagged activated DAPK (ΔCaM). Beclin 1 was immunoprecipitated using Flag antibodies, and the co‐immunoprecipitated proteins, as well as the total cell extracts, were blotted using the indicated antibodies. Levels of Bcl‐XL were quantified using NIH image software, and the ratio between immunoprecipitated and expressed Bcl‐XL was calculated.(E) Transmission electron micrographs of HEK293 cells 24 h after they were transfected with Bcl‐XL, Flag‐tagged beclin 1 and ΔCaM or pcDNA3‐luciferase as a control. The images in (c) and (d) were taken at higher magnifications of the ΔCaM treatment (see the scale bars). 'AV' indicates autophagic vacuoles. CaM, calmodulin; DAPK, death‐associated protein kinase; GFP, green fluorescent protein; GST, glutathione S‐transferase; HEK, human embryonic kidney; NIH, National Institutes of Health.
Figure 1(A) Expression levels of NAT4 analyzed by qRT-PCR using total RNA extracted from a wild-type (BY4741) and Pste5-NAT4 strain grown in 2% (NCR), or 0.1% glucose (CR). NAT4 levels were normalized to TAF10, whose expression remains unchanged. Error bars, SEM (Standard Error of the Mean) of 3 independent experiments. ** p ≤ 0.01; calculated by unpaired two-tailed Student's t-test.(B) ChIP analysis performed in BY4741 wild-type, nat4Δ and Pste5-NAT4 strains grown in the same conditions as in (A). Chromatin was immunoprecipitated using an antibody against N-acH4 and analyzed by qRT-PCR using primers for the different rDNA regions. Enrichment was normalized to Histone H4 levels. Error bars, SEM of 3 independent experiments. Statistical significance was determined by unpaired two-tailed Student's t test: * p ≤ 0.05; ** p ≤ 0.01 compared values to wild-type NCR conditions; arrowhead p ≤ 0.05 compared values to wild-type CR conditions(C) Replicative lifespan (RLS) for BY4741 wild-type and nat4Δ strains. Values in parenthesis (here and hereafter) indicate mean lifespan. Statistical significance (here and hereafter) was determined by one-way ANOVA test: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.(D) Mean RLS for wild-type and nat4∆ strains using different genetic backgrounds. Values outside of parenthesis indicate mean lifespan, while values in parenthesis indicate the number of cells examined.(E) Mean RLS for BY4741 wild-type and nat4Δ strains under NCR and CR conditions.(F) Mean RLS for BY4741 wild-type and Pste5-NAT4 strains under NCR and CR conditions.
Figure 2(B) ChIP experiments were performed in SF10 wild-type, nat4∆, NET1-TAP and NET1-TAP nat4∆ strains using an anti-IgG antibody. The enrichment from the antibody was normalized to input. Error bars, SEM of 3 independent experiments. Statistical significance was determined by unpaired two-tailed Student's t test: ** p ≤ 0.01 compared nat4∆ to WT values.(C) Quantitative real-time PCR analysis of rDNA copy number for young and old cells in BY4741 wild-type, nat4Δ and fob1Δ strains. Error bars, SEM of 3 independent experiments. Statistical significance was determined by unpaired two-tailed Student's t test: ** p ≤ 0.01 compared to corresponding WT values.(D) Polysome profiling analysis performed in BY4741 wild-type and nat4Δ strains. The plots are representative of two independent experiments.
Figure 3(C) Gene expression analysis of selected upregulated stress-response genes (according to results in 3A) in BY4741 wild-type and nat4∆ strains grown under NCR and CR conditions. Expression levels were normalized to TAF10, whose expression remains unchanged. Error bars, SEM of 3 independent experiments. p-values were obtained comparing results to wild-type under NCR conditions, and calculated by unpaired two-tailed Student's t-test: * p ≤ 0.05; ** p ≤ 0.01.(D) Gene expression analysis showing fold induction levels (Log2) of selected genes comparing BY4741 wild-type and STE5p-NAT4 strains under NCR (2% glucose) and CR (0.1% glucose) conditions. Expression levels were normalized to TAF10, whose expression remains unchanged.
Figure 4(A) RLS analysis for BY4741 wild-type and nat4-E186A strains. Values in parenthesis (here and hereafter) indicate mean lifespan. Statistical significance (here and hereafter) was determined by one-way ANOVA test: *** p ≤ 0.001; **** p ≤ 0.0001.(B) Mean RLS for YSC5106 wild-type, nat4∆ and H4S1D strains.(C) Gene expression analysis of the indicated stress-induced genes in YSC5106 wild-type, nat4∆ and H4S1D strains. Expression levels were normalized to RPP0 whose expression remains unchanged. Error bars, SEM of 3 independent experiments. p-values were obtained by comparing nat4∆ and H4S1D to wild-type values and calculated by unpaired two-tailed Student's t test: * p ≤ 0.05; ** p ≤ 0.01.(D) Gene expression analysis of the indicated stress-induced genes in BY4742 wild-type, nat4∆ and nat4∆[hNAA40] strains. Expression levels were normalized to ACT1, whose expression remains unchanged.
