( d - f ) The percentage of EGFP - positive cells with EGFP - HDQ74 aggregates ( above ) and cell death ( below ) in COS - 7 cells , as in Figure 1c - treated with DMSO ( control ) , or with 0 . 2 μM rapamycin alone , SMER alone ( 140 μM SMER10 ( d ) , 43 μM SMER18 ( e ) or 47 μM SMER28 ( f ) ) or both for 24 h - were expressed as odds ratios . Error bars : 95 % confidence interval . Aggregation ( top panels ) : P = 0 . 248 ( Rap ) , P = 0 . 217 ( SMER10 ) , P 0 . 0001 ( SMER10 + Rap ) , P 0 . 001 ( Rap or SMER10 versus SMER10 + Rap ) ( d ) ; P = 0 . 248 ( Rap ) , P = 0 . 543 ( SMER18 ) , P 0 . 0001 ( SMER18 + Rap ) , P = 0 . 008 ( Rap versus SMER18 + Rap ) , P = 0 . 002 ( SMER18 versus SMER18 + Rap ) ( e ) ; P = 0 . 248 ( Rap ) , P = 0 . 002 ( SMER28 ) , P 0 . 0001 ( SMER28 + Rap ) , P 0 . 0001 ( Rap versus SMER28 + Rap ) , P = 0 . 012 ( SMER28 versus SMER28 + Rap ) ( f ) . Cell death ( bottom panels ) : P = 0 . 002 ( Rap ) , P 0 . 0001 ( SMER10 , SMER10 + Rap , Rap or SMER10 versus SMER10 + Rap ) ( d ) ; P = 0 . 002 ( Rap ) , P = 0 . 948 ( SMER18 ) , P 0 . 0001 ( SMER18 + Rap ) , P = 0 . 015 ( Rap versus SMER18 + Rap ) , P 0 . 0001 ( SMER18 versus SMER18 + Rap ) ( e ) ; P = 0 . 002 ( Rap ) , P 0 . 0001 ( SMER28 , SMER28 + Rap , Rap or SMER28 versus SMER28 + Rap ) ( f ) . Note that we have treated cells for a shorter time in this experiment ( 24 h ) , compared with Figure 1c ( 48 h ) . This probably accounts for the failure of the protective trends of rapamycin and some of the SMERs to reach significance for aggregation on their own in some of the experiments . * * * P 0 . 001 ; * * P 0 . 01 ; * P 0 . 05 ; NS , nonsignificant .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O 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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(d-f) The percentage of EGFP-positive cells with EGFP-HDQ74 aggregates (above) and cell death (below) in COS-7 cells, as in Figure 1c-treated with DMSO (control), or with 0.2 μM rapamycin alone, SMER alone (140 μM SMER10 (d), 43 μM SMER18 (e) or 47 μM SMER28 (f)) or both for 24 h-were expressed as odds ratios. Error bars: 95% confidence interval. Aggregation (top panels): P = 0.248 (Rap), P = 0.217 (SMER10), P 0.0001 (SMER10 + Rap), P 0.001 (Rap or SMER10 versus SMER10 + Rap) (d); P = 0.248 (Rap), P = 0.543 (SMER18), P 0.0001 (SMER18 + Rap), P = 0.008 (Rap versus SMER18 + Rap), P = 0.002 (SMER18 versus SMER18 + Rap) (e); P = 0.248 (Rap), P = 0.002 (SMER28), P 0.0001 (SMER28 + Rap), P 0.0001 (Rap versus SMER28 + Rap), P = 0.012 (SMER28 versus SMER28 + Rap) (f). Cell death (bottom panels): P = 0.002 (Rap), P 0.0001 (SMER10, SMER10 + Rap, Rap or SMER10 versus SMER10 + Rap) (d); P = 0.002 (Rap), P = 0.948 (SMER18), P 0.0001 (SMER18 + Rap), P = 0.015 (Rap versus SMER18 + Rap), P 0.0001 (SMER18 versus SMER18 + Rap) (e); P = 0.002 (Rap), P 0.0001 (SMER28, SMER28 + Rap, Rap or SMER28 versus SMER28 + Rap) (f). Note that we have treated cells for a shorter time in this experiment (24 h), compared with Figure 1c (48 h). This probably accounts for the failure of the protective trends of rapamycin and some of the SMERs to reach significance for aggregation on their own in some of the experiments. ***P 0.001; **P 0.01; *P 0.05; NS, nonsignificant.


