Journal: Clinical Cancer Research

Article Title: The Investigational Aurora Kinase A Inhibitor MLN8237 Induces Defects in Cell Viability and Cell-Cycle Progression in Malignant Bladder Cancer Cells In Vitro and In Vivo

doi: 10.1158/1078-0432.ccr-12-2383

Figure Lengend Snippet: Figure 1. Mitotic spindle checkpoint genes are broadly overexpressed in human urothelial carcinoma. A, human samples of normal urothelium (N ¼ 10) and urothelial carcinoma of the bladder (N ¼ 8) were subjected to RNA microarray. A subset of 13 gene transcripts related to the mitotic spindle checkpoint, including Aurora A and B, were upregulated at least 5-fold in the urothelial carcinoma (UCC) compared with the normal urothelium. B, upregulation of these genes was validated by 2-step quantitative real-time PCR on a separate set of human samples of urothelial carcinoma (N ¼ 3) and normal urothelium (N ¼ 3). Ten of 13 genes (asterisked) showed statistical significance (t test; P < 0.05) for differential expression in urothelial carcinoma compared with normal urothelium.

Article Snippet: Cell culture and drug treatments Human urothelial carcinoma cell lines T24, UM-UC-3, and RT4 were purchased from the American Type Culture Collection and cultured at 37 C and 5%CO2 in RPMI-1640 media (Gibco) supplemented with 10% FBS (Gibco).

Techniques: Microarray, Real-time Polymerase Chain Reaction, Quantitative Proteomics

Journal: Clinical Cancer Research

Article Title: The Investigational Aurora Kinase A Inhibitor MLN8237 Induces Defects in Cell Viability and Cell-Cycle Progression in Malignant Bladder Cancer Cells In Vitro and In Vivo

doi: 10.1158/1078-0432.ccr-12-2383

Figure Lengend Snippet: Figure 2. MLN8237 (MLN) induces cell-cycle arrest and aneuploidy of bladder cancer cell lines. A, MLN8237 inhibited expression of phospho-Aurora A-T288 at mitotic spindles. B, MLN8237 showed specificity for inhibiting Aurora A, as expression of histone-H3 and phospho-histone-H3, markers of Aurora B function, was maintained. C, PI staining with flow cytometry analysis was conducted to assess cell-cycle changes. T24, UM-UC- 3, and RT4 cells were treated with 10 nmol/L to 1 mmol/L MLN8237 for 48 hours. All 3 cell lines showed dramatic cell-cycle arrest and increase in the 4N cell population in a dose-dependent manner. T24 and UM-UC-3 cells also showed a considerable increase in aneuploidy, whereas RT4 cells did not.

Article Snippet: Cell culture and drug treatments Human urothelial carcinoma cell lines T24, UM-UC-3, and RT4 were purchased from the American Type Culture Collection and cultured at 37 C and 5%CO2 in RPMI-1640 media (Gibco) supplemented with 10% FBS (Gibco).

Techniques: Expressing, Staining, Cytometry

Journal: Clinical Cancer Research

Article Title: The Investigational Aurora Kinase A Inhibitor MLN8237 Induces Defects in Cell Viability and Cell-Cycle Progression in Malignant Bladder Cancer Cells In Vitro and In Vivo

doi: 10.1158/1078-0432.ccr-12-2383

Figure Lengend Snippet: Figure 3. Cellular phenotypes of T24 and RT4 cells differ after MLN8237 (MLN) treatment. A, MLN8237 induces a dramatic increase in cell size in T24 cells but not RT4 cells. B, immunocytochemistry and fluorescence microscopy of T24 and RT4 cells revealed the formation of aberrant spindle figures upon MLN8237 treatment, with multipolar spindle apparatuses and failure of localization of chromatids to a single metaphase plate. C, T24 cells show a phenotype of increased cell size and ploidy, whereas RT4 cells do not. D, T24 cells also exhibit a subpopulation of cells exhibiting marked cytoplasmic Aurora A expression, whereas RT4 cells lacked cytoplasmic Aurora A expression. E, real-time imaging of T24 and RT4 cells treated with MLN8237 was conducted over 48 hours. T24 cells exhibited dramatic increases in cell size as a result of repeated cell-cycle progressions without separation of daughter cells. RT4 cells seemed to become arrested after one failed mitotic attempt, preventing repeated cell-cycle progressions that could otherwise result in increased ploidy.

