产品介绍
酪酰胺信号放大技术（Tyramide Signal Amplification, TSA）主要是利用酪胺的过氧化物酶反应。酪胺非活性荧光素底物在HRP和过氧化氢的作用下，会被激活产生活化荧光底物，同时形成共价键结合位点，共价结合在蛋白抗原表面或附近的酪氨酸残基上，抗原和抗体的结合部位就会有大量的酪胺荧光素沉积，使抗原位点处的荧光信号增强。
酪胺荧光素底物-抗原酪氨酸共价稳定结合，故TSA信号不会受微波影响，可用热修复法清除第一轮与抗原非共价结合的抗体复合物(冰冻切片，细胞爬片样本请试用抗体洗脱液洗脱法清除)，并能在抗体去除后保留与抗原相关的荧光信号。然后，再用第二种一抗进行第二轮孵育，同时更换另一种酪胺荧光素底物，多次循环反复，不同的酪胺荧光素进行标记就可实现多重免疫组化染色。
TSA原理示意图
产品组成成分
单标双色 | 10T | 50T | 100T | 保存条件 |
TSA单标荧光染料 | 0.5 ml | 2.5 ml | 5 ml | -20℃，避光 |
内源性过氧化物酶阻断剂 | 0.5 ml | 2.5 ml | 5 ml | 4℃，避光 |
超敏酶标山羊抗小鼠/兔 IgG 聚合物 | 0.5 ml | 2.5 ml | 5 ml | 4℃，避光 |
双标三色 | 20T | 100T | 保存条件 |
TSA-520 荧光染料 | 1 ml | 5 ml | -20℃，避光 |
TSA-570 荧光染料 | 1 ml | 5 ml | -20℃，避光 |
抗体洗脱液（仅冰冻切片、细胞爬片有） | 6 ml | 30 ml | RT |
内源性过氧化物酶阻断剂 | 4 ml | 15 ml | 4℃，避光 |
超敏酶标山羊抗小鼠/兔 IgG 聚合物 | 2 ml | 10 ml | 4℃，避光 |
三标四色 | 20T | 100T | 保存条件 |
TSA-520 荧光染料 | 1 ml | 5 ml | -20℃，避光 |
TSA-570 荧光染料 | 1 ml | 5 ml | -20℃，避光 |
TSA-690 荧光染料 | 1 ml | 5 ml | -20℃，避光 |
抗体洗脱液（仅冰冻切片、细胞爬片有） | 6 ml | 30 ml | RT |
内源性过氧化物酶阻断剂 | 5 ml | 25 ml | 4℃，避光 |
超敏酶标山羊抗小鼠/兔 IgG 聚合物 | 3 ml | 15 ml | 4℃，避光 |
四标五色 | 20T | 100T | 保存条件 |
TSA-520 荧光染料 | 1 ml | 5 ml | -20℃，避光 |
TSA-570 荧光染料 | 1 ml | 5 ml | -20℃，避光 |
TSA-620 荧光染料 | 1 ml | 5 ml | -20℃，避光 |
TSA-690 荧光染料 | 1 ml | 5 ml | -20℃，避光 |
抗体洗脱液（仅冰冻切片、细胞爬片有） | 6 ml | 30 ml | RT |
内源性过氧化物酶阻断剂 | 6 ml | 30 ml | 4℃，避光 |
超敏酶标山羊抗小鼠/兔 IgG 聚合物 | 4 ml | 20ml | 4℃，避光 |
五标六色 | 20T | 100T | 保存条件 |
TSA-520 荧光染料 | 1 ml | 5 ml | -20℃，避光 |
TSA-570 荧光染料 | 1 ml | 5 ml | -20℃，避光 |
TSA-620 荧光染料 | 1 ml | 5 ml | -20℃，避光 |
TSA-690 荧光染料 | 1 ml | 5 ml | -20℃，避光 |
TSA-780 荧光染料 | 1 ml | 5 ml | -20℃，避光 |
抗体洗脱液（仅冰冻切片、细胞爬片有） | 6 ml | 30 ml | RT |
内源性过氧化物酶阻断剂 | 8 ml | 35 ml | 4℃，避光 |
超敏酶标山羊抗小鼠/兔 IgG 聚合物 | 5 ml | 25 ml | 4℃，避光 |
六标七色 | 20T | 100T | 保存条件 |
TSA-480 荧光染料 | 1 ml | 5 ml | -20℃，避光 |
TSA-520 荧光染料 | 1 ml | 5 ml | -20℃，避光 |
