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Supplementary figure 1.
JHH-7 cells were seeded at 2 X 105 cells per 35 mm
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glass based dish coated with Type I-C collagen. The cells were
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transiently transfected with 0.8 μg of TG2 (ABCD). Six hours
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after, the cells were treated with 10 μM ACR. The transfected
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cells that expressed exogenously overexpressed TG2 (as judged
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by EGFP fluorescence intensity) at similar extent were
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monitored under time lapse for the next 12 hours. Change in
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numbers of cells, having the different cellular distribution of
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the overexpressed TG2 categorized as cytoplasmic or nuclear,
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and cell fate as dead or alive was measured. Number of cells in
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each category out of total number of cells are expressed in
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percentages and plotted as bar graphs. A hundred % represents
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sum of the number of cells in four categories under observation
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expressing exogenous EGFP tagged TG2. Quantitated data
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presented as mean ± SD of three independent experiments (n =
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3-7).
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Supplementary figure 2.
JHH-7 cells were seeded at 2 X 105 cells per 35 mm
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dish containing a glass cover-slip coated with Type-IC collagen
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and were transiently transfected with 0.8 μg expressing vector.
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A) and C) Twenty four hours after the transfection with EGFP
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tagged TG2 mutants, the cells were treated with the indicated
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treatments for next 10 hours. The cells were then fixed and
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stained with H33258. B) Forty eight hours after the transfection
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with GAPDH myc-HIS fused with either SV40 NLS or a novel
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TG2 NLS, the cells were fixed and immunostained using a
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FITC tagged antibody against myc and co-stained with H33258.
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Green fluorescence intensities derived from EGFP or FITC
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along with blue fluorescence from H33258 were monitored
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under confocal microscope. Transfected cells expressing
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nuclear TG2 mutants observed in 320 µm x 320 µm
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microscopic field area were classified into four categories as
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we indicated in figure 2 in the main text, and cell numbers in
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each category under each condition were counted and
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expressed in percentages calculated against total number of
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counted cells expressing exogenous TG2 mutants in the area.
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The percentage obtained from 4-6 microscopic fields in the
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same experiment is presented as mean ± SD. *p-value <0.05,
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**p-value <0.01. A representative result from 3 independent
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experiments with similar results is presented.
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Supplementary figure 3.
Recombinant human TG2 (1.5 p mole) was incubated
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for 1 hour at room temperature with glutathione Sepharose 4B
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beads conjugated with six times molar excess of GST-
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importins-α3/HA tagged importin-β complex in the presence or
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absence of ATP, EtOH, ACR or Z-DON as indicated. After
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spin-down, proteins were eluted with SDS-PAGE sample
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buffer and TG2 levels in each co-precipitate obtained under
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each condition were determined by western blotting using an
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antibody against TG2.
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Supplementary figure 4.
JHH-7 cells were seeded at 2 X 105 cells per 35 mm
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glass based dish coated with Type I-C collagen. The cells were
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transiently transfected with 0.8 μg of (A) TG2 (AB) or (B) TG2
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(CD). Six hours after, the cells were treated with 0.1% EtOH or
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10 μM ACR when indicated. The transfected cells that
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expressed exogenously overexpressed TG2 (as judged by
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EGFP fluorescence intensity) at similar extent were monitored
63
under time lapse for the next 12 hours. Change in numbers of
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cells having the different cellular distribution of the
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overexpressed TG2 categorized as cytoplasmic or nuclear, and
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cell fate as dead or alive. Number of cells in each category out
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of total number of cells are expressed in percentages and
68
plotted as bar graphs. A hundred % represents sum of the
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number of cells in four categories under observation expressing
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exogenous EGFP tagged TG2. Quantitated data presented as
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mean ± SD of three independent experiments (n = 3-7). (C)
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Time course changes in each category of (B) are plotted as Box
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and Whisker diagrams showing each sample value as a dot. The
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lower side and upper side of boxes represent 25 and 75
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percentiles in each distribution, respectively. The line and plus
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sign in the box represents median and mean value respectively.
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Ends of the whiskers represent either the highest or lowest
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sample values.
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Supplementary figure 5.
NCBI Align Sequences Protein BLAST was used for
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aligning the newly identified NLS sequence of human TG2
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with A) other members of human transglutaminase family
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proteins B) TG2 proteins of other mammalian species. Multiple
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sequence alignment columns with no gaps are colored in blue
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or red. The red color indicates highly conserved amino acids
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and blue indicates less conserved ones.
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Supplementary figure 6.
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NCBI Align Sequences Protein BLAST was used for
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aligning the computationally predicted putative and currently
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confirmed NES sequence of human TG2 with A) other
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members of human transglutaminase family proteins B) TG2
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proteins of other mammalian species. Multiple sequence
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alignment columns with no gaps are colored in blue or red. The
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red color indicates highly conserved amino acids and blue
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indicates less conserved ones.
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Supplementary figure 7.
JHH-7 cells were seeded at 1 X 106 cells per 10 cm dish
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for overnight. The cells were then treated with 0.1% EtOH
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(column 1, 3, and 5) or 10 µM ACR (column 2, 4, and 6) for
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next 5 hours. The cells were lysed using Tris buffer (pH7.4)
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containing 1% Triton X-100, 0.1mg/mL PMSF and the protease
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inhibitor cocktail. Exportin-1 was co-immunoprecipitated using
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CRM1 antibody from samples containing equal amount of total
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protein determined by bicinchoninic acid (BCA) protein assay
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method. After precipitation, proteins were eluted with SDS-
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PAGE sample buffer and TG2 level in each co-precipitation
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obtained under each condition was determined by western
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blotting using an antibody indicated.
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Supplementary table 1.
JHH-7 cells were seeded at 1 X 104 cells per well in a
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96 well plate coated with Type I-C collagen. The cells were
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transiently transfected with 0.04 μg of A) EGFP, B) EGFP-
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TG2 (ABCD), C) EGFP-(ABC), D) 2x EGFP TG2 (C), E) 2x
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EGFP TG2 (CD), F) 2xEGFP TG2 (465-479), G) 2xEGFP TG2
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(1-479). Eighteen hours after the transfection, fluorescence
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intensity from each the mutant was monitored till 60 hours in
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an interval of 6 hours using Image express. Fluorescence half-
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life of the overexpressed protein was calculated using formula
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t1/2 = [{(ln(2)*t}/ln(N0/Nt)], where No is the initial
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fluorescence intensity and Nt is the fluorescence intensity at
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time t. No was the highest fluorescence intensity observed
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during the time course and Nt was the fluorescence intensity
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observed either at 6, 12, 24 or 36 hours after No. An average
130
value ± SD of three calculated t1/2 values is given.
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