Fig 2: Analysis of AIMP2-DX2 in AML.a RNA-smFISH images of ML-1 cells. Bars indicate 20 µm. Box plot of the quantified red-to-green signal is shown on the right with the number in the bottom denotes the average ratio of 51 images. b AIMP2-DX2 is positively correlated with MAPK and JAK-STAT signaling pathways in ML-1 cells. The RNA expression profiles of representative genes for p53, MAPK, and JAK-STAT signaling pathways are visualized. GAPDH normalized fold change and its P values are indicated as color and size of the corresponding circle, respectively. n = 6 for p53 signaling pathway genes, and n = 7 for other signaling pathway genes. c Electroporation with siAIMP2-DX2 enhances apoptosis induced by paclitaxel, etoposide, and cisplatin in ML-1 cells. n = 7 for paclitaxel, n = 5 for etoposide, n = 6 for cisplatin. A solid red line indicates cell viability relative to siLuc, while a black dashed line indicates cell viability of control. For all data presented, the average is shown with error bars indicate s.e.m. d, e Knockdown of AIMP2-DX2 can activate the p53 signaling pathway upon etoposide treatment. In d, the RNA expression of the representative genes for the p53 signaling pathway is examined. For all data presented, an average of three biological replicates is shown with error bars indicate s.e.m. The numbers under the graph denote the P values. In e, protein expression of phosphorylated p53 and PUMA are examined via western blotting. The black arrow indicates the band for the AIMP2-DX2 protein. GAPDH and TUBB were used as loading control. Box plot features: center line, median; box limits, upper and lower quartiles; whiskers, 1.5× interquartile range; points, individual red-to-green ratio. *P<0.05, **P<0.01. For statistical analysis, a one-tailed t test with unequal variance was used. AML acute myeloid leukemia, smFISH single-molecule fluorescence ISH, ETO etoposide.

Fig 3: RNA-smFISH to quantify the expression ratio of AIMP2-DX2/AIMP2.a The design of RNA-smFISH probes. C1-tagged ZZ pairs target the exon 2 of AIMP2, and C2-tagged ZZ pairs target exons 1, 3, and 4 of AIMP2 and AIMP2-DX2. By using two different fluorophores for visualization, both probes can be hybridized and analyzed simultaneously. b C1 and C2 probes of the RNA-smFISH yield foci-like signal patterns in HeLa cells. Without the hybridization of probes, no fluorescent signal was detected. Bars indicate 20 µm. Box plot of the quantified red-to-green signal is shown on the right with the average ratio indicated in the bottom. Thirty images were analyzed. c Transfecting siRNAs specific to the full-length AIMP2 or AIMP2-DX2 effectively reduced target protein expressions in HeLa cells. The black arrow indicates the true-positive band for AIMP2-DX2. TUBB was used as loading control. d RNA-smFISH images upon transfecting HeLa cells with siAIMP2 or siAIMP2-DX2. Bars indicate 20 µm. e Box plot of RNA-smFISH red-to-green signal ratios in siRNA-transfected cells. Numbers on the box plots specify the average ratio of 30 images. f The knockdown efficiency of siRNAs was confirmed using RT-qPCR. The average of three replicates is shown with error bars indicating s.e.m. Numbers on the bar specify the average values. RNA expressions were measured relative to the control GAPDH. g RNA-smFISH images of siRNA-transfected A549 cells. Bars indicate 20 µm. h Box plot of red-to-green signal ratios in A549 cells. Numbers on the box plots specify the average ratio of 30 images. i A comparison of the red-to-green signal ratio between HeLa and A549. Numbers on the bottom indicate average ratios of 30 images. The data for HeLa is the same as the one shown in b. Box plot features: center line, median; box limits, upper and lower quartiles; whiskers, 1.5× interquartile range; points, individual red-to-green ratio. *P < 0.05, ***P < 0.001. For statistical analysis, a one-tailed t test with unequal variance was used. smFISH single-molecule fluorescence ISH.