Expression level of Shox2 , Tbx15 , HoxC8 , HoxC5 , mRNA was compared using quantitative real - time PCR ( qPCR ) of isolated from different fat depots . Two subcutaneous depots ( inguinal scapular ) , three intra - abdominal fat depots ( perigonadal , perirenal , mesenteric ) , interscapular brown adipose tissue of 8 week - old C57BL / 6 mice . Data are shown as mean ± of six samples . Expression level of Shox2 , Tbx15 , HoxC8 , HoxC5 , mRNA was compared using qPCR of isolated from cultured stromovascular derived from adipose tissue of Immortomouse ™ . depot - specific lines were made from inguinal , scapular , perigonadal , perirenal , mesenteric , interscapular brown adipose tissue of 8 week - old mice . Data are shown as mean ± of lines from three mice .

B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O B-TISSUE O O B-TISSUE I-TISSUE O O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O O B-TISSUE O B-TISSUE O B-TISSUE O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O O O O O B-ORGANISM O O O O O O O O O O O B-ORGANISM O

Expression level of Shox2, Tbx15, HoxC8, and HoxC5, mRNA was compared using quantitative real-time PCR (qPCR) of RNA isolated from different fat depots. Two subcutaneous depots (inguinal and scapular white), three intra-abdominal fat depots (perigonadal, perirenal, and mesenteric), and interscapular brown adipose tissue of 8 week-old male C57BL/6 mice. Data are shown as mean ± SEM of six samples. Expression level of Shox2, Tbx15, HoxC8, and HoxC5, mRNA was compared using qPCR of RNA isolated from cultured stromovascular cells derived from adipose tissue of the Immortomouse™. The depot-specific cell lines were made from inguinal, scapular white, perigonadal, perirenal, mesenteric, and interscapular brown adipose tissue of 8 week-old male mice. Data are shown as mean ± SEM of cell lines from three individual mice.


Bright - field image of depot - derived lines after in vitro adipogenic differentiation Oil Red O ( Lee et al . , 2017a ) . Original were taken at 10x magnification ( left panel ) . Scale bar = 100 μm .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O

Bright-field image of depot-derived cell lines after in vitro adipogenic differentiation and Oil Red O staining (Lee et al., 2017a). Original photographs were taken at 10x magnification (left panel). Scale bar = 100 μm.


qPCR for adiponectin ( Adipoq ) , peroxisome proliferator - activated receptor gamma ( Pparγ2 ) , fatty acid synthase ( FAS ) in isolated from depot - derived lines after in vitro differentiation . Data are shown as mean ± of 3 lines / group .

B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O

qPCR analysis for adiponectin (Adipoq), peroxisome proliferator-activated receptor gamma (Pparγ2), and fatty acid synthase (FAS) in RNA isolated from depot-derived cell lines after in vitro differentiation. Data are shown as mean ± SEM of 3 cell lines/group.


Representative picture of preadipocyte clonal lines showing media color 48 hours after last media change . in wells were 100 % confluent when media was changed .

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Representative picture of preadipocyte clonal cell lines showing media color 48 hours after last media change. Cells in all wells were 100% confluent when media was changed.


depicting 50 most variable genes among preadipocyte clonal lines . Red represents high expression of genes , while blue represents low expression of genes . depicting 50 most variable genes among Type 2 3 preadipocyte clonal lines . Red represents high expression of genes , while blue represents low expression of genes .

O O O O O O B-CELL_TYPE O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-CELL_TYPE O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

Heatmap depicting the 50 most variable genes among all preadipocyte clonal cell lines. Red represents high expression of genes, while blue represents low expression of genes. Heatmap depicting the 50 most variable genes among Type 2 and 3 preadipocyte clonal cell lines. Red represents high expression of genes, while blue represents low expression of genes.


Expression of Wt1 , leucine rich repeat neuronal 4 ( Lrrn4 ) , Uroplakin 3b ( Upk3b ) , in Immortomouse clonal clusters as preadipocytes ( d0 ) during six days of adipogenic differentiation .

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Expression of Wt1, leucine rich repeat neuronal 4 (Lrrn4), Uroplakin 3b (Upk3b), in Immortomouse clonal cell line clusters as preadipocytes (d0) and during six days of adipogenic differentiation.


Bright - field of preadipocyte lines under standard culture conditions . were taken at 10x magnification .

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Bright-field images of preadipocyte cell lines under standard culture conditions. The photographs were taken at 10x magnification.


Expression of Tagln , connective tissue growth factor ( Ctgf ) , Keratin 19 ( Krt19 ) , Mx1 , caspase 1 ( Casp1 ) , C - X - C Motif Chemokine Ligand 12 ( Cxcl12 ) in Immortomouse clonal clusters as preadipocytes ( d0 ) during six days of adipogenic differentiation .

B-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O B-CELL_TYPE O O O O O O O O O O

Expression of Tagln, connective tissue growth factor (Ctgf), Keratin 19 (Krt19), Mx1, caspase 1 (Casp1), and C-X-C Motif Chemokine Ligand 12 (Cxcl12) in Immortomouse clonal cell line clusters as preadipocytes (d0) and during six days of adipogenic differentiation.


Triglyceride content of cultures after six days of differentiation using protocol described in Methods . Data are shown as mean ± of 3 - 7 samples , entire experiment was repeated twice .

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Triglyceride content of cell cultures after six days of differentiation using the protocol described in Methods. Data are shown as mean ± SEM of 3-7 samples, and the entire experiment was repeated twice.


[ 3H ] - labeled 2 - deoxyglucose uptake in preadipocyte lines in basal state after pretreatment for 20 minutes with 100 nM insulin . Uptake was measured for one hour , data are shown as mean ± of 3 - 7 samples repeated twice . [ 3H ] - labeled 2 - deoxyglucose uptake in adipocytes after six days of adipogenic differentiation in basal state after stimulation for 20 minutes with 100 nM insulin . Uptake was measured for one hour , data are shown as mean ± of 3 - 7 samples . experiment was repeated twice .

O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O B-CELL_TYPE O O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O B-CELL_TYPE O O O O O O O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O

[3H]-labeled 2-deoxyglucose uptake in preadipocyte cell lines in the basal state and after pretreatment for 20 minutes with 100 nM insulin. Uptake was measured for one hour, and data are shown as mean ± SEM of 3-7 samples and repeated twice. [3H]-labeled 2-deoxyglucose uptake in adipocytes after six days of adipogenic differentiation in the basal state and after stimulation for 20 minutes with 100 nM insulin. Uptake was measured for one hour, and data are shown as mean ± SEM of 3-7 samples. The experiment was repeated twice.


[ 14C ] D - glucose incorporation into lipids in adipocytes after six days of adipogenic differentiation in basal state after stimulation for 20 minutes with 100 nM insulin . [ 14C ] D - glucose incorporation was measured after three hours , data are shown as mean ± of 3 - 7 samples . experiment was repeated twice .

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[14C] D-glucose incorporation into lipids in adipocytes after six days of adipogenic differentiation in the basal state and after stimulation for 20 minutes with 100 nM insulin. [14C] D-glucose incorporation was measured after three hours, and data are shown as mean ± SEM of 3-7 samples. The experiment was repeated twice.


Lipolysis rates after stimulation with 1 nM isoproterenol as measured by glycerol release from adipocytes after six days of adipogenic differentiation . Data are shown as mean ± of 3 - 7 replicates normalized by lipid content of . entire experiment was repeated twice .

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Lipolysis rates after stimulation with 1 nM isoproterenol as measured by glycerol release from adipocytes after six days of adipogenic differentiation. Data are shown as mean ± SEM of 3-7 replicates and normalized by lipid content of the cells. The entire experiment was repeated twice.


Extracellular acidification rate ( ECAR ) of preadipocytes adipocytes after 4 days of in vitro differentiation was determined by calculating under curve ( AUC ) during measurements of basal respiration . whole experiment was repeated three times . Data are shown as mean ± of 3 - 7 lines per group .

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Extracellular acidification rate (ECAR) of preadipocytes and adipocytes after 4 days of in vitro differentiation was determined by calculating the area under the curve (AUC) during measurements of basal respiration. The whole experiment was repeated three times. Data are shown as mean ± SEM of 3-7 cell lines per group.


Basal respiration of preadipocytes adipocytes after 4 days of in vitro differentiation was determined by calculating under curve ( AUC ) during measurements of oxygen consumption rate ( OCR ) . whole experiment was repeated three times . Data are shown as mean ± of 3 - 7 lines per group .

B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O

Basal respiration of preadipocytes and adipocytes after 4 days of in vitro differentiation was determined by calculating the area under the curve (AUC) during measurements of oxygen consumption rate (OCR). The whole experiment was repeated three times. Data are shown as mean ± SEM of 3-7 cell lines per group.


of oxygen consumption rate ( OCR ) to extracellular acidification rate ( ECAR ) of preadipocytes adipocytes after 4 days of in vitro differentiation . Data are shown as mean ± of 3 - 7 lines per group .

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Ratio of oxygen consumption rate (OCR) to extracellular acidification rate (ECAR) of preadipocytes and adipocytes after 4 days of in vitro differentiation. Data are shown as mean ± SEM of 3-7 cell lines per group.


Representative Western blot of three independent adipocyte lines after 6 days of in vitro differentiation for : pJnk T183 / Y185 total Jnk after treatment with 10 nM TNFα for 15 minutes ( Inflammation ) ; pAkt S473 total Akt after treatment with 100 nM insulin for 15 minutes ( Insulin Signaling ) ; pStat5 Y694 total Stat5 after treatment with 500 nM GH for 20 minutes ( Growth Hormone ) .

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Representative Western blot analysis of three independent adipocyte cell lines after 6 days of in vitro differentiation for: pJnk T183/Y185 and total Jnk after treatment with 10 nM TNFα for 15 minutes (Inflammation); pAkt S473 and total Akt after treatment with 100 nM insulin for 15 minutes (Insulin Signaling); pStat5 Y694 and total Stat5 after treatment with 500 nM GH for 20 minutes (Growth Hormone).


