PCR I
M1 1 2 3 4 5 6 7 8 9 10 11 / PCR IIM2 1 2 3 4 5 6 7 8 9 10 11
Supplement 1. Genotyping of mice offspring by PCR
PCR products were separated using 1 % agarose / TBE gel at 150 V, visualized by SYBR safe DNA gel stain (Invitrogen), and detected at 320nm. PCR I was used for detection of ERp29 knock out gene (2.5 kb), PCRII– for wild type gene (0.7 kb). M1 – 1 kb DNA gene rulerTM (Fermentas), M2 - 100 bp DNA gene rulerTM (Fermentas). Here shown: the mice 1-5 are knock out, the mice 6-7, 9, 11 are heterozygous, the mice 8, 10 are wild type.
ERp29+/+ mouse ERp29-/- mouse
Supplement 2. Comparison of wild type and mutant mice from strain C5BL/6N.
The knock out of ERp29 gene did not cause any phenotypical differences.
day 0 /day 1
/day 2
/ day 7Supplement 3. Primary culture of ERp29-/- mouse thyrocytes at different times after seeding.
Day 0: After enzymatic digestion the follicles will be resuspended in culture medium. They sink to the bottom, and attach oneself; day 1: follicles get flat and spread on the bottom; day 2: thyrocytes grown in the middle as a multilayer culture; day 7: the thyrocytes reach 80 % confluence, and are suitable for experiments. Phase-contrast microscopy, 200x magnification.
Culture plate Ø 10 cm
skin pieces / Supplement 4. Isolation of adult dermal fibroblasts from mouse skin.
Left: the scheme of experimental setup; right: shows a border of skin piece with migrated fibroblasts. Phase-contrast microscopy, 200x magnification.
ERp29+/+
/ERp29-/-
Supplement 5. Staining of vimentin cytoskeleton of dermal fibroblasts.
Immortalized cells were fixed with acetone/methanol (1:1) and following double stained: nuclei are blue (HOECHST44432) (a); Vimentin filaments are shown in red (Cy3) (b); merged (c). 400-times magnified by CLSM.
Supplement 6. Quantitative analysis of cell death measured by FACS
Treatment / Genotype / Cell population, % of total cell number(average ± standard deviation)
vital / apoptotic / necrotic
Control / ERp29 +/+ / 90.2 ± 3.2 / 2.6 ± 0.1 / 7.15 ± 3.3
ERp29 -/- / 89.3 ± 1.4 / 2.5 ± 0.5 / 8.15 ± 0.9
1 mM H2O2 / ERp29 +/+ / 27.65 ± 3.0 / 63.0 ± 1.4 / 9.35 ± 4.4
ERp29 -/- / 43.8 ± 1.5 / 42.5 ± 3.5 / 13.6 ± 5.0
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