Supplemental Material - Comparison of Posterior Approach and Combined Anterior-Posterior Approach in the Treatment of Ankylosing Spondylitis Combined With Cervical Spine Fracture: A Systematic Review and Meta-Analysis

Supplemental Material for Comparison of Posterior Approach and Combined Anterior-Posterior Approach in the Treatment of Ankylosing Spondylitis Combined With Cervical Spine Fracture: A Systematic Review and Meta-Analysis by Cong Peng, Haopeng Luan, Kai Liu, and Xinghua Song in Global Spine Journal</p

Nuclear envelope-distributed <i>CD147</i> interacts with and inhibits the transcriptional function of <i>RING1</i> and promotes melanoma cell motility

<div><p>Melanoma accounts for nearly 80% of all deaths associated with skin cancer.<i>CD147</i> plays a very important role in melanoma progression and the expression level may correlate with tumor malignancy. <i>RING1</i> can bind DNA and act as a transcriptional repressor, play an important role in the aggressive phenotype in melanoma. The interactions between <i>CD147</i> and <i>RING1</i> were identified with a yeast two-hybrid and <i>RING1</i> interacted with <i>CD147</i> through the transmembrane domain. <i>RING1</i> inhibits <i>CD147</i>’s capability promoting melanoma cell migration. In conclusion, the study identified novel interactions between <i>CD147</i> and <i>RING1</i>, recovered <i>CD147</i> nuclear envelope distribution in melanoma cells, and suggested a new mechanism underlying how cytoplasmic <i>CD147</i> promotes melanoma development.</p></div

Additional file 1 of Association of lymphocyte subsets and cytokines with bone metabolism: a retrospective, cross-sectional study

Additional file 1

<i>CD147</i> gene knockdown in A375 cells affected many gene translation pathways, thus leading to the differential expression of hundreds of genes.

<p>(A) Heatmap representing the expression levels of genes; red (normal) and green (siCD147) colors indicate the up- and down-regulated genes. Transcriptome analysis demonstrated 67 up-regulated and 115 down-regulated genes in A375 cells transfected with siCD147. (B) Several genes referred to in A were shown to be up- or down-regulated in A375 cells by RT-PCR The result was consistent with the transcriptome analysis.</p

<i>CD147</i> colocalizes with <i>RING1</i> dependent on transmembrane domain.

<p>(A) An illustration of the <i>CD147</i> mutants (left) used in CO-IP and immunofluorescence experiments;34–87: 1st IgG (IgG1); 105–199: 2nd IgG (IgG2); 207–230: transmembrane (M); 231–269: cytoplasmic domain(C). (B) The A375 cells co-transfected with Flag-<i>RING1</i>and different <i>CD147</i>-MYC deletion mutants were stained with fluorophore-conjugated anti-Flag (red) and anti-Myc (green). The <i>CD147</i> mutants without the transmembrane domain (D207-230 and D207-269) showed no co-localization with <i>RING1</i>. (C) Cells (293T) were co-transfected with plasmids encoding Flag-RING1 and different CD147-MYC deletion mutants. Transfected 293T cells were subjected to IP with anti-Flag Ab. The immunoprecipitates and the whole cell lysates were further analyzed by IB with anti-Flag Ab and anti-Myc Ab.</p

The interaction between <i>RING1</i> and <i>CD147</i> in vitro.

<p><i>RING1</i> colocalizes with <i>CD147</i> proteins on the nuclear envelope of melanoma cells. (A) Clones were abundant in both -2 SD and -4 SD culture media when the yeast cells were transfected with <i>RING1</i> and <i>CD147</i> compared with yeast cells transfected with <i>CD147</i> and EV. In these colonies, the ONPG-containing medium became a yellow color, thus confirming the interaction between <i>RING1</i> and <i>CD147</i>. By comparison, the yeast co-transfected with <i>CD147</i> and EV produced no viable colonies, and the OPNG assay was negative. (B) Exogenous <i>CD147</i> and <i>RING1</i> were co-localized in regions surrounding the nuclear envelope in 293T cells, as identified by immunofluorescence staining.(C) 293T cells were harvested and subjected to immunoprecipitation (IP) with mouse normal IgG and anti-CD147 Ab. The membranes were further probed with the indicated antibodies.</p

<i>RING1</i> depletion led to impaired migration ability in A375 cells transfected with shCD147.