( a - c ) Clearance of A53T α - synuclein in stable PC12 cells as in Figure 1b - treated for 24 h with either DMSO ( control ) , or with 47 μM SMER10 and its analogs ( SMER10a - c ) ( a ) , 43 μM SMER18 and its analogs ( SMER18a - l ) ( b ) , or 47 μM SMER28 and its analogs ( SMER28a - k ) ( c ) - was analyzed by immunoblotting with anti - HA antibody ( above ) and densitometry analysis relative to actin ( below ) . All the analogs were used in the cell culture medium at 1 : 400 dilution of 5 mg ml − 1 stock solution ( in DMSO ) . Error bars denote s . e . m . P = 0 . 0058 ( SMER10a ) , P = 0 . 6736 ( SMER10b ) , P = 0 . 9507 ( SMER10c ) , P = 0 . 0481 ( SMER10 ) ( a ) ; P = 0 . 0006 ( SMER18a ) , P = 0 . 0249 ( SMER18b ) , P = 0 . 0167 ( SMER18c ) , P = 0 . 0117 ( SMER18d ) , P = 0 . 0269 ( SMER18e ) , P = 0 . 0165 ( SMER18f ) , P = 0 . 0148 ( SMER18g ) , P = 0 . 0011 ( SMER18h ) , P = 0 . 7369 ( SMER18i ) , P = 0 . 0012 ( SMER18j ) , P = 0 . 1531 ( SMER18k ) , P = 0 . 0006 ( SMER18l ) , P = 0 . 0001 ( SMER18 ) ( b ) ; P = 0 . 0014 ( SMER28a ) , P = 0 . 0002 ( SMER28b ) , P = 0 . 0001 ( SMER28d ) , P = 0 . 0048 ( SMER28h ) , P = 0 . 0002 ( SMER28i ) , P = 0 . 0162 ( SMER28j ) , P 0 . 0001 ( SMER28c , SMER28e - g , SMER28k , SMER28 ) ( c ) .

O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O

(a-c) Clearance of A53T α-synuclein in stable PC12 cells as in Figure 1b-treated for 24 h with either DMSO (control), or with 47 μM SMER10 and its analogs (SMER10a-c) (a), 43 μM SMER18 and its analogs (SMER18a-l) (b), or 47 μM SMER28 and its analogs (SMER28a-k) (c)-was analyzed by immunoblotting with anti-HA antibody (above) and densitometry analysis relative to actin (below). All the analogs were used in the cell culture medium at 1:400 dilution of 5 mg ml−1 stock solution (in DMSO). Error bars denote s.e.m. P = 0.0058 (SMER10a), P = 0.6736 (SMER10b), P = 0.9507 (SMER10c), P = 0.0481 (SMER10) (a); P = 0.0006 (SMER18a), P = 0.0249 (SMER18b), P = 0.0167 (SMER18c), P = 0.0117 (SMER18d), P = 0.0269 (SMER18e), P = 0.0165 (SMER18f), P = 0.0148 (SMER18g), P = 0.0011 (SMER18h), P = 0.7369 (SMER18i), P = 0.0012 (SMER18j), P = 0.1531 (SMER18k), P = 0.0006 (SMER18l), P = 0.0001 (SMER18) (b); P = 0.0014 (SMER28a), P = 0.0002 (SMER28b), P = 0.0001 (SMER28d), P = 0.0048 (SMER28h), P = 0.0002 (SMER28i), P = 0.0162 (SMER28j), P 0.0001 (SMER28c, SMER28e-g, SMER28k, SMER28) (c).


( d - f ) The percentage of EGFP - positive cells with EGFP - HDQ74 aggregates in COS - 7 cells as in Figure 1c - treated for 48 h with either DMSO ( control ) , or with 47 μM SMER10 and its analog ( SMER10a ) ( d ) , 43 μM SMER18 and its analogs ( SMER18a , SMER18c - h , SMER18j , SMER18l ) ( e ) , or 47 μM SMER28 and its analogs ( SMER28a - k ) ( f ) - were expressed as odds ratios . All the analogs were used in the cell culture medium at 1 : 400 dilution of 5 mg ml − 1 stock solution ( in DMSO ) . Error bars : 95 % confidence interval . P = 0 . 003 ( SMER10a ) , P = 0 . 004 ( SMER10 ) ( d ) ; P 0 . 0001 ( SMER18a , SMER18c , SMER18d , SMER18f , SMER18h ) , P = 0 . 009 ( SMER18e ) , P = 0 . 001 ( SMER18g ) , P = 0 . 382 ( SMER18j ) , P = 0 . 067 ( SMER18l ) , P = 0 . 031 ( SMER18 ) ( e ) ; P 0 . 0001 ( SMER28a , SMER28c , SMER28e - g , SMER28i - k , SMER28 ) , P = 0 . 574 ( SMER28b ) , P = 0 . 041 ( SMER28d ) , P = 0 . 002 ( SMER28h ) ( f ) . * * * P 0 . 001 ; * * P 0 . 01 ; * P 0 . 05 ; NS , nonsignificant .

