Journal: Cancer Cell International

Article Title: Rho-related GTP-binding protein RhoE (RND3) regulates multiple myeloma bone disease

doi: 10.1186/s12935-025-04166-2

Figure Lengend Snippet: RND3-ROCK1/2 axis regulate MMP9 expression in myeloma cells. A ) The qPCR array showed the up-regulation of MMP9 and DKK1 in RN-KD MM cells compared with CT-KD ARD MM cells. B ) According to the qPCR results, it was evident that the MMP-9 transcription level in RN-KD ARD MM was elevated. C ) The culture supernatants of CT-KD and RN-KD were detected by ELISA. The content of MMP-9 in the culture supernatant of RN-KD ARD MM was higher. D ) The dual-luciferase reporter gene assays showed that the transcriptional level of MMP-9 in RN-KD ARD MM was higher than that in CT-KD. E ) The Ch-IP assays demonstrated that Smad2/3 could bind to the MMP-9 promoter, and compared with CT-KD, RN-KD ARD MM cells exhibited a greater amount of such binding. F ) Western blot results showed that compared with CT-KD MM cells, the expression of RhoA, ROCK1, ROCK2, MMP9, and pSMAD2/3 (Thr 8) was upregulated in RN-KD MM cells (AMO.1 and ARD). G ) The Western blot results showed that, compared with CT-KD, both the nucleus and cytoplasm of RN-KD MM cells contained more pSMADsmad2/3 (Thr 8). H ) After ARD MM cells were treated with the pSMAD2/3 inhibitor NCB-0846 (1 µM) for 48 h, the expression level of MMP9 was significantly downregulated. (* p < 0.05 (Student’s t test). RN-KD = RND3 knockdown; CT-KD = control knockdown.)

Article Snippet: The antibodies utilized included RND3 (ProMab, USA, #30838), actin (Proteintech, China, #2D4H5), GAPDH (Proteintech, China, #1E6D9), ROCK1 (Proteintech, China, #21850-1-AP), ROCK2 (Proteintech, China, #21645-1-AP), Phospho-Smad2/3 (Affinity, China, #AF3367), DKK1 (Proteintech, China, # 21112-1-AP), STAT3 (Proteintech, China, # 10253-2-AP), Phospho-Stat3 (CST, USA, #9134), TBP (Proteintech, China, # 66166-1-Ig), and MMP-9 (Hua Bio, China, #ET1704-69).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Luciferase, Binding Assay, Western Blot, Knockdown, Control

Journal: Cancer Cell International

Article Title: Rho-related GTP-binding protein RhoE (RND3) regulates multiple myeloma bone disease

doi: 10.1186/s12935-025-04166-2

Figure Lengend Snippet: RND3-ROCK2 axis regulate DKK1 expression in myeloma cells. A ) The culture supernatants of CT-KD ARD cells and RN-KD ARD cells were detected by ELISA. The content of DKK1 in the culture supernatant of RN-KD MM was higher. B ) The Western blot results showed that, compared with CT-KD, the expression levels of DKK1 and p-STAT3 (Ser 727) in RN-KD were upregulated using ARD and AMO.1 MM experiment. C ) The Co-IP results showed that there was a protein-protein interaction between STAT3 and ROCK2 using ARD MM experiment. D ) The Western blot results showed that the expression of p-STAT3 (Ser 727) was upregulated in both the cytoplasm and nucleus of RN-KD ARD MM cells. E ) The results of the Ch-IP experiment demonstrated that STAT3 could bind to the DKK1 promoter using AMO.1 MM experiment. F ) After treatment with the ROCK2 inhibitor KD025 (10 μm, 24 h), the expression of DKK1 and other signaling molecules were examined by western blotting in AMO.1 MM cells. G ) After KD025treatment, DKK1 in the culture supernatant was examined by ELISA in AMO.1 MM cells. H ) Treatment with the pSTAT3 inhibitor HY-13818 (20 μm, 24 h) significantly downregulated DKK1 expression in AMO.1 MM cells. (* p < 0.05 (Student’s t test). RN-KD = RND3 knockdown; CT-KD = control knockdown.)

Article Snippet: The antibodies utilized included RND3 (ProMab, USA, #30838), actin (Proteintech, China, #2D4H5), GAPDH (Proteintech, China, #1E6D9), ROCK1 (Proteintech, China, #21850-1-AP), ROCK2 (Proteintech, China, #21645-1-AP), Phospho-Smad2/3 (Affinity, China, #AF3367), DKK1 (Proteintech, China, # 21112-1-AP), STAT3 (Proteintech, China, # 10253-2-AP), Phospho-Stat3 (CST, USA, #9134), TBP (Proteintech, China, # 66166-1-Ig), and MMP-9 (Hua Bio, China, #ET1704-69).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Co-Immunoprecipitation Assay, Knockdown, Control

Journal: Cancer Cell International

Article Title: Rho-related GTP-binding protein RhoE (RND3) regulates multiple myeloma bone disease

doi: 10.1186/s12935-025-04166-2

Figure Lengend Snippet: Pan-ROCK inhibitor Y-27632 alleviate myeloma bone disease . A ) The Western blot results showed that after treatment with the ROCK inhibitor Y-27632 (5μM) 48 h, the downstream molecules including MMP-9, p-smad2/3 (Thr 8), p-STAT3 (Ser 727) and DKK1 were inhibited in ARD. B ) After treatment with Y-27632, the content of MMP-9 and DKK1 in the culture supernatant decreased significantly in ARD. C ) In the co-culture system of pre-OCs and ARD MM, the number of mature OCs significantly decreased after the addition of Y-27632. However, in the co-culture system of pre-OBs and ARD MM, the number of mature OBs increased after the addition of Y-27632. D ) Alizarin red staining results showed that Y-27632 significantly increased the number of osteoblasts (OBs) in vitro after treatment. E )The bone disease in mice was alleviated after treatment with Y-27632 (5 mg/kg). F ). Y-27632 had no obvious toxic and side effects on the heart, liver and kidney of mice. G ) Graphic model of RND3 function in MM bone diseases. (*p<0.05 (Student’s t test). RN-KD=RND3 knockdown; CT-KD=control knockdown)

Article Snippet: The antibodies utilized included RND3 (ProMab, USA, #30838), actin (Proteintech, China, #2D4H5), GAPDH (Proteintech, China, #1E6D9), ROCK1 (Proteintech, China, #21850-1-AP), ROCK2 (Proteintech, China, #21645-1-AP), Phospho-Smad2/3 (Affinity, China, #AF3367), DKK1 (Proteintech, China, # 21112-1-AP), STAT3 (Proteintech, China, # 10253-2-AP), Phospho-Stat3 (CST, USA, #9134), TBP (Proteintech, China, # 66166-1-Ig), and MMP-9 (Hua Bio, China, #ET1704-69).

Techniques: Western Blot, Co-Culture Assay, Staining, In Vitro, Knockdown, Control