TSA-570 荧光染料 | 1 ml | 5 ml | -20℃，避光 |
TSA-620 荧光染料 | 1 ml | 5 ml | -20℃，避光 |
TSA-690 荧光染料 | 1 ml | 5 ml | -20℃，避光 |
TSA-780 荧光染料 | 1 ml | 5 ml | -20℃，避光 |
抗体洗脱液（仅冰冻切片、细胞爬片有） | 6 ml | 30 ml | RT |
内源性过氧化物酶阻断剂 | 10 ml | 45ml | 4℃，避光 |
超敏酶标山羊抗小鼠/兔 IgG 聚合物 | 6 ml | 30 ml | 4℃，避光 |
保存条件
1、荧光染料（TSA-480/520/570/620/690/780）： -20℃避光保存，12个月
2、内源性过氧化物酶阻断剂、超敏酶标山羊抗小鼠/兔 IgG 聚合物 ：4℃避光保存，有效期 12 个月。
3、抗体洗脱液 ：RT保存，有效期 12 个月。
实验前材料准备
1、石蜡或冰冻切片，细胞爬片。建议配合 Abiowell 带有 IF-T 应用的一抗使用。
石蜡切片：建议一抗应用中注明 IHC-P/IF-T；
冰冻切片：建议一抗应用中注明 IHC-F/IF-T；
细胞爬片：建议一抗应用中注明 IF/ICC/IF-C；
2、缓冲液与修复液：PBS 缓冲液（货号：AWC0215）、柠檬酸钠抗原修复液（货号：AWI0206）（或其他适配修复液）。
3、DAPI 染色液（推荐浓度：5μg/mL 货号：AWC0293）
4、其他辅助材料：组化笔、避光湿盒、慢速摇床、抗荧光淬灭封片剂（货号：AWI0197）等。
5、封闭山羊血清（货号：AWI0115）
操作步骤
（一）操作前注意事项
1、TSA 染色灵敏度高于常规的荧光显色，建议开展正式实验前，进行单色预实验，选择最适合的一抗稀释比例；
2、在进行多重染色时，通常建议先标记预实验中阳性丰度低的抗体。
3、针对四标染色，推荐的染色顺序为：TSA-520 → TSA-570 → TSA-690 → TSA-620。
4、由于 TSA-570 与 TSA-620 染料的发射波长较为接近，使用宽带通成像设备易出现信号串色，若需同时使用，需注意：
*建议将 TSA-570 与 TSA-620 搭配弱阳性一抗使用；
*尽量避免将其用于同一细胞中共同表达的靶标（例如 CD3 与 CD4 的共染组合）；
*图像采集时，请务必使用配备窄带通滤光片的成像设备，以有效区分相邻通道信号，确保成像质量。
5、加入荧光染料及后续步骤需进行避光操作。
6、操作过程中需保持载玻片组织的湿润，如出现干片情况，会导致非特异性的染色结果。
7、持续加热修复的过程中，只需中小火维持沸腾，切勿高火加热让容器内试剂蒸发导致干片，且高火易导致组织脱片。
8、使用小鼠组织实验时，建议选用兔源一抗搭配 HRP 标记的抗兔二抗，可减少非特异性染色。
9、对富含脂肪的组织（如肝脏、乳腺等）染色时，建议在第 11 步 DAPI 染色前增加苏丹黑处理步骤，可有效消除脂肪的自发荧光及非特异性着色，避免干扰目标抗原信号检测，提升目标信号清晰度与检测准确性。
（二）石蜡切片操作步骤（四标为例）
1、脱蜡复水：
(1)将石蜡玻片在 60 ℃ 烘烤 1-2 小时。
(2)依次将切片放入二甲苯Ⅰ 15 分钟→二甲苯Ⅱ 15 分钟→二甲苯Ⅱ 15 分钟→无水乙醇Ⅰ 10分钟→无水乙醇Ⅱ 10分钟→ 95% 乙醇 5 分钟→ 85% 乙醇 5 分钟→ 75% 乙醇 5 分钟→蒸馏水洗。
2、抗原修复：
(1)向容器中加入约 2/3 体积的柠檬酸修复液或 EDTA 修复液，加盖后用微波炉高火加热至沸腾（修复液及条件需根据组织、抗原类型调整）；
(2)取出容器，待液体停止沸腾后，将切片间隔插入切片架并放入容器；
(3)容器放回微波炉，低火加热 20 分钟，加热结束后在微波炉内静置 20 分钟后，取出自然冷却；
(4)将玻片转移至 PH7.4 的 PBS 中，用慢速摇床洗涤 3 次，每次 5 分钟；
(5)擦干组织周围水分（保持组织湿润），用组化笔在组织周围画封闭圈。
3、内源性过氧化物酶阻断：
向组织滴加内源性过氧化物酶阻断剂，在湿盒内室温孵育 10-15 分钟；随后将切片放入 PBS中，用慢速摇床洗涤 3 次，每次 3 分钟。
4、封闭：
