1. ZKSCAN3 Is a Master Transcriptional Repressor of Autophagy
Santosh Chauhan, Jinesh G. Goodwin, Swati Chauhan, Ganiraju Manyam, Jing Wang, Ashish M. Kamat, Douglas D. Boyd 
Molecular Cell 
Volume 50, Issue 1, Pages (April 2013)
DOI: /j.molcel
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2. Molecular Cell 2013 50, 16-28DOI: (10.1016/j.molcel.2013.01.024)
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3. Figure 1
Silencing ZKSCAN3 Promotes Membrane Blebbing, Represses Cell Growth, and Induces Senescence and Autophagy
(A) MTT assays performed at different time points with NTLV cells and ZKSCAN3-shRNA cells. Data shown represent mean ±SD from triplicate experiments.
(B) Clonogenic assays performed with NTLV and ZKSCAN3-shRNA cells. A total of 1,000 cells were seeded in six-well plates and grown for 21 days.
(C) Scanning electron microscopy with NTLV and ZKSCAN3-shRNA cells at the indicated magnifications.
(D) SA-β-gal staining.
(E) The graph shows the percentage of SA-β-gal-stained cells as measured from ten different fields from two independent experiments (mean ±SD).
(F) Western blotting with an anti-p16INK4A (senescence marker) antibody.
(G) Live cell imaging of NTLV and ZKSCAN3-shRNA cells stained with acridine orange. Acridine orange stains both nucleus (green) and acidic vesicles (red).
(H) A typical ultrastructure of a NTLV and ZKSCAN3-shRNA cell visualized using transmission electron microscopy. ZKSCAN3-shRNA cells showed increased autophagic vesicles of different stages of maturation (also see Figures S2C and S3). Autophagosomes are indicated by “A”, lysosomes are indicated by “L”, and autophagolysosome are indicated by a red arrow.
(I) NTLV and ZKSCAN3-shRNA cells (40 cells each) were visualized and cells with more than ten autophagosome-like structures were considered positive. Graph shows the percentage of cells with autophagic organelles (AOs) from three different experiments (mean ±SD).
(J) Western blotting using anti-LC3 and actin antibodies was performed with varying protein inputs. The graph shows quantification (mean ±SD) of LC3-II band intensity relative to actin from three different blots.
(K) Representative live cell images of NTLV and ZKSCAN3-shRNA cells stably expressing the DsRed-LC3 fusion protein. Student’s unpaired t test was used to test for statistical significance: ∗∗p < See also Figures S1, S2, S3, S4, and S5.
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4. Figure 2 Modulated ZKSCAN3 Expression Regulates Autophagic Flux
(A) Protein extracted from bafilomycin-treated or untreated NTLV and ZKSCAN3-shRNA cells was subjected to western blotting using anti-LC3 and actin antibodies. The graph shows quantification (mean ±SD) of LC3-II band intensity relative to actin. ∗p < 0.05.
(B) Representative live cell images of cells stably expressing a GFP-RFP-LC3 fusion protein.
(C) NTLV and ZKSCAN3-shRNA cells were transiently transfected with a plasmid encoding GFP-Neo fusion protein. After 48 hr, ZKSCAN3-shRNA cells expressing GFP-Neo were treated or untreated with bafilomycin (5 nM, lane 3) for 24 hr. Subsequently after total 72 hr, protein was extracted and western blotted using anti-GFP and anti-actin antibodies.
(D) UC13 and RKO cells were either transiently transfected with an empty vector (pcDNA3.1) or a streptavidin-FLAG-tagged-ZKSCAN3-expressing vector. After 72 hr, cells were treated, where indicated, with rapamycin (6 hr/200 nM), extracted, and immunoblotted for LC3 and actin.
Molecular Cell  , 16-28DOI: ( /j.molcel )
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5. Figure 3 Silencing ZKSCAN3 Enhances Lysosome Biogenesis
(A) Live cell imaging of NTLV and ZKSCAN3-shRNA cells stained with LysoTracker red, which fluoresces red in acidic compartments.
(B and C) Western blotting of protein extracts from UC13 NTLV and ZKSCAN3-shRNA cells (B) or transiently ZKSCAN3 repressed RKO and SKOV3 cells (C) using anti-Lamp1 and anti-actin antibodies.
(D) Representative live cell images of NTLV and ZKSCAN3-shRNA cells stably expressing a construct encoding the Lamp1-RFP fusion protein.
(E) Hexosaminidase assay was performed with total protein extract of the indicated cells. The experiment was performed in triplicate and data are shown as mean ±SD. ∗∗p < 0.05.
(F) Live cell imaging of LysoTracker red-stained SKOV3 cells transiently transfected with a ZKSCAN3-GFP expressing vector. ZKSCAN3-GFP expressing (yellow arrow) and nonexpressing (white arrows) are indicated.