O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(d-f) The percentage of EGFP-positive cells with EGFP-HDQ74 aggregates in COS-7 cells as in Figure 1c-treated for 48 h with either DMSO (control), or with 47 μM SMER10 and its analog (SMER10a) (d), 43 μM SMER18 and its analogs (SMER18a, SMER18c-h, SMER18j, SMER18l) (e), or 47 μM SMER28 and its analogs (SMER28a-k) (f)-were expressed as odds ratios. All the analogs were used in the cell culture medium at 1:400 dilution of 5 mg ml−1 stock solution (in DMSO). Error bars: 95% confidence interval. P = 0.003 (SMER10a), P = 0.004 (SMER10) (d); P 0.0001 (SMER18a, SMER18c, SMER18d, SMER18f, SMER18h), P = 0.009 (SMER18e), P = 0.001 (SMER18g), P = 0.382 (SMER18j), P = 0.067 (SMER18l), P = 0.031 (SMER18) (e); P 0.0001 (SMER28a, SMER28c, SMER28e-g, SMER28i-k, SMER28), P = 0.574 (SMER28b), P = 0.041 (SMER28d), P = 0.002 (SMER28h) (f). ***P 0.001; **P 0.01; *P 0.05; NS, nonsignificant.


( B ) Immunofluorescence ( IF ) validation of mintbody specificity . Mouse MC12 cells , which stably express H3K27me3 - mintbody ( sfGFP ) , are labelled with antibodies specific for H3K27me3 ( Cy5 ) and H3K9me3 ( Cy3 ) . DNA is stained with Hoechst33342 . Single confocal sections are shown . Arrows mark Xi . Scale bar = 10 μm .

O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O

(B) Immunofluorescence (IF) validation of mintbody specificity. Mouse MC12 cells, which stably express H3K27me3-mintbody (sfGFP), are labelled with antibodies specific for H3K27me3 (Cy5) and H3K9me3 (Cy3). DNA is stained with Hoechst33342. Single confocal sections are shown. Arrows mark Xi. Scale bar = 10 μm.


( D ) ESCs as in ( B ) were engineered to express H3K27me3 - mintbody ( SNAP / JF646 ) and H4K20me1 - mintbody ( mCherry ) . Cells were cultured in the absence of LIF for 2 days . Time - lapse images for 12 z - plane confocal stack were acquired every 1 h . Maximum intensity projection images are shown with elapse time ( hh : mm ) . Arrowheads mark Xi . Scale bar = 10

O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) ESCs as in (B) were engineered to express H3K27me3-mintbody (SNAP/JF646) and H4K20me1-mintbody (mCherry). Cells were cultured in the absence of LIF for 2 days. Time-lapse images for 12 z-plane confocal stack were acquired every 1 h. Maximum intensity projection images are shown with elapse time (hh:mm). Arrowheads mark Xi. Scale bar = 10 μm


( E ) H4K20me1 ( blue ) and H3K27me3 ( red ) accumulation across the Xi after 12 h of DOX treatment . The black line is a locally estimated scatterplot smoothing ( LOESS ) regression on all 10 - kb windows ( dots ) . Below each plot shown is the Xist locus ( green bar ) and Xist entry sites ( black bars ) .

O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O

(E) H4K20me1 (blue) and H3K27me3 (red) accumulation across the Xi after 12 h of DOX treatment. The black line is a locally estimated scatterplot smoothing (LOESS) regression on all 10-kb windows (dots). Below each plot shown is the Xist locus (green bar) and Xist entry sites (black bars).


( A ) Genome browser tracks showing H3K27me3 ( top ) and H4K20me1 ( bottom ) accumulation at a gene silenced rapidly ( Rnf12 ) or more slowly ( Pgk1 ) . Allele - specific tracks were overlaid ( B6 in red and Cast in blue ) . Note strong H4K20me1 bi - allelic pre - marking at gene bodies .

O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O

(A) Genome browser tracks showing H3K27me3 (top) and H4K20me1 (bottom) accumulation at a gene silenced rapidly (Rnf12) or more slowly (Pgk1). Allele-specific tracks were overlaid (B6 in red and Cast in blue). Note strong H4K20me1 bi-allelic pre-marking at gene bodies.


( B ) Average H3K27me3 ( top ) and H4K20me1 ( bottom ) enrichment at the B6 allele over initially active or inactive genes ± 30kb at the X chromosome . Shown is data for all time points . Data information : TSS : transcription start site ; TES : transcript end site .