用正常山羊血清均匀覆盖组织，室温封闭30min。
5、一抗孵育：
沥干玻片（约几秒），稀释一抗后滴加至完全覆盖组织；室温或 37 ℃ 孵育 3-4 小时，或 4 ℃ 湿盒过夜后 37 ℃ 复温 1-2 小时；最后用 PBST 冲洗 3 次，每次 2分钟。
6、二抗孵育：
沥干玻片，滴加 50-100μL 超敏酶标山羊抗小鼠/兔 IgG聚合物（以覆盖组织为宜），室温孵育 30 分钟；用 PBST 冲洗 3 次，每次 2 分钟。
7、TSA 反应：
再次沥干玻片，滴加 50-100μL TSA-520 荧光染料（覆盖组织即可），孵育 5-15 分钟；PBST冲洗 3 次，每次 2分钟。
8、抗体洗脱：
将切片间隔插入切片架，放入含煮沸修复液的容器（煮沸修复液需能没过组织）；微波炉低火加热 20 分钟后，在微波炉内静置 20 分钟，取出自然冷却；将玻片转移至 PH7.4 的 PBS 中，慢速摇床洗涤 3 次，每次 5 分钟。
9、第二轮染色：
重复步骤 4-8，一抗更换为第二种目标一抗，TSA荧光染料换为 TSA-570。
10、第三轮染色：
重复步骤 4-8，一抗更换为第三种目标一抗，TSA荧光染料换为 TSA-690。
11、第四轮染色：
重复步骤 4-7（无需抗体洗脱），一抗更换为第四种目标一抗，TSA荧光染料换为 TSA-620。
（注：多重免疫荧光染色的最后一轮均无需抗体洗脱）
12、DAPI 复染细胞核：
将玻片放入 PH7.4 的 PBS 中，慢速摇床洗涤 3 次，每次 5分钟；稍甩干后，向封闭圈内滴加DAPI 染液，避光室温孵育 10 分钟。
13、封片：
PBS 洗涤 3 次，每次 5 分钟；稍甩干切片，用抗荧光淬灭封片剂封片。
14、镜检拍照：
在荧光显微镜、共聚焦显微镜、多通道荧光扫描仪或多光谱成像系统下观察并采集图像。
（三）细胞爬片/冰冻切片使用步骤（四标为例）
1、固定（选做）：
冰冻切片：复温至室温后，滴加 4% 多聚甲醛（试剂盒未提供）孵育 10-15 分钟，室温晾干后用 PBS 浸泡脱胶（3 缸，每缸 10 分钟）；若 OCT 包埋前未固定，建议必做此步骤。
细胞爬片：直接加入 4% 多聚甲醛固定 10-15 分钟，PBS 洗涤 3 次，每次 5 分钟。
2、破膜（选做）：
用 0.1-0.3% Triton X-100（需自备，浓度不建议超过 0.3%）室温通透 20 分钟；免疫原在胞外段的一抗可省略此步骤；后续用 PBS 洗涤 3 次，每次 5 分钟。
3、内源性过氧化物酶阻断：
(1)擦干组织周围水分（保持组织湿润），用组化笔在组织周围画封闭圈。
(2)向组织滴加内源性过氧化物酶阻断剂，在湿盒内室温孵育 10-15 分钟；随后将切片放入PBS中，用慢速摇床洗涤 3 次，每次 3 分钟。
4、封闭：
用正常山羊血清均匀覆盖组织，室温封闭30min。
5、一抗孵育：
沥干玻片（约几秒），稀释一抗后滴加至完全覆盖组织；室温或 37 ℃ 孵育 3-4 小时，或 4 ℃ 湿盒过夜后 37 ℃ 复温 1-2 小时；最后用 PBST 冲洗 3 次，每次 2 分钟。
6、二抗孵育：
沥干玻片，滴加 50-100μL 超敏酶标山羊抗小鼠/兔 IgG聚合物（以覆盖组织为宜），室温孵育 30 分钟；用 PBST 冲洗 3 次，每次 2 分钟。
7、TSA 反应：
滴加 50-100μL TSA-520 荧光染料（覆盖组织即可），孵育 5-15 分钟 PBST 冲洗 3 次，每次 2 分钟。
8、抗体洗脱：
滴加适量 37 ℃ 预热的抗体洗脱液覆盖组织，37 ℃ 放置 5-20 分钟，甩干后无需洗涤，再次滴加适量抗体洗脱液覆盖组织 37 ℃ 放置 5-20 分钟(通常两次洗脱合计20分钟），PBST 洗三次，每次 5 分钟。
9、第二轮染色：
重复步骤 4-8，一抗更换为第二种目标一抗，TSA荧光染料换为 TSA-570。
10、第三轮染色：
重复步骤 4-8，一抗更换为第三种目标一抗，TSA荧光染料换为 TSA-690。
11、第四轮染色：
重复步骤 4-7（无需抗体洗脱），一抗更换为第四种目标一抗，TSA荧光染料换为 TSA-620。
（注：多重免疫荧光染色的最后一轮均无需抗体洗脱）
12、DAPI 复染细胞核：
