rapalog  (TaKaRa)

Journal: The Journal of Cell Biology

Article Title: Optogenetic and chemical genetic tools for rapid repositioning of vimentin intermediate filaments

doi: 10.1083/jcb.202504004

Figure Lengend Snippet: Repositioning of the vimentin network by rapalog-induced recruitment of kinesin motors. (A) A scheme of rapalog-induced heterodimerization constructs. A truncated motor domain of P. patens kinesin-14b (ppKin14, amino acids 861–1321) is fused to the FRB domain, along with a GCN4 leucine zipper for tetramerization and a BFP tag for detection. A fragment of human KIF5A (amino acids 1–560) is fused to an HA-tagged dimeric motor domain and the FRB domain. The FKBP domain and mCherry were fused to the C terminus of vimentin. Flexible glycine-serine linkers separate protein domains. (B and C) Schemes illustrating vimentin repositioning triggered by rapalog-induced recruitment of kinesins at the level of a single microtubule (B) and in whole cells (C). Without rapalog, Vim-mCh-FKBP does not interact with either FRB-BFP-GCN4-ppKin14 or HA-KIF5A-FRB. Upon rapalog addition, FRB and FKBP heterodimerize, triggering motor attachment to vimentin filaments and their movement along microtubules. Minus-end-directed kinesins trigger vimentin clustering around the MTOC, whereas plus end–directed kinesins cause the formation of small peripheral clusters and a large perinuclear one. (D and E) Representative fluorescence images of COS-7 cells co-transfected with Vim-mCh-FKBP and FRB fusions of the indicated motors, with or without 1 h of rapalog treatment. The top panels show untreated cells, whereas the bottom panels display rapalog-treated cells. Transfected cells are outlined with dashed lines, and non-transfected cells serve as controls. Anti-vimentin and anti-HA antibodies detect total vimentin and HA-KIF5A-FRB, respectively. (F) Quantifications of the fraction of the cell area occupied by vimentin in untransfected and transfected cells with either minus- or plus-end motor constructs, with and without rapalog treatment. (G–I) Mean fluorescence intensities of Vim-mCh-FKBP intensity (G), FRB-BFP-GCN4-ppKin14 (H), and HA-KIF5A-FRB, detected with anti-HA antibody (I) per cell, normalized to untransfected cells. In (F–I), n = 19–34 cells per condition across three independent experiments. Plots indicate mean ± SD, with individual cell measurements shown as dots. ns, not significant; ∗, P < 0.05; ∗∗∗∗, P < 0.0001. Statistical significance was assessed using the Mann–Whitney t test for (H) and (I), while the Kruskal–Wallis test followed by Dunn’s multiple comparisons test was applied for (F) and (G). ( J and L) Live-cell imaging reveals the morphology of the vimentin network immediately before and at 10, 20, and 60 min after rapalog addition. Cells co-transfected with ppKin14 (J) or KIF5A constructs (L) show vimentin reorganization over time. (K and M) Cells shown in J and L, fixed and stained with antibodies against vimentin 60 min after rapalog treatment.

Article Snippet: A/C heterodimerizer (rapalog) , Takara , AP21967.

Techniques: Construct, Fluorescence, Transfection, MANN-WHITNEY, Live Cell Imaging, Staining

Journal: The Journal of Cell Biology

Article Title: Optogenetic and chemical genetic tools for rapid repositioning of vimentin intermediate filaments

doi: 10.1083/jcb.202504004

Figure Lengend Snippet: Motor recruitment to the filaments with the rapalog system and repositioning of the vimentin network in U2OS cells. (A) Fluorescence images of COS-7 cells expressing Vim-mCh-FKBP. The overexpressed vimentin is visualized by mCherry fluorescence, while total vimentin intensity is detected via anti-vimentin immunostaining. The dashed box indicates the region of the cell enlarged in the zoom image. Images were captured using Airyscan microscopy. (B and C) Fluorescence images of COS-7 cells co-transfected with Vim-mCh-FKBP and either FRB-GFP-GCN4-ppKin14 (B) or HA-KIF5A-FRB (C), with or without 5-min rapalog treatment. The top panels show untreated cells, while the bottom panels display rapalog-treated cells. FRB-GFP-GCN4-ppKin14 and HA-KIF5A-FRB are detected using anti-GFP and anti-HA antibodies respectively. Images were captured using Airyscan microscopy. (D and E) Representative fluorescence images of U2OS cells co-transfected with Vim-mCh-FKBP and FRB fusions of the indicated motors, with or without 1 h of rapalog treatment. Transfected cells are outlined with dashed lines, and non-transfected cells serve as controls. Anti-vimentin and anti-HA antibodies detect total vimentin and HA-KIF5A-FRB, respectively.