Article Snippet: Cell culture and drug treatments Human urothelial carcinoma cell lines T24, UM-UC-3, and RT4 were purchased from the American Type Culture Collection and cultured at 37 C and 5%CO2 in RPMI-1640 media (Gibco) supplemented with 10% FBS (Gibco).

Techniques: Immunocytochemistry, Microscopy, Expressing, Imaging

Journal: Clinical Cancer Research

Article Title: The Investigational Aurora Kinase A Inhibitor MLN8237 Induces Defects in Cell Viability and Cell-Cycle Progression in Malignant Bladder Cancer Cells In Vitro and In Vivo

doi: 10.1158/1078-0432.ccr-12-2383

Figure Lengend Snippet: Figure 4. MLN8237 (MLN) induces cytotoxicity and differential apoptotic processes. A, MTS assay was used to calculate IC50 values for each cell line following treatment over a range of MLN8237 concentrations for 48 hours. MLN8237 exhibited highest potency in T24 and UM-UC-3 cells (IC50 of 31 and 45 nmol/L, respectively) and lowest potency in RT4cells (IC50 of 120 nmol/L). B, Western blot analysis of T24 and RT4 cells for apoptotic markers revealed induction of p53 expression in RT4 cells, and induction of p73, but not p53, expression in T24 cells. Both cell lines showed increased expression of the apoptotic marker cleaved PARP starting 24 hours after initiation of treatment. C, annexin V staining with flow cytometry analysis of T24 and RT4 cells revealed an increased apoptotic cell fraction at 48 and 72 hours after initiation of MLN8237 treatment. D, clonogenic assays of T24 and RT4 cells showed 90% inhibition of long-term clone forming capability at 100 nmol/L MLN8237.

Article Snippet: Cell culture and drug treatments Human urothelial carcinoma cell lines T24, UM-UC-3, and RT4 were purchased from the American Type Culture Collection and cultured at 37 C and 5%CO2 in RPMI-1640 media (Gibco) supplemented with 10% FBS (Gibco).

Techniques: MTS Assay, Western Blot, Expressing, Marker, Staining, Cytometry, Inhibition

Journal: Clinical Cancer Research

Article Title: The Investigational Aurora Kinase A Inhibitor MLN8237 Induces Defects in Cell Viability and Cell-Cycle Progression in Malignant Bladder Cancer Cells In Vitro and In Vivo

doi: 10.1158/1078-0432.ccr-12-2383

Figure Lengend Snippet: Figure 6. Interactions of MLN8237 with paclitaxel and gemcitabine in vitro are schedule-dependent. MLN8237 (MLN) was combined with either paclitaxel (PTX) or gemcitabine (Gem) in T24 cells. Drugs were administered either simultaneously for 48 hours (left), or sequentially, with one drug for 48 hours, followed by washout and the other drug for 48 hours (middle and right). MTS assay was used to quantify the effect on cell viability of these combination treatments. MLN8237 showed synergistic effects with paclitaxel and gemcitabine when dosed sequentially (middle and right), and antagonistic effects when dosed simultaneously (left).

Article Snippet: Cell culture and drug treatments Human urothelial carcinoma cell lines T24, UM-UC-3, and RT4 were purchased from the American Type Culture Collection and cultured at 37 C and 5%CO2 in RPMI-1640 media (Gibco) supplemented with 10% FBS (Gibco).

Techniques: In Vitro, MTS Assay

Journal: International journal of molecular sciences

Article Title: Discovery of Small Molecule COX-1 and Akt Inhibitors as Anti-NSCLC Agents Endowed with Anti-Inflammatory Action.

doi: 10.3390/ijms24032648

Figure Lengend Snippet: Figure 5. Flow cytometric analysis of A549 cells treated with IC50/2 and IC50 concentrations of compounds 3b, 4a, and cisplatin. At least 10,000 cells were analyzed per sample, and quadrant analysis was performed. Q1-LR, Q1-UR, Q1-LL, and Q1-UL quadrants represent early apoptosis, late apoptosis, viability, and necrosis, respectively. (a) Control; (b) Control; (c) Compound 3b at IC50/2 concentration; (d) Compound 3b at IC50 concentration; (e) Compound 4a at IC50/4 concentration; (f) Compound 4a at IC50/2 concentration; (g) Cisplatin at IC50/2 concentration; (h) Cisplatin at IC50 concentration.

