Fig 1: TCAB1 is excluded from the nucleolus(A) Western blot probed for TCAB1 of parental and genome-edited HeLa cells expressing HaloTag-TCAB1.(B) Maximum intensity projection of a single-molecule imaging movie of HaloTag-TCAB1 (JFX650) in cells expressing BFP-coilin and GFP-NPM1 to mark Cajal bodies and nucleoli, respectively (scale bar, 5 µm).(C) Western blots probed with TCAB1, fibrillarin, and lamin B1 antibodies of samples of cellular fractionation experiments (left to right: input, cytoplasm, nucleus, nucleoplasm, nucleolus) from HeLa cells.(D) Probability density functions of the step sizes derived from nuclear HaloTag-TCAB1 trajectories and the corresponding three-state model fit using the Spot-On tool (pooled data from two replicates of two independent HaloTag-TCAB1 clones, >10 cells per replicate).(E) Kymograph of nucleolar HaloTag-TCAB1 particles over time.(F) Live-cell fluorescence images of single 3xFLAG-HaloTag-dyskerin particles, nucleoli marked by GFP-NPM1 (scale bar, 5 µm).(G) Kymograph of nucleolar 3xFLAG-HaloTag-dyskerin particles over time.
Fig 2: The specific activity of telomerase is unchanged in the absence of TCAB1(A and B) Direct telomerase extension assay (300 mM KCl) of endogenous telomerase purified from parental (WT) and TCAB1 knockout (A) HeLa and (B) Halo-TERT cell lines (LC1 and LC2 loading controls, n = 2 and 3, mean).(C) Quantification of endogenous telomerase activity in samples from TCAB1 knockout cells relative to parental controls (n = 3, mean). TKO, TCAB1 knockout.(D and E) Direct telomerase extension assay (150 mM KCl) of telomerase purified from parental (WT) and TCAB1 knockout overexpressing TERT and TR (–Ab is a no-TERT antibody control). (D) HeLa and (E) Halo-TERT cell lines (LC1 and LC2 loading controls). TKO, TCAB1 knockout.(F) Quantification of telomerase activity in samples from TCAB1 knockout cells relative to parental controls (n = 4–7, mean, t test).(G) Specific activity (total activity/TR in sample; see Figure 3F) of overexpressed telomerase purified from TCAB1 knockout cells relative to parental controls normalized to 1 (n = 4–7, mean, t test).
Fig 3: TR is localized to nucleoli in TCAB1 knockout cells(A and B) Western blot probed with TCAB1 antibody of TCAB1 knockouts from (A) HeLa and (B) Halo-TERT cells.(C) IF with anti-dyskerin and anti-TCAB1 antibodies and FISH using TR probes (scale bar, 5 µm).(D) Growth rate of parental and TCAB1 knockout cell lines.(E) IF with anti-dyskerin and anti-TCAB1 antibodies and FISH with TR probes (scale bar, 5 µm).(F) Maximum intensity projection (1,000 frames) of HaloTag-TERT (JFX650) in control and TCAB1 knockout cells expressing GFP-NPM1 (scale bar, 5 µm).(G) Quantification of the fraction of TERT trajectories that overlap with the nucleolus in control and TCAB1 knockout cells (n = 16 and 20 cells, mean).
Fig 4: WDR79 knockdown inhibited aerobic glycolysis of pancreatic cancer cells. (a) Immunoblot showed the expression of GLUT1, HK2, LDHA in HPAC, and Capan-1 cells upon the indicated transfection. (b) Glucose uptake assays showed the relative glucose uptake capacity of HPAC and Capan-1 cells upon the indicated transfection. (c) OCR and ECAR levels of HPAC and Capan-1 cells upon the indicated transfection. *p < 0.05, **p < 0.01, ***p < 0.01, WDR79 vs control; # p < 0.01, ## p < 0.05, ### p < 0.01. shWDR79 vs shControl.
Fig 5: Depletion of SIRT4 expression reversed the effects of WDR79. (a) Immunoblot showed the expression of SIRT4 in HPAC and Capan-1 cells upon the indicated transfection. (b) CCK-8 assays showed the OD value at 450 nm wavelength of HPAC and Capan-1 cells upon the indicated transfection at 24, 48, 72, and 96 h. (c) Transwell showed the invasive pancreatic cancer cells upon the transfection of indicated plasmids or shRNAs. (d) Immunoblot showed the expression of E-cadherin and N-cadherin in HPAC and Capan-1 cells upon the indicated transfection. *p < 0.05, **p < 0.01, ***p < 0.01, shWDR79 vs shControl; # p < 0.01, ## p < 0.05, ### p < 0.01. shWDR79 + shSIRT4 vs shWDR79 + shControl.
Supplier Page from Abcam for Anti-WDR79 antibody