将玻片放入 PH7.4 的 PBS 中，慢速摇床洗涤 3 次，每次 5分钟；稍甩干后，向封闭圈内滴加 DAPI 染液，避光室温孵育 10 分钟。
13、封片：
PBS 洗涤 3 次，每次 5 分钟；稍甩干切片，用抗荧光淬灭封片剂封片。
14、镜检拍照：
在荧光显微镜、共聚焦显微镜、多通道荧光扫描仪或多光谱成像系统下观察并采集图像。
荧光染料参数表：
染料 | 激发波长 | 发射波长 | 荧光强度 |
DAPI | 350nm | 420nm | - |
TSA-480 | 450nm | 480nm | + |
TSA-520 | 490nm | 520nm | ++ |
TSA-570 | 550nm | 570nm | +++ |
TSA-620 | 590nm | 620nm | + |
TSA-690 | 630nm | 690nm | +++ |
TSA-780 | 750nm | 780nm | ++++ |
常见问题
1、染色过深：一抗浓度过高，时间过长；TSA孵育时间过长，导致非特异性结合。
2、染色过浅或无染色：一抗浓度过低，时间过短；抗原修复不够；HRP是否失效。
3、无特异性染色：切片脱蜡不彻底。可适当延长烤片时间。
4、有明显串色现象：上一支一抗未洗脱干净。
5、抗体洗脱液使用三轮及以上易致组织 / 细胞核损伤，为降低核破碎风险，前两轮染色后的一抗洗脱可采用以下方法：将切片间隔置于切片架，浸入 50-60 ℃热水中浸泡 5-20 分钟，重复 2-3 次；若一抗残留未净，可少量使用抗体洗脱液并缩短孵育时长。第三轮开始使用抗体洗脱液正常洗脱。
注意事项
1、试剂初次使用前请置于4℃解冻，解冻后于4℃短期保存，避免反复冻融，请尽快使用。
2、为了您的安全和健康，请穿好实验服并佩戴一次性手套和口罩操作。
3、本产品仅限于专业人员的科学研究用，不得用于临床诊断或治疗，不得用于食品或药品。
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AWI0197 | 抗荧光淬灭封片剂 |
AWI0603 | 免疫染色通透液(Triton X-100) |
参考文献 (8)
Pseudomonas aeruginosa pneumonia poses a significant therapeutic challenge. Nanoparticles serve as an effective tool for nucleic acid delivery to efficiently alleviate pneumonia. This study develops a hyaluronic acid (HA)-coated peptide nanoparticle system for targeted delivery of small interfering RNA (siRNA) against Tudor domain-containing protein 9 ( TDRD9 ), identified via RNA sequencing of bronchoalveolar lavage fluid-derived neutrophils from 21 recruited patients (11 males/10 females). Adoptive transfer of TDRD9-silenced polymorphonuclear neutrophils into neutrophil-depleted male mice attenuates lung inflammation and edema. Mechanistically, TDRD9 suppresses neutrophil cuproptosis by upregulating programmed death ligand 1 (PD-L1) through interaction with CD80 to activate p38 mitogen-activated protein kinase (MAPK) signaling. HA-si-TDRD9 nanoparticles enhance neutrophil