O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O 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) Average H3K27me3 (top) and H4K20me1 (bottom) enrichment at the B6 allele over initially active or inactive genes ± 30kb at the X chromosome. Shown is data for all time points. Data information: TSS : transcription start site ; TES : transcript end site.


( B ) Representative image of IF / RNA FISH for Xist RNA and H4K20me1 in cells expressing XistFL or XistΔBC . Arrowheads point to the Xist RNA domains . Scale bar = 5 μm .

O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O

(B) Representative image of IF/RNA FISH for Xist RNA and H4K20me1 in cells expressing XistFL or XistΔBC. Arrowheads point to the Xist RNA domains. Scale bar = 5 μm.


( C ) Graph represents the mean % + / - StDev of Xist RNA domains enriched for H4K20me1 ( left ) or H3K27me3 ( right ) in cells expressing XistWT or XistΔBC . Shown are averages from at least 2 independent experiments ; minimum of 50 Xist RNA domains were counted per experiment ; only P - values corresponding to significant differences from unpaired Student ' s t - test comparing mutants to Xist FL are indicated as * ( P - value < 0 . 05 ) . Data for H3K27me3 was extracted from published data ( Bousard et al . , 2019 ) .

O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O

(C) Graph represents the mean % +/- StDev of Xist RNA domains enriched for H4K20me1 (left) or H3K27me3 (right) in cells expressing XistWT or XistΔBC. Shown are averages from at least 2 independent experiments; minimum of 50 Xist RNA domains were counted per experiment; only P-values corresponding to significant differences from unpaired Student's t-test comparing mutants to Xist FL are indicated as * (P-value < 0.05). Data for H3K27me3 was extracted from published data (Bousard et al., 2019).


( A ) Morphological phenotypes of Wild type ( WT ) , mkk1 mkk2 and summ3 - 1 mkk1 mkk2 plants . Photos were taken on three - week - old soil - grown plants .

O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O

(A) Morphological phenotypes of Wild type (WT), mkk1 mkk2 and summ3-1 mkk1 mkk2 plants. Photos were taken on three-week-old soil-grown plants.


( B ) Trypan Blue staining of cell death in the indicated genotypes .

(B) Trypan Blue staining of cell death in the indicated genotypes.


( C ) DAB staining of H2O2 accumulation in the indicated genotypes .

(C) DAB staining of H2O2 accumulation in the indicated genotypes.


( D - E ) Expression of PR1 ( D ) and PR2 ( E ) in the indicated genotypes as determined by quantitative RT - PCR . Values were normalized to the expression levels of ACTIN1 . The data are shown as mean ± SD ( n = 3 ) with one - way ANOVA analysis and Tukey test ( P < 0 . 01 ) . Different letters indicate significant differences . The experiments were repeated three times with similar results .

O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D-E) Expression of PR1 (D) and PR2 (E) in the indicated genotypes as determined by quantitative RT-PCR. Values were normalized to the expression levels of ACTIN1. The data are shown as mean ± SD (n=3) with one-way ANOVA analysis and Tukey test (P < 0.01). Different letters indicate significant differences. The experiments were repeated three times with similar results.


( A ) Morphological phenotypes of WT , summ3 - 17 , mekk1 - 1 and summ3 - 17 mekk1 - 1 plants grown on soil for three weeks .

O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O

(A) Morphological phenotypes of WT, summ3-17, mekk1-1 and summ3-17 mekk1-1 plants grown on soil for three weeks.


( B - C ) Expression levels of PR1 ( B ) and PR2 ( C ) in the indicated genotypes as determined by quantitative RT - PCR . Values were normalized to the expression levels of ACTIN1 . The data are shown as mean ± SD ( n = 3 ) with one - way ANOVA analysis and Tukey test ( P < 0 . 01 ) . Different letters indicate significant differences . The experiments were repeated three times with similar results . Two - week - old seedlings grown on half MS plates were used for assays in ( B ) - ( C ) .

O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C) Expression levels of PR1 (B) and PR2 (C) in the indicated genotypes as determined by quantitative RT-PCR. Values were normalized to the expression levels of ACTIN1. The data are shown as mean ± SD (n=3) with one-way ANOVA analysis and Tukey test (P < 0.01). Different letters indicate significant differences. The experiments were repeated three times with similar results. Two-week-old seedlings grown on half MS plates were used for assays in (B)-(C).


( D ) Morphological phenotypes of WT , mpk4 - 3 , summ3 - 1 mpk4 - 3 and summ3 - 2 mpk4 - 3 plants . Photos were taken on three - week - old soil grown plants .

