Supplementry informtion for: Low one mss nd chnges in the osteocyte network in mice lcking utophgy in the osteolst linege Mrilin Piemontese, Meld Onl, Jinhu Xiong, Li Hn, Jeff D. Thostenson, Mri Almeid, nd Chrles A. O Brien
% of Atg7 genomic DNA deletion 1 0 80 60 Femur Spleen Kidney Liver LC3-I LC3-II ctin % of LC3-II/LC3-I 2.0 1.5 1.0 0.5 c LC3-I LC3-II % of LC3-II/LC3-I 1.2 0.8 0.4 Figure S1. Confirmtion tht deletes Atg7 nd suppresses utophgy in osteolst-linege cells. () Quntittive PCR of loxp-flnked Atg7 exon in genomic DNA from femorl corticl one nd the indicted soft tissues. Tissues were otined from (n = 6) nd (n = 5) littermtes. () Immunolot of LC3 in protein extrcted from humerl corticl one of (n = 6) nd (n = 5) littermtes. Ech lne is from single mouse. Quntifiction of the rtio of LC3-II to LC3-I nd intensity is shown t the right. (c) Immunolot of LC3 in protein extrcted from primry one mrrow cultures of (n = 4) nd (n = 4) littermtes. Ech lne represents protein extrcted from the culture on n individul mouse. Quntifiction of the rtio of LC3-II to LC3-I nd intensity is shown t the right. All mice were 9-month-old mle littermtes. Vlues re the men ±sd. P < 0.05 y Student s t-test.
wt Atg7-f/f 30 BODY WEIGHT 25 g 15 8 12 16 24 Figure S2. Femle mice lcking Atg7 in osteolsts hve norml ody weight. Body weight ws mesured monthly in the sme cohort of mice eginning t 8 of ge until 24 of ge. Femle littermtes of the following genotypes were used: wt (n = 7), Atg7-f/f (n = 16), (n = 8), nd (n = 11). Vlues re the men ± sd.
wt Atg7-f/f 0.08 FEMUR 0.08 SPINE 8 12 16 24 8 12 16 24 c 0.08 TOTAL d BODY WEIGHT 35 g 30 25 15 8 12 16 24 8 12 16 24 Figure S3. Mle mice lcking Atg7 in osteolsts hve low one mss. (-c) BMD ws mesured monthly in the sme cohort of mice y DXA eginning t 8 of ge until 24 of ge. Regions of interest were the right femur, the lumr spine (T12-L6), nd whole ody excluding the hed nd neck. Mle littermtes of the following genotypes were used: wt (n = 9), Atg7-f/f (n = 13), Osx1- Cre(n=5),nd (n = 12). (d) Body weight ws mesured in the sme mice descried in. Vlues re the men ± sd. P < 0.05 y two-wy ANOVA t ech time point.
wt Atg7-f/f Femur Length Lod c Stress d Strin e Toughness f Stiffness mm 16 12 8 4 15 5 N MP 1 80 % 6 4 2 mj 3 2 1 N/mm 1 80 4 th lumr verter g Stress h Lod 25 50 MP 15 5 N 30 Figure S4. Loss of osteolst utophgy lowers one strength. () Femur length ws mesured in 6-month-old femle mice of the following genotypes using micrometer: wt (n = 6), Atg7-f/f (n = 13), (n = 5), nd (n = 9). (-f) Femurs descried in were sujected to three-point ending using mteril testing instrument to yeild the indicted mesurements. (g-h) L4 verter from the mice descried in were sujected to compression testing using mteril testing instrument to yeild the indicted mesurements. wt (n = 7), Atg7-f/f (n = 14), Osx1- Cre (n = 7), nd (n = ). Vlues re the men ± sd. P < 0.05 y one-wy ANOVA.
reltive mrna expression 000 00 0 1 col11 ocn osx runx2 V PTH reltive mrna expression 0 80 60 c d e TRAP clcitonin receptor 80 0.12 60 0.08 Cthepsin K Figure S5. Loss of osteolst utophgy does not lter osteolst or osteoclst differentition in vitro. () Quntittive RT-PCR of collgen 11 (col11), osteoclcin (ocn), osterix 1 (osx), nd runx2 mrna in primry one mrrow osteolst cultures. Cultures for ech genotype were performed in triplicte using one mrrow cells pooled from 3 mice of ech genotype. P < 0.05 using Student s t- test () Alizrin Red stining of primry one mrrow cells cultured for 21 dys in osteolst differentition medium (n = 3 wells/genotype). (c-e) Quntittive RT-PCR of TRAP, clcitonin receptor, nd cthepsin K mrna in one mrrow cultures treted with vehicle or PTH for 11 dys to induce osteoclstformtion(n=4wellspergroup). P < 0.05 using two-wy ANOVA
ROS AFU/ug (x 3 ) Bone mrrow 25 15 5 p-p66shc/ -ctin 6 th lumr verter 14 12 8 6 4 2 Figure S6. Loss of osteolst utophgy cused oxidtive stress. () ROSin one mrrow isolted from tii of 6-month-old femle (n = 5) nd Atg7 O (n = 6) mice. () Quntifiction of phospho-p66shc in immunolot of protein from L6 verter 6-month-old femle (n = 4) nd Atg7 O (n = 5) mice. P < 0.05 using Student s t-test.
mcat; mcat; 0.08 FEMUR SPINE 0.02 c 8 12 16 24 TOTAL d 25.00 8 12 16 24 BODY WEIGHT.00 g 15.00 0.00 8 12 16 24 8 12 16 24 Figure S7. mcat expression does not rescue low BMD of mice. (-d) BMD ws mesured monthly in the sme cohort of mice y DXA eginning t 8 of ge until 24 of ge. Regions of interest were the right femur, the lumr spine (T12-L6), nd whole ody excluding the hed nd neck. Femle littermtes of the following genotypes were used: : (n = 7), mcat; (n = 6), (n = 6), nd mcat; (n = 6). () Body weight ws mesured in the sme mice descried in. Vlues re the men ± sd. P <0.05ytwo-wy ANOVA t ech time point.
Atg7-f/f Atg7 Ot Averge Intensity A.U. 8e+7 6e+7 4e+7 2e+7 Atg7-f/f Atg7 Ot Figure S8. Loss of utophgy in osteocytes does not disrupt the osteocyte network. () Averge intensity of osteocyte projections in tiil corticl one sections from Atg7-f/f (n = 3) nd Atg7 Ot (n = 5) mice stined with phllodin-alex488. () Representtive imges of sections used to otin vlues shown in. Ech imge is of one section from different mice nd size r = μm. All mesurements were performed in 6-month-old femle littermtes. Vlues re the men ± sd.
μm c 18 16 14 12 8 6 4 2 Osteocyte lcun dimeter A.U d Averge intensity 2.5e+8 2.0e+8 1.5e+8 1.0e+8 5.0e+7 Atg7 Ot Figure S9. Loss of utophgy in osteolsts does not lter the cnliculr network. () Low power imge of femorl cross section stined with FITC. White dotted ox outlines representtive region of interest showed in. Size r = 0 μm. () Representtive imges of corticl femorl sections stined with FITC used for osteocyte cnliculr mesurements. Size r = μm. (c-d) Osteocyte lcun dimeter nd verge intensity of osteocyte cnliculr network in femorl cross sections from (n = 3) nd Atg7 Ot (n = 4) mice stined with FITC from sections descried in. All mesurements were performed in 6-month-old femle littermtes. Vlues re the men ± sd. P < 0.05 y Nested nlysis.
Tle S1 Rte of spontneous frctures mcat; Animls 17 Animls with tii frctures 12 5 % 70 50