Article Snippet: Cell Culture and Drug Treatment A549 human lung adenocarcinoma and L929 mouse fibroblast cell lines were obtained from American Type Culture Collection (ATCC) (Manassas, VA, USA).

Techniques: Control, Concentration Assay

Journal: Experimental & molecular medicine

Article Title: PEX5 regulates autophagy via the mTORC1-TFEB axis during starvation.

doi: 10.1038/s12276-017-0007-8

Figure Lengend Snippet: Fig. 1 PEX5 depletion suppresses starvation-induced autophagy. a HepG2 cells were treated with CTL siRNA (100 nM) and PEX5 siRNA #1 or #2 (100 nM) for 24 h. Then, cells were incubated in the presence or absence of serum. After 24 h, cells were harvested and immunoblotted with antibodies against PEX5, LC3I/II, p62, and β-actin. b Immunoblot analysis of p-70S6K, p-S6R, and p-4E-BP-1, and total 70S6K, S6R, and 4E-BP-1. c Cells were transfected with GFP-LC3 plasmid. After 6 h of transfection, cells were treated with CTL and PEX5 siRNAs. Visualization and quantification of GFP- LC3 puncta were performed by fluorescence microscopy after 48 h of incubation. Representative images from three independent determinations are shown (*P < 0.05). Scale bar: 10 μm. d Immunoblot analysis of TSC2 and PEX5 protein levels. e Transcript levels of TSC2 were measured by Q-PCR (**P < 0.01)

Article Snippet: Cell culture and drug treatments HepG2 (human hepatocyte carcinoma cell line) and HeLa (human epithelial carcinoma cell line) cells were originally purchased from the American Type Culture Collection (ATCC, Manassa, VA, USA).

Techniques: Incubation, Western Blot, Transfection, Plasmid Preparation, Microscopy

Journal: Experimental & molecular medicine

Article Title: PEX5 regulates autophagy via the mTORC1-TFEB axis during starvation.

doi: 10.1038/s12276-017-0007-8

Figure Lengend Snippet: Fig. 3 PEX5 depletion inhibits nuclear translocation of TFEB in response to starvation. a Cells were treated with CTL and PEX5 siRNAs (100 nM) for 24 h. Then, cells were incubated in the presence or absence of serum. After 24 h, cells were stained with an antibody against TFEB and analyzed by fluorescence microscopy. Scale bar: 10 μm. b After HepG2 cells were treated as indicated, cells were subjected to nuclear and cytosol fractionation and immunoblotted with antibodies against TFEB, PCNA, and β-actin. PCNA and β-actin were used as nuclear and cytosolic markers, respectively. c, d Cells were transfected with CTL or TFEB siRNA (100 nM) for 24 h, and then incubated in the presence or absence of serum for 24 h. Protein expression of TFEB, LC3 and SQSTM1/p62 (autophagy proteins), and PMP70, DBP, and catalase (peroxisomal proteins) were measured by immunoblotting. e Q-PCR analysis of peroxisomal genes was performed (**P < 0.01, *P < 0.05)

Article Snippet: Cell culture and drug treatments HepG2 (human hepatocyte carcinoma cell line) and HeLa (human epithelial carcinoma cell line) cells were originally purchased from the American Type Culture Collection (ATCC, Manassa, VA, USA).

Techniques: Translocation Assay, Incubation, Staining, Microscopy, Fractionation, Transfection, Expressing, Western Blot

Journal: Experimental & molecular medicine

Article Title: PEX5 regulates autophagy via the mTORC1-TFEB axis during starvation.

doi: 10.1038/s12276-017-0007-8

Figure Lengend Snippet: Fig. 5 Rapamycin restored TFEB localization and autophagy in PEX5 depletion during serum starvation. Cells transfected with CTL siRNA (100 nM) and PEX5 siRNA (100 nM) for 24 h were pretreated with DMSO or rapamycin (1 μM) for 6 h, and then incubated in the absence of serum for 24 h. (a) After HepG2 cells were treated as indicated, cells were stained with an antibody against TFEB and analyzed by fluorescence microscopy. Scale bar: 10 μm. Data are presented as mean ± SD from three independent experiments (**P < 0.01). (b) After HepG2 cells were treated as indicated, cells were subjected to nuclear and cytosol fractionation and immunoblotted with antibodies against TFEB, PCNA, and β-actin. PCNA and β-actin were used as nuclear and cytosolic markers, respectively. (c) Immunoblot analysis of LC3 and SQSTM1/p62. (d) Immunoblot analysis of PEX5, p-70S6K, p-S6R, and p-4E-BP-1

Article Snippet: Cell culture and drug treatments HepG2 (human hepatocyte carcinoma cell line) and HeLa (human epithelial carcinoma cell line) cells were originally purchased from the American Type Culture Collection (ATCC, Manassa, VA, USA).

