Raw data files associated with Figure 4 in RBL2 represses the transcriptional activity of Multicilin to inhibit multiciliogenesis.

<b>Immunoprecipitation, immunoblotting analysis, and silver staining</b>For identifying interacting proteins of FLAG-tagged Multicilin by MASS analysis, MEFs were infected by pAd/CMV/V5 vector encoding 3xFLAG-Multicilin, NLS-6xmyc-E2f4ΔCt-VP16-T2A-Multicilin, and NLS-6xmyc-E2f4WT-T2A-Multicilin. Non-infected MEFs were used as the negative control. Two days after infection, the non-infected MEFs or infected MEFs were lysed in lysis buffer (50mM HEPES-KOH [pH 7.5], 500NaCl, 5mM EDTA [pH 8.0]), 2% Triton, DNase I (10ug/ml)) by incubation on ice for 20min. Lysates were cleared by centrifugation at 12,000 rpm for 20 min at 4 °C, then precleared by incubation with Protein G agarose (Invitrogen #20397) agitating for 1hr at 4°C. Spun-down beads were incubated with FLAG M2 agarose (Sigma #A2220) agitating for 2.5 hours at 4°C. The beads were washed twice with wash buffer (1:1 mix of 50mM HEPES and Lysis buffer), washed with lysis buffer twice, then washed twice again with wash buffer. Spun-down beads were incubated with 40uL of FLAG peptide (500ng/mL) for bound protein elution overnight at 4°C. Elutes were subjected to protein sampling for further immunoblot analysis and silver staining. Eluted protein samples and experimental protein lysates were subjected to SDS-PAGE and transferred to a PVDF membrane that was then blocked with 0.5% casein blocker (#161-0783, Bio-Rad) in PBS for 30 min followed by incubation with the indicated primary antibodies overnight at 4 °C in 0.1% Tween-20 in blocking buffer. After extensive washing in 0.1% Tween-20 in PBS, the blots were incubated with Alexa 680 or 800-conjugated anti- mouse or rabbit secondary antibodies (Invitrogen, A21058, A21076, A32735) for 45 min at room temperature, washed with 0.1% Tween-20 in PBS, and imaged using Odyssey (LI-COR). Silver staining was performed with Pierce Silver Stain Kit (Thermo #24612) based on the manufacturer’s instructions.<b>Immunocytochemistry and image processing</b>MEF centriolar and cilia imaging was performed as previously described¹⁰. HBECs were grown on Corning 6.5mm Transwell inserts (#3470) or Nunc Lab-Tek chamber slides (#177445) and fixed in 4% paraformaldehyde in PBS overnight at 4°C. Cells were concurrently permeabilized and blocked-in antibody dilution buffer (5% bovine serum albumin, 0.3% Triton X-100, PBS) before incubation with antibodies (<b>Supplemental Table S4</b>) in antibody dilution buffer. Samples were incubated at room temperature with primary antibodies for 2 hours and secondary antibodies for 45 minutes. Counterstains in PBS were performed directly after secondary antibody incubation (DAPI 1µg/mL 5 minutes, Phalloidin (Biolegend#424203) 5 units/mL), with (3x) 5-minute PBS washes performed after primary antibody and counterstain incubations. Samples were mounted with No. 1.5 coverslips using Fluoromount-G mounting medium (Invitrogen #00-4958-02).Fluorescent image tile scans were performed on either the DMi8 (Leica) or Revolution (Echo) fluorescent microscope systems and processed in Fiji³⁸. TP73 positive cells were quantified by thresholding for positive DAPI and TP73 signal based on control samples, water shedding and then analyzing particle counts to give the number of nuclei positive for each stain. Cilia area coverage was calculated by thresholding for positive acetylated α-tubulin staining and then dividing by the area covered by counterstain (Phalloidin), with a thresh hold set high enough to give positive signal for the entire cell coverage area.