O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

(D) Morphological phenotypes of WT, mpk4-3, summ3-1 mpk4-3 and summ3-2 mpk4-3 plants. Photos were taken on three-week-old soil grown plants.


( E - F ) Expression levels of PR1 ( E ) and PR2 ( F ) in the indicated genotypes as determined by quantitative RT - PCR . Values were normalized to the expression levels of ACTIN1 . The data are shown as mean ± SD ( n = 3 ) with one - way ANOVA analysis and Tukey test ( P < 0 . 01 ) . Different letters indicate significant differences . The experiments were repeated three times with similar results . Two - week - old seedlings grown on half MS plates were used for assays in ( E ) - ( F ) .

(E-F) Expression levels of PR1 (E) and PR2 (F) in the indicated genotypes as determined by quantitative RT-PCR. Values were normalized to the expression levels of ACTIN1. The data are shown as mean ± SD (n=3) with one-way ANOVA analysis and Tukey test (P < 0.01). Different letters indicate significant differences. The experiments were repeated three times with similar results. Two-week-old seedlings grown on half MS plates were used for assays in (E)-(F).


( A ) Morphological phenotypes of transgenic plants over - expressing MEKK2 - FLAG protein in WT and summ3 - 17 background . Line 1 and 2 express MEKK2 - FLAG in WT background . Line 3 - 5 express MEKK2 - FLAG in the summ3 - 17 mutant background . The photograph shows four - week - old soil grown plants . Expression of MEKK2 - FLAG was detected by western blot using an anti - FLAG antibody .

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(A) Morphological phenotypes of transgenic plants over-expressing MEKK2-FLAG protein in WT and summ3-17 background. Line 1 and 2 express MEKK2-FLAG in WT background. Line 3-5 express MEKK2-FLAG in the summ3-17 mutant background. The photograph shows four-week-old soil grown plants. Expression of MEKK2-FLAG was detected by western blot using an anti-FLAG antibody.


( B - C ) Trypan Blue staining of cell death ( B ) in the indicated genotypes .

(B-C) Trypan Blue staining of cell death (B) in the indicated genotypes.


( B - C ) DAB staining of H2O2 accumulation ( C ) in the indicated genotypes .

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(B-C) DAB staining of H2O2 accumulation (C) in the indicated genotypes.


( D - E ) Expression levels of PR1 ( D ) and PR2 ( E ) in the indicated genotypes as determined by quantitative RT - PCR . Values were normalized to the expression levels of ACTIN1 . The data are shown as mean ± SD ( n = 3 ) with one - way ANOVA analysis and Tukey test ( P < 0 . 01 ) . Different letters indicate significant differences . The experiments were repeated three times with similar results .

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(D-E) Expression levels of PR1 (D) and PR2 (E) in the indicated genotypes as determined by quantitative RT-PCR. Values were normalized to the expression levels of ACTIN1. The data are shown as mean ± SD (n=3) with one-way ANOVA analysis and Tukey test (P < 0.01). Different letters indicate significant differences. The experiments were repeated three times with similar results.


( A ) Quantification of luciferase activity in N . benthamiana leaves expressing CRCK3NLuc and MPK4CLuc or MPK6CLuc . Data were collected three days after infiltration . The data are shown as mean ± SD ( n = 8 ) with one - way ANOVA analysis and Tukey test ( P < 0 . 01 ) . Different letters indicate significant differences . The experiments were repeated twice with similar results .

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(A) Quantification of luciferase activity in N. benthamiana leaves expressing CRCK3NLuc and MPK4CLuc or MPK6CLuc. Data were collected three days after infiltration. The data are shown as mean ± SD (n=8) with one-way ANOVA analysis and Tukey test (P < 0.01). Different letters indicate significant differences. The experiments were repeated twice with similar results.


( B ) Phosphorylation of CRCK3G390R by MPK4 . MPK4 was immunoprecipitated from wild type or MPK4 : : MPK4 - FLAG . After incubation with [ γ - 32P ] ATP and the immunoprecipitated MPK4 - FLAG protein in protein kinase buffer , CRCK3G390R was separated by SDS - PAGE . The autoradiograph of the gel is shown in the top panel , and immunoblot analysis of MPK4 - FLAG levels is shown in the bottom panel . Myelin basic protein ( MBP ) was used as a positive control . This experiment was repeated three times with similar results .