Techniques: Transfection, Incubation, Staining, Microscopy, Fractionation, Western Blot

Journal: The Journal of cell biology

Article Title: INF2-mediated actin polymerization at the ER stimulates mitochondrial calcium uptake, inner membrane constriction, and division.

doi: 10.1083/jcb.201709111

Figure Lengend Snippet: Figure 1. The stimulus-induced actin burst precedes the mitochondrial calcium spike. (A and D) Quantification of cytoplasmic calcium (Cyto-R-GECO), cytoplasmic actin (GFP-Ftractin), or mitochondrial calcium (Mito-R-GECO) in U2OS cells after 4 µM ionomycin (A) or 100 µM histamine (D) stimulation. Rapid acquisition mode (4.8 frames/s). Values on the y axis represent the value at time X normalized to the value at time 0 (F/F0); n = 10–16 cells (also given in C). Error bars represent SEM. Corresponds to Videos 1 and 2. (B and E) Zoom of early time points from graphs in A (ionomycin) and D (histamine) showing the distinct kinetics of cytoplasmic calcium, actin, and mitochondrial calcium. Error bars have been removed for clarity. (C and F) Box-and-whiskers plots of stimulation half-times based on the curves in A and D. Number of cells for each reading is 14 (cytoplasmic calcium, ionomycin), 10 (actin, ionomycin), 14 (mitochondrial calcium, ionomycin), 12 (ER calcium, ionomycin), 14 (cytoplasmic calcium, histamine), 11 (actin, histamine), 16 (mitochondrial calcium, histamine), and 10 (ER calcium, histamine). Each point represents one cell. Error bars represent SD (G and H) Time-lapse montages of actin (green, GFP-Ftractin) and mitochondrial calcium (red, mito-R-GECO) changes in the same cell after 4 µM ionomycin (G) or 100 µM histamine (H) stimulation. Mitochondrial calcium alone shown in the top panels and merge in the bottom panels; taken at one frame per 1.03 s (G) and 1.2 s (H). Arrows in the merged panel indicate regions of the cytoplasm displaying the cytoplasmic actin burst. Bars: (G) 5 µm; (H) 10 µm. Corresponds to Videos 3 and 4.

Article Snippet: Cell culture, transfection, and drug treatments Human osteosarcoma U2OS cells (HTB96; American Type Culture Collection) were grown in DMEM (Invitrogen) supplemented with 10% calf serum (Atlanta Biologicals).

Techniques:

Journal: The Journal of cell biology

Article Title: INF2-mediated actin polymerization at the ER stimulates mitochondrial calcium uptake, inner membrane constriction, and division.

doi: 10.1083/jcb.201709111

Figure Lengend Snippet: Figure 2. Calcium release from the ER triggered by ionomycin and histamine. (A) Time-lapse montages of U2OS cells transfected with the ER calcium probe (ER-GCaMP6-150) and treated with DMSO (top), 4 µM ionomycin (middle), or 100 µM histamine (bottom). Time in seconds. Bar, 10 µm. Corre- sponds to Video 5. (B) Graph quantifying changes in ER calcium upon the treatments described in A; n = 12, 15, and 15 cells for DMSO, ionomycin, and histamine, respectively. Error bars represent SEM. (C) Effect of thapsigargin on ER calcium (black curves), actin burst (green curves), and mitochondrial calcium (red curves) before and after ionomycin stimulation. U2OS cells transfected with ER calcium probe and mApple-Ftractin. DMSO (top graph) or 1 µM thapsigargin (bottom graph) was applied at 0 s (bold line) and 4 µM ionomycin was applied at the time indicated by the dashed line; n = 10 cells (DMSO) or 13 cells (thapsigargin). Error bars represent SEM. (D) Effect of thapsigargin on histamine-induced cytoplasmic calcium and ER calcium changes. U2OS cells transfected with ER calcium probe and Cyto-R-GECO; n = 15 cells (DMSO) or 15 cells (thapsigargin). (E) Effect of thapsigargin on histamine-induced actin burst and ER calcium. U2OS cells were transfected with ER calcium probe and mApple-Ftractin; n = 10 cells (DMSO) or 11 cells (thapsigargin). Error bars represent SEM. (F) Effect of thapsigargin on histamine-induced mitochondrial calcium and ER calcium. U2OS cells transfected with ER calcium probe and mito-R-GECO; n = 12 cells (DMSO) or 14 cells (Thapsigargin). Error bars represent SEM. (G) Effect of thapsigargin on iono- mycin-induced cytoplasmic calcium and ER calcium. U2OS cells transfected with both the Cyto-R-Geco and ER-GCaMP6-150; n = 17 cells (DMSO) or 16 cells (thapsigargin). Error bars represent SEM.