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6. Figure 4 ZKSCAN3 Regulates Autophagy and Lysosomal Gene mRNA Levels
(A–F) Quantitative real-time PCR validation of selected candidate downstream autophagy targets of ZKSCAN3 identified in expression profiling. RNA isolated from NTLV and ZKSCAN3-shRNA cells were subjected to quantitative real-time PCR with TaqMan probes against (A and B) autophagy and (C) lysosome genes upregulated and (D) downregulated in ZKSCAN3-repressed and control cells (NTLV). The autophagosome marker gene, Map1cl3b/Atg8f is upregulated in various cell lines (F) on transient repression of ZKSCAN3 (E). Data represent mean ±SD of duplicate experiments performed in triplicate (six values). ∗p < 0.05; ∗∗p < See also Tables S1, S2, and S3.
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7. Figure 5
ZKSCAN3 Directly Interacts with the Regulatory Region of Autophagy and Lysosome Genes
(A) Quantitative real-time PCR followed by ChIP assay was performed using primers mapping to regulatory or intron regions of autophagy and lysosome genes and DNA precipitated with ZKSCAN3 or IgG antibodies. The graph displays the amount of the immunoprecipitated DNA expressed as a percentage of the total input DNA. Gapdh and uPAR gene promoters were used as control. Quantitative real-time PCR with each primer set was performed in triplicate in two separate experiments.
(B) Expression of Map1lc3b and Gabarapl2 promoter-driven luciferase reporters was induced in ZKSCAN3-shRNA and compared to control NTLV cells in dual luciferase assays. The data represent mean ±SD of two experiments performed in triplicate (six values). ∗∗p < 0.05.
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8. Figure 6
Nutritional Starvation and mTOR Regulates Subcellular Relocalization of ZKSCAN3
(A) Immunofluorescence of SKOV3 cells using an anti-ZKSCAN3 antibody (Origene) starved for 12 hr or treated with Torin 1 (500 nM) for 12 hr.
(B) Western blot analysis of nuclear and cytoplasmic subcellular fractions from HeLa cells subjected to starvation or treated with Torin 1 (500 nM) for 12 hr using an anti-ZKSCAN3 antibody.
(C) Liver tissues from fed/starved/refed mice were subjected to nuclear-cytoplasmic fractionation and western blotting was carried out using anti-ZKSCAN3 antibody (Sigma).
(D) RNA isolated from starved and fed; control and ZKSCAN3-overexpressing cells were subjected to quantitative real-time PCR with TaqMan probes against map1lc3b.
(E) Starved and fed, control, and ZKSCAN3-overexpressing cells were western blotted with LC3 and actin antibody.
(F) Densitometric analysis of western blots was done using ImageJ software.
(G) Starved and fed NTLV and ZKSCAN3-shRNA cells were western blotted with LC3 and actin antibody.
(H) RNA isolated from starved and fed NTLV and ZKSCAN3-shRNA cells were subjected to quantitative real-time PCR with TaqMan probes against map1lc3b and ulk1. For western blotting quantification, the data represent mean ±SD of three experiments performed. For quantitative real-time PCR, the data represent mean ±SD of two experiments performed in triplicate (six values). ∗p < See also Figure S7.
Molecular Cell  , 16-28DOI: ( /j.molcel )
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9. Figure 7 TFEB and ZKSCAN3 Regulate Autophagy Oppositely
(A) RNA isolated from control HeLa cells and stable TFEB-expressing HeLa cells (TFEB cells) transfected with control siRNA and ZKSCAN3-siRNA were subjected to quantitative real-time PCR with TaqMan probes against map1lc3b.
(B–E) Cell lysate from control and TFEB cells transfected with control siRNA and ZKSCAN3-siRNA were subjected to western blotting using (B) LC3 antibody and (D) Lamp-1 antibody. (C and E) Densitometric analysis of western blots using ImageJ software.
(F–H) Live cell imaging of (F) acridine orange-stained and (G) LysoTracker-stained control and TFEB cells transfected with control siRNA and ZKSCAN3-siRNA. (H) Graph represents corrected total cell fluorescence of LysoTracker-stained cells measured using Image J.
(I) RNA isolated from NTLV and ZKSCAN3-shRNA cells that were transiently transfected with pcDNA 3.1 plasmid or FLAG-TFEB-expressing plasmid were subjected to quantitative real-time PCR with TaqMan probes against map1lc3b.
(J) Cell lysate from NTLV and ZKSCAN3-shRNA cells transiently transfected with pcDNA 3.1 vector or FLAG-TFEB-expressing vector were subjected to western blotting using LC3 and actin antibody.
(K) Densitometric analysis of western blots.
(L and M) Cell lysate from control and TFEB cells transiently transfected with pcDNA 3.1 vector or FLAG-ZKSCAN3-expressing vector were subjected to western blotting using LC3 and actin antibody. (M) Densitometric analysis of western blots. For all western blotting quantification, the data represent mean ±SD of three experiments performed. For quantitative real-time PCR, the data represent mean ±SD of two experiments performed in triplicate (six values). ∗p < 0.05.
Molecular Cell  , 16-28DOI: ( /j.molcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions