Ab188161

A total of 93 paired Formalin-fixed, paraffin-embedded (FFPE) tissue samples were stained using primary anti-RRS1 antibody (ab188161, Abcam). The intensity of RRS1 expression was graded as follows: negative = score 0, weak = score 1, moderated = score 2 and strong = score 3. Extent of staining was grouped according to the percentage of high-staining cells in the cancer nest: negative = score 0, 1% to 25% = score 1, 26% to 50% = score 2, 51% to 75% = score 3 and 76% to 100% = score 4. The final quantitation of each staining was obtained by multiplying the two scores. Low expression means refers to a RRS1 score ing≤6, and high expression means refers to a RRS1 scoring score >6. Immuno-reactivity was assessed independently by two expert pathologists blind to all clinical data.

The cells were lysed in RIPA buffer (Sparkjade, Jinan, China) supplemented with protease inhibitor cocktail (Servicebio, Wuhan, China) and phosphatase inhibitors A and B on ice for 30 min. The lysates were centrifuged for 15 min at 12,000 rpm, and their protein content was measured. Equal amounts of protein per sample were resolved on 10% SDS-PAGE gels and transferred to a PVDF membrane. After blocking with 5% BSA in TBST for 2 h at room temperature, the membranes were incubated overnight with antibodies targeting GAPDH (Abclonal, Wuhan, China, AC002), RRS1 (Abcam, Cambridge, UK, AB188161), AEG-1 (Abcam, Cambridge, UK, ab227981), ABCG2 (ZenBio, Research Triangle Park, NC, USA, R26465), MDR1 (Proteintech, Wuhan, China, 22336-1-AP), ERK (ZenBio, NC, USA, 340373), p-ERK (ZenBio, NC, USA, 340767), BAX (CST, Danvers, MA, USA, #89477), Bcl-2 (CST, Danvers, MA, USA, #3498), BAD (Abcam, Cambridge, UK, ab32445), p-BAD (Abcam, Cambridge, UK, ab129192) and ubiquitin (Proteintech, Wuhan, China, 10201-2-AP) at 4 °C. The membranes were washed thrice with TBST and then incubated with HRP-conjugated secondary antibody (1:1000; Bioss, Beijing, China, bs-0295G-HRP), followed by three more washes with TBST. The positive bands were visualized via ECL (MDBio, Taiwan, China).

Animal experiments were conducted with approval from the Qingdao University Laboratory Animal Welfare Ethics Committee (approval number: 202207BALB/cnude12202208082, date: 5 June 2022). All procedures adhered to relevant guidelines and regulations and were reported in accordance with ARRIVE guidelines.
BALB/c nude mice (4 weeks old) were supplied by Beijing Vital River Laboratory Animal Technology (China). Sh-SNHG1 or negative control MCF-7 cells (5 × 10⁶) were resuspended in a 200 μL mixture of 100 μL PBS with 100 μL Matrigel (Corning) and subcutaneously injected into the anterior part of mice (n = 5 per group). Tumor volume was calculated using the formula V = (length × width²)/2 every 3 days after tumor formation. On day 24, mice were euthanized using the cervical dislocation method under anesthesia (0.7% sodium pentobarbital), and tumors were harvested and weighed. Tumor tissues were fixed with paraformaldehyde, embedded in paraffin, and cut into 5-μm sections for immunohistochemistry (IHC). The primary antibody RRS1 (1:200, ab188161; Abcam) was used for IHC, with nuclei stained blue with hematoxylin and RRS1-positive level appearing brown or brownish yellow with diaminobenzidine (Supplementary Information).