Article Snippet: Cell culture, transfection, and drug treatments Human osteosarcoma U2OS cells (HTB96; American Type Culture Collection) were grown in DMEM (Invitrogen) supplemented with 10% calf serum (Atlanta Biologicals).

Techniques: Transfection

Journal: The Journal of cell biology

Article Title: INF2-mediated actin polymerization at the ER stimulates mitochondrial calcium uptake, inner membrane constriction, and division.

doi: 10.1083/jcb.201709111

Figure Lengend Snippet: Figure 3. Actin requirement for stimulus-induced mitochondrial calcium spike. (A and B) Effect of 2 μM LatA treatment on ionomycin-induced (A) or histamine-induced (B) actin polymerization burst and mitochondrial calcium spike in U2OS cells; n = 10–15 cells. Error bars represent SEM. (C and D) Mitochondrial calcium spike after ionomycin (C) or histamine (D) stimulus in U2OS-WT, U2OS-INF2-KO, or INF2-KO cells reexpressing GFP-INF2-CAAX or GFP-INF2-nonCAAX. Control U2OS-INF2-KO cells are expressing GFP-Sec61β as an ER marker instead of INF2. Error bars represent SEM. (E) Actin polymerization burst after ionomycin stimulation in U2OS-WT and U2OS-INF2-KO cells. Rescue by transfection of GFP-INF2-CAAX or GFP-INF2-nonCAAX is also shown; n = 15–18 cells. Error bars represent SEM. (F) Ionomycin-induced actin morphology in INF2-KO cells reexpressing GFP-INF2-CAAX or GFP-INF2-nonCAAX. Whole-cell view shown prestimulation. Insets shown prestimulation (0”, left) and after 30 s stimulation (30”, right). Taken in a single confocal plane in an apical region of the cell body, which reduces actin background from stress fibers but causes the ER to appear fragmented. Arrows show examples of actin filament accumulation. Bars: (main) 5 µm; (inset) 2 µm. Corresponds to Video 6.

Article Snippet: Cell culture, transfection, and drug treatments Human osteosarcoma U2OS cells (HTB96; American Type Culture Collection) were grown in DMEM (Invitrogen) supplemented with 10% calf serum (Atlanta Biologicals).

Techniques: Control, Expressing, Marker, Transfection

Journal: The Journal of cell biology

Article Title: INF2-mediated actin polymerization at the ER stimulates mitochondrial calcium uptake, inner membrane constriction, and division.