Article Snippet: A/C heterodimerizer (rapalog) , Takara , AP21967.

Techniques: Fluorescence, Expressing, Immunostaining, Microscopy, Transfection

Journal: The Journal of Cell Biology

Article Title: Optogenetic and chemical genetic tools for rapid repositioning of vimentin intermediate filaments

doi: 10.1083/jcb.202504004

Figure Lengend Snippet: Rapalog-induced vimentin repositioning by minus end–directed kinesin. COS-7 cell co-expressing Vim-mCh-FKBP and FRB-BFP-GCN4-ppKin14. Imaging was conducted for a total of 70 min; rapalog was added at the 10-min mark. Time is shown in hh:mm:ss.

Article Snippet: A/C heterodimerizer (rapalog) , Takara , AP21967.

Techniques: Expressing, Imaging

Journal: The Journal of Cell Biology

Article Title: Optogenetic and chemical genetic tools for rapid repositioning of vimentin intermediate filaments

doi: 10.1083/jcb.202504004

Figure Lengend Snippet: Rapalog-induced vimentin repositioning by plus end–directed kinesin. COS-7 cell co-expressing Vim-mCh-FKBP and HA-KIF5A-FRB. Total imaging time: 70 min; rapalog is added at the 10-min mark. Time is shown in hh:mm:ss.

Article Snippet: A/C heterodimerizer (rapalog) , Takara , AP21967.

Techniques: Expressing, Imaging

Journal: The Journal of Cell Biology

Article Title: Optogenetic and chemical genetic tools for rapid repositioning of vimentin intermediate filaments

doi: 10.1083/jcb.202504004

Figure Lengend Snippet: Rapalog-induced vimentin repositioning by plus end–directed kinesin. COS-7 cell co-expressing Vim-mCh-FKBP and HA-KIF5A-FRB. The movie starts from the moment the rapalog is added. Time is shown in hh:mm:ss.

Article Snippet: A/C heterodimerizer (rapalog) , Takara , AP21967.

Techniques: Expressing

Journal: The Journal of Cell Biology

Article Title: Optogenetic and chemical genetic tools for rapid repositioning of vimentin intermediate filaments

doi: 10.1083/jcb.202504004

Figure Lengend Snippet: Effects of vimentin repositioning on the microtubule cytoskeleton. (A and B) COS-7 cells were co-transfected with Vim-mCh-FKBP and either FRB-BFP-GCN4-ppKin14 (A) or HA-KIF5A-FRB (B), with non-transfected cells as controls. Transfected cells are outlined with a dashed line. After 1 h of treatment with or without rapalog, cells were fixed for analysis. Representative fluorescent images display the microtubule network, labeled with antibodies against tyrosinated α-tubulin (Tyr-tubulin). Microtubules are further color-coded to indicate radial (cyan) and non-radial (yellow) orientations. (C) Quantification of tyrosinated α-tubulin intensity in cells expressing Vim-mCh-FKBP along with either FRB-BFP-GCN4-ppKin14 or HA-KIF5A-FRB, normalized to non-transfected cells, with or without rapalog treatment. n = 21–23 cells analyzed across two independent experiments. (D) Quantification of the ratio of non-radial to radial tyrosinated microtubules in non-transfected cells and cells expressing Vim-mCh-FKBP with either FRB-BFP-GCN4-ppKin14 or HA-KIF5A-FRB, with or without rapalog treatment. n = 20–22 cells were analyzed across two independent experiments. (E and F) Representative images of U2OS cells stained for acetylated microtubules (Ac-tubulin), showing either untransfected cells or cells co-expressing Vim-mCh-FKBP with either FRB-BFP-GCN4-ppKin14 (E) or HA-KIF5A-FRB (F). Transfected cells are outlined with a dashed line. After 1 h of treatment with or without rapalog, cells were fixed for analysis. (G) Quantification of normalized acetylated microtubule intensity in untransfected U2OS cells and cells co-expressing Vim-mCh-FKBP with either FRB-BFP-GCN4-ppKin14 or HA-KIF5A-FRB, in the presence and absence of rapalog. n = 37–49 cells were analyzed across three independent experiments. Plots indicate mean ± SD, with individual cell measurements shown as dots. ns, not significant; ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001; ∗∗∗∗, P < 0.0001 as assessed by Kruskal–Wallis test.

