Fig 2: Nuclear Pfn1 inhibits SEC function(A) Western blot of NLS- or NES-tagged YFP-Pfn1 relative to endogenous Pfn1 by GFP or Pfn1 antibodies. S137D, residing in the epitope of the Pfn1 antibody, abolishes the detection.(B) qRT-PCR of MYC in MCF-7 cells in (A). p values were based on unpaired t test relative to YFP control.(C) qRT-PCR of XPO6 and MYC in MCF-7 cells expressing XPO6(WT) or XPO6(RNAi-Res) and infected with shLUC or shXPO6 (#3). p values were based on unpaired t test by comparing shLUC versus shXPO6 #3.(D) qRT-PCR of MYC in XPO6 KD/rescue MCF-7 xenograft samples (10 tumors/group, 2 technical replicates/tumor) from Figure 3. p values were based on unpaired t test.(E) qRT-PCR of MYC in Pfn1-null chondrocytes infected first with vector or Pfn1 and subsequently with shLUC or shXPO6 #2 (recognizing mouse XPO6). p values were based on unpaired t test.(F) qRT-PCR of MYC and Pfn1 in MCF-7 cells expressing Pfn1(WT) or Pfn1(RNAi-Res) and infected with scrambled shCTRL or shPFN1 #1 or #2. p values were based on unpaired t test by comparing shCTRL versus shPFN1.(G) Correlations of MYC mRNA levels with XPO6, BRD4, and NCL in different TCGA datasets. The y axis represents Spearman’s correlation coefficients (?). Fifteen cancer types in which statistically significant positive correlations between XPO6 and MYC expression are shown (? > 0; FDR q values < 0.05).(H) ChIP using antibodies for ENL, cyclin T1, Cdk9, p-Rpb1(Ser2), and H3K36me3 from MCF-7 cells expressing YFP or YFP-NLS-Pfn1(WT or S137D) followed by MYC qPCR. p values were based on unpaired t test.(I) ChIP using antibodies for ENL, p-Rpb1(Ser2), and H3K36me3 from MCF-7 cells infected with shLUC or shXPO6 #3 followed by MYC qPCR.p values were based on unpaired t test. All data (except A and G) represent mean ± SEM of representative experiments, which were confirmed at least three times. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. See also Figure S7 and Table S3.

Fig 3: Nuclear Pfn1 interacts with the SEC(A) IP of YFP or YFP-NLS-Pfn1(WT or S137D) from nuclear extracts of MDA-MB-231 stable cells by a GFP antibody, followed by silver staining.(B) Eluates from (A) were analyzed by liquid chromatography-MS. Proteins specifically bound to NLS-Pfn1(WT) were identified using label-free quantification (LFQ) intensity and a threshold of >1.5-fold-higher intensity over those bound to YFP and NLS-Pfn1(S137D). LFQ intensities of detectable SEC components are shown.(C) Confirmation of the interaction between NLS-Pfn1(WT) and SEC by anti-GFP co-IP as in (B) using nuclear extracts of stable MCF-7 cells, followed by western blot.(D) Interaction between the SEC and endogenous Pfn1 in MCF-7 cells. ENL and cyclin T1 were pulled down followed by western blot.(E) Cyclin T1 pulldown from shLUC- versus shENL-infected MCF-7 cells, followed by western blot for endogenous Pfn1 and other proteins. In total, 50-fold-less input was used for the Pfn1 blot given the small fraction of nuclear Pfn1 (most in the cytoplasm) interacting with the SEC.(F) Size-exclusion chromatography and western blot analyses of nuclear extracts of MDA-MB-231 cells for endogenous Pfn1 and SEC components.Results in (C)–(F) were confirmed by three independent experiments. Lanes in (A), (D), and (E) were cropped and rearranged from the same blots for clarity of presentation (indicated by the black lines). See also Table S2.