<p>(A) Cell migration was assessed with a wound healing assay by measuring the distance between the wound edges at the indicated time points in both A375 cells transfected with shCD147 and A375 cells transfected with shCD147 and shRING1. (B) Five random experiments were performed. The gap was measured by Image plus 6.0 software. The ratio of the gap at 48 h to 0 h was analyzed using GraphPad software; t-tests were performed, and the p values are represented by a double asterisk (**, p<0.01).</p

Reduction of <i>MGP</i> caused by <i>CD147</i> depletion was rescued by <i>RING1</i> depletion.

<p>(A) <i>CD147</i> knockdown in A375 cells (SiCD147-A375) led to the decreased expression of MGP mRNA. Knockdown of <i>RING1</i> in siCD147-A375 cells rescued the expression of MGP mRNA. (B) <i>RING1</i> knockdown in siCD147-A375 cells rescued the expression of MGP, as shown by western blotting analysis.</p

Irradiation and 5-FU treatment promote cellular transition of LSK to LSK⁻ cells.

<p>(<b>A</b>) Lethal irradiation causes an increase in the percentage of apoptotic LSK⁻ cells. Mice were treated by two split doses of 550-cGy gamma irradiation (separated by 3 hours), and the apoptotic cells (7AAD⁺Annexin V⁺) in the LSK⁻ population were analyzed by FACS at different time points (n = 4). **: <i>p</i><0.01. (<b>B</b>) Total number and percentages of LSK and LSK⁻ cells in bone marrow of lethally irradiated WT mice were determined at different time points (n = 4). *: <i>p</i><0.05; **: <i>p</i><0.01. (<b>C</b>) Total number and percentages of LSK and LSK⁻ in bone marrow of 5-FU treated WT mice at different time points. The mice were treated with 5-FU (200 mg/kg) by intravenous injection, and the percentages of LSK and LSK⁻ cells were monitored at different time points (n = 4). *: <i>p</i><0.05; **: <i>p</i><0.01. (<b>D</b>) The apoptotic rate of LSK⁻ cells in 5-FU treated mice. WT mice were treated with 5-FU, and apoptotic rate of LSK⁻ cells were monitored at different time points (n = 4). *: <i>p</i><0.05; **: <i>p</i><0.01.</p

The <i>Icsbp</i>-<i>Lyn</i> pathway controls the transition of LSK to LSK⁻ cells.

<p>(<b>A</b>) The percentages and numbers of LSK, LSK⁻ and Lin⁻Sca1⁻c-Kit⁺ were compared between WT and <i>Icsbp^−/−</i> mice (n = 4 for each group). *: <i>p</i><0.05; **: <i>p</i><0.01. (<b>B</b>) The percentages of LSK, LSK⁻ and Lin⁻Sca1⁻c-Kit⁺ were compared between WT and BXH2 mice. (<b>C</b>) Cell cycle analysis of progenitor and stem cells in <i>Icsbp^−/−</i> mice. Bone marrow cells were stained with Hoechst blue, and DNA contents, represented by the percentages of progenitor and stem cells in the S+G2M phase of the cell cycle, were examined by FACS (n = 4). **: <i>p</i><0.01. (<b>D</b>) Apoptotic rates of progenitor and stem cells in <i>Icsbp^−/−</i> mice. The cells were labeled with 7AAD and Annexin V, and the percentages of progenitor and stem cells were determined by FACS (n = 4). **: <i>p</i><0.01. (<b>E</b>) <i>Lyn</i> expression in bone marrow cells was compared between WT and <i>Icsbp^−/−</i> mice. Bone marrow cells were collected from WT and <i>Icsbp</i>^−/− mice, respectively, and <i>Lyn</i> expression was detected by real-time PCR (n = 3). *: <i>p</i><0.05. (<b>F</b>) The percentages of LSK and LSK⁻ were compared between WT and <i>Lyn^−/−</i> mice. <i>Lyn</i> deletion caused an increase in the percentage of LSK and decrease in the percentage of LSK⁻ cells. (<b>G</b>) Overexpression of <i>Lyn</i> caused an increase in the LSK⁻ population. <i>Lyn</i>^−/− bone marrow cells were transduced with <i>MSCV-GFP</i> or <i>MSCV-Lyn-GFP</i> retrovirus, followed by transplantation of the transduced cells into lethally irradiated WT recipient mice. At day 60 after the transplantation, the percentages of LSK and LSK⁻ cells were determined by FACS.</p