Article Snippet: A/C heterodimerizer (rapalog) , Takara , AP21967.

Techniques: Transfection, Labeling, Expressing, Staining

Journal: The Journal of Cell Biology

Article Title: Optogenetic and chemical genetic tools for rapid repositioning of vimentin intermediate filaments

doi: 10.1083/jcb.202504004

Figure Lengend Snippet: Effects of vimentin repositioning on the actin cytoskeleton, cell spreading, and focal adhesions. (A–F) COS-7 (A–C) or U2OS (D–F) cells were co-transfected with Vim-mCh-FKBP and FRB-BFP-GCN4-ppKin14, while non-transfected cells served as controls. Transfected cells are outlined with a dashed line. (A and D) After 1 h of treatment with or without rapalog, the cells were fixed and stained for total vimentin, paxillin, and actin using phalloidin. (B and E) Quantification of the total cell area based on the phalloidin staining in transfected (T) and untransfected (U) cells, with and without rapalog treatment. Dashed boxes show regions enlarged in the zoom panels. (C and F) Quantification of the total focal adhesion number based on paxillin staining, normalized to cell area as determined by phalloidin staining in both transfected and untransfected cells, with or without rapalog treatment. Measurements were collected from n = 28–31 cells in B and C and from n = 27–29 cells in E and F across three independent experiments. The plots display the mean ± SD, with individual cell measurements represented as dots. ns, not significant, determined by Kruskal–Wallis analysis.

Article Snippet: A/C heterodimerizer (rapalog) , Takara , AP21967.

Techniques: Transfection, Staining

Journal: The Journal of Cell Biology

Article Title: Optogenetic and chemical genetic tools for rapid repositioning of vimentin intermediate filaments

doi: 10.1083/jcb.202504004

Figure Lengend Snippet: Effects of vimentin clustering on the keratin-8 network across cell lines. (A, C, and E) Indicated cell lines were co-transfected with Vim-mCh-FKBP and FRB-BFP-GCN4-ppKin14 constructs (transfected cells are outlined), while non-transfected cells served as controls. After 1 h of treatment with or without rapalog, the cells were fixed for analysis. Total vimentin and keratin-8 network intensities were measured after staining with anti-vimentin and anti–keratin-8 antibodies, respectively. The white dashed box indicates the region of the cell enlarged in zoom images. Images were obtained with Airyscan microscopy. (B, D, and F) Colocalization analysis in COS-7 (B), U2OS (D), and HeLa (F) cells. The graphs represent Manders’ coefficients of thresholded images, measured from 50.2 × 50.2-µm ROIs per COS-7 cell (B), 45.11 × 45.11-µm ROIs per U2OS cell (D), and 35.03 × 35.03-µm ROIs per HeLa cell (F). Graphs show mean ± SD, with individual cell measurements represented by dots. In B, data were collected from n = 25–29 cells; in D, n = 27–29 cells; and in F, n = 27–34 cells across three independent experiments. ns, not significant; ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗∗, P < 0.0001 based on Kruskal–Wallis statistical analysis.

Article Snippet: A/C heterodimerizer (rapalog) , Takara , AP21967.

Techniques: Transfection, Construct, Staining, Microscopy

Journal: The Journal of Cell Biology

Article Title: Optogenetic and chemical genetic tools for rapid repositioning of vimentin intermediate filaments

doi: 10.1083/jcb.202504004

Figure Lengend Snippet: Effects of vimentin repositioning on cell stiffness. (A and B) Schematic depiction of a control and a rapalog-treated U2OS cell co-transfected with Vim-mCh-FKBP and FRB-BFP-GCN4-ppKin14, before and during indentation in the perinuclear region using a spherical tip of 3.5-µm radius. Each cell was indented at three separate locations in the perinuclear region. (C) The graph presents the Young’s modulus of cells co-transfected with Vim-mCh-FKBP and FRB-BFP-GCN4-ppKin14 constructs, with and without rapalog treatment. Data were collected from n = 19–22 cells across two independent experiments. A total of 56 measurements were obtained from 19 cells without rapalog, and 63 measurements from 22 cells treated with rapalog. The plots display the mean ± SD, with each dot representing an individual cell measurement. ****, P < 0.0001 via Mann–Whitney test.

Article Snippet: A/C heterodimerizer (rapalog) , Takara , AP21967.

Techniques: Control, Transfection, Construct, MANN-WHITNEY

Journal: The Journal of Cell Biology

Article Title: Optogenetic and chemical genetic tools for rapid repositioning of vimentin intermediate filaments

doi: 10.1083/jcb.202504004

Figure Lengend Snippet: Effects of vimentin repositioning on ER morphology. (A and B) Representative image of a COS-7 cell stained for endogenous calnexin (ER) and vimentin, and (B) intensity profile along the indicated line. Images were collected using STED microscopy. (C–E) U2OS cells co-transfected with Vim-mCh-SspB, iLID-GFP-GCN4-ppKin14, and Halo-KDEL were first imaged for 5 min without 488-nm pulsing (−5 min, 0 min), then imaged for 25 min with whole-cell 488-nm pulsing to induce optogenetic vimentin clustering (5, 10, 25 min). (C) Spinning disc confocal images from the experiment, with black dashed boxes indicating the region of the cell enlarged in the zoom panels. (D) Plot showing ratio of perinuclear fluorescence intensity to intensity at the cell periphery. (E) Plot showing loss of total fluorescence signal over time. (F) Images of COS-7 cells expressing Vim-mCh-FKBP and FRB-BFP-GCN4-ppKin14. Transfected cells are outlined with dashed lines. After 1 h of treatment with or without rapalog, cells were fixed and stained for vimentin and calnexin. The images show the distribution of the ER network, visualized by calnexin staining, in both transfected (T) and untransfected (U) cells, in the presence and absence of rapalog. Dashed boxes show the regions that have been enlarged and shown as masks in the zoom images.

Article Snippet: A/C heterodimerizer (rapalog) , Takara , AP21967.

Techniques: Staining, Microscopy, Transfection, Fluorescence, Expressing

Journal: The Journal of Cell Biology

Article Title: Optogenetic and chemical genetic tools for rapid repositioning of vimentin intermediate filaments

doi: 10.1083/jcb.202504004

Figure Lengend Snippet: Effects of vimentin clustering on the distribution of mitochondria and lysosomes. (A and D) U2OS cell, co-transfected with Vim-mCh-SspB, iLID-GFP-GCN4-ppKin14, and Halo-MitoTag to label mitochondria (A) or LAMP1-Halo to label lysosomes (D), locally illuminated with 488-nm pulses inside an ROI (blue dashed box) for 10 min (A) or with global illumination for 20 min (D). The cell is shown before application of blue light (0 min) and at the indicated time points after blue light pulsing. (B and E) Imaging of U2OS cells transfected with Vim-mCh-FKBP and FRB-BFP-GCN4-ppKin14 constructs. After 1 h of treatment with or without rapalog, cells were subjected to immunostaining for total vimentin and cytochrome C to label mitochondria (B) or LAMTOR4 to label lysosomes (E). Transfected cells are indicated by a dotted outline. Images were acquired with Airyscan mode. (C and F) The distribution of mitochondria (C) or lysosomes (F) relative to the MTOC. Organelles were detected using the ComDet plugin, and their distance from the MTOC was calculated using a radius plugin. The graph shows the percentage of mitochondria or lysosomes located in the peripheral region, which was defined as an area beyond a 13.81-μm radius from the MTOC, with each dot representing data from an individual cell. Measurements were collected from n = 20–29 cells in C and from n = 20–22 cells in F across three independent experiments. The plots display the mean ± SD, with individual cell measurements represented as dots. ns, not significant; ∗∗, P < 0.01 by Mann–Whitney test.

Article Snippet: A/C heterodimerizer (rapalog) , Takara , AP21967.

Techniques: Transfection, Imaging, Construct, Immunostaining, MANN-WHITNEY

Journal: Nature Cell Biology

Article Title: Reconstitution of BNIP3/NIX-mitophagy initiation reveals hierarchical flexibility of the autophagy machinery

doi: 10.1038/s41556-025-01712-y

Figure Lengend Snippet: a , Schematic of the experimental approach and effect of rapalog treatment, leading to the tethering of WIPI proteins to the outer mitochondrial membrane. IMS, intermembrane space; OMM, outer mitochondrial membrane. b , Mitophagy flux measured by flow cytometry in WT HeLa cells expressing Fis1-FRB, FKBP–GFP–WIPI1/2/3/4 and mt-mKeima, not induced or induced for 24 h by rapalog treatment. Data are presented as mean ± s.d. ( n = 4 biologically independent experiments). Two-way ANOVA with Šídák’s multiple comparisons test. c , As in b but with or without the addition of the VPS34-inhibitor VPS34-IN1. Data are presented as mean ± s.d. ( n = 5 biologically independent experiments). Dunnett’s multiple comparisons test. d , As in b but in NIX/BNIP3 2KO HeLa cells expressing Fis1-FRB, FKBP–GFP–WIPI2 and mt-mKeima. Data are presented as mean ± s.d. ( n = 3 biologically independent experiments). Unpaired t -test. e , Immunoblotting for LC3B lipidation (LC3B-II) and turnover of the mitochondrial protein COXII in WT or NIX/BNIP3 2KO HeLa cells before and after tethering of FKBP–WIPI1/2/3 with rapalog. Results are representative of three biological replicates. f – h , Quantitative proteome analysis of WT HeLa cells treated for 24 h with DFP ( f ) or quantitative proteome analysis of WT ( g ) and NIX/BNIP3 2KO ( h ) HeLa cells treated for 24 h with rapalog to artificially tether FKBP–WIPI2 to the outer mitochondrial membrane. Mitochondrial proteins as annotated in MitoCarta 3.0 are marked in red ( n = 3 biologically independent experiments). Moderated t -statistics were calculated and multiple testing correction was applied using the Benjamini–Hochberg method. **** P < 0.0001. NS, not significant. Source numerical data, including exact P values, and unprocessed blots are available in the source data. Schematic generated with BioRender.

Article Snippet: The following chemicals were used in this study: rapalog A/C hetero-dimerizer (635057, Takara), bafilomycin A1 (sc-201550, Santa Cruz Biotech), TBK1 inhibitor GSK8612 (S8872, Selleck Chemicals), ULK1/2 inhibitor (MRT68921, BLDpharm), Vps34-IN1 inhibitor (APE-B6179, ApexBio), CK2 kinase inhibitor (CX4945, Selleck Chemicals), deferiprone (DFP; 379409, Sigma-Aldrich), oligomycin A (A5588, ApexBio), antimycin A1 (A8674, Sigma-Aldrich), Q-VD-OPh (A1901, ApexBio) and dimethylsulfoxide (DMSO; D2438, Sigma).

Techniques: Membrane, Flow Cytometry, Expressing, Western Blot, Generated

Journal: Nature Cell Biology

Article Title: Reconstitution of BNIP3/NIX-mitophagy initiation reveals hierarchical flexibility of the autophagy machinery

doi: 10.1038/s41556-025-01712-y

Figure Lengend Snippet: a , Representative maximum intensity projection images of WT HeLa cells stably expressing Fis1-FRB and FKBP–GFP–WIPI2. Cells were left untreated or treated with rapalog for 16 h and immunostained for ATG13. Scale bars, 20 µm and 10 µm (zooms). Results are representative of two biologically independent replicates. b , Immunoblotting for phosphorylated ATG13 in HeLa cells overexpressing Fis1-FRB and FKBP–EGFP–WIPI2d, treated with rapalog for the indicated time. Results are representative of three biologically independent replicates. c , Mitophagy flux measured by flow cytometry in WT HeLa cells transfected with siRNAs targeting FIP200 or ATG13, and expressing Fis1-FRB, FKBP–GFP–WIPI1/2/3 and mt-mKeima, not induced or induced for 24 h by rapalog treatment. Data are presented as mean ± s.d. ( n = 4 biologically independent experiments). Two-way ANOVA with Dunnett’s multiple comparisons test. d , e , As in c , but with or without the addition of the ULK1/2 inhibitor MRT68921 ( d ) or the VPS34-inhibitor VPS34-IN1 ( e ). Data are presented as mean ± s.d. ( n = 6 biologically independent experiments in d and n = 3 in e ). Two-way ANOVA with Dunnett’s multiple comparisons test. f , g , As in c , but with WT HeLa cells expressing mt-mKeima and transfected with siRNAs targeting ATG13, FIP200 or ULK1, and treated with DFP for 24 h. Data are presented as mean ± s.d. ( n = 3 biologically independent experiments). One-way ANOVA with Dunnett’s multiple comparisons test. h , i , As in f , but with the kinase inhibitors GSK8612 for TBK1, MRT68921 for ULK1/2, VPS34-IN1 for VPS34, or bafilomycin A1 (BafA1). Data are presented as mean ± s.d. ( n = 3 biologically independent experiments in h and n = 4 in i ). Two-way ANOVA with Dunnett’s multiple comparisons test ( i ) or one-way ANOVA with Dunnett’s multiple comparisons test ( h ). j , Immunofluorescence images of WIPI2 and mitochondrial HSP60 in WT or FIP200 KO HeLa cells, untreated or treated for 24 h with DFP. All cells were depleted for PPTC7, aiding the visualization of mitophagy events. Scale bars, 20 µm and 5 µm (zoom). Data are representative of two biologically independent experiments. k , Quantification of the percentage of cells in each field of view that contained autophagosome-like cup structures that colocalized with the mitochondrial marker HSP60. Data are presented as mean ± s.d. ( n = 4 biologically independent biological samples). One-way ANOVA with Dunnett’s multiple comparisons test. ** P < 0.005, *** P < 0.001, **** P < 0.0001. NS, not significant. Source numerical data, including exact P values, and unprocessed blots are available in the source data.

Article Snippet: The following chemicals were used in this study: rapalog A/C hetero-dimerizer (635057, Takara), bafilomycin A1 (sc-201550, Santa Cruz Biotech), TBK1 inhibitor GSK8612 (S8872, Selleck Chemicals), ULK1/2 inhibitor (MRT68921, BLDpharm), Vps34-IN1 inhibitor (APE-B6179, ApexBio), CK2 kinase inhibitor (CX4945, Selleck Chemicals), deferiprone (DFP; 379409, Sigma-Aldrich), oligomycin A (A5588, ApexBio), antimycin A1 (A8674, Sigma-Aldrich), Q-VD-OPh (A1901, ApexBio) and dimethylsulfoxide (DMSO; D2438, Sigma).

Techniques: Stable Transfection, Expressing, Western Blot, Flow Cytometry, Transfection, Immunofluorescence, Marker

Journal: Nature Cell Biology

Article Title: Reconstitution of BNIP3/NIX-mitophagy initiation reveals hierarchical flexibility of the autophagy machinery

doi: 10.1038/s41556-025-01712-y

Figure Lengend Snippet: a , Mitophagy flux measured by flow cytometry in WT HeLa cells expressing Fis1-FRB, FKBP–GFP–WIPI2 WT or ATG16L1-binding mutant R108E/R125E, and mt-mKeima, not induced or induced for 24 h by rapalog treatment. Data are presented as mean ± s.d. ( n = 5 biologically independent experiments). Two-way ANOVA with Šídák’s multiple comparisons test. b , Microscopy-based bead assay of agarose beads coated with GFP-tagged ULK1 complex (consisting of FIP200-GFP, ULK1, ATG13, ATG101) and incubated with mCherry-tagged WIPI proteins. c , As in b , but with GFP-tagged ATG13/101 subcomplex and incubated with mCherry-tagged WIPI proteins. d , As in b , but with GFP-tagged FIP200-coated beads and incubated with mCherry-tagged WIPI proteins. e , As in b , but with GFP-tagged ULK1-coated beads and incubated with mCherry-tagged WIPI proteins. f , As in b , but with GFP-tagged ATG13/101-coated agarose beads incubated with mCherry-tagged full-length (FL) or IDR only (residues 364–425) WIPI2d. g , Mitophagy flux measured by flow cytometry in WT HeLa cells expressing Fis1-FRB, FL or IDR only (364–425 aa) FKBP–GFP–WIPI2, and mt-mKeima, not induced or induced for 24 h by rapalog treatment. Data are presented as mean ± s.d. ( n = 4 biologically independent experiments). Two-way ANOVA with Šídák’s multiple comparisons test. **** P < 0.0001. NS, not significant. Results are representative of three biologically independent replicates ( b – f ). Scale bars, 100 µm. Source numerical data, including exact P values, are available in the source data.

Article Snippet: The following chemicals were used in this study: rapalog A/C hetero-dimerizer (635057, Takara), bafilomycin A1 (sc-201550, Santa Cruz Biotech), TBK1 inhibitor GSK8612 (S8872, Selleck Chemicals), ULK1/2 inhibitor (MRT68921, BLDpharm), Vps34-IN1 inhibitor (APE-B6179, ApexBio), CK2 kinase inhibitor (CX4945, Selleck Chemicals), deferiprone (DFP; 379409, Sigma-Aldrich), oligomycin A (A5588, ApexBio), antimycin A1 (A8674, Sigma-Aldrich), Q-VD-OPh (A1901, ApexBio) and dimethylsulfoxide (DMSO; D2438, Sigma).

Techniques: Flow Cytometry, Expressing, Binding Assay, Mutagenesis, Microscopy, Incubation