Relative changes in body weight, clinical score and survival in ALS mice.

<p><b>(A-F)</b> Hind limb extension of WT mouse, IL-6(-/-) mouse (160–180 days of age), SOD1 TG/IL-6(+/+) mice and SOD1 TG/IL-6(-/-) mice (the early stage and the end stage), when suspended by their tail. <b>(G)</b> Kaplan-Meier time–to-failure plot for onset of symptomatic neurological disease in ALS mice. Neurological symptoms of SOD1 TG/IL-6(-/-) (n = 17), SOD1 TG/IL-6(+/-) (n = 18) and SOD1 TG/IL-6(+/+) (n = 11) were monitored. The age at which mice attain a neurological severity score 2 is taken to be definitive onset of symptomatic neurological disease (p = 0.597 by log rank test for onset of symptomatic neurological disease). <b>(H)</b> The clinical score of SOD1 TG/IL-6(-/-) mice in comparison with those of SOD1 TG/IL-6(+/-) mice and SOD1 TG/IL-6(+/+) mice. Score criteria (severity from 0 to 4) are shown in materials and methods. SOD1 TG/IL-6 (-/-) (n = 17), SOD1 TG/IL-6(+/-) (n = 18), SOD1 TG /IL-6(+/+) (n = 11), IL-6(-/-) (n = 8), WT (n = 15), IL-6(+/-) (n = 5). WT, IL-6(-/-) or IL-6(+/-) mice showed no disease symptoms. <b>(I)</b> Relative changes in body weight of SOD1 TG/IL-6(-/-) mice (n = 17), SOD1 TG /IL-6(+/-) mice (n = 18) and SOD1 TG /IL-6(+/+) mice (n = 11). Peak body weight of each mouse was calculated as 1. (<b>J</b>) Kaplan-Meier time–to-failure plot for onset of body weight loss of SOD1 TG/IL-6(-/-), SOD1 TG/IL-6(+/-) and SOD1 TG/IL-6(+/+) mice. The age at which mice attained a peak body weight was analyzed (p = 0.595 by log rank test). (<b>K</b>) Kaplan-Meier time–to-failure plot for survival of SOD1 TG/IL-6(-/-), SOD1 TG/IL-6(+/-) and SOD1 TG/IL-6(+/+) mice (p = 0.430 by log rank test). The survival of SOD1 TG/IL-6(-/-) (n = 17), SOD1 TG/IL-6(+/-) (n = 18) and SOD1 TG/IL-6(+/+) (n = 11) was monitored. The mean (± SD) life spans were; SOD1 TG/IL-6(-/-) 167.55 ± 11.52 days, SOD1 TG/IL-6(+/-) 161.82 ± 12.69 days, TG/IL-6(+/+) 164.31 ± 12.16 days.</p

Histological analysis of motor neuron loss and muscle atrophy in ALS mice.

<p>(A, B, C, D, E) HE staining of hamstring muscles from the early stage and the end stage of ALS mice and control mice with indicated genotype. Scale bars: 50 μm. (F, G, H, I, J) Lumber spinal cords from mice with indicated genotypes at the early and the end stage were stained with Cresyl violet solution (Nissl staining). Scale bars: 50μm. (K) A representative image of Nissl-stained mouse spinal cord. Boxed area is a region defined as the anterior horn for motor neuron counting. (L) Quantitative cell counts of motor neurons in the anterior horn section. Spinal cord sections from mice with indicated genotype at the early and the end stage were stained with Cresyl violet solution and motor neurons were counted (*p<0.05 and **p<0.01 by Tukey Kramer post-hoc test).</p

Quantitative Real Time PCR of IL-1β and TNF-α in spinal cords of SOD1 TG/IL-6(-/-) mice and control mice.

<p>Messenger RNA obtained from spinal cord of SOD1 TG mice (n = 7), SOD1 TG/IL-6(-/-) mice (n = 7), IL-6 knockout mice (n = 6) and WT mice (n = 8) was subjected to real-time PCR analysis of IL-1β <b>(A)</b>, TNF-α <b>(B)</b> expression (* p≤0.05 by Scheffe’s multiple comparison).</p

Suppression of cell growth by combined KPNA knockdown.