Figure 5(A) RLS analysis for YSC5106 wild-type, nat4∆, H4R3K and double mutant strains. Values in parenthesis indicate mean lifespan. Statistical significance was determined by one-way ANOVA test: **** p ≤ 0.0001.(B) Gene expression analysis of the indicated stress-induced genes in YSC5106 WT, nat4∆, H4R3K and double mutant strains. Expression levels were normalized to RPP0, whose expression remains unchanged.
Figure 6(A) Gene expression analysis of the indicated stress-induced genes in young and old cells isolated from YSC5106 WT, nat4∆ and H4R3K nat4∆ strains. Expression levels were normalized to ACT1, whose expression remains unchanged. Error bars, SEM of 3 independent experiments. P-values were calculated by unpaired two-tailed Student's t test: * p ≤ 0.05 compared to young wild-type; arrowhead p ≤ 0.05 compared to old wild-type.(B) RLS analysis for WT, nat4∆, pnc1∆ and double mutant strains in BY4741 genetic background. Values in parenthesis (here and hereafter) indicate mean lifespan. Statistical significance (here and hereafter) was determined by one-way ANOVA test: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001. ns = non-significant.(C) Mean RLS in BY4741 wild-type, msn2∆ msn4∆ double and msn2∆ msn4∆ nat4∆ triple mutant strains.(D) Mean RLS in BY4741 wild-type, nat4∆, fob1∆ sir2∆ double and fob1∆ sir2∆ nat4∆ triple mutant strains.(E) ChIP analysis performed in BY4741 wild-type, nat4Δ and Pste5-NAT4 strains grown under NCR and CR conditions. Chromatin was immunoprecipitated using antibodies against N-acH4 and Histone H4, and analyzed by qRT-PCR with the indicated primers. PNC1-P (Promoter), PNC1-S (Transcriptional Start), PNC1-E (End of gene). The enrichment from the antibody was normalized to Histone H4.
Figure 1MDM were incubated with 1,25D3 for 4 h before infection with HIV and/or M. tuberculosis (TB) for 3 h, washed then incubated with or without 1,25D3 for 7 days. (A) Top, extracellular release of HIV p24 antigen into the cell supernatant at days 0, 4 and 7 was detected by ELISA. Bottom, MDM were harvested and stained for HIV p17. Histograms are shown for a representative donor.(B) Top, cells were lysed after 3 h exposure to infectious agents or at the completion of the infection phase. Intracellular mycobacteria were harvested and assayed for mycobacterial growth at day 0 and day 7. Bottom, MDM were harvested and stained for mycobacterium. Histograms are shown for a representative donor.(C) Cell lysates from panel B were diluted and run in a MGIT 960. Bar and line graphs are reported as mean ± s.e.m. of three independent experiments performed in triplicate. § P<0.05; * P<0.001.
Figure 2HIV and/or M. tuberculosis (TB) infected MDM were treated for 7 days with 100 pmol/L 1,25D3. (A) Cells were lysed and immunoblots of LC3B isoforms using antibody to LC3B or β-actin performed.B) Cells were incubated with 10 µg/mL pepstatin A for 4 h on day 7 prior to lysis. Immunoblots of LC3B isoforms using antibody to LC3B or β-actin.C) Flow cytometry analysis of saponin-resistant LC3B-II in macrophages after 1,25D3 treatment for 7 d. Representative histograms of cells displaying saponin-resistant LC3B-II from three donors are shown.(D) Flow cytometry analysis of saponin-resistant LC3B-II in macrophages after 1,25D3 treatment for 7 d followed by 10 µg/mL pepstatin A for a further 4 h. Representative histograms of cells displaying saponin-resistant LC3B-II from three donors are shown.(E) Immunoblots of p62 using antibody to p62 or β-actin 7 d after macrophages treated with 1,25D3.(F) At 7 d post-infection, aliquots of supernatant taken before the addition of WST-1 were tested for lactate dehydrogenase (LDH) spectrophotometrically using the LDHPLUS assay. For the last hour cells were incubated with WST-1, and the reduction of the WST-1 reagent to its formazan product was monitored spectrophotometrically.
Figure 31,25D3 induces autophagy in human macrophages co-infected with HIV and M. tuberculosis.HIV (A), M. tuberculosis (TB) (B), and HIV/TB dual infected MDM (C) were treated for 7 days with 100 pmol/L 1,25D3, harvested and stained for HIVp17, mycobacteria, and saponin-resistant LC3B-II. Representative density plots from three donors are shown. (D) HIV/M. tuberculosis infected MDM (C) were treated for 7 days with 100 pmol/L 1,25D3, harvested and stained for HIVp17, mycobacteria, and saponin-resistant LC3B-II. Left, representative density plots from three donors from HIV/M. tuberculosis infected MDM are shown for HIV/M. tuberculosis co-infection. Right, histograms of saponin-resistant LC3B-II in macrophages that were HIV+M. tuberculosis− or HIV+day 7+ at day 7 post-infection.
