CME/clathrin-mediated endocytosis

Ikarugamycin's half-life in H1299 cells is 2.7 μM [1].
Ikarugamycin (IKA) is a previously discovered antibiotic, which has been shown to inhibit the uptake of oxidized low-density lipoproteins in macrophages. Furthermore, several groups have previously used IKA to inhibit clathrin-mediated endocytosis (CME) in plant cell lines. However, detailed characterization of IKA has yet to be performed. Consequently, we performed biochemistry and microscopy experiments to further characterize the effects of IKA on CME. We show that IKA has an IC50 of 2.7 μm in H1299 cells and acutely inhibits CME, but not other endocytic pathways, in a panel of cell lines. Although long-term incubation with IKA has cytotoxic effects, the short-term inhibitory effects on CME are reversible. Thus, IKA can be a useful tool for probing routes of endocytic trafficking[1].

TIRF microscopy[1]
Total internal reflection fluorescence (TIRF) microscopy was performed using ARPE-19 cells stably expressing eGFP-CLCa and imaged using a 100 × 1.49 NA Apo TIRF objective mounted on a Ti-Eclipse inverted microscope equipped with the Perfect Focus System. During imaging, cells were maintained in medium lacking phenol red containing 4 µm Ikarugamycin (IKA) or not (control). Time-lapse image sequences from different cells were acquired at a frame rate of 1 frame/second and exposure time of 150 ms using a pco-edge 5.5 sCMOS camera with 6.5 µm pixel size. Cells were either treated with 4 µm of Ikarugamycin (IKA) for 3 h before imaging or not (control) and then immediately imaged.
Image and data analyses of CCP dynamics were carried out in Matlab (MathWorks), using custom-written software. Up-to-date versions of the software will be made available at http://lccb.hms.harvard.edu/software.html.

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Electron microscopy[1]
Rip-off[1]
Cells were applied to EM grids (carbon film 200 mesh gold – Electron Microscopy Sciences) coated with collagen and allowed to adhere overnight under normal culture conditions. Ikarugamycin (IKA) was added to the cells at the desired concentration and incubated for 3 h, after which the grids with adhered cells were washed and processed for ‘rip-off’ images. After washing with cold PBS (20 mm sodium phosphate pH 7.4, 150 mm sodium chloride), grids with adhered cells were fixed lightly with 0.5% (v/v) paraformaldehyde (PFA) in PBS for 2′. Grids were then washed with PBS and hypotonic buffer (25 mm HEPES, pH7.2 with 25 mm potassium chloride and 2.5 mm magnesium acetate). The last wash of hypotonic buffer was left on the cells to swell slightly for 10′ –15′. The grids were then quickly placed, cell side down, onto cover slips previously coated with poly-l-lysine. A small piece of filter paper was used to wick off the moisture as the grid was pulled quickly and laterally away from the cover slip. The grids were then fixed in 2% (v/v) PFA in PBS for 10′; 3 × 5′ washes in H2O; 10′ fixation in 2% (v/v) glutaraldehyde in H2O; 2 × 5′ washes in H2O; 1′ in 1% tannic acid (w/v) in H2O; 3 × 5′ washes in H2O; 1′ in 1% (w/v) uranyl acetate; 2 × 5′ washes in H2O. Droplets were wicked away from the grids with filter paper pieces and finally the grids were laid out on filter paper to air dry until imaged.

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Thin-sectioning[1]
Cells were applied to collagen-coated glass-bottomed dishes and allowed to adhere overnight under normal culture conditions. Ikarugamycin (IKA) was added to the cells at the desired concentrations and incubated for 3 h. Cells were then washed with cold PBS and fixed in 2.5% (v/v) glutaraldehyde in 0.1 m sodium cacodylate buffer. Processing for embedding and sectioning continued as follows: after three rinses in 0.1 m sodium cacodylate buffer, they were post-fixed in 1% osmium in 0.1 m sodium cacodylate buffer for 1 h. Cells were rinsedin0.1 m sodium cacodylate buffer and en bloc stained with 0.5% tannic acid in 0.05 m sodium cacodylate buffer for 30 min. After two rinses in 1%sodium sulfate in 0.1 m sodium cacodylate buffer, samples were rinsed three times in 0.1m sodium cacodylate buffer and five times in water. Samples were then dehydrated through a series of increasing concentrations of ethanol, and infiltrated and embedded in Embed-812 resin. Enough resin was added into the MatTek dishes to just fill the well and polymerized at 60°C. Polymerized samples were dropped into liquid nitrogen to pop out the resin disks from center of the MatTek dish. Two resin disks containing the same sample were sandwiched together with fresh Embed-812, monolayers were facing each other. Resin disks were polymerized at 60°C over-night and sectioned along the longitudinal axis of the two monolayers of cells with a diamond knife on a Leica Ultracut UCT 6 ultramicrotome. Sections were post-stained with 2% (w/v) uranyl acetate in water and lead citrate.

