Additional file 2: Table S1. of Expression and knockdown of zebrafish folliculin suggests requirement for embryonic brain morphogenesis

Absolute numbers of embryos after injection with control, mismatch or flcn morpholinos. Columns show: injection group - where each group was injected on a different day, treatment group – where embryos were either uninjected, injected with control MO, flcn ATG MO, flcn sp1 MO, flcn sp1 MM, flcn sp2 MO or flcn sp2 MM, classification of embryos – showing the number of embryos which are dead, normal or abnormal. (XLSX 8 kb

Loss of <i>flcn-1</i> increases glycogen content, which mediates resistance to hyperosmotic stress.

<p>(A) Representative electron micrographs from longitudinal sections of the hypodermis in indicated nematodes strains exposed or not to 400mM NaCl for 16 hours. Arrows represent glycogen stores. Scale bars: 2μm. (B, C) Iodine staining (B) and quantification of staining intensities (C) of indicated worm strains treated or not with 400mM NaCl for 16 hours. Data represent mean ± SEM, n≥ 3. (D, E) Percent survival to 400mM NaCl of indicated worm strains treated with indicated RNAi. (F) Relative mRNA levels of indicated target genes in indicated strains with or without 400mM NaCl treatment for 2 hours. Data represent the mean ± SEM, n≥ 3.</p

Glycogen degradation heightens glycerol levels and protects animals from hyperosmotic stress.

<p>(A) Representative scheme of glycogen metabolism and osmolyte production in worms. (B-D) Relative mRNA levels of <i>gpdh-1</i> and <i>gpdh-2</i> (B, C) and glycerol content (D) in wt and <i>flcn-1(ok975)</i> L4/young adult animals treated with or without 400mM NaCl for 2 hours. Data represent mean ± SEM, n ≥3. (E) Percent survival of indicated worm strains exposed to 400mM NaCl.</p

Loss of <i>flcn-1</i> confers resistance to hyperosmotic stress.

<p>(A-C, F) Percent survival (A, B, F) and mean survival (C) of indicated worm strains exposed to 400mM and 500mM NaCl. (D) Percent recovery from paralysis of wt and <i>flcn-1(ok975)</i> animals after 2 hours from exposure to 400mM NaCl. Data represent mean ± SEM, n≥ 3. (E) Representative images of wt and <i>flcn-1(ok975)</i> animals treated with 400mM NaCl for 48 hours.</p

Graphical representation of FLCN-1/AMPK hyperosmotic stress resistance pathway.

<p>Loss of <i>flcn-1</i> chronically activates AMPK and leads to glycogen accumulation under normal conditions. Upon exposure to hyperosmotic stress, glycogen is rapidly degraded leading to the production of glycerol and animal survival.</p

The increased survival to hyperosmotic stress and the accumulation of glycogen in <i>flcn-1</i> mutant worms require AMPK.

<p>(A-D) Percent survival of indicated worm strains exposed to 400mM NaCl. (A) <i>aak-2(ok524)</i>, (B) <i>aak-2(gt33)</i>, (C) <i>aak-1(tm1944)</i>, (D) <i>aak-1(tm1944)</i>; <i>aak-2(ok524)</i>. (E, F) Iodine staining (E) and quantification of staining intensities (F) of indicated worm strains. Scale bar:100μm. Data represent the mean ± SEM of at least 3 independent experiments.</p

The FLCN-dependent glycogen accumulation is conserved from <i>C</i>. <i>elegans</i> to humans.

<p>(A-B) PAS staining of kidney sections from wt and <i>Flcn</i> kidney-specific KO mice (A) and human BHD kidney tumor in comparison with an adjacent region from the same individual (B). Scale bars:100μm. (C) Table indicating the upregulated glycogen metabolism genes in kidney tumors (KIRC, KIRP, and KICH) as compared to normal kidney. The sign (+) indicates genes that are upregulated in these tumors. The values are indicated in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005520#pgen.1005520.s011" target="_blank">S5 Table</a>. (D) Heat map indicating correlation of expression between glycogen metabolism genes and <i>FLCN</i> in KIRC, KIRP, and KICH tumors. Green and red colors indicate genes that are negatively or positively correlated with FLCN expression, respectively.</p