Expression of Wt1 , Lrrn4 , Upk3b , Tagln , Ctgf , Krt19 , Mx1 , Casp1 , Cxcl12 in preadipocytes isolated from adipose tissue of 5 - 6 month old Wt1 - creERT2 ; Rosa26mT / mG , Tagln - cre ; Rosa26mT / mG , Mx1 - cre ; Rosa26mT / mG mice . mGFP mTomato positive preadipocytes were isolated from pooled visceral fat depots ( perigonadal , perirenal , mesenteric , pericardial ) of Wt1 - creERT2 ; Rosa26mT / mG mice , pooled subcutaneous fat depots ( subcutaneous scapular ) of Mx1 - cre ; Rosa26mT / mG , both pooled visceral ( Tagln - V ) subcutaneous depots ( Tagln - S ) from Tagln - cre ; Rosa26mT / mG mice . Data are shown as mean ± of 4 - 6 mice / group .

B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O B-TISSUE I-TISSUE O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O O O B-CELL_TYPE O O O O B-TISSUE I-TISSUE O O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O B-TISSUE I-TISSUE O O B-TISSUE I-TISSUE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-TISSUE O B-GENEPROD O O O B-TISSUE O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O B-ORGANISM O O O

Expression of Wt1, Lrrn4, Upk3b, Tagln, Ctgf, Krt19, Mx1, Casp1, and Cxcl12 in primary preadipocytes isolated from white adipose tissue of 5-6 month old Wt1-creERT2;Rosa26mT/mG, Tagln-cre;Rosa26mT/mG, and Mx1-cre;Rosa26mT/mG male mice. mGFP and mTomato positive preadipocytes were isolated from the pooled visceral fat depots (perigonadal, perirenal, mesenteric, and pericardial) of Wt1-creERT2;Rosa26mT/mG mice, the pooled subcutaneous fat depots (subcutaneous and scapular white) of Mx1-cre;Rosa26mT/mG, and both the pooled visceral (Tagln-V) and subcutaneous depots (Tagln-S) from Tagln-cre;Rosa26mT/mG mice. Data are shown as mean ± SEM of 4-6 mice/group.


Bright - field image of immortalized mGFP mTomato positive preadipocytes after in vitro adipogenic differentiation . Neutral lipoids were stained with Oil Red O original were at 10x magnification ( left panel ) . Scale bar = 100 μm .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-CELL_TYPE O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O

Bright-field image of immortalized mGFP and mTomato positive preadipocytes after in vitro adipogenic differentiation. Neutral lipoids were stained with Oil Red O and original photographs were at 10x magnification (left panel). Scale bar = 100 μm.


Triglyceride content of immortalized mGFP mTomato positive preadipocytes after six days of differentiation . Data are shown as mean ± of 5 - 8 replicates , entire experiment was repeated with three independent lines .

B-SMALL_MOLECULE O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O

Triglyceride content of immortalized mGFP and mTomato positive preadipocytes after six days of differentiation. Data are shown as mean ± SEM of 5-8 replicates, and the entire experiment was repeated with three independent cell lines.


Basal extracellular acidification rate ( ECAR ) of immortalized mGFP mTomato positive preadipocytes was determined by calculating under curve ( AUC ) in basal conditions . whole experiment was repeated twice . Data are shown as mean ± of 3 lines per group .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Basal extracellular acidification rate (ECAR) of immortalized mGFP and mTomato positive preadipocytes was determined by calculating the area under the curve (AUC) in basal conditions. The whole experiment was repeated twice. Data are shown as mean ± SEM of 3 cell lines per group.


Basal respiration of preadipocytes was of immortalized mGFP mTomato positive preadipocytes determined by calculating under curve ( AUC ) during measurements of oxygen consumption rate ( OCR ) . whole experiment was repeated twice . Data are shown as mean ± of 3 lines per group .

B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O O O B-CELL_TYPE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O

Basal respiration of preadipocytes was of immortalized mGFP and mTomato positive preadipocytes determined by calculating the area under the curve (AUC) during measurements of oxygen consumption rate (OCR). The whole experiment was repeated twice. Data are shown as mean ± SEM of 3 cell lines per group.


of basal oxygen consumption rate ( OCR ) to basal extracellular acidification rate ( ECAR ) of immortalized mGFP mTomato positive preadipocytes . Data are shown as mean ± of 3 lines per group .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O B-CELL_TYPE O O O O O O O O O O O O O

Ratio of basal oxygen consumption rate (OCR) to basal extracellular acidification rate (ECAR) of immortalized mGFP and mTomato positive preadipocytes. Data are shown as mean ± SEM of 3 cell lines per group.


Number of mGFP mTomato positive preadipocytes isolated by FACS from indicated adipose depots from 5 - 6 month old Wt1 - creERT2 ; Rosa26mT / mG , Tagln - cre ; Rosa26mT / mG , Mx1 - cre ; Rosa26mT / mG mice . Data are shown as mean ± of 4 - 9 mice .

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Number of mGFP and mTomato positive preadipocytes isolated by FACS from the indicated white adipose depots from 5-6 month old male Wt1-creERT2;Rosa26mT/mG, Tagln-cre;Rosa26mT/mG, and Mx1-cre;Rosa26mT/mG mice. Data are shown as mean ± SEM of 4-9 mice.


Representative of whole - mount preparations of indicated adipose tissue depots of 5 - 6 month old Wt1 - creERT2 ; Rosa26mT / mG , Tagln - cre ; Rosa26mT / mG , Mx1 - cre ; Rosa26mT / mG mice . were taken at 10x magnification . Scale bar = 100 μm .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-TISSUE I-TISSUE O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O

Representative images of whole-mount preparations of the indicated white adipose tissue depots of 5-6 month old Wt1-creERT2;Rosa26mT/mG, Tagln-cre;Rosa26mT/mG, and Mx1-cre;Rosa26mT/mG male mice. The photographs were taken at 10x magnification. Scale bar = 100 μm.


Quantitation of mGFP mTomato positive adipocytes from each of indicated adipose depots from 5 - 6 month old Wt1 - creERT2 ; Rosa26mT / mG , Tagln - cre ; Rosa26mT / mG , Mx1 - cre ; Rosa26mT / mG mice . Adipocytes were counted from four non - overlapping per depot per mouse .

O O O O O B-CELL_TYPE O O O O B-TISSUE O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-ORGANISM O B-CELL_TYPE O B-EXP_ASSAY O O O O O O O O B-ORGANISM O

Quantitation of mGFP and mTomato positive adipocytes from each of the indicated white adipose depots from 5-6 month old male Wt1-creERT2;Rosa26mT/mG, Tagln-cre;Rosa26mT/mG, and Mx1-cre;Rosa26mT/mG mice. Adipocytes were counted from four non-overlapping images per depot per mouse.


Number of mGFP positive preadipocytes isolated by FACS from each of indicated adipose depots from 5 - 6 month - old Wt1 - creERT2 , Tagln - cre , Mx1 - cre crossed to Rosa26mT / mG mice from experiment shown in Figure 6B . Data are shown as mean ± of 4 - 9 mice .

O O O O B-CELL_TYPE O O B-EXP_ASSAY O O O O B-TISSUE O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O B-ORGANISM O

Number of mGFP positive preadipocytes isolated by FACS from each of the indicated white adipose depots from 5-6 month-old Wt1-creERT2, Tagln-cre, and Mx1-cre crossed to Rosa26mT/mG mice from the experiment shown in Figure 6B. Data are shown as mean ± SEM of 4-9 mice.


Number of mGFP positive adipocytes from each of indicated adipose depots from 5 - 6 month - old Wt1 - creERT2 , Tagln - cre , Mx1 - cre crossed to Rosa26mT / mG mice . Four separate non - overlapping / depot / mouse from experiment shown in Figure 6C . Data are shown as mean ± of 4 - 9 mice .

O O O O B-CELL_TYPE O O O O B-TISSUE O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-ORGANISM O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O B-ORGANISM O

Number of mGFP positive adipocytes from each of the indicated white adipose depots from 5-6 month-old Wt1-creERT2, Tagln-cre, and Mx1-cre crossed to Rosa26mT/mG mice. Four separate non-overlapping images/depot/mouse from the experiment shown in Figure 6C. Data are shown as mean ± SEM of 4-9 mice.


( F ) TIRF of Ramos transduced with endophilin A2 - GFP mCherry - ClathrinLC on antigen - loaded PMS . Scale bar = 5 μm . arrowheads highlight endophilin A2 colocalization with antigen clusters .

O O O B-EXP_ASSAY O B-CELL_LINE O O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O

(F) TIRF images of Ramos cells transduced with endophilin A2-GFP and mCherry-ClathrinLC on antigen-loaded PMS. Scale bar = 5 μm. White arrowheads highlight endophilin A2 colocalization with antigen clusters.


( J ) IgM - BCR immunoprecipitation to investigate recruitment of endophilin A2 to activated BCRs . Anti - endophilin A2 blots detect overexpressed endophilin A2 - GFP in streptavidin immunoprecipitates total supernatants from anti - IgM - biotin - stimulated Ramos DG75 . Representative experiment from 3 independent pulldowns in WT , BLNK - or GRB2 - knockout ( KO ) DG75 . Pulldown is calculated as a of total endophilin A2 normalized to WT in each experiment . Data show mean ; p values calculated using one - way ANOVA .

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(J) IgM-BCR immunoprecipitation to investigate recruitment of endophilin A2 to activated BCRs. Anti-endophilin A2 blots detect overexpressed endophilin A2-GFP in streptavidin immunoprecipitates and total supernatants from anti-IgM-biotin-stimulated Ramos and DG75 cells. Representative experiment and quantification from 3 independent pulldowns in WT, BLNK- or GRB2-knockout (KO) DG75 cells. Pulldown intensity is calculated as a percentage of total endophilin A2 protein and normalized to WT in each experiment. Data show mean and SEM; p values calculated using one-way ANOVA.


( E ) of surface CD86 CD69 mean ( MFI ) . Data show mean from 6 mice across 2 experiments .

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(E) Quantification of surface CD86 and CD69 mean fluorescence intensity (MFI). Data show mean and SEM from 6 mice across 2 experiments.


( F ) Fractions of mCherry + in GC , PC , IgG1 + class - switched follicular B at 4 - , 8 - 14 - days post SRBC - HEL immunization . N = 8 - 10 mice ; data shows mean . * p < 0 . 05 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 using 2 - way ANOVA with multiple comparisons .