doi: 10.1083/jcb.201709111

Figure Lengend Snippet: Figure 4. INF2-mediated actin polymerization is required for stimulation of ER–mitochondrial contact. (A) Change in calcium levels at the cytoplasmic face of the OMM upon 4 µM ionomycin stimulation in U2OS-WT and INF2-KO cells. Cells were transfected with the Mass70-LAR-GECO1.2 construct, a low-affinity calcium probe (Kd of 12 µM) tethered to the cytoplasmic face of the OMM. For rescue, INF2-KO cells were transfected with plasmid expressing GFP-INF2-CAAX or GFP- INF2-nonCAAX. Ionomycin was added at 0 s in n = 24 cells (WT), 19 cells (INF2-KO), 22 cells (INF2-CAAX), and 16 cells (INF2-non CAAX). Error bars represent SEM. (B) Electron micrographs showing examples of the relationship between the ER and mitochondria in WT cells (top images) and INF2-KO cells (bottom images) in either the unstimulated condition [(−) ionomycin, left images] or after 60 s stimulation with 4 µM ionomycin [(+) ionomycin, right images]. Two zooms shown for each panel (regions indicated by numbered arrows on the main panels). Examples of ER–mitochondrial contacts of <30 nm are indicated by red arrows, whereas examples of more distant contacts indicated by black arrows. Bars: (main micrographs) 500 nm; (zooms) 100 nm. (C) Quantification from electron micrographs of the percentage of mitochondria with close ER contacts in WT cells (n = 244 mitochondria), WT cells stimulated with ionomycin (n = 176 mitochondria), INF2-KO cells (n = 245 mitochondria), and INF2-KO cells stimulated with ionomycin (n = 204 mitochondria). Values represent mean ± SEM; ***, P < 0.001 (unpaired Stu- dent’s t test). Each point represents one imaged field. (D) Rescue of mitochondrial calcium response in INF2-KO cells by overexpression of ER–mitochondrial tethers. INF2-KO U2OS cells were transfected with a mitochondrial matrix calcium probe (Mito-R-GECO) along with either CFP-VAPB or GFP-PTPIP51 and then stimulated with 4 µM ionomycin. The effects of either GFP-INF2-CAAX or GFP-INF2-nonCAAX reexpression are shown for comparison; n = 10 cells (WT), 8 cells (INF2-KO), 11 cells (INF2 non-CAAX), 15 cells (INF2-CAAX), 10 cells (PTPIP51), or 14 cells (VAPB). Error bars represent SEM.

Article Snippet: Cell culture, transfection, and drug treatments Human osteosarcoma U2OS cells (HTB96; American Type Culture Collection) were grown in DMEM (Invitrogen) supplemented with 10% calf serum (Atlanta Biologicals).

Techniques: Transfection, Construct, Plasmid Preparation, Expressing, Over Expression, Comparison

Journal: The Journal of cell biology

Article Title: INF2-mediated actin polymerization at the ER stimulates mitochondrial calcium uptake, inner membrane constriction, and division.

doi: 10.1083/jcb.201709111

Figure Lengend Snippet: Figure 5. MCU suppression inhibits the mi- tochondrial calcium spike without inhibiting the actin burst. U2OS cells were transfected with either scrambled siRNA (control) or siRNA against MCU (MCU KD). After 48 h, cells were transfected with mito-R-GECO (mitochon- drial calcium) and GFP-Ftractin (polymerized actin) or cyto-R-GECO (cytoplasmic calcium) and imaged at 72 h after siRNA treatment. (A) Confocal image montages of cells at indi- cated times after 4 µM ionomycin stimulation. For MCU KD, the mitochondrial calcium signal is enhanced to reveal the faint mitochondrial outline. Arrowheads show mitochondrial cal- cium rise, and arrows denote polymerized actin increases. Bars, 5 µm. (B) Quantification of the mitochondrial and cytosolic calcium spikes upon stimulation with 4 µM ionomycin (at time 0); n = 10 cells (Mito calcium con- trol), 18 cells (Mito calcium MCU KD), 10 cells (Cyto calcium control), or 15 cells (Cyto calcium MCU KD). Error bars represent SEM. (C) Quantification of the actin burst upon stimulation with 4 µM ionomycin (at time 0); n = 10 cells (control) or 15 cells (MCU KD). Error bars represent SEM.

Article Snippet: Cell culture, transfection, and drug treatments Human osteosarcoma U2OS cells (HTB96; American Type Culture Collection) were grown in DMEM (Invitrogen) supplemented with 10% calf serum (Atlanta Biologicals).