cuproptosis, reduce pulmonary neutrophil accumulation, and ameliorate lung injury via PD-L1/CD80/MAPK. Importantly, HA-si-TDRD9 nanoparticles reduce bacterial growth, apoptosis, and inflammation in human lung organoids. This work demonstrates that targeting TDRD9 with siRNA nanoparticle platform presents a promising therapeutic strategy for treating bacterial lung injury.
In South and Southeast Asia, the habit of chewing betel nuts is prevalent, which leads to oral submucous fibrosis (OSF). OSF is a well-established precancerous lesion, and a portion of OSF cases eventually progress to oral squamous cell carcinoma (OSCC). However, the specific molecular mechanisms underlying the malignant transformation of OSCC from OSF are poorly understood. In this study, the leading-edge techniques of Spatial Transcriptomics (ST) and Spatial Metabolomics (SM) are integrated to obtain spatial location information of cancer cells, fibroblasts, and immune cells, as well as the transcriptomic and metabolomic landscapes in OSF-derived OSCC tissues. This work reveals for the first time that some OSF-derived OSCC cells undergo partial epithelial–mesenchymal transition (pEMT) within the in situ carcinoma (ISC) region, eventually acquiring fibroblast-like phenotypes and participating in collagen deposition. Complex interactions among epithelial cells, fibroblasts, and immune cells in the tumor microenvironment are demonstrated. Most importantly, significant metabolic reprogramming in OSF-derived OSCC, including abnormal polyamine metabolism, potentially playing a pivotal role in promoting tumorigenesis and immune evasion is discovered. The ST and SM data in this study shed new light on deciphering the mechanisms of OSF-derived OSCC. The work also offers invaluable clues for the prevention and treatment of OSCC.