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(F) Fractions of mCherry+ cells in GC, PC, IgG1+ class-switched and follicular B cells at 4-, 8- and 14-days post SRBC-HEL immunization. N = 8-10 mice; data shows mean and SEM. *p<0.05; ***p<0.001; ****p<0.0001 using 2-way ANOVA with multiple comparisons.


( C ) Surface of MHCII , BAFFR , CD40 LPAM1 in Sh3gl1 - / - WT . N = 4 - 5 mice . P , statistical significance from unpaired t . Data show mean ± .

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(C) Surface levels of MHCII, BAFFR, CD40 and LPAM1 in Sh3gl1-/- and WT littermates. N = 4-5 mice. P, statistical significance from unpaired t tests. Data show mean ± SEM.


( D ) of B220 + Fas + CD38 - GC population as of total splenic B NP - specific as a of GC population 14 days after NP - CGG immunization in alum . Data show mean ± from N = 6 mice , p , significance in unpaired t .

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(D) Quantification of B220+Fas+CD38- GC population as percentage of total splenic B cells and NP-specific cells as a percentage of the GC population 14 days after NP-CGG immunization in alum. Data show mean ± SEM from N = 6 mice, p, significance in unpaired t tests.


( E - F ) Serum antibodies collected pre - immunization 7 - or 14 - days post NP - CGG immunization , detected by NP7 - BSA ELISA using isotype - specific secondary antibodies . Data show mean ± from N = 3 experiments with statistical significance from 2 - way ANOVA with multiple comparisons .

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(E-F) Serum antibodies collected pre-immunization and 7- or 14-days post NP-CGG immunization, detected by NP7-BSA ELISA using isotype-specific secondary antibodies. Data show mean ± SEM from N = 3 experiments with statistical significance from 2-way ANOVA with multiple comparisons.


( H ) Numbers of Tfh 14 days post - immunization , identified as a CD4 + CD44 + PD - 1 + CXCR5 + population . Mean of N = 4 mice .

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(H) Numbers of Tfh cells 14 days post-immunization, identified as a CD4+CD44+PD-1+CXCR5+ population. Mean and SEM of N = 4 mice.


( A ) Soluble anti - IgM internalization in CRISPR - targeted follicular B . N = 11 - 12 mice . Data show mean ± . P , statistical significance from two - way ANOVA . ( B ) Soluble anti - IgD internalization . N = 3 mice . Data show mean ± . P , statistical significance from two - way ANOVA . ( C ) Soluble anti - IgG internalization in GC B from SRBC - immunized mice . N = 4 mice . Data show mean ± . P , statistical significance from two - way ANOVA .

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(A) Soluble anti-IgM internalization in CRISPR-targeted follicular B cells. N = 11- 12 mice. Data show mean ± SEM. P, statistical significance from two-way ANOVA. (B) Soluble anti-IgD internalization. N = 3 mice. Data show mean ± SEM. P, statistical significance from two-way ANOVA. (C) Soluble anti-IgG internalization in GC B cells from SRBC-immunized mice. N = 4 mice. Data show mean ± SEM. P, statistical significance from two-way ANOVA.


( F ) IgM degradation following total surface biotinylation , measured as loss of biotin - labelled IgM ( mIgM ) in lysates over time . ( G ) Western densitometry quantifying IgM degradation . Proportion of biotin detected is normalized to initial WT condition ( left ) or to initial amount for each genotype ( right ) . Data show mean ± from three independent . P , statistical significance from two - way ANOVA .

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(F) IgM degradation following total cell surface biotinylation, measured as loss of biotin-labelled membrane IgM (mIgM) in cell lysates over time. (G) Western densitometry quantifying IgM degradation assay. Proportion of biotin detected is normalized to the initial WT condition (left) or to the initial amount for each genotype (right). Data show mean ± SEM from three independent tests. P, statistical significance from two-way ANOVA.


( K ) Proliferation ( CFSE dilution ) of OTII CD4 in co - cultures with anti - Igκ - OVA pulsed B ; from 6 - 8 mice . Data show mean ± .

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(K) Proliferation (CFSE dilution) of OTII CD4 cells in co-cultures with anti-Igκ-OVA pulsed B cells; and quantification from 6-8 mice. Data show mean ± SEM.


( C - D ) Isolated naïve B from CRISPR - targeted chimeras after 3 - day culture in stated cytokines . ( C ) mCherry in targeted cultures . N = 15 mice . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 using two - way ANOVA . ( D ) AnnexinV surface stain in targeted . N = 3 - 4 mice . Data information : data show means ± .

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(C-D) Isolated naïve B cells from CRISPR-targeted chimeras after 3-day culture in stated cytokines. (C) mCherry percentage in targeted primary cell cultures. N = 15 mice. *p<0.05, **p<0.01, ****p<0.0001 using two-way ANOVA. (D) AnnexinV surface stain in targeted cells. N = 3-4 mice. Data information: All data show means ± SEM.


( G ) Oxygen consumption rate ( OCR ) in CRISPR - targeted Ramos . N = 2 independent infections . P , two - way ANOVA of basal maximum OCR . ( H ) OCR in B isolated from Sh3gl1 - / - WT . N = 9 mice . P , two - way ANOVA of basal maximum OCR .

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(G) Oxygen consumption rate (OCR) in CRISPR-targeted Ramos cells. N = 2 independent infections. P, two-way ANOVA of basal and maximum OCR. (H) OCR in B cells isolated from Sh3gl1-/- and WT littermates. N = 9 mice. P, two-way ANOVA of basal and maximum OCR.


cultures with full RPMI media supplemented with 50 μg / ml FeSO4 or 5 μM hemin chloride . ( J ) SH3GL1 - targeted Ramos . N = 4 independent infections . P , statistical significance from two - way ANOVA from both treatments versus RPMI . ( K ) Control sgRNA - targeted Ramos .

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Cell cultures with full RPMI media supplemented with 50 μg/ml FeSO4 or 5 μM hemin chloride. (J) SH3GL1-targeted Ramos cells. N = 4 independent infections. P, statistical significance from two-way ANOVA from both treatments versus RPMI. (K) Control sgRNA-targeted Ramos cells.


( O ) TfR internalization in Ramos using biotinylated anti - CD71 . 4 independent CRISPR infections in 2 experiments . P , significance calculated using two - way ANOVA indicated in corresponding color .

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(O) TfR internalization assay in Ramos cells using biotinylated anti-CD71. 4 independent CRISPR infections in 2 experiments. P, significance calculated using two-way ANOVA and indicated in corresponding color.


( P , Q ) Mouse transferrin internalization in B from SRBC - immunized WT Sh3gl1 - / - . N = 7 mice per genotype in 2 experiments . P , statistical significance analyzed using two - way ANOVA . ( P ) Naïve B . ( Q ) GC B .

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(P,Q) Mouse transferrin internalization in B cells from SRBC-immunized WT and Sh3gl1-/- littermates. N = 7 mice per genotype in 2 experiments. P, statistical significance analyzed using two-way ANOVA. (P) Naïve B cells. (Q) GC B cells.


A Robust immunofluorescence detection of ubiquitination on LAMP1 - positive compartments with pan - ubiquitin FK2 or K48 chain - specific antibodies . HeLa were treated with 250 µM LLOMe or EtOH ( untreated ) for 3 h , fixed stained with indicated antibodies . Confocal microscopy was followed by automated image . Graphs represent data from three independent experiments with ≥ 50 per condition ( mean ± SD ) . Scale bar : 20 µm .

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A Robust immunofluorescence detection of ubiquitination on LAMP1-positive compartments with pan-ubiquitin FK2 or K48 chain-specific antibodies. HeLa cells were treated with 250 µM LLOMe or EtOH (untreated) for 3 h, fixed and stained with indicated antibodies. Confocal microscopy was followed by automated image analysis. Graphs represent data from three independent experiments with ≥ 50 cells per condition (mean ± SD). Scale bar: 20 µm.


C screen using pools of four single siRNAs targeting 37 human E2 enzymes . HeLa were seeded in 384 - well plates , transfected with a siRNA library in quadruplicates for 72 h . were LLOMe - treated processed for imaging Plates were imaged automatically with a spinning disk confocal microscope . Automated image was done using Acapella software of ubiquitinated lysosomes was determined . Robust z - scores were calculated for LLOMe - treated samples for each depletion against values of whole plate . Samples exceeding threshold of - 2 of robust z - score ( highlighted in red ) were considered for secondary screen .

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C Primary screen using pools of four single siRNAs targeting 37 human E2 enzymes. HeLa cells were seeded in 384-well plates, transfected with a siRNA library in quadruplicates for 72 h. Cells were LLOMe-treated and processed for imaging Plates were imaged automatically with a spinning disk confocal microscope. Automated image analysis was done using the Acapella software and the percentage of ubiquitinated lysosomes was determined. Robust z-scores were calculated for LLOMe-treated samples for each depletion against the values of the whole plate. Samples exceeding the threshold of -2 of robust z-score (highlighted in red) were considered for the secondary screen.


D Selected candidates were re - screened with same work pipeline but with four single siRNAs of pools of each candidate from screen . Candidates exceeding threshold in both stainings are highlighted in red .

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D Selected candidates were re-screened with the same work pipeline but with the four single siRNAs of pools of each candidate from the primary screen. Candidates exceeding the threshold in both stainings are highlighted in red.


E Damage - induced translocation of UBE2QL1 to lysosomes . three best candidates ( from D ) were transiently expressed with N - terminal HA - tag in HeLa . were treated with LLOMe or EtOH ( untreated ) for 3 h , fixed stained for HA LAMP1 as indicated imaged by confocal scanning microscopy . Scale bar : 10 µm . F Automated of ( E ) . Shown are Pearson correlation coefficients ( P . C . C ) of HA LAMP1 signals . Graph represents data from three independent experiments with ≥ 30 per condition ( mean ± SD ) . * * * * P < 0 . 0001 ( One - way - ANOVA with Bonferroni ' s multiple comparison ) .

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E Damage-induced translocation of UBE2QL1 to lysosomes. The three best candidates (from D) were transiently expressed with N-terminal HA-tag in HeLa cells. Cells were treated with LLOMe or EtOH (untreated) for 3 h, fixed and stained for HA and LAMP1 as indicated and imaged by confocal laser scanning microscopy. Scale bar: 10 µm. F Automated quantification of (E). Shown are Pearson correlation coefficients (P.C.C) of HA and LAMP1 signals. Graph represents data from three independent experiments with ≥ 30 cells per condition (mean ± SD). ****P<0.0001 (One-way-ANOVA with Bonferroni's multiple comparison test).