Figure 4MDM were transduced with non-specific scrambled shRNA (shNS) or Beclin-1 shRNA (shBCLN1) and selected using puromycin resistance. Five days later, cells were incubated with 100 pmol/L 1,25D3 or vehicle control for 4 h before infection with HIV and/or M. tuberculosis (TB) for 3 h. Cells were then washed and incubated with 100 pmol/L 1,25D3 or vehicle control for 7 days. (A) Immunoblot analysis performed using antibodies raised to Beclin-1 or β-actin after initial pathogen exposure (Day 0) or after 7 days.(B) ELISA performed for HIV p24 antigen release over 7 d.(C) Intracellular mycobacteria were harvested and assayed for mycobacterial growth by cfu enumeration at day 0 and day 7.D) Intracellular mycobacteria harvested and assayed for viability based on time to positivity (75 growth units) at day 0 and day 7 using the MGIT 960. All bar and line graphs are reported as mean ± s.e.m. of three independent experiments performed in triplicate. § P<0.05; * P<0.001.
Figure 5MDM were transduced with non-specific scrambled shRNA (shNS) or ATG5 shRNA (shATG5) and selected using puromycin resistance. Five days later, cells were incubated with 100 pmol/L 1,25D3 or vehicle control for 4 h before infection with HIV and/or M. tuberculosis (TB) for 3 h. Cells were then washed and incubated with 100 pmol/L 1,25D3 or vehicle control for 7 days. (A) Immunoblot analysis performed using antibodies raised to ATG5 or β-actin after initial pathogen exposure (Day 0) or after 7 days(B) ELISA performed for HIV p24 antigen release over 7 d.(C) Intracellular mycobacteria were harvested and assayed for mycobacterial growth by cfu enumeration at day 0 and day 7. ((D) Intracellular mycobacteria harvested and assayed for viability based on time to positivity (75 growth units) at day 0 and day 7 using the MGIT 960. All bar and line graphs are reported as mean ± s.e.m. of three independent experiments performed in triplicate. § P<0.05; * P<0.001.
Figure 6MDM were pretreated with bafilomycin A1 (Baf A1) before treatment with 1,25D3 and subsequent infection with HIV and/or M. tuberculosis (TB). (A) ELISA performed for HIV p24 antigen release at days 0, 4 and 7.(B) Intracellular mycobacteria were harvested and assayed for mycobacterial growth by cfu enumeration at day 0 and 7.(C) Intracellular mycobacteria harvested and assayed for viability using the MGIT 960 based on time to positivity at day 0 and 7. All bar and line graphs are reported as mean ± s.e.m. of three independent experiments performed in triplicate. § P<0.05; * P<0.01.
Figure 7SID 26681509 inhibits the 1,25D3 mediated inhibition of HIV and M. tuberculosis replication.MDM were pretreated with SID 26681509 (SID) before treatment with 1,25D3 and subsequent infection with HIV and/or M. tuberculosis (TB). (A) ELISA performed for HIV p24 antigen release at days 0, 4 and 7.B) Intracellular mycobacteria were harvested and assayed for mycobacterial growth by cfu enumeration at day 0 and 7.(C) Intracellular mycobacteria harvested and assayed for viability using the MGIT 960 based on time to positivity at day 0 and 7. All bar and line graphs are reported as mean ± s.e.m. of three independent experiments performed in triplicate. § P<0.05; * P<0.01.
Figure 8Inhibition of HIV and M. tuberculosis by 1,25D3 is CAMP and autophagy dependent.MDM were transduced with non-specific scrambled shRNA (shNS) or CAMP shRNA (shCAMP) and selected using puromycin resistance. Five days later, cells were incubated with 100 pmol/L 1,25D3 or vehicle control for 4 h before infection with HIV and/or M. tuberculosis (TB) for 3 h. Cells were then washed and incubated with 100 pmol/L 1,25D3 or vehicle control for 7 days. (A) Immunoblot analysis performed using antibodies raised to CAMP or β-actin after initial pathogen exposure (Day 0) or after 7 days.(B) Day 7 HIV-1Ba-L and TB co-infected cells were harvested and stained for saponin-resistant LC3B-II at day 7. A representative histogram from three donors are shown.C) ELISA performed for HIV p24 antigen release over 7 d.(D) Intracellular mycobacteria were harvested and assayed for mycobacterial growth by cfu enumeration at day 0 and day 7. All bar and line graphs are reported as mean ± s.e.m. of three independent experiments performed in triplicate. * P<0.001.