Background: Hepatic inflammatory cell accumulation and the subsequent systematic inflammation drive acute-on-chronic liver failure (ACLF) development. Previous studies showed that the vagus nerve exerts anti-inflammatory activity in many inflammatory diseases. Here, we aimed to identify the key molecule mediating the inflammatory process in ACLF and reveal the neuroimmune communication arising from the vagus nerve and immunological disorders of ACLF. Methods: Proteomic analysis was performed and validated in ACLF model mice or patients, and intervention animal experiments were conducted using neutralizing antibodies. PNU-282987 (acetylcholine receptor agonist) and vagotomy were applied for perturbing vagus nerve activity. Single-cell RNA sequencing (scRNA-seq), flow cytometry, immunohistochemical and immunofluorescence staining, and CRISPR/Cas9 technology were used for in vivo or in vitro mechanistic studies. Results: The unbiased proteomics identified C-X-C motif chemokine ligand 9 (CXCL9) as the greatest differential protein in the livers of mice with ACLF and its relation to the systematic inflammation and mortality were confirmed in patients with ACLF. Interventions on CXCL9 and its receptor C-X-C chemokine receptor 3 (CXCR3) improved liver injury and decreased mortality of ACLF mice, which were related to the suppressing of hepatic immune cells’ accumulation and activation. Vagus nerve stimulation attenuated while vagotomy aggravated the expression of CXCL9 and the severity of ACLF. Blocking CXCL9 and CXCR3 ameliorated liver inflammation and increased ACLF-associated mortality in ACLF mice with vagotomy. scRNA-seq revealed that hepatic macrophages served as the major source of CXCL9 in ACLF and were validated by immunofluorescence staining and flow cytometry analysis. Notably, the expression of CXCL9 in macrophages was modulated by vagus nerve-mediated cholinergic signaling. Conclusions: Our novel findings highlighted that the neuroimmune communication of the vagus nerve–macrophage–CXCL9 axis contributed to ACLF development. These results provided evidence for neuromodulation as a promising approach for preventing and treating ACLF.
Background and aims Increasing evidence indicates that modulating pyroptosis in endothelial cells (ECs) can alleviate atherosclerosis (AS) progression; however, despite reports that nucleolin (NCL) regulates vascular smooth muscle cell proliferation in AS, the potential mechanism by which cell surface NCL mediates pyroptosis in ECs during AS remains poorly understood. Methods AS was induced in ApoE -/- mice by feeding a high-fat diet, after which aortic lesions were evaluated. Pyroptosis, inflammatory status, and NCL expression in ECs of the aortic root were then assessed. The effects of NLRP3 inflammasome inhibition and NCL modulation on atherosclerotic lesion severity in AS mice, as well as on pyroptosis in ox-LDL-stimulated ECs, were systematically investigated. In addition, the mechanistic role of NCL in AS was further explored using approaches including immunoprecipitation-mass spectrometry (IP-MS). Results AS model mice developed severe aortic lesions accompanied by pronounced EC pyroptosis and inflammation, together with elevated NCL expression in ECs of the aortic root. Both inhibition of NLRP3 and NCL knockdown alleviated atherosclerotic lesion severity in ApoE -/- mice and attenuated ox-LDL-induced EC pyroptosis. Mechanistically, cell-surface NCL interacted with RASSF2 via its RNA-binding domain, and suppression of NCL decreased nuclear RASSF2 expression. NCL facilitated the translocation of RASSF2 into the nucleus, thereby exacerbating EC pyroptosis and amplifying inflammatory responses. Conclusions This study demonstrates that, in AS, NCL exacerbates EC pyroptosis and promotes disease progression by facilitating nuclear transport of RASSF2. This study defines the mechanistic roles of NCL in AS, thereby identifying a new molecular pathway and suggesting potential therapeutic targets.