G Western blot detection of endogenous UBE2QL1 depletion efficiency . were transfected with control siRNA ( Ctrl ) , two UBE2QL1 siRNAs from screen ( # 2 or # 4 ) or an additional siRNA ( # 5 ) , lysates probed with an antibody to UBE2QL1 as indicated . GAPDH was probed as loading control .

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G Western blot detection of endogenous UBE2QL1 and depletion efficiency. Cells were transfected with control siRNA (Ctrl), two UBE2QL1 siRNAs from the screen (#2 or #4) or an additional siRNA (#5), and lysates probed with an antibody to UBE2QL1 as indicated. GAPDH was probed as loading control.


A HeLa were transfected with different UBE2QL1 siRNAs for 60 h , LLOMe or control - treated ( untreated ) for 3 h . were fixed immuno - stained for LAMP1 along with antibodies specific for K63 or K48 - linked ubiquitin chains as indicated imaged with a scanning confocal microscope . Note strong reduction of damage - induced K48 ubiquitination decreased of K63 signal . Scale bar : 20 µm . B , C Automated image of ( A ) . Shown are percentages of K48 or K63 - positive LAMP1 vesicles per . Graphs represent data from three independent experiments with ≥ 70 per condition ( mean ± SD ) . * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 ( One - way - ANOVA with Bonferroni ' s multiple comparison ) .

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A HeLa cells were transfected with different UBE2QL1 siRNAs for 60 h, and LLOMe or control-treated (untreated) for 3 h. Cells were fixed and immuno-stained for LAMP1 along with antibodies specific for K63 or K48-linked ubiquitin chains as indicated and imaged with a laser scanning confocal microscope. Note the strong reduction of damage-induced K48 ubiquitination and decreased intensity of the K63 signal. Scale bar: 20 µm. B, C Automated image quantification of (A). Shown are percentages of K48 or K63-positive LAMP1 vesicles per cell. Graphs represent data from three independent experiments with ≥ 70 cells per condition (mean ± SD). **P<0.01; ***P<0.001; ****P<0.0001 (One-way-ANOVA with Bonferroni's multiple comparison test).


A HeLa expressing UBE2QL1 - HA were fixed at indicated time points after LLOMe treatment processed for confocal microscopy . were stained with antibodies specific for LAMP1 ( not shown ) , combinations of HA with K48 chains , K63 chains or p62 as indicated . Note delayed co - emergence of K48 chains UBE2QL1 vesicles , compared to earlier appearance of K63 chains p62 . Arrows indicate colocalizing or non - colocalizing vesicles . Scale bars : 10 µm , 2 µm for inlays . B Automated of ( A ) . Shown are Pearson correlation coefficients ( P . C . C . ) representing colocalization of UBE2QL1 - HA , K48 chains , K63 chains or p62 with LAMP1 - positive vesicles . Graphs represent data from three independent experiments with ≥ 30 per condition ( mean ± SD ) . C Automated of ( A ) . P . C . C . ' s representing colocalization of K48 chains , K63 chains or p62 with UBE2QL1 - HA in treated with LLOMe or vehicle alone ( untreated ) for 180 min . Graphs represent data from three independent experiments with ≥ 30 per condition ( mean ± SD ) .

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A HeLa cells expressing UBE2QL1-HA were fixed at indicated time points after LLOMe treatment and processed for confocal microscopy. Cells were stained with antibodies specific for LAMP1 (not shown), and combinations of HA with K48 chains, K63 chains or p62 as indicated. Note the delayed co-emergence of K48 chains and UBE2QL1 vesicles, compared to the earlier appearance of K63 chains and p62. Arrows indicate colocalizing or non-colocalizing vesicles. Scale bars: 10 µm, 2 µm for inlays. B Automated quantification of (A). Shown are the Pearson correlation coefficients (P.C.C.) representing colocalization of UBE2QL1-HA, K48 chains, K63 chains or p62 with LAMP1-positive vesicles. Graphs represent data from three independent experiments with ≥ 30 cells per condition (mean ± SD). C Automated quantification of (A). P.C.C.'s representing colocalization of K48 chains, K63 chains or p62 with UBE2QL1-HA in cells treated with LLOMe or vehicle alone (untreated) for 180 min. Graphs represent data from three independent experiments with ≥ 30 cells per condition (mean ± SD).


Immuno - electron microscopy of LLOMe - treated HeLa . in ( A ) overexpress mCherry - Gal3 a dominant - negative C160S mutant of GFP - YOD1 , were immunostained for p62 ( A ) p62 ( 10 nm gold particles ) localizes as a cap ( arrowheads ) to outer limiting of lysosomes ( L ) . Scale bars : 200 nm .

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Immuno-electron microscopy of LLOMe-treated HeLa cells. Cells in (A) overexpress mCherry-Gal3 and a dominant-negative C160S mutant of GFP-YOD1, and were immunostained for p62 (A) p62 (10 nm gold particles) localizes as a cap (arrowheads) to the outer limiting membrane of lysosomes (L). Scale bars: 200 nm.


Immuno - electron microscopy of LLOMe - treated HeLa . in ( B ) overexpress mCherry - Gal3 a dominant - negative C160S mutant of GFP - YOD1 , were immunostained for p62 K48 . ( B ) K48 ubiquitin chains ( 15 nm gold particles , arrows ) are found in lumen of lysosomes , that also contain p62 ( 10 nm gold particles , arrowheads ) at their outer . Scale bars : 200 nm .

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Immuno-electron microscopy of LLOMe-treated HeLa cells. Cells in (B) overexpress mCherry-Gal3 and a dominant-negative C160S mutant of GFP-YOD1, and were immunostained for p62 and K48. (B) K48 ubiquitin chains (15 nm gold particles, arrows) are found in the lumen of lysosomes, that also contain p62 (10 nm gold particles, arrowheads) at their outer membrane. Scale bars: 200 nm.


Immuno - electron microscopy of LLOMe - treated HeLa . ( C ) Overexpressed UBE2QL1 - GFP ( 10 nm gold particles ) is found in late endosomes ( LE , Inset ) lysosomes . M = mitochondrion . Scale bars : 200 nm .

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Immuno-electron microscopy of LLOMe-treated HeLa cells. (C) Overexpressed UBE2QL1-GFP (10 nm gold particles) is found in late endosomes (LE, Inset) and lysosomes. M = mitochondrion. Scale bars: 200 nm.


D , E Comparative UBE2QL1 proximity mapping before after lysosome damage using SILAC mass spectrometry . HeLa expressing a UBE2QL1 - APEX2 fusion were EtOH ( - labelled , L ) - or LLOMe ( heavy - labelled , H ) - treated pulsed with biotin phenol ( 30 min ) H2O2 ( 1 min ) . detected in replicative streptavidin purifications are depicted in a Venn diagram ( D ) displayed high correlation coefficients ( E ) .

O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O O O O O

D, E Comparative UBE2QL1 proximity mapping before and after lysosome damage using SILAC mass spectrometry. HeLa cells expressing a UBE2QL1-APEX2 fusion were EtOH (light-labelled, L)- or LLOMe (heavy-labelled, H)-treated and pulsed with biotin phenol (30 min) and H2O2 (1 min). Proteins detected in replicative streptavidin purifications are depicted in a Venn diagram (D) and displayed high correlation coefficients (E).


, F Comparative UBE2QL1 proximity mapping before after lysosome damage using SILAC mass spectrometry . HeLa expressing a UBE2QL1 - APEX2 fusion were EtOH ( - labelled , L ) - or LLOMe ( heavy - labelled , H ) - treated pulsed with biotin phenol ( 30 min ) H2O2 ( 1 min ) . ( F ) Results from four experiments are summarized in a volcano plot . Horizontal dotted represents significance threshold ( p > 0 . 05 ) . vertical lines indicate fold change cutoff ( log2 ( H / L ) ≥ 1 . 5 ) . Red dots indicate above significance fold - change thresholds . names are color - coded in blue ( endosome / lysosome - associated ) , green ( endolysosomal damage / autophagy - associated ) orange ( VCP / p97 its cofactors ) .

O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O B-GENEPROD O B-GENEPROD O O O O

, F Comparative UBE2QL1 proximity mapping before and after lysosome damage using SILAC mass spectrometry. HeLa cells expressing a UBE2QL1-APEX2 fusion were EtOH (light-labelled, L)- or LLOMe (heavy-labelled, H)-treated and pulsed with biotin phenol (30 min) and H2O2 (1 min). (F) Results from the four experiments are summarized in a volcano plot. Horizontal dotted line represents the significance threshold (p > 0.05). The vertical lines indicate the fold change cutoff (log2 (H/L) ≥ 1.5). Red dots indicate proteins above the significance and fold-change thresholds. Protein names are color-coded in blue (endosome/lysosome-associated), green (endolysosomal damage/autophagy-associated) and orange (VCP/p97 and its cofactors).


A HeLa stably expressing p97 - GFP were control or UBE2QL1 - depleted for 48 h expression of p97 - GFP was induced for last 24 h . were LLOMe or control treated for 1 h , fixed after a chase of 2 h , stained with antibodies to K48 ubiquitin chains LAMP1 as indicated . Note reduction of K48 p97 - GFP signals on damaged lysosomes in UBE2QL1 - depleted . Arrows indicate colocalizing or non - colocalizing vesicles . Scale bars : 20 µm , 10 µm for inlays .

O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR O B-GENEPROD O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY B-SUBCELLULAR O O O O O O O O O O O O

A HeLa cells stably expressing p97-GFP were control or UBE2QL1-depleted for 48 h and expression of p97-GFP was induced for the last 24 h. Cells were LLOMe or control treated for 1 h, fixed after a chase of 2 h, and stained with antibodies to K48 ubiquitin chains and LAMP1 as indicated. Note the reduction of the K48 and p97-GFP signals on damaged lysosomes in UBE2QL1-depleted cells. Arrows indicate colocalizing or non-colocalizing vesicles. Scale bars: 20 µm, 10 µm for inlays.