O O O O O B-MEASURED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O 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) Phosphorylation of CRCK3G390R by MPK4. MPK4 was immunoprecipitated from wild type or MPK4::MPK4-FLAG. After incubation with [γ-32P] ATP and the immunoprecipitated MPK4-FLAG protein in protein kinase buffer, CRCK3G390R was separated by SDS-PAGE. The autoradiograph of the gel is shown in the top panel, and immunoblot analysis of MPK4-FLAG levels is shown in the bottom panel. Myelin basic protein (MBP) was used as a positive control. This experiment was repeated three times with similar results.


( C ) Western blot analysis of CRCK3 - FLAG proteins in wild type and mpk4 - 3 . Total protein was extracted from two - week - old CRCK3 - FLAG transgenic plants in WT and mpk4 background grown on ½ MS medium plates . The protein extracts were treated with or without λ - PMPase phosphatase . The proteins were separated by Phos - tagTM acrylamide gel electrophoresis and CRCK - FLAG proteins were detected by western blot analysis using an anti - FLAG antibody . Similar results were obtained in two independent experiments . pCRCK3 , phosphorylated CRCK3 - FLAG protein .

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(C) Western blot analysis of CRCK3-FLAG proteins in wild type and mpk4-3. Total protein was extracted from two-week-old CRCK3-FLAG transgenic plants in WT and mpk4 background grown on ½ MS medium plates. The protein extracts were treated with or without λ-PMPase phosphatase. The proteins were separated by Phos-tagTM acrylamide gel electrophoresis and CRCK-FLAG proteins were detected by western blot analysis using an anti-FLAG antibody. Similar results were obtained in two independent experiments. pCRCK3, phosphorylated CRCK3-FLAG protein.


( D ) Western blot analysis of CRCK3 - FLAG and CRCK35S - 5A - FLAG proteins in transgenic plants of wild type background . Total protein was extracted from two - week - old plants grown on ½ MS medium plates . The protein extracts were treated with or without λ - PMPase phosphatase . The proteins were separated by Phos - tagTM acrylamide gel electrophoresis and the FLAG - tagged proteins were detected by western blot analysis using an anti - FLAG antibody . Similar results were obtained in two independent experiments . 5S - 5A stands for a change in five amino acids ( S172A , S181A , S188A , S195A , S202A ) .

O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Western blot analysis of CRCK3-FLAG and CRCK35S-5A-FLAG proteins in transgenic plants of wild type background. Total protein was extracted from two-week-old plants grown on ½ MS medium plates. The protein extracts were treated with or without λ-PMPase phosphatase. The proteins were separated by Phos-tagTM acrylamide gel electrophoresis and the FLAG-tagged proteins were detected by western blot analysis using an anti-FLAG antibody. Similar results were obtained in two independent experiments. 5S-5A stands for a change in five amino acids (S172A, S181A, S188A, S195A, S202A).


( A ) In vivo pull - down of HA - tagged SUMM2 by FLAG - tagged CRCK3 . FLAG - tagged full - length or truncated CRCK3 and HA - tagged SUMM2 proteins were transiently expressed in N . bethamiana by agro - infiltration . FLAG - tagged CRCK3 proteins were immunoprecipitated with anti - FLAG conjugated beads ( Sigma ) and detected by Western blot using anti - FLAG antibody . HA - tagged SUMM2 protein co - immunoprecipitated with FLAG - tagged CRCK3 was detected by Western blot using anti - HA antibody . Similar results were obtained in two independent experiments .

O O O O O O O O O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O 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-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O

(A) In vivo pull-down of HA-tagged SUMM2 by FLAG-tagged CRCK3.FLAG-tagged full-length or truncated CRCK3 and HA-tagged SUMM2 proteins were transiently expressed in N. bethamiana by agro-infiltration. FLAG-tagged CRCK3 proteins were immunoprecipitated with anti-FLAG conjugated beads (Sigma) and detected by Western blot using anti-FLAG antibody. HA-tagged SUMM2 protein co-immunoprecipitated with FLAG-tagged CRCK3 was detected by Western blot using anti-HA antibody. Similar results were obtained in two independent experiments.


( B ) In vivo pull - down of HA - tagged SUMM2 by FLAG - tagged CRCK3 kinase domain . KD represents a truncation of SUMM3 with only the kinase domain ( amino acid 201 - 510 ) . FLAG - tagged full - length or truncated CRCK3 and HA - tagged SUMM2 proteins were transiently expressed in N . bethamiana by agro - infiltration . FLAG - tagged CRCK3 proteins were immunoprecipitated with anti - FLAG conjugated beads ( Sigma ) and detected by Western blot using anti - FLAG antibody . HA - tagged SUMM2 protein co - immunoprecipitated with FLAG - tagged CRCK3 was detected by Western blot using anti - HA antibody . Similar results were obtained in two independent experiments .