Pancreatic cancer is highly challenging, with most patients developing intrinsic or acquired resistance to first-line chemotherapy drug gemcitabine (GEM). Although Matrix Metalloproteinase 28 (MMP28) is upregulated in pancreatic cancer and predicts a poor prognosis, its role in GEM resistance and molecular mechanism remain unclear. Here, we aimed to investigate the role of MMP28 in GEM resistance and molecular mechanism. First, differentially expressed genes in pancreatic cancer were identified through bioinformatics and validated in clinical samples and cells. MMP28 was significantly overexpressed in pancreatic cancer tissues and Capan-1 and PANC-1 cells, correlating with poor prognosis. Then, MMP28 knockdown was performed in Capan-1 and PANC-1 cells, followed by GEM treatment. Furthermore, in vivo experiments evaluated GEM sensitivity after MMP28 knockdown. The results showed that MMP28 knockdown enhanced GEM sensitivity both in vitro , reducing cell proliferation and survival, and in vivo , where tumor growth was significantly suppressed. Additionally, glycolysis-related changes were assessed. We revealed that glycolysis was implicated as a key pathway in this process, with reduced glucose uptake and lactate production observed after MMP28 knockdown. Protein-protein interaction analysis identified Staphylococcal nuclease domain-containing protein 1 (SND1) as a key interactor, and SND1 expression was upregulated in pancreatic cancer tissues. Moreover, MMP28 interacted with SND1 to regulate SND1′s recruitment of HK2 mRNA to promote glycolysis. However, overexpression of SND1 reversed the effects of MMP28 knockdown, restoring glycolysis and GEM resistance. In conclusion, MMP28 promoted tumor growth and GEM resistance in pancreatic cancer by regulating glycolysis via interaction with SND1.
Background Inhibiting cholesterol metabolism has shown great potential in non-small cell lung cancer (NSCLC). However, the regulatory mechanism of the lipid metabolism key factor Sect. 14-like lipid binding 2 (SEC14L2) in NSCLC remains unclear. This study investigates the effects of differentially expressed genes related to cholesterol metabolism on the development of NSCLC.Methods Cox regression and survival analysis were performed to screen cholesterol metabolism-related genes and predict survival prognosis in NSCLC patients. The proliferation and migration of NSCLC cells were assessed by CCK-8, EdU, colony formation and wound-healing assay. Cholesterol depletion and rescue trials were used to evaluate the effect of SEC14L2 on cholesterol transport in NSCLC cells. IF and Co-IP were used to analyze the targeting relationship between SEC14L2 and scavenger receptor class B member 1 (SCARB1).Results SEC14L2 was a key gene related to prognosis in NSCLC patients and was highly expressed in A549 and Calu-1 cells. Subsequent studies demonstrated that knockdown of SEC14L2 significantly reduced the proliferation and migration of NSCLC cells, resulting in inhibited tumor growth. Furthermore, both in vitro and in vivo experiments indicated that SEC14L2 regulated cholesterol uptake. Silencing SEC14L2 partially counteracted the promotion of cholesterol content by MβCD-chol in A549 and Calu-1 cells. We then verified that there was a protein interaction between SEC14L2 and SCARB1.Conclusion SEC14L2 promoted cholesterol uptake in NSCLC cells by up-regulating SCARB1 expression, thereby promoting NSCLC development.