B Automated Shown are fold increases of Pearson correlation coefficients ( P . C . C . ) of p97 - GFP LAMP1 signals , normalized to untreated siCtrl . Data are from four independent experiments with ≥ 30 per condition . Data information : data are presented as mean ± SD . * P < 0 . 05 ; * * * P < 0 . 001 ( One - way - ANOVA with Bonferroni ' s multiple comparison ) .

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B Automated quantification Shown are fold increases of Pearson correlation coefficients (P.C.C.) of p97-GFP and LAMP1 signals, normalized to untreated siCtrl. Data are from four independent experiments with ≥ 30 cells per condition. Data information: data are presented as mean ± SD. *P<0.05; ***P<0.001 (One-way-ANOVA with Bonferroni's multiple comparison test).


C HeLa were control or UBE2QL1 - depleted for 60 h , fixed after 1 h of LLOMe or control treatment stained for LAMP1 p62 analyzed by confocal microscopy . Arrows indicate colocalizing or non - colocalizing vesicles . Scale bars : 20 µm , 5 µm for inlays .

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C HeLa cells were control or UBE2QL1-depleted for 60 h, fixed after 1 h of LLOMe or control treatment and stained for LAMP1 and p62 and analyzed by confocal microscopy. Arrows indicate colocalizing or non-colocalizing vesicles. Scale bars: 20 µm, 5 µm for inlays.


D Shown are fold increases of Pearson correlation coefficients ( P . C . C . ) of p62 on LAMP1 signals , normalized to untreated siCtrl . Graph represents data from three independent experiments with ≥ 30 per condition . Data information : data are presented as mean ± SD . * P < 0 . 05 ; * * * P < 0 . 001 ( One - way - ANOVA with Bonferroni ' s multiple comparison ) .

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D Quantification Shown are fold increases of Pearson correlation coefficients (P.C.C.) of p62 on LAMP1 signals, normalized to untreated siCtrl. Graph represents data from three independent experiments with ≥ 30 cells per condition. Data information: data are presented as mean ± SD. *P<0.05; ***P<0.001 (One-way-ANOVA with Bonferroni's multiple comparison test).


E HeLa were treated but fixed after additional 2 h of chase . Samples were stained for Galectin - 3 ( Gal3 ) to detect damaged lysosomes , LC3B as marker for autophagosomes . Note reduction of LC3B colocalization with Gal3 indicating impaired autophagosome formation . Arrows indicate colocalizing or non - colocalizing vesicles . Scale bars : 20 µm , 5 µm for inlays .

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E HeLa cells were treated but fixed after additional 2 h of chase. Samples were stained for Galectin-3 (Gal3) to detect damaged lysosomes, and LC3B as marker for autophagosomes. Note the reduction of LC3B colocalization with Gal3 indicating impaired autophagosome formation. Arrows indicate colocalizing or non-colocalizing vesicles. Scale bars: 20 µm, 5 µm for inlays.


F Shown are fold increases of Pearson correlation coefficients ( P . C . C . ) of LC3B Gal3 signals , normalized to untreated siCtrl , representing LC3B colocalization with Gal3 . Graph represents data from three independent experiments with ≥ 50 per condition . Data information : data are presented as mean ± SD . * P < 0 . 05 ; * * * P < 0 . 001 ( One - way - ANOVA with Bonferroni ' s multiple comparison ) .

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F Quantification Shown are fold increases of Pearson correlation coefficients (P.C.C.) of LC3B and Gal3 signals, normalized to untreated siCtrl, representing LC3B colocalization with Gal3. Graph represents data from three independent experiments with ≥ 50 cells per condition. Data information: data are presented as mean ± SD. *P<0.05; ***P<0.001 (One-way-ANOVA with Bonferroni's multiple comparison test).


G Western blot of lysates of transfected with UBE2QL1 or control siRNA for 72 h , treated with 200 nM Bafilomycin A1 for 5 h or 250 µM LLOMe for 3 h as indicated probed with an antibody specific for LC3A / B . GAPDH was probed as loading control .

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G Western blot analysis of lysates of cells transfected with UBE2QL1 or control siRNA for 72 h, treated with 200 nM Bafilomycin A1 for 5 h or 250 µM LLOMe for 3 h as indicated and probed with an antibody specific for LC3A/B. GAPDH was probed as loading control.


A Gal3 for damaged lysosomes . HeLa were depleted of UBE2QL1 with two siRNAs for 60 h treated with LLOMe or EtOH alone ( untreated ) for 1 h . were fixed at indicated times after washout , stained with DAPI Gal3 LAMP1 antibodies , processed for confocal microscopy . Note increased number of Gal3 in untreated UBE2QL1 - depleted their persistence 10 h after LLOMe - induced damage . Scale bars : 10 µm , 2 µm for inlays . Arrows indicate Gal3 LAMP1 colocalizing vesicles .

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A Gal3 puncta assay for damaged lysosomes. HeLa cells were depleted of UBE2QL1 with two siRNAs for 60 h and treated with LLOMe or EtOH alone (untreated) for 1 h. Cells were fixed at indicated times after washout, stained with DAPI and Gal3 and LAMP1 antibodies, and processed for confocal microscopy. Note increased number of Gal3 puncta in untreated UBE2QL1-depleted cells and their persistence 10 h after LLOMe-induced damage. Scale bars: 10 µm, 2 µm for inlays. Arrows indicate Gal3 and LAMP1 colocalizing vesicles.


B Automated of with more than three Gal3 is shown . Graph represents data from three independent experiments with ≥ 50 per condition ( mean ± SD ) . * P < 0 . 05 ; * * P < 0 . 01 ( One - way - ANOVA with Dunnett ' s multiple comparison ) .

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B Automated quantification The percentage of cells with more than three Gal3 puncta is shown. Graph represents data from three independent experiments with ≥ 50 cells per condition (mean ± SD). *P<0.05; **P<0.01 (One-way-ANOVA with Dunnett's multiple comparison test).


C Survival . Control or UBE2QL1 - depleted HeLa ( 48 h ) were treated with increasing concentrations of LLOMe as indicated . viability was measured with MTS . Graph represents data from one experiment including three replicates ( mean ± SD ) . * P < 0 . 05 ; * * * * P < 0 . 0001 ( One - way - ANOVA with Bonferroni ' s multiple comparison ) .

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C Survival assay. Control or UBE2QL1-depleted HeLa cells (48 h) were treated with increasing concentrations of LLOMe as indicated. Cell viability was measured with the MTS assay. Graph represents data from one experiment including three replicates (mean ± SD). *P<0.05; ****P<0.0001 (One-way-ANOVA with Bonferroni's multiple comparison test).


A Control or UBE2QL1 - depleted ( for 60 h ) HeLa were stained with DAPI an antibody against TFEB automatically analyzed by confocal microscopy . Note translocation of TFEB upon loss of UBE2QL1 . Scale bar : 20µm .

O O O B-GENEPROD O O O O O O O B-CELL_LINE O B-EXP_ASSAY O B-SMALL_MOLECULE O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O O

A Control or UBE2QL1-depleted (for 60 h) HeLa cells were stained with DAPI and an antibody against TFEB and automatically analyzed by confocal microscopy. Note nuclear translocation of TFEB upon loss of UBE2QL1. Scale bar: 20µm.


B Automated of TFEB signals was determined in both conditions . Graph represents data from four independent experiments with ≥ 100 per condition ( mean ± SD ) . * * * P < 0 . 001 ; * * * * P < 0 . 0001 ( One - way - ANOVA with Bonferroni ' s multiple comparison ) .

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B Automated quantification The intensity ratio of the cytoplasmic and nuclear TFEB signals was determined in both conditions. Graph represents data from four independent experiments with ≥ 100 cells per condition (mean ± SD). ***P<0.001; ****P<0.0001 (One-way-ANOVA with Bonferroni's multiple comparison test).


C were stained for LAMP1 mTOR . Note increase in lysosomes lysosomal dissociation of mTOR in UBE2QL1 depleted . Scale bar : 20 µm .

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C Cells were stained for LAMP1 and mTOR. Note the increase in lysosomes and lysosomal dissociation of mTOR in UBE2QL1 depleted cells. Scale bar: 20 µm.


D Automated colocalization of mTOR LAMP1 is shown as Pearson correlation coefficients ( P . C . C . ) of signals . Graph represents data from three independent experiments with ≥ 70 per condition ( mean ± SD ) . * P < 0 . 05 ( One - way - ANOVA with Bonferroni ' s multiple comparison ) .

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D Automated quantification The colocalization of mTOR and LAMP1 is shown as Pearson correlation coefficients (P.C.C.) of the signals. Graph represents data from three independent experiments with ≥ 70 cells per condition (mean ± SD). *P<0.05 (One-way-ANOVA with Bonferroni's multiple comparison test).


E Automated Number of LAMP1 - positive vesicles per is shown . Graph represents data from three independent experiments with ≥ 70 per condition ( mean ± SD ) .

O O B-EXP_ASSAY O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O

E Automated quantification Number of LAMP1-positive vesicles per cell is shown. Graph represents data from three independent experiments with ≥ 70 cells per condition (mean ± SD).


F Western blot of lysates of treated As a positive control , were treated with mTOR - inhibitor Torin1 ( 1 µM ) for 2 h . GAPDH was probed as loading control .

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F Western blot analysis of lysates of cells treated As a positive control, cells were treated with the mTOR-inhibitor Torin1 (1 µM) for 2 h. GAPDH was probed as loading control.


G C . elegans wild - type ( N2 ) mutant strains for ubc - 25 ( ok1732 ) , for scav - 3 ( qx193 ) or ubc - 25 scav - 3 double mutant ( ok1732 ; qx193 ) carrying a EGFP - Gal3 transgene were imaged in adult worms ( L4 + 24 h ) . 3D reconstructions of hypodermis . Small big units represent 1 5 µm , respectively .

O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O O B-ORGANISM O O O O O O O O O O B-TISSUE O O O O O O O O O O O

G C.elegans wild-type (N2) and mutant strains for ubc-25(ok1732), for scav-3(qx193) or the ubc-25 and scav-3 double mutant (ok1732;qx193) carrying a EGFP-Gal3 transgene were imaged in adult worms (L4 + 24 h). 3D reconstructions of the hypodermis. Small and big units represent 1 and 5 µm, respectively.


