Fig 2: PRMT3 inhibits the ALDH1A1 activity in vitro.a Purification of GST-tagged ALDH1A1-FL, GST-tagged PRMT3 full-length protein, GST-tagged N-terminal region of PRMT3, and GST-tagged catalytic domain of PRMT3 proteins. Coomassie-stained gel of purified GST-tagged ALDH1A1-FL (left panel) and GST-tagged PRMT3 full-length protein (PR-FL), GST-tagged N-terminal region of PRMT3 (PR-NTR), and GST-tagged catalytic domain of PRMT3 proteins (PR-CTR) (right panel). b PRMT3 inhibits the ALDH1A1 activity in vitro. ALDH1A1 activity was measured in the presence and absence of GST-tagged PRMT3 full-length protein or GST-tagged N-terminal region of PRMT3 or GST-tagged catalytic domain of PRMT3. The representative activity of the ALDH1A1 of the three independent experiments is shown in the graph (top panel), and the mean initial slopes of the three independent reactions were plotted as the rate of the reactions (bottom panel). Error bar indicates the standard deviations of the mean. The statistical significance was assessed by a two-tailed t test (Supplementary Fig. 10 and Supplementary Data 1).

Fig 3: Cancer stemness markers in cells treated with chemotherapeutic agents in vivo, and downregulation of lncRNA-LET enhances CSC-like properties. (A) In vivo GEM chemotherapy simulates clinical regimen with multiple GEM treatment cycles (dashed boxes) and gap periods. Tumor sizes of T24 xenografts were measured for GEM treatment and vehicle control group (n = 6 per group). (B) Sphere formation assay of primary cells derived from T24 xenografts of control group (Veh) and GEM (n=3 per group). (C) Representative H&E and IHC data showing the expression levels of CSC markers (CK5 and CK14) in T24 xenografts of control and GEM groups (n = 6 per group). (D) Western blotting of CSC markers (ALDH1A1, CD44, KLF4, and HMGA2) in T24 xenografts of control and GEM groups. (E) The levels of lncRNA-LET in T24 and 5637 cells treated with GEM or vehicle. (F) Knockdown efficiency of lncRNA-LET in T24 cells and forced expression of lncRNA-LET in J82 cells. (G-H) ALDHhigh population (G) and CSC markers (H) were determined by flow cytometer and Western blotting in T24 with knockdown and J82 cells with overexpression of lncRNA-LET (n=3 per group). (I) Knockdown efficiency of lncRNA-LET in T24 cells infected with shRNA targeting lncRNA-LET virus (shLET), compared to control (shCTL). (J) Tumor volume of shCTL and shLET T24 cells with limiting dilution. Each dot represents an individual mouse (n=6 per group). Data are shown as mean ± SD and represent three independent experiments with similar results. * P < 0.05; ** P < 0.01, *** P < 0.001 (Student's unpaired two-tailed t-test). (K) CSC frequency was calculated using extreme limiting dilution analysis (ELDA). There was a significant difference in CSC frequency between two groups, with a P value of 0.025.

Fig 4: PRMT3 inhibits the ALDH1A1 activity in vivo.a ALDEFLUOR assay reveals that PRMT3 inhibits the ALDH1A1 activity in vivo. HEK293 cells were transfected with HA vector or HA-tagged PRMT3 construct or control siRNA or PRMT3 siRNA. The efficiency of PRMT3 overexpression (upper left blots) and knockdown (upper right blots) was quantified by immunoblotting with PRMT3 antibody. ALDEFLUOR assay was performed in the PRMT3 perturbed cells by incubating with ALDEFLUOR reagent. Representative graphs show the ALDEFLUOR fluorescence intensities of the cells which were transfected with HA vector or HA-tagged PRMT3 construct (middle left panel) or control siRNA or PRMT3 siRNA (middle right panel). The lower panel graphs represent the relative mean fluorescence intensities of three biologically independent ALDEFLUOR experiments. Error bars indicate the standard deviations of the mean. The statistical significance was assessed by a two-tailed t test. b PRMT3 reduces the expression of the RARE-luciferase reporter gene. HEK293 cells were co-transfected with HA vector or HA-tagged PRMT3 construct or HA-tagged PRMT3-E338Q construct or control siRNA or PRMT3 siRNA with pGL3-RARE-luciferase and pRL-Renilla-luciferase reporter vector and treated with 2.5 µM of all-trans-retinal (RALD). The efficiency of PRMT3 or PRMT3-E338Q overexpression (upper left blots) and knockdown (upper right blots) was quantified by immunoblotting with PRMT3 antibody. Luciferase reporter activities were quantified in these cells. The firefly luciferase activities were normalized to Renilla luciferase activities and presented relative to the control sample in the graphs (lower panel). The values in the graphs represent the mean of three biologically independent experiments, with error bars representing standard deviations. The statistical significance was assessed by a two-tailed t test (Supplementary Fig. 10 and Supplementary Data 1).

Fig 5: Schema illustrating the functional significance of PRMT3–ALDH1A1 interaction.PRMT3 interacts with ALDH1A1 and inhibits the enzymatic activity of ALDH1A1. This interaction negatively regulates retinoic acid signaling-mediated gene expression.