Glioblastoma stem cells (GSCs) have been implicated in the self-renewal and treatment resistance of glioblastoma (GBM). Our previous study found that 4,5-dimethoxycanthin-6-one has the potential to inhibit GBM cell proliferation. This current study aims to elucidate the molecular mechanism underlying the effects of 4,5-dimethoxycanthin-6-one in GBM development. The effect of 4,5-dimethoxycanthin-6-one on GSC formation and differentiation was explored in human GBM cell lines U251 and U87. Subsequently, 4,5-dimethoxycanthin-6-one binding to tetraspanin 1 (TSPAN1) / transmembrane 4 L six family member 1 (TM4SF1) was analyzed by molecular simulation docking. Co-immunoprecipitation (Co-IP) and immunofluorescence (IF) were used to assess the interactions between TSPAN1 and TM4SF1 in GSCs. Cell proliferation was detected by cell counting kit-8 (CCK-8) and colony formation assay. To evaluate cell migration, invasion and apoptosis, we employed wound healing assay, transwell and flow cytometry, respectively. Furthermore, subcutaneous xenograft tumor models in nude mice were constructed to evaluate the impact of 4,5-dimethoxycanthin-6-one on GSCs in vivo by examining tumor growth and histological characteristics. 4,5-Dimethoxycanthin-6-one inhibited GSC formation and promoted stem cell differentiation in a concentration-dependent manner. Molecular docking models of 4,5-dimethoxycanthin-6-one with TM4SF1 and TSPAN1 were constructed. Then, the interaction between TSPAN1 and TM4SF1 in GSC was clarified. Moreover, 4,5-dimethoxycanthin-6-one significantly inhibited the expressions of TM4SF1 and TSPAN1 in vitro and in vivo. Overexpression of TSPAN1 partially reversed the inhibitory effects of 4,5-dimethoxycanthin-6-one on GSC formation, proliferation, migration and invasion. 4,5-Dimethoxycanthin-6-one inhibited GBM progression by inhibiting TSPAN1/TM4SF1 axis. 4,5-Dimethoxycanthin-6-one might be a novel and effective drug for the treatment of GBM.
Ferroptosis is a critical contributor to ischemia-reperfusion (I/R) injury and subsequent organ failure. While ENPP2 has been implicated in regulating ferroptosis in cardiomyocytes, its specific role in myocardial I/R injury remains unclear. This study aims to elucidate the function of ENPP2-mediated ferroptosis in myocardial ischemia-reperfusion injury (MI/RI), to provide novel insights into potential therapeutic strategies. A mouse model of MI/RI was established and subjected to interventions with ENPP2 overexpression and/or SIRT1 knockdown. In vitro, cardiomyocytes were treated with palmitate and subjected to hypoxia/reoxygenation (H/R) to simulate I/R injury. These cells received treatments with ENPP2 overexpression (oe-ENPP2), SIRT1 overexpression (oe-SIRT1), PGC-1α silencing (si-PGC-1α), and/or SIRT1 knockdown (sh-SIRT1). Additionally, Erastin-induced ferroptosis in cardiomyocytes was used to assess the protective effects of oe-ENPP2. Ferroptosis was assessed through the lipid peroxidation (MDA, 4-HNE), iron and Fe 2+ assays, GPx4 and SLC7A11 expression, and transmission electron microscope. Overexpression of ENPP2 significantly alleviated myocardial infarction in MI/RI mice, as indicated by the upregulation of GPx4 and SLC7A11 protein levels. In cardiomyocytes subjected to hypoxia/reoxygenation (H/R) or erastin-induced ferroptosis, oe-ENPP2 reduced apoptosis rates, preserved Fe 2+ content, and restored GPx4 and SLC7A11 expression. Silencing PGC-1α blocked the protective effect of oe-ENPP2 against H/R-induced ferroptosis in HL-1 cells. Additionally, SIRT1 overexpression inhibited PGC-1α acetylation, whereas SIRT1 knockdown similarly reversed the anti-ferroptotic effects of oe-ENPP2 in H/R-treated HL-1 cells. SIRT1 silencing blocked the protective effects of oe-ENPP2 against myocardial infarction and fibrosis in MI/RI mice via the PGC-1α/NRF1 pathway. ENPP2 overexpression protects the mouse myocardium from I/R-induced ferroptosis injury via the SIRT1/PGC-1α/NRF1 pathway. These findings suggest a novel gene therapy strategy for mitigating myocardial I/R injury.