H Average volume of permeabilized lysosomes within region of hypodermis corresponding to 100 µm of was quantified . At least 20 were scored in each . * P < 0 . 05 ; * * P < 0 . 01 ; * * * * P < 0 . 0001 ( One - way - ANOVA with Dunnett ' s multiple comparison or Student ' s two - unpaired t ) .

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H Average volume of permeabilized lysosomes within the region of hypodermis corresponding to 100 µm of body length was quantified. At least 20 animals were scored in each strain. *P<0.05; **P<0.01; ****P<0.0001 (One-way-ANOVA with Dunnett's multiple comparison test or Student's two-tailed unpaired t test).


I Populations of worms with different extent of lysosome permeabilization were analyzed . Graph represents of worms with less than 5 , 5 to 20 or more than 20 permeabilized lysosomes in region of hypodermis corresponding to 100 µm of .

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I Populations of worms with different extent of lysosome permeabilization were analyzed. Graph represents percentage of worms with less than 5, 5 to 20 or more than 20 permeabilized lysosomes in the region of hypodermis corresponding to 100 µm of body length.


RT - PCR ( top ) representative direct sequencing ( bottom ) of Ttn exon 326 transcripts from HL - 1 cardiomyocytes transiently transfected with different 2OMePS AONs . Only 2OMePS - AON1 2OMePS - AON1 + 3 ( * ) lead to a correct excision of exon 326 as confirmed by direct sequencing .

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RT-PCR analysis (top) and representative direct sequencing (bottom) of Ttn exon 326 transcripts from HL-1 cardiomyocytes transiently transfected with different 2OMePS AONs. Only 2OMePS-AON1 and 2OMePS-AON1 + 3 (*) lead to a correct excision of exon 326 as confirmed by direct sequencing.


Immunofluorescence of sarcomeric α - actinin in untransfected 2OMePS - AON1 + 3 - transfected HL - 1 cardiomyocytes . Scale bars , 10 μm .

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Immunofluorescence images of sarcomeric α-actinin in untransfected and 2OMePS-AON1 + 3-transfected HL-1 cardiomyocytes. Scale bars, 10 μm.


RT - PCR of TTN exon 326 transcripts from DCM cardiomyocytes transiently transfected with 2OMePS - AON1 , 2OMePS - AON3 , 2OMePS - AON1 + 3 .

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RT-PCR analysis of TTN exon 326 transcripts from DCM cardiomyocytes transiently transfected with 2OMePS-AON1, 2OMePS-AON3, and 2OMePS-AON1 + 3.


RT - PCR ( left ) representative direct sequencing ( right ) of TTN exon 326 transcripts from DCM cardiomyocytes infected with U7snRNA - TTNAONs - IRES - GFP lentiviral vector carrying AON1 3 sequences ( TTN - AON ) or with a control vector ( U7snRNA - ScrAONs - IRES - GFP ) .

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RT-PCR analysis (left) and representative direct sequencing (right) of TTN exon 326 transcripts from DCM cardiomyocytes infected with the U7snRNA-TTNAONs-IRES-GFP lentiviral vector carrying the AON1 and 3 sequences (TTN-AON) or with a control vector (U7snRNA-ScrAONs-IRES-GFP).


Mass spectrometry - of titin peptides in infected with U7snRNA - ScrAONs - IRES - GFP ( Scr - AON ) U7snRNA - TTNAONs - IRES - GFP ( TTN - AON ) vectors . Unsupervised hierarchical clustering identified a cluster significantly enriched in peptides mapping to exon 326 that was down - regulated in DCM Scr - AON cardiomyocytes compared to CTR Scr - AON cardiomyocytes ( n = 3 , P = 9 . 03E − 8 , Fisher ' s exact , FDR = 0 . 04 , top ) . Down - regulation of exon 326 was also detected in DCM TTN - AON when compared to DCM Scr - AON ( n = 3 , P = 0 . 02 , Fisher ' s exact , bottom ) .

B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mass spectrometry-based analysis of titin peptides in cells infected with the U7snRNA-ScrAONs-IRES-GFP (Scr-AON) and U7snRNA-TTNAONs-IRES-GFP (TTN-AON) vectors. Unsupervised hierarchical clustering identified a cluster significantly enriched in peptides mapping to exon 326 that was down-regulated in DCM Scr-AON cardiomyocytes compared to CTR Scr-AON cardiomyocytes (n = 3, P = 9.03E−8, Fisher's exact test, FDR = 0.04, top). Down-regulation of exon 326 was also detected in DCM TTN-AON cells when compared to DCM Scr-AON cells (n = 3, P = 0.02, Fisher's exact test, bottom).


of iPSC - derived cardiomyocytes with cross - striated myofibrils occupying a fraction ( partially organized , gray ) or whole cytoplasm ( fully organized , gray ) from two control two clones . Statistical difference was tested using two - sided chi - squared ( CTR1 basal : n = 283 , CTR2 basal : n = 250 , DCM1 basal : n = 255 , DCM2 basal : n = 236 ; * * * P = 1 . 93E − 16 ) .

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Percentage of iPSC-derived cardiomyocytes with cross-striated myofibrils occupying a fraction (partially organized, dark gray) or the whole cytoplasm (fully organized, light gray) from two control and two patient clones. Statistical difference was tested using the two-sided chi-squared test (CTR1 basal: n = 283, CTR2 basal: n = 250, DCM1 basal: n = 255, DCM2 basal: n = 236; ***P = 1.93E−16).


Immunofluorescence of titin ( Z - disk portion ) cardiac troponin T ( cTNT , A - band ) in well - organized sarcomeres from CTR DCM single cardiomyocytes under basal conditions . Scale bars , 5 μm .

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Immunofluorescence images of titin (Z-disk portion) and cardiac troponin T (cTNT, A-band) in well-organized sarcomeres from CTR and DCM single cardiomyocytes under basal conditions. Scale bars, 5 μm.


Immunofluorescence ( left ) of α - actinin cTNT in CTR DCM single cardiomyocytes , illustrating 3 different of sarcomeric organization ( perinuclear , fully peripherally organized ) . of with different of sarcomeric organization ( right ) under basal stress conditions . Statistical difference was tested using chi - squared ( CTR1 basal : n = 283 , CTR2 basal : n = 250 , DCM1 basal : n = 255 , DCM2 basal : n = 236 , CTR1 Iso : n = 245 , CTR2 Iso : n = 230 ; DCM1 Iso : n = 242 DCM2 Iso : n = 269 ; * * * P = 1 . 93E − 16 CTR basal versus DCM basal ; * * * P = 9 . 61E − 34 CTR Iso versus DCM Iso , * * P = 0 . 001 DCM basal versus DCM Iso ) . No significant differences were observed comparing CTR basal CTR Iso groups . Scale bars , 25 μm .

B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O B-CELL_TYPE O O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O

Immunofluorescence images (left) of α-actinin and cTNT in CTR and DCM single cardiomyocytes, illustrating 3 different levels of sarcomeric organization (perinuclear, fully and peripherally organized). Percentage of cells with different levels of sarcomeric organization (right) under basal and stress conditions. Statistical difference was tested using the chi-squared test (CTR1 basal: n = 283, CTR2 basal: n = 250, DCM1 basal: n = 255, DCM2 basal: n = 236, CTR1 Iso: n = 245, CTR2 Iso: n = 230; DCM1 Iso: n = 242 and DCM2 Iso: n = 269; ***P = 1.93E−16 CTR basal versus DCM basal; ***P = 9.61E−34 CTR Iso versus DCM Iso, **P = 0.001 DCM basal versus DCM Iso). No significant differences were observed comparing CTR basal and CTR Iso groups. Scale bars, 25 μm.


of perinuclear , fully , peripherally organized single cardiomyocytes from two CTR two DCM iPSC clones after infection with U7snRNA - ScrAONs - IRES - GFP U7snRNA - TTNAONs - IRES - GFP lentiviruses . Statistical difference was tested using two - sided chi - squared ( CTR1 Scr - AON : n = 190 , CTR2 Scr - AON : n = 200 , DCM1 Scr - AON : n = 221 , DCM2 Scr - AON : n = 115 , CTR1 TTN - AON : n = 223 , CTR2 TTN - AON : n = 187 , DCM1 TTN - AON : n = 171 , DCM2 TTN - AON : n = 243 ; * * * P = 4 . 22E − 15 CTR Scr - AON versus DCM Scr - AON ; * * * P = 4 . 61E − 02 DCM Scr - AON versus DCM TTN - AON ) . No significant differences were observed comparing CTR Scr - AON CTR TTN - AON groups .

O O O O O O O O B-CELL_TYPE O O O O O B-CELL_TYPE O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O

Percentage of perinuclear, fully, and peripherally organized single cardiomyocytes from two CTR and two DCM iPSC clones after infection with the U7snRNA-ScrAONs-IRES-GFP and U7snRNA-TTNAONs-IRES-GFP lentiviruses. Statistical difference was tested using the two-sided chi-squared test (CTR1 Scr-AON: n = 190, CTR2 Scr-AON: n = 200, DCM1 Scr-AON: n = 221, DCM2 Scr-AON: n = 115, CTR1 TTN-AON: n = 223, CTR2 TTN-AON: n = 187, DCM1 TTN-AON: n = 171, DCM2 TTN-AON: n = 243; ***P = 4.22E−15 CTR Scr-AON versus DCM Scr-AON; ***P = 4.61E−02 DCM Scr-AON versus DCM TTN-AON). No significant differences were observed comparing CTR Scr-AON and CTR TTN-AON groups.


qRT - PCR of SRF target genes ( MYH6 , MYH7 ACTC1 ) in CTR DCM single cardiomyocytes under basal condition ( no infection , NI ) after infection with control U7snRNA - ScrAONs - IRES - GFP ( Scr - AON ) U7snRNA - TTNAONs - IRES - GFP ( TTN - AON ) lentiviruses . Statistical difference was tested using two - sided Student ' s t - ( * * P = 0 . 009 , CTR Scr - AON versus DCM Scr - AON ; * P = 0 . 04 , DCM Scr - AON versus DCM TTN - AON for MYH6 ; * * P = 0 . 002 , CTR Scr - AON versus DCM Scr - AON ; * * P = 0 . 002 , DCM Scr - AON versus DCM TTN - AON for MYH7 ; * * P = 0 . 004 , CTR Scr - AON versus DCM Scr - AON ; * P = 0 . 02 , DCM Scr - AON versus DCM TTN - AON for ACTC1 ) . No significant differences were observed comparing CTR NI , CTR Scr - AON CTR TTN - AON groups comparing DCM NI DCM Scr - AON groups . Expression values were relative to CTR Scr - AON , normalized to GAPDH , presented as mean ± , n = 3 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O

qRT-PCR analysis of SRF target genes (MYH6, MYH7 and ACTC1) in CTR and DCM single cardiomyocytes under basal condition (no infection, NI) and after infection with control U7snRNA-ScrAONs-IRES-GFP (Scr-AON) and the U7snRNA-TTNAONs-IRES-GFP (TTN-AON) lentiviruses. Statistical difference was tested using the two-sided Student's t-test (**P = 0.009, CTR Scr-AON versus DCM Scr-AON; *P = 0.04, DCM Scr-AON versus DCM TTN-AON for MYH6; **P = 0.002, CTR Scr-AON versus DCM Scr-AON; **P = 0.002, DCM Scr-AON versus DCM TTN-AON for MYH7; **P = 0.004, CTR Scr-AON versus DCM Scr-AON; *P = 0.02, DCM Scr-AON versus DCM TTN-AON for ACTC1). No significant differences were observed comparing the CTR NI, CTR Scr-AON and CTR TTN-AON groups and comparing the DCM NI and DCM Scr-AON groups. Expression values were relative to CTR Scr-AON, normalized to GAPDH, and presented as mean ± SEM, n = 3.