O O O O O O O O O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O 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-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O

(B) In vivo pull-down of HA-tagged SUMM2 by FLAG-tagged CRCK3 kinase domain. KD represents a truncation of SUMM3 with only the kinase domain (amino acid 201-510).FLAG-tagged full-length or truncated CRCK3 and HA-tagged SUMM2 proteins were transiently expressed in N. bethamiana by agro-infiltration. FLAG-tagged CRCK3 proteins were immunoprecipitated with anti-FLAG conjugated beads (Sigma) and detected by Western blot using anti-FLAG antibody. HA-tagged SUMM2 protein co-immunoprecipitated with FLAG-tagged CRCK3 was detected by Western blot using anti-HA antibody. Similar results were obtained in two independent experiments.


RT - qPCR of Abca1 transcripts following 1 μM T0901317 stimulation of LXR + / + and LXR − / − primary BMMs . Fold changes are shown relative to LXR + / + 0 h . Error bars represent ± SEM for n = 4 - 8 ( * * P = 0 . 001 at 8 h and * * P = 0 . 00004 at 16 h versus LXR − / − ; * * P = 0 . 0003 at 0 h versus LXR + / + ) ; bottom , proposed model explaining the ligand ‐ dependent induction of Abca1 . Following ligand stimulation , LXR - corepressor complexes are exchanged for LXR - coactivator complexes , which promote gene expression . The LXR - cofactor interactions responsible for the attenuation of expression beginning after 16 h remain unknown .

O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O

RT-qPCR of Abca1 transcripts following 1 μM T0901317 stimulation of LXR+/+ and LXR−/− primary BMMs. Fold changes are shown relative to LXR+/+ 0 h. Error bars represent ± SEM for n = 4-8 (**P = 0.001 at 8 h and **P = 0.00004 at 16 h versus LXR−/−; **P = 0.0003 at 0 h versus LXR+/+); bottom, proposed model explaining the ligand‐dependent induction of Abca1. Following ligand stimulation, LXR-corepressor complexes are exchanged for LXR-coactivator complexes, which promote gene expression. The LXR-cofactor interactions responsible for the attenuation of expression beginning after 16 h remain unknown.


Luciferase reporter assays from RAW 264 . 7 macrophages expressing reporter alone , or together with full ‐ length SND1 , SART1 , or HMBOX1 . LXR ligand stimulations with 1 μM T0901317 were performed for 18 h . A diagram of the reporter construct is shown above . Fold changes are shown relative to vehicle ‐ stimulated reporter alone ( first lane ) . Error bars represent ± SEM for n = 9 ( * * P = 0 . 001 , * P = 0 . 011 for SND1 , * P = 0 . 046 for SART1 ) .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O

Luciferase reporter assays from RAW 264.7 macrophages expressing reporter alone, or together with full‐length SND1, SART1, or HMBOX1. LXR ligand stimulations with 1 μM T0901317 were performed for 18 h. A diagram of the reporter construct is shown above. Fold changes are shown relative to vehicle‐stimulated reporter alone (first lane). Error bars represent ± SEM for n = 9 (**P = 0.001, *P = 0.011 for SND1, *P = 0.046 for SART1).


Luciferase reporter assays from RAW 264 . 7 macrophages expressing reporter alone , or together with full ‐ length NCOA5 or an NCOA5 mutant lacking the NH2 ‐ terminus . LXR ligand stimulations with 1 μM T0901317 were performed for 18 h . Diagrams of the reporter constructs are shown above . Fold changes are shown relative to vehicle ‐ stimulated reporter alone ( first lane ) . Error bars represent ± SEM for n = 6 - 12 ( * * P 0 . 001 , * P = 0 . 035 ) .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Luciferase reporter assays from RAW 264.7 macrophages expressing reporter alone, or together with full‐length NCOA5 or an NCOA5 mutant lacking the NH2‐terminus. LXR ligand stimulations with 1 μM T0901317 were performed for 18 h. Diagrams of the reporter constructs are shown above. Fold changes are shown relative to vehicle‐stimulated reporter alone (first lane). Error bars represent ± SEM for n = 6-12 (**P 0.001, *P = 0.035).


RT - qPCR of Abca1 transcripts following infection of primary BMMs with Ncoa5 or control retrovirus . LXR ligand stimulations were performed with 1 μM T0901317 for 18 h . Fold changes are shown relative to vehicle ‐ stimulated control . Error bars represent ± SEM for n = 4 ( * P = 0 . 029 ) .

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RT-qPCR of Abca1 transcripts following infection of primary BMMs with Ncoa5 or control retrovirus. LXR ligand stimulations were performed with 1 μM T0901317 for 18 h. Fold changes are shown relative to vehicle‐stimulated control. Error bars represent ± SEM for n = 4 (*P = 0.029).