Ikarugamycin (IKA) inhibition[1]
For inhibition by Ikarugamycin (IKA), cells were preincubated in the absence (control) or presence of defined concentrations of Ikarugamycin (IKA) for varied time points at 37°C before internalization assays were performed as described above except the PBS4+ contained IKA. IKA was stored in 1000× stock solutions in 100% dimethyl sulphoxide (DMSO) at −20°C for up to 2 months per the manufactures instructions. All experiments were preformed with equal DMSO concentrations per condition and a DMSO control.

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Immunofluorescence[1]
ARPE-19 cells expressing enhanced green fluorescent protein, fused of the N-terminus of clathrin light chain a (eGFP-CLCa) grown overnight on glass cover slips, were preincubated in the absence (control) or presence of 4 µm of Ikarugamycin (IKA) for 3 h at 37°C, washed with PBS and fixed in 4% PFA in PBS for 10 min at room temperature, permeabilized with 0.1% Triton X-100 for 2 min and further blocked with Q-PBS (0.01% saponin, 2% BSA, 0.1% lysine, pH7.4) for 1 h. After three washes with PBS, cells were incubated with the indicated primary antibody in Q-PBS for 1 h using the recommended dilution. Cells were washed three times with PBS and further incubated with suitable AlexaFluor®-labeled secondary antibodies for 1 h. After three additional washes with PBS, samples were mounted on Fluoromount G on glass slides and examined using either 60× or 100× 1.49 NA objectives (Nikon) mounted on an epifluorescence Ti-Eclipse inverted microscope.

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Cell viability and caspase 3/8/9 activation[1]
Measurement of cell viability/cytotoxicity was performed by using the CCK-8 Counting Kit, which measures dehydrogenase activity in viable cells. Briefly, 1 × 105 cells/well cells were treated with either 4 or 32 µm of Ikarugamycin (IKA) in 96-well plates for increasing time periods at 37°C, and analyzed according to the manufacturer’s instructions. To measure viability in parallel to uptake assays cells were plated at the same density under the same conditions as the uptake conditions.
Caspase-3, -8 and -9 activations were assessed using western blotting. Briefly, ARPE-19 cells were treated with 4 µm Ikarugamycin (IKA) for either 2, 4, or 8 h, or no treatment (control), washed three times with PBS and harvested/ resuspended in 200 µL of reducing Laemmli sample buffer. The cell lysate was boiled for 10 min and loaded onto an SDS gel. After transferring to a nitrocellulose membrane, membranes were probed with antibodies against the following proteins: caspase-3, -8 and -9.

[1]. Ikarugamycin: A Natural Product Inhibitor of Clathrin-Mediated Endocytosis. Traffic. 2016 Oct;17(10):1139-49.

Ikarugamycin is a polyketide macrolactam containing a tetramic acid (pyrrolidine-2,4-dione) ring system. It is isolated from Streptomyces as an antibiotic with antiprotozoal and cytotoxic activities. It has a role as an antimicrobial agent, an antiprotozoal drug, an antineoplastic agent, an apoptosis inducer and a bacterial metabolite. It is a lactam, an azamacrocycle, an enone, a polyketide and an organic heteropentacyclic compound.
Ikarugamycin has been reported in Streptomyces, Streptomyces harbinensis, and other organisms with data available.

C29H38N2O4

478.62302

478.283

C, 72.77; H, 8.00; N, 5.85; O, 13.37

36531-78-9

54680304

White to light yellow solid powder

1.2±0.1 g/cm3

741.9±60.0 °C at 760 mmHg

402.5±32.9 °C

0.0±5.6 mmHg at 25°C

1.604

5.05

3

4

1

35

1010

9

CC[C@@H]1[C@@H](C[C@H]2[C@H]1C=C[C@H]3[C@@H]2C[C@@H]/4[C@@H]3C/C=C\C(=O)NCCC[C@H]5C(=O)/C(=C(\C=C4)/O)/C(=O)N5)C

GHXZHWYUSAWISC-KZRBWAKNSA-N

InChI=1S/C29H38N2O4/c1-3-18-16(2)14-22-20(18)10-11-21-19-6-4-8-26(33)30-13-5-7-24-28(34)27(29(35)31-24)25(32)12-9-17(19)15-23(21)22/h4,8-12,16-24,32H,3,5-7,13-15H2,1-2H3,(H,30,33)(H,31,35)/b8-4-,12-9+,27-25-/t16-,17-,18-,19+,20+,21-,22+,23+,24+/m1/s1

(1Z,3E,5S,7R,8R,10R,11R,12S,15R,16S,18Z,25S)-11-ethyl-2-hydroxy-10-methyl-21,26-diazapentacyclo[23.2.1.05,16.07,15.08,12]octacosa-1,3,13,18-tetraene-20,27,28-trione

Ikarugamycin; 36531-78-9; (1Z,3E,5S,7R,8R,10R,11R,12S,15R,16S,18Z,25S)-11-ethyl-2-hydroxy-10-methyl-21,26-diazapentacyclo[23.2.1.05,16.07,15.08,12]octacosa-1(2),3,13,18-tetraene-20,27,28-trione; EIA; CHEMBL4283254; CHEBI:75276; MFCD01722005; NSC789948;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)