Immunofluorescence showing normal ( a , b , e , f , i , l , o , p ) altered ( c , d , g , h , m , n , q , r ) intracellular distribution of SRF ( a b , ; c d , ) , MURF2 ( e f , sarcomeric ; g h , ) , Nbr1 ( i l , sarcomeric ; m n , diffused ) , SQSTM1 / p62 ( o p , sarcomeric ; q r , diffused ) in representative single cardiomyocytes ( left ) . Sarcomeres are marked by α - actinin . On right , of CTR DCM cardiomyocytes showing expression of SRF , accumulation of MURF2 , diffused expression of Nbr1 of SQSTM1 / p62 after infection with control U7snRNA - ScrambleAONs - IRES - GFP ( Scr - AON ) U7snRNA - TTNAONs - IRES - GFP ( TTN - AON ) lentiviruses ( right ) . Data represent mean values ± from two control two DCM clones . Statistical difference was tested using two - sided chi - squared ( CTR Scr - AON : n = 874 , n = 874 , n = 882 n = 890 , CTR TTN - AON : n = 880 , n = 990 , n = 878 n = 890 , DCM Scr - AON : n = 890 , n = 887 , n = 884 n = 886 , DCM TTN - AON : n = 900 , n = 875 , n = 899 n = 891 for SRF , MURF2 , Nbr1 SQSTM1 / p62 , respectively ; * P = 0 . 01 , CTR Scr - AON versus DCM Scr - AON ; * P = 0 . 04 , DCM Scr - AON versus DCM TTN - AON for SRF ; * P = 0 . 02 , CTR Scr - AON versus DCM Scr - AON ; * P = 0 . 04 , DCM Scr - AON versus DCM TTN - AON for MURF2 ; * P = 0 . 03 , CTR Scr - AON versus DCM Scr - AON ; * P = 0 . 03 , DCM Scr - AON versus DCM TTN - AON for Nbr1 ; * * P = 0 . 009 , CTR Scr - AON versus DCM Scr - AON ; * P = 0 . 01 , DCM Scr - AON versus DCM TTN - AON for SQSTM1 / p62 ) . No significant differences were observed comparing CTR Scr - AON CTR TTN - AON groups . Scale bars , 50 μm .

B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O O B-CELL_TYPE O O O O B-SUBCELLULAR O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-CELL_TYPE O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O

Immunofluorescence images showing normal (a, b, e, f, i, l, o, p) and altered (c, d, g, h, m, n, q, r) intracellular distribution of SRF (a and b, nuclear; c and d, cytoplasmic), MURF2 (e and f, sarcomeric; g and h, nuclear), Nbr1 (i and l, sarcomeric; m and n, diffused), and SQSTM1/p62 (o and p, sarcomeric; q and r, diffused) in representative single cardiomyocytes (left). Sarcomeres are marked by α-actinin. On the right, percentage of CTR and DCM cardiomyocytes showing cytoplasmic expression of SRF, nuclear accumulation of MURF2, and diffused expression of Nbr1 and of SQSTM1/p62 after infection with control U7snRNA-ScrambleAONs-IRES-GFP (Scr-AON) and the U7snRNA-TTNAONs-IRES-GFP (TTN-AON) lentiviruses (right). Data represent mean values ± SEM from two control and two DCM clones. Statistical difference was tested using the two-sided chi-squared test (CTR Scr-AON: n = 874, n = 874, n = 882 and n = 890, CTR TTN-AON: n = 880, n = 990, n = 878 and n = 890, DCM Scr-AON: n = 890, n = 887, n = 884 and n = 886, DCM TTN-AON: n = 900, n = 875, n = 899 and n = 891 for SRF, MURF2, Nbr1 and SQSTM1/p62, respectively; *P = 0.01, CTR Scr-AON versus DCM Scr-AON; *P = 0.04, DCM Scr-AON versus DCM TTN-AON for SRF; *P = 0.02, CTR Scr-AON versus DCM Scr-AON; *P = 0.04, DCM Scr-AON versus DCM TTN-AON for MURF2; *P = 0.03, CTR Scr-AON versus DCM Scr-AON; *P = 0.03, DCM Scr-AON versus DCM TTN-AON for Nbr1; **P = 0.009, CTR Scr-AON versus DCM Scr-AON; *P = 0.01, DCM Scr-AON versus DCM TTN-AON for SQSTM1/p62). No significant differences were observed comparing CTR Scr-AON and CTR TTN-AON groups. Scale bars, 50 μm.


B , C Immunofluorescence of sarcomeric α - actinin myosin ( B ) assessment of cardiac function ( C ) in homozygous Ttn knock - in cultured for 24 h after mScrAON mTtnAON transient transfection ( n = 3 per group ) or lentivirus delivery ( n = 7 per group ) . Scale bars , 10 μm .

O O O B-EXP_ASSAY O B-SUBCELLULAR B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-TISSUE O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O

B, C Immunofluorescence images of sarcomeric α-actinin and myosin (B) and assessment of cardiac function (C) in homozygous Ttn knock-in embryos cultured for 24 h after mScrAON and mTtnAON transient transfection (n = 3 per group) or lentivirus delivery (n = 7 per group). Scale bars, 10 μm.


D Electron microscopy in Ttn knock - in cultured for 24 h after mScrAON mTtnAON transient transfection . mTtnAON treatment of homozygous rescued myofibril formation ( left , black arrows ) , resulting in thicker filaments ( right ) .

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D Electron microscopy analysis in Ttn knock-in embryos cultured for 24 h after mScrAON and mTtnAON transient transfection. mTtnAON treatment of homozygous embryos rescued myofibril formation (left, black arrows), resulting in thicker filaments (right).


Echocardiographic of adult knock - in mice . Statistical difference was tested using two - sided Student ' s t - ( WT : n = 6 , HET : n = 6 ; HET + vPMO - mScrAON : n = 10 HET + vPMO - mTtnAON : n = 14 ; * P = 0 . 013 , HET versus HET + vPMO - mTtnAON * P = 0 . 02 , HET + vPMO - mScrAON versus HET + vPMO - mTtnAON for LVEDD differences ; * * P = 0 . 006 , HET versus HET + vPMO - mTtnAON * P = 0 . 02 , HET + vPMO - mScrAON versus HET + vPMO - mTtnAON for LV - EF differences ; * P = 0 . 03 , HET versus HET + vPMO - mTtnAON * P = 0 . 04 , HET + vPMO - mScrAON versus HET + vPMO - mTtnAON for IVSd differences ; * * P = 0 . 001 , HET versus HET + vPMO - mTtnAON * P = 0 . 03 , HET + vPMO - mScrAON versus HET + vPMO - mTtnAON for PWd differences ) . No significant differences were observed among groups at day 0 day 7 . Data represent mean values ± .

B-EXP_ASSAY O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Echocardiographic analysis of adult knock-in mice. Statistical difference was tested using the two-sided Student's t-test (WT: n = 6, HET: n = 6; HET + vPMO-mScrAON: n = 10 and HET + vPMO-mTtnAON: n = 14; *P = 0.013, HET versus HET + vPMO-mTtnAON and *P = 0.02, HET + vPMO-mScrAON versus HET + vPMO-mTtnAON for LVEDD differences; **P = 0.006, HET versus HET + vPMO-mTtnAON and *P = 0.02, HET + vPMO-mScrAON versus HET + vPMO-mTtnAON for LV-EF differences; *P = 0.03, HET versus HET + vPMO-mTtnAON and *P = 0.04, HET + vPMO-mScrAON versus HET + vPMO-mTtnAON for IVSd differences; **P = 0.001, HET versus HET + vPMO-mTtnAON and *P = 0.03, HET + vPMO-mScrAON versus HET + vPMO-mTtnAON and for PWd differences). No significant differences were observed among groups at day 0 and day 7. Data represent mean values ± SEM.


Masson ' s trichrome of heart sections from HET injected with vPMO - mScrAONs vPMO - mTtnAON . Scale bars , 1 mm .

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Masson's trichrome staining of heart sections from HET animals injected with vPMO-mScrAONs and vPMO-mTtnAON. Scale bars, 1 mm.


in situ hybridization ( FISH ) of heart muscle tissue from adult knock - in mice with a probe complementary to vPMO - mTtnAON . Scale bars , 250 50 μm ( magnification ) .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-TISSUE I-TISSUE I-TISSUE O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O

Fluorescence in situ hybridization (FISH) of heart muscle tissue from adult knock-in mice with a probe complementary to vPMO-mTtnAON. Scale bars, 250 and 50 μm (magnification).


RT - PCR of Ttn exon 326 transcripts from heart tissue of untreated WT HET vPMO - mScrAON - vPMO - mTtnAON - treated mice ( left ) . Representative direct sequencing of Ttn exon 326 transcripts from vPMO - mTtnAON - treated HET heart tissue ( right ) .