Techniques: Transfection, Control

Journal: The Journal of cell biology

Article Title: INF2-mediated actin polymerization at the ER stimulates mitochondrial calcium uptake, inner membrane constriction, and division.

doi: 10.1083/jcb.201709111

Figure Lengend Snippet: Figure 6. MCU suppression inhibits mitochondrial division, but not Drp1 oligomerization. (A) U2OS cells were transfected with scrambled siRNA (control), MCU siRNA (MCU KD), and Drp1 siRNA (Drp1 KD) for 72 h Cells were then fixed and mitochondria stained using anti-Tom20 (red) and DAPI (blue). ROIs of fixed dimension were analyzed for mitochondrial length and number as described in Materials and methods. Images of control (left), Drp1 KD (middle), or MCU KD cells (right) are shown. Bars: (main) 5 µm; (insets) 2 µm. (B) Mean mitochondrial length quantification represented as area (square micrometers) per mitochondrion (left) and mean mitochondrial number quantification (right) for 80, 53, and 50 cells for control, MCU KD, and Drp1 KD cells, respectively. Errors bars represent SEM. P-values were obtained from an unpaired Student’s t test. (C) Quantification of mitochondrial division rate in control and MCU KD U2OS cells by measuring the number of division events in peripheral ROIs from live-cell videos of mitochondrial matrix marker (mito-BFP), either in unstimulated cells or cells in the first 10 min of 4 µM ionomycin stimulation; n = 19 cells [control (−) ionomycin], 21 cells [control (+) ionomycin], 20 cells [MCU KD (−) ionomycin], or 22 cells [MCU KD (+) ionomycin]. Each point represents one ROI per cell. P-values were obtained from an unpaired Student’s t test. *, P < 0.015; ***, P < 0.0001. Error bars represent SD. (D) Quantification of mitochondrially associated Drp1 oligomers and mitochondrial calcium in response to 4 µM ionomycin. GFP-Drp1 knock-in cells were treated with scrambled siRNA or MCU siRNA for 48 h and then transfected with mito-R-GECO and mito-BFP. Cells were imaged at 72 h after siRNA treatment; n = 20 cells in each case. Ionomycin addition at 0 s. Error bars represent SEM. (E) Micrographs from GFP-Drp1 knock-in U2OS cells (control and MCU siRNA treated) transfected with mito-BFP (red) and treated with 4 µM ionomycin as in B. Prestimulation (0”) and 300-s stimulations are shown. Bar, 10 µm. (F) Drp1 oligomerization kinetics in MCU KD cells, as measured in the whole cell. GFP-Drp1 knock-in U2OS cells were treated with control siRNA or MCU siRNA for 72 h and then stimulated with 4 µM ionomycin while acquiring GFP images. The amount of oligomerized Drp1 was assessed as GFP signal above the cytoplasmic background signal, as described in Materials and methods; n = 20 cells in each case. Error bars represent SEM. (G) Quantification of mitochondrial calcium increase in control or Drp1 KD U2OS cells upon 4 µM ionomycin stimulation; n = 14 cells in each case. Error bars represent SEM.

Article Snippet: Cell culture, transfection, and drug treatments Human osteosarcoma U2OS cells (HTB96; American Type Culture Collection) were grown in DMEM (Invitrogen) supplemented with 10% calf serum (Atlanta Biologicals).

Techniques: Transfection, Control, Staining, Marker, Knock-In

Journal: The Journal of cell biology

Article Title: INF2-mediated actin polymerization at the ER stimulates mitochondrial calcium uptake, inner membrane constriction, and division.

doi: 10.1083/jcb.201709111

Figure Lengend Snippet: Figure 7. Mitochondrial constrictions are MCU dependent and Drp1 independent. (A) Quantification of mitochondrial constriction frequency in the unstim- ulated (blue) or ionomycin-stimulated (red) conditions, represented as constrictions per mm mitochondrial length in U2OS-control, Drp1-KD, and MCU-KD cells. P-values were obtained from an unpaired Student’s t test. Error bars represent SEM. Control:, 16 ROI, 2,508 µm mitochondrial length; Drp1 KD, 10 ROI, 1,000 µm; MCU KD: 20 ROI, 2,600 µm. (B) Representative confocal montages of control, Drp1-KD, and MCU-KD U2OS cells transfected with mito- R-GECO (mitochondrial calcium, red) and mitoBFP (mitochondrial matrix, blue) and then treated with 4 µM ionomycin at time 0. Time in seconds. White arrows point to constrictions and yellow arrows to fission events. Bar, 5 µm. Corresponds to Video 8. (C) U2OS cell transfected with GFP-Sec61β (green, ER marker) and mito-R-GECO (red, mitochondrial calcium) and imaged live before ionomycin stimulation (top) and at 35 s after 4 µM ionomycin addition (bottom). Image on the right is an expanded view of the (+) ionomycin condition. Bars: (regular panels) 2 µm; (expanded panel) 1 µm. Arrows denote ER presence at mitochondrial constrictions. Corresponds to Video 9. (D) Quantification of percent mitochondrial constrictions corresponding to ER contact from live-cell confocal microscopy of ionomycin-stimulated cells such as in C and Video 9. 204 constrictions from 26 ROIs from 13 cells assessed, totaling a 461-µm2 mitochondrial area.

