*2.1. Animals*

Male hemizygous hSOD1G93A transgenic mice and female B6SJL mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA) and maintained as described previously [30]. hSOD1G93A mice have a glycine-to-alanine base-pair mutation at the 93rd codon of the cytosolic Cu/Zn superoxide dismutase gene. Male hSOD1G93A mice were housed at 3–4 per cage under specific pathogen-free conditions and had *ad libitum* access to food and water. The facilities were maintained under a constant temperature (21 ± 3 ◦C) and humidity (50 ± 10%) with a 12 hours light/dark cycle (lights on 07:00–19:00). All mice were treated in accordance with the animal care guidelines of the Korea Institute of Oriental Medicine (protocol number: 13–109).

#### *2.2. Hochu-Ekki-To (HET) Treatment*

Hochu-ekki-to (HET) was purchased from TSUMURA Co. Ltd (TSUMURA, Osaka, Japan) and diluted at 1 mg/g with autoclaved distilled water. The mice were randomly divided into three groups, as follows: a non-transgenic mice group (nTg, *n* = 8), a hSOD1G93A transgenic mice group (Tg, *n* = 11), and a HET treated hSOD1G93A transgenic mice group (Tg-HET, *n* = 11) (Figure 1). HET (1 mg/g) was orally administered with a disposable oral gavage syringe (FUCHIGAMI, Kurume, Japan) once a day for 6 weeks from the age of 2 months (the pre-symptomatic stage). The dose was translated from human to animal based on a previous study [31].

#### *2.3. Rota-Rod Test*

The rota-rod test is used to assess motor activity and balance in rodents. Mice were trained every other day for 2 weeks to adapt to the apparatus (Rotarod, B.S Technolab Inc., Korea). During training, the rota-rod was maintained at a constant speed of 10 rpm for 180 seconds. After the last administration of HET, mice performed the test, and we recorded the time mice remained on the rod before falling. Each mouse performed three trials and the average time spent on the rod was determined for each group.

#### *2.4. Foot Print Test*

The day before mice were sacrificed, the footprint test was used to measure gait. To record stride length, mice hind paws were stained with nontoxic water-soluble black ink, and the alley floor (70 cm length, 6 cm width, and 16 cm height) was covered with white paper to absorb the ink. Each mouse performed three trials and the average of stride length was determined for each group.

#### *2.5. Survival Test*

To measure lifespan, male transgenic mice were randomly divided into the following treatment groups: distilled water-treated ALS mice (*n* = 8) and ALS mice treated with HET for 6 weeks (*n* = 8/group). Death was defined according to our previous paper [32].

#### *2.6. Tissue Preparation*

Body weight of mice was measured and mice were anesthetized using pentobarbital sodium (Entobar, Hanlim Pharm, Co., Ltd., Seoul, Korea) and perfused with phosphate-buffered saline (PBS). The gastrocnemius muscle and spinal cord of the mice were dissected and stored at −80 ◦C until use. The gastrocnemius muscle weight was recorded and the average value for each group recorded. For hematoxylin and eosin (H&E) staining, the gastrocnemius muscle of the mice was fixed in 4% paraformaldehyde at 4 ◦C before embedding in paraffin. The tissues were cut into transverse sections (5 μm thick) using a microtome (Leica biosystems, IL, USA) and mounted on glass slides.

## *2.7. Western Blotting*

For Western blotting, the gastrocnemius muscle and lumbar (L4–5) spinal cord of mice were homogenized in radioimmunoprecipitation assay buffer (50 mM, Tris-HCl (pH 7.4); 1% Nonidet P−40; 0.1% sodium dodecyl sulfate; 150 mM NaCl) containing protease and a phosphatase inhibitor cocktail (Thermo, Waltham, MA, USA). Homogenized tissues were centrifuged at 20,800 × g for 15 minutes at 4 ◦C. The protein concentration was determined using the Bicinchoninic Acid Assay Kit (Pierce, IL, USA). The samples (20 μg of protein) were denatured with sodium dodecyl sulfate sampling buffer, separated using SDS-PAGE electrophoresis, and transferred to a Polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA, USA). Membranes were incubated in a blocking solution (5% skim milk in TBS) for 1 hour at room temperature then incubated in the various primary antibodies (anti-iba-1, anti-GFAP, anti-TLR4, anti-BAX, anti-HO1, anti-transferrin, anti-CD11b, anti-Ferritin, anti-tubulin, and anti-actin) overnight at 4 ◦C. The next day, blots were washed and incubated with horseradish peroxidase-conjugated secondary antibodies, and then visualized using the SuperSignal West Femto Substrate Maximum Sensitivity Substrate (Thermo Fisher Scientific, Waltham, MA, USA). For detection of the other antibodies, membranes were stripped in a stripping buffer (Thermo Fisher Scientific, Waltham, MA, USA). The blots were analyzed using the ChemiDoc imaging system (Bio-Rad, Hercules, CA, USA), which were then quantified using the NIH ImageJ program (National Institutes of Health, Bethesda, MD, USA).

#### *2.8. H&E Staining and Immunohistohcemistry*

For H&E staining, the paraffin sections were de-paraffinized in xylene and rehydrated in a graded alcohol series (100%, 95%, 80% ethanol), followed by deionized H2O. Slices were incubated in hematoxylin (Sigma-Aldrich Corp., St. Louis, MO, USA) for 6 minutes and washed under flowing distilled water for 5 minutes, then incubated in eosin for 45 seconds, dehydrated (95%, 100%, xylene), and mounted using a Histomount medium (Sigma-Aldrich Corp.). Immunohistochemistry was performed with previous paper described [32]. In brief, de-paraffinized slides were incubated with 3% hydrogen peroxide (H2O2) and 5% bovine serum albumin (BSA) in 0.01% PBS-Triton X–100 (Sigma-Aldrich, Oakville, ON, Canada). The sections were incubated with anti-IL-1β (Abcam, Cambridge, UK) and then secondary antibody. For observation, the ABC kit and 3,3-diaminobenzidine (DAB)/H2O2 substrate were used with a hematoxylin counterstain. Immunostained tissues were observed with a light microscope (Olympus, Tokyo, Japan). The central nuclei (as a marker of abnormal nuclei) were counted and expressed as a percentage: the number of myocytes with central nuclei divided by the total number of myocytes in each captured image. For the quantification of myocyte cross-sectional area (CSA), the average area of individual myocytes was measured using the NIH ImageJ program.

#### *2.9. Statistical Analysis*

All values are expressed as the mean ± SEM. The results were analyzed using a one-way analysis of variance (ANOVA) followed by the Newman-Keuls's *post hoc* test for multiple comparisons. For survival test, the data were analyzed by Kaplan–Meier survival curves. Data were analyzed using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA). Statistical significance was set at *p* < 0.05.