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RT-PCR analysis of Ttn exon 326 transcripts from heart tissue of untreated WT and HET animals and vPMO-mScrAON- and vPMO-mTtnAON-treated mice (left). Representative direct sequencing of Ttn exon 326 transcripts from vPMO-mTtnAON-treated HET heart tissue (right).


Mass spectrometry - of titin peptides in adult knock - in mice injected with vPMO - mScrAONs vPMO - mTtnAONs . Unsupervised hierarchical clustering identified a cluster enriched in exon 326 peptides that was down - regulated in vPMO - mTtnAON - treated compared to vPMO - mScrAON - treated . Another cluster enriched in C - terminal peptides was up - regulated in vPMO - mTtnAON group compared to vPMO - mScrAON group ( n = 3 , P = 0 . 02 , Fisher ' s exact , FDR = 0 . 04 ) .

B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mass spectrometry-based analysis of titin peptides in adult knock-in mice injected with vPMO-mScrAONs and vPMO-mTtnAONs. Unsupervised hierarchical clustering identified a cluster enriched in exon 326 peptides that was down-regulated in vPMO-mTtnAON-treated animals compared to vPMO-mScrAON-treated littermates. Another cluster enriched in C-terminal peptides was up-regulated in the vPMO-mTtnAON group compared to the vPMO-mScrAON group (n = 3, P = 0.02, Fisher's exact test, FDR = 0.04).


A - B ) Western - blot for GAPDH LC3 of lysates obtained from HeLa treated with DMSO or 1 µM rapamycin ( Rapa ) for indicated time periods ( A ) ( n = 7 ) or for 5 h with indicated concentrations ( B ) ( n = 6 ) . One hour before harvesting , 100 nM bafilomycin A1 was added . Upper panels : representative Western blots ; lower panels : of LC3 - II / GAPDH .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O B-CELL_LINE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O

A-B) Western-blot analysis for GAPDH and LC3 of protein lysates obtained from HeLa cells treated with DMSO or 1 µM rapamycin (Rapa) for the indicated time periods (A) (n = 7) or for 5 h with the indicated concentrations (B) (n = 6). One hour before harvesting, 100 nM bafilomycin A1 was added. Upper panels: representative Western blots; lower panels: quantification of the LC3-II/GAPDH ratio.


C ) GFP - LC3 - in HeLa treated for 5 h with different concentrations of rapamycin . Left : representative pictures . scale bar represents 10 µm . Concentrations are mentioned in right lower corner . Right : of number of per ( n = 3 ) . * p < 0 . 05 , repeated measurements ANOVA .

O O B-GENEPROD O B-GENEPROD O O B-CELL_LINE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C) GFP-LC3-punctae quantification in HeLa cells treated for 5 h with different concentrations of rapamycin. Left: representative pictures. The scale bar represents 10 µm. Concentrations are mentioned in the right lower corner. Right: Quantification of the number of punctae per cell (n = 3). * p<0.05, repeated measurements ANOVA.


A ) Representative measurements ( n = 4 ) of Ca2 + signals , displayed as Fura2 ( F340 / F380 ) , showing effect of 0 . 3 µM 100 µM ATP , 1 µM thapsigargin ( Tg ) or 10 µM ionomycin ( Iono ) in intact HeLa treated with different concentrations of rapamycin ( Rapa ) for 5 h . 45 s prior to addition of ATP , Tg or Iono , EGTA ( 3 mM ) was given to buffer extracellular Ca2 + as indicated .

O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O B-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O

A) Representative measurements (n = 4) of cytosolic Ca2+ signals, displayed as Fura2 ratio (F340/F380), showing the effect of 0.3 µM and 100 µM ATP, 1 µM thapsigargin (Tg) or 10 µM ionomycin (Iono) in intact HeLa cells treated with different concentrations of rapamycin (Rapa) for 5 h. 45 s prior to the addition of ATP, Tg or Iono, EGTA (3 mM) was given to buffer extracellular Ca2+ as indicated.


B ) of average amplitude of response ( F − F0 ) ( n = 4 ) . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 , repeated measurements ANOVA .

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B) Quantification of the average amplitude of the response (F−F0) (n = 4). * p<0.05; ** p<0.01; *** p<0.001, repeated measurements ANOVA.


C ) Mean resting [ Ca2 + ] , measured in Fura2 - loaded HeLa treated with indicated concentrations of rapamycin for 5 h , as well as in absence ( Ctrl ) or presence of DMSO ( n = 3 ) . * p < 0 . 05 , repeated measurements ANOVA .

O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-EXP_ASSAY O O B-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O

C) Mean resting cytosolic [Ca2+], measured in Fura2-loaded HeLa cells treated with the indicated concentrations of rapamycin for 5 h, as well as in the absence (Ctrl) or presence of DMSO (n = 3). * p<0.05, repeated measurements ANOVA.


D ) Unidirectional 45Ca2 + - flux experiments in permeabilized pretreated with 1 µM rapamycin for 5 h or with DMSO ( Ctrl ) . Mean fractional 45Ca2 + release ( % / 2 min ) is shown as a function of time with effect of 0 . 7 µM IP3 ( circles ) or no addition ( squares ) . horizontal bar indicates presence of IP3 .

O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O

D) Unidirectional 45Ca2+-flux experiments in permeabilized cells pretreated with 1 µM rapamycin for 5 h or with DMSO (Ctrl). Mean fractional 45Ca2+ release (%/2 min) is shown as a function of time with the effect of 0.7 µM IP3 (circles) or no addition (squares). The horizontal bar indicates the presence of IP3.


E ) Quantitative of IP3 - induced 45Ca2 + release in pretreated for 5 h with 1 µM rapamycin or DMSO ( Ctrl ) ( n = 8 ) . * * * p < 0 . 001 , paired Student ' s t - .

O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E) Quantitative analysis of the IP3-induced 45Ca2+ release in cells pretreated for 5 h with 1 µM rapamycin or DMSO (Ctrl) (n = 8). *** p<0.001, paired Student's t-test.


A - B ) Western - blot for luminal Ca2 + - binding in HeLa treated with indicated concentrations of rapamycin ( Rapa ) for 5 h : calreticulin ( CRT ) ( A ) BiP / Grp78 ( BiP ) ( B ) . Upper panels : representative Western blots ; lower panels : of / GAPDH ( n = 4 ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O

A-B) Western-blot analysis for luminal Ca2+-binding proteins in HeLa cells treated with the indicated concentrations of rapamycin (Rapa) for 5 h: calreticulin (CRT) (A) and BiP/Grp78 (BiP) (B). Upper panels: representative Western blots; lower panels: quantification of the protein/GAPDH ratio (n = 4).


C ) Western - blot for SERCA2 in HeLa treated with indicated concentrations of rapamycin for 5 h . Upper panels : representative Western blots ; lower panel : of SERCA2 / GAPDH ( n = 4 ) .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O

C) Western-blot analysis for SERCA2 in HeLa cells treated with the indicated concentrations of rapamycin for 5 h. Upper panels: representative Western blots; lower panel: quantification of the SERCA2/GAPDH ratio (n = 4).


D ) Representative plot showing decrease in ER 45Ca2 + content ( logarithmic scale ) in a Ca2 + - free efflux medium without ATP as a function of time in permeabilized HeLa pretreated for 5 h with 1 µM rapamycin or with DMSO . passively bound Ca2 + was determined by loading with 45Ca2 + in presence of 10 µM of Ca2 + ionophore A23187 then subtracted from stored 45Ca2 + . ER Ca2 + - leak rate can be estimated as rate of decline of ER 45Ca2 + - store content as a function of time .

O O O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-CELL_LINE O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O O O

D) Representative plot showing the decrease in ER 45Ca2+ content (logarithmic scale) in a Ca2+-free efflux medium without ATP as a function of time in permeabilized HeLa cells pretreated for 5 h with 1 µM rapamycin or with DMSO. The passively bound Ca2+ was determined by loading the cells with 45Ca2+ in the presence of 10 µM of the Ca2+ ionophore A23187 and then subtracted from the stored 45Ca2+. The ER Ca2+-leak rate can be estimated as the rate of decline of the ER 45Ca2+-store content as a function of time.


E ) of mean 45Ca2 + - store content at beginning of measurement ( t0 ) ( n = 5 ) .

O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O

E) Quantification of the mean 45Ca2+-store content at the beginning of the measurement (t0) (n = 5).


F ) of mean slope of curve in D after transformation to a linear scale , which is a measure of 45Ca2 + - leak rate ( n = 5 ) . * p < 0 . 05 ; * * p < 0 . 01 , paired Student ' s t - .

O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F) Quantification of the mean slope of the curve in D after transformation to a linear scale, which is a measure of the 45Ca2+-leak rate (n = 5). * p<0.05; ** p<0.01, paired Student's t-test.


A ) Representative Western - blot for Atg12 ( showing autophagic Atg12 - Atg5 ) , GAPDH LC3 of lysates obtained from MEF pretreated with ( + Dox ) or without ( - Dox ) doxycycline treated with DMSO or 0 . 1 , 1 or 5 µM rapamycin ( Rapa ) for 5 h ( n = 3 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD O O O O B-CELL_TYPE O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O

A) Representative Western-blot analysis for Atg12 (showing the autophagic Atg12-Atg5 complex), GAPDH and LC3 of protein lysates obtained from MEF cells pretreated with (+Dox) or without (-Dox) doxycycline and treated with DMSO or 0.1, 1 or 5 µM rapamycin (Rapa) for 5 h (n = 3).


B - C ) Representative measurements of Ca2 + signals , displayed as Fura2 ( F340 / F380 ) , showing effect of 1 mM ATP ( B ) or 10 µM ionomycin ( Iono ) in intact MEF pretreated with or without doxycycline treated with different concentrations of rapamycin for 5 h . Prior to addition of ATP or Iono , EGTA ( 3 mM ) was added to chelate extracellular Ca2 + as indicated .

O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-CELL_TYPE O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O

B-C) Representative measurements of cytosolic Ca2+ signals, displayed as Fura2 ratio (F340/F380), showing the effect of 1 mM ATP (B) or 10 µM ionomycin (Iono) in intact MEF cells pretreated with or without doxycycline and treated with different concentrations of rapamycin for 5 h. Prior to the addition of ATP or Iono, EGTA (3 mM) was added to chelate the extracellular Ca2+ as indicated.