Article Snippet: Cell culture, transfection, and drug treatments Human osteosarcoma U2OS cells (HTB96; American Type Culture Collection) were grown in DMEM (Invitrogen) supplemented with 10% calf serum (Atlanta Biologicals).

Techniques: Control, Transfection, Marker, Confocal Microscopy

Journal: The Journal of cell biology

Article Title: INF2-mediated actin polymerization at the ER stimulates mitochondrial calcium uptake, inner membrane constriction, and division.

doi: 10.1083/jcb.201709111

Figure Lengend Snippet: Figure 8. IMM division before OMM division during mitochondrial division. (A) U2OS cells transfected with mito-R-GECO (mitochondrial matrix calcium, red) and GFP-Tom20 (OMM, green) were imaged live by Airyscan microscopy at 9-s intervals after 4 µM ionomycin treatment. Representative time-lapse montage of a mitochondrial division event shown here. Bar, 2 µm. (B) Zoom of the division site (boxed in A). The mito-R-GECO levels have been enhanced to detect the existence of any thin matrix tether. Line scans indicate that the OMM tether persists in the absence of a matrix tether. Bar, 500 nm. (C) Quanti- fication of time between apparent matrix separation and apparent OMM separation in mitochondrial division events after CGP37157 stimulation (example in Fig. S4 E). 29 division events from 23 cells. Mean time, 32.3 ± 29.1 s (SD). Each point represents one division event.

Article Snippet: Cell culture, transfection, and drug treatments Human osteosarcoma U2OS cells (HTB96; American Type Culture Collection) were grown in DMEM (Invitrogen) supplemented with 10% calf serum (Atlanta Biologicals).

Techniques: Transfection, Microscopy

Journal: The Journal of cell biology

Article Title: INF2-mediated actin polymerization at the ER stimulates mitochondrial calcium uptake, inner membrane constriction, and division.

doi: 10.1083/jcb.201709111

Figure Lengend Snippet: Figure 9. IMM constrictions do not require Oma1 activity but do require the ETC. (A) Western blot of Opa1 isoform pattern in U2OS cells during 4 µM ionomycin stimulation during the period of optimum calcium-induced constrictions. Mass is in kilodaltons. L1, L2, and S3–5 refer to Opa1 isoforms are as defined previously (Anand et al., 2014; Otera et al., 2016). (B) Western blot of Opa1 after live-cell cross-linking with BMH to reveal Opa1 oligo- merization changes during 4 µM ionomycin stimulation. Oligomers are labeled with arrowheads and monomers with an arrow. Mass is in kilodaltons. (C) Constrictions in U2OS cells that had been transfected with Oma1 siRNA for 72 h, followed by transfection of mito-R-GECO and mitoBFP for 24 h and stimulation by 4 µM ionomycin for 60 s. Bars: (main) 5 µm; (insets) 2 µm. (D) Effect of 5 µM antimycin A or 2.5 µM rotenone on (left to right) mitochondrial constrictions, actin burst, and mitochondrial calcium spike induced by 4 µM ionomycin. Antimycin A or rotenone was added simultaneously to ionomycin. For mitochondrial constriction assessment: n = 10 ROIs, 200 µm2 mitochondrial area (Iono), 11 ROIs, 136 µm2 (Iono + rotenone); and 16 ROIs, 300 µm2 (Iono + Antimycin A). For actin filament assessment, 10 cells were used for each condition. For mitochondrial calcium assessment, 15–20 cells were used for each condition. P-values are from an unpaired Student’s t test. ***, P < 0.0011. Error bars represent SD (constriction quantification) or SEM (actin filament and mitochondrial calcium assessment).

Article Snippet: Cell culture, transfection, and drug treatments Human osteosarcoma U2OS cells (HTB96; American Type Culture Collection) were grown in DMEM (Invitrogen) supplemented with 10% calf serum (Atlanta Biologicals).

Techniques: Activity Assay, Western Blot, Labeling, Transfection