<b>免疫沉淀、免疫印迹与银染实验</b>为通过质谱分析鉴定带有FLAG标签的Multicilin的互作蛋白，本实验使用携带3xFLAG-Multicilin、NLS-6xmyc-E2f4ΔCt-VP16-T2A-Multicilin及NLS-6xmyc-E2f4WT-T2A-Multicilin的pAd/CMV/V5载体感染小鼠胚胎成纤维细胞（Mouse Embryonic Fibroblasts, MEFs），以未感染的MEFs作为阴性对照。感染两天后，将未感染及感染的MEFs置于裂解缓冲液（50mM HEPES-KOH [pH 7.5]、500mM NaCl、5mM EDTA [pH 8.0]、2% Triton X-100、DNase I [10μg/mL]）中，冰浴孵育20分钟完成裂解。裂解液于4℃下以12000 rpm离心20分钟以去除不溶沉淀，随后加入Protein G琼脂糖磁珠（Invitrogen #20397），4℃下振荡孵育1小时进行预澄清。离心收集磁珠后，与FLAG M2琼脂糖树脂（Sigma #A2220）混合，4℃下振荡孵育2.5小时。之后用洗涤缓冲液（50mM HEPES与裂解缓冲液按1:1体积比混合）洗涤磁珠2次，再用裂解缓冲液洗涤2次，最后再次用洗涤缓冲液洗涤2次。再次离心收集磁珠后，加入40μL FLAG肽段（500ng/mL），4℃下孵育过夜以洗脱结合的蛋白。洗脱液取部分样本用于后续免疫印迹分析与银染。将洗脱的蛋白样本及实验裂解液进行SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE），随后转印至聚偏氟乙烯（Polyvinylidene Fluoride, PVDF）膜；膜用含0.5%酪蛋白封闭液（#161-0783, Bio-Rad）的PBS溶液封闭30分钟，随后在含0.1% Tween-20的封闭缓冲液中，与指定的一抗于4℃下孵育过夜。用含0.1% Tween-20的PBS溶液充分洗涤膜后，于室温下与Alexa Fluor 680或Alexa Fluor 800标记的抗小鼠/兔二抗（Invitrogen, A21058、A21076、A32735）孵育45分钟，再用含0.1% Tween-20的PBS洗涤，最后使用Odyssey红外成像系统（LI-COR）进行成像。银染实验使用Pierce银染试剂盒（Thermo #24612），严格按照制造商说明书操作完成。<b>免疫细胞化学与图像处理</b>MEFs的中心粒与纤毛成像实验参照已发表文献¹⁰进行。人支气管上皮细胞（Human Bronchial Epithelial Cells, HBECs）接种于Corning 6.5mm Transwell小室插入膜（#3470）或Nunc Lab-Tek腔室玻片（#177445）上，用含4%多聚甲醛的PBS溶液于4℃下固定过夜。细胞先用抗体稀释缓冲液（5%牛血清白蛋白、0.3% Triton X-100、PBS）同时进行透化与封闭，随后在抗体稀释缓冲液中与对应一抗（见<b>补充表S4</b>）孵育。样本于室温下分别与一抗孵育2小时、二抗孵育45分钟。二抗孵育结束后直接进行复染：使用1μg/mL DAPI染色5分钟，或使用5 units/mL 鬼笔环肽（Phalloidin, Biolegend #424203）染色；一抗孵育及复染完成后，均用PBS洗涤3次，每次5分钟。样本使用Fluoromount-G封片剂（Invitrogen #00-4958-02）与1.5号盖玻片进行封片。荧光图像平铺扫描使用DMi8（Leica）或Revolution（Echo）荧光显微镜系统完成，图像后期处理使用Fiji软件³⁸。TP73阳性细胞的定量分析方法为：以对照样本为基准，对DAPI及TP73阳性信号进行阈值分割，去除背景噪声后统计颗粒数量，从而得到每种染色的阳性细胞核数目。纤毛面积覆盖率的计算方法为：对乙酰化α-微管蛋白阳性染色进行阈值分割，将得到的面积除以鬼笔环肽复染标记的细胞总面积，阈值设置需确保可覆盖整个细胞区域的阳性信号。