<p>Under starvation conditions (0.1% FBS), siRNA-mediated knockdown of KPNA2, 1, 3, and 4 suppressed cell growth after 120 h (*p<0.05). Only KPNA2 siRNA subtraction produced no change in proliferation.</p

Lists of proteins analyzed by pathway analysis.

<p>Lists of proteins analyzed by pathway analysis.</p

Suppression of protein synthesis by combined KPNA knockdown.

<p>Under starvation conditions (0.1% fetal bovine serum), siRNA-mediated knockdown of KPNA2, 1, 3, and 4 significantly suppressed protein synthesis after 48 h (*p<0.05) and 72 h (***p<0.01), as demonstrated by metabolic labeling with ³⁵S-methionine.</p

Detection and analysis of proteins that interact with KPNA2 and localization of KPNA2 in the nucleolus.

<p>Proteins that interact with KPNA2 in the cytoplasm and nucleus were purified using the TAP method and detected by silver staining. Proteins marked with arrows were analyzed by LC/MS/MS. HaCaT cells expressing GFP-TAP were used to detect nonspecific interactions. <b>a)</b> The results of LC/MS/MS were analyzed by pathway analysis using reactome (<a href="http://www.reactome.org" target="_blank">http://www.reactome.org</a>). The categories of “mRNA processing”, “ribonucleoprotein complex biogenesis”, “chromatin modification,” and “transcription” were the most significantly represented pathways. <b>b)</b> Immunohistochemistry revealed KPNA2 co-localization with UBF, a nucleolar marker.</p

Overexpression of KPNA2 in proliferating cells.

<p>Immunohistochemistry showed KPNA2 was uniformly expressed throughout the epidermis in healthy skin, although KPNA2 overexpression was observed in the basal layer in psoriasis. In contrast, very few cells exhibited KPNA2 staining in the basal cells of atopic dermatitis. KPNA2 overexpression was observed in the tumor cells of Bowen’s disease, actinic keratosis, squamous cell carcinoma, Paget’s disease, Merkel cell carcinoma, and mycosis fungoides.</p

Suppression of ribosomal RNA synthesis by combined KPNA knockdown.

<p>Under starvation conditions (0.1% fetal bovine serum), siRNA-mediated knockdown of KPNA2, 1, 3, and 4 significantly suppressed ribosomal RNA synthesis analyzed by reverse transcription-quantitative polymerase chain reaction (***p<0.01). The amount of pre-ribosomal RNA was reduced by about 37% after 72 h.</p

Identification of Sialylated Glycoproteins in Doxorubicin-Treated Hepatoma Cells with Glycoproteomic Analyses

Sialylation is one of the most important types of glycosylation involved in carcinogenesis and establishment of cancer stemness. We previously showed that increased sialylation is a characteristic glycan change in cancer stem cells (CSCs) from hepatocellular carcinoma. However, the identities of glycoproteins targeted for sialylation remain unknown. In the present study, we identified glycoproteins targeted for sialylation in doxorubicin (DXR)-treated hepatocarcinoma cell line, Huh7, using glycoproteomic analyses. Since CSCs constitute a small subset of cells within carcinoma cell lines, it is difficult to identify sialylated proteins using general glycoproteomic strategies. It is known that treatment with anticancer drug can condense CSCs, we used DXR to concentrate CSCs. In DXR-treated Huh7 cells, isobaric tag for relative and absolute quantitation (iTRAQ) analysis identified 17 sialylated glycoproteins. Most of the identified glycoproteins were cancer-associated proteins. Furthermore, two proteins of approximately 70 kDa were detected using <i>Sambucus sieboldoana</i> agglutinin (SSA) blot analysis and identified as beta-galactosidase and alpha-2-HS-glycoprotein (fetuin-A) by SSA precipitation followed by liquid chromatography-tandem mass spectrometry analyses. Sialylation levels of fetuin-A were increased in DXR-treated Huh7 cell lysates. These changes in sialylation of glycoproteins might be involved in the establishment of cancer stemness