In vitro pulldown assays using recombinant GST ‐ LXRα to isolate the indicated in vitro translated NCOA5 constructs . Assays were performed for 2 h in the presence or absence of 2 μM T0901317 . Samples were immunoblotted as indicated . Note the requirement of the NCOA5 NH2 ‐ terminus for interacting with LXRα .

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In vitro pulldown assays using recombinant GST‐LXRα to isolate the indicated in vitro translated NCOA5 constructs. Assays were performed for 2 h in the presence or absence of 2 μM T0901317. Samples were immunoblotted as indicated. Note the requirement of the NCOA5 NH2‐terminus for interacting with LXRα.


Immunoprecipitation of Protein C ‐ tagged LXRα from stable RAW 264 . 7macrophagenuclear extracts stimulated with 1 μM T0901317 or vehicle control for 18 h , followed by immunoblotting for endogenous NCOA5 or over ‐ expressed LXRα . Protein immunoblots of nuclear extracts are shown below .

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Immunoprecipitation of Protein C‐tagged LXRα from stable RAW 264.7macrophagenuclear extracts stimulated with 1 μM T0901317 or vehicle control for 18 h, followed by immunoblotting for endogenous NCOA5 or over‐expressed LXRα. Protein immunoblots of nuclear extracts are shown below.


NCOA5 ChIP time course from primary BMMs stimulated with 1 μM T0901317 or vehicle control . qPCR was performed for the Abca1 proximal LXRE . Note the ligand ‐ stimulated association of NCOA5 . Error bars represent ± SEM for n = 4 - 9 ( * P = 0 . 02 ) .

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NCOA5 ChIP time course from primary BMMs stimulated with 1 μM T0901317 or vehicle control. qPCR was performed for the Abca1 proximal LXRE. Note the ligand‐stimulated association of NCOA5. Error bars represent ± SEM for n = 4-9 (*P = 0.02).


NCOA5 ChIP assays from LXR − / − BMMs stimulated with 1 μM T0901317 or vehicle control for 18 h . qPCR was performed for the Abca1 proximal LXRE . Error bars represent ± SEM for n = 5 - 6 ( * * P = 0 . 0003 for vehicle and * * P = 0 . 002 for T0901317 ) .

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NCOA5 ChIP assays from LXR−/− BMMs stimulated with 1 μM T0901317 or vehicle control for 18 h. qPCR was performed for the Abca1 proximal LXRE. Error bars represent ± SEM for n = 5-6 (**P = 0.0003 for vehicle and **P = 0.002 for T0901317).


RT - qPCR of Abca1 expression following 1 μM T0901317 treatment in primary BMMs infected with a non ‐ silencing or Ncoa5 ‐ specific shRNA . Fold changes are shown relative to shControl 0 h . Note the elevated Abca1 expression at 24 - 30 h following loss of NCOA5 . Error bars represent ± SEM for n = 4 - 6 ( * * P = 0 . 0007 at 24 h and * * P = 0 . 004 at 30 h , * P = 0 . 02 ) .

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RT-qPCR of Abca1 expression following 1 μM T0901317 treatment in primary BMMs infected with a non‐silencing or Ncoa5‐specific shRNA. Fold changes are shown relative to shControl 0 h. Note the elevated Abca1 expression at 24-30 h following loss of NCOA5. Error bars represent ± SEM for n = 4-6 (**P = 0.0007 at 24 h and **P = 0.004 at 30 h, *P = 0.02).


Quantification of ABCA1 immunoblots from primary BMMs infected as in ( A ) . Representative immunoblot is shown in Supplementary Fig S7E . LXR ligand stimulation with 1 μM T0901317 or vehicle control was performed for 8 h or 30 h . Error bars represent ± SEM for n = 2 ( * * P = 0 . 007 ) .

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Quantification of ABCA1 immunoblots from primary BMMs infected as in (A). Representative immunoblot is shown in Supplementary Fig S7E. LXR ligand stimulation with 1 μM T0901317 or vehicle control was performed for 8 h or 30 h. Error bars represent ± SEM for n = 2 (**P = 0.007).


A , B RT - qPCR of Abca1 expression from primary BMMs infected with non ‐ silencing or Ncoa5 ‐ specific shRNAs . Ligand stimulations were performed for 4 h with vehicle control , 1 μM T0901317 alone or together with 6 μg / ml PolyIC ( A ) or 10 ng / ml LPS ( B ) . Fold changes are shown relative to vehicle ‐ stimulated shControl . Note only the loss of TLR3 ‐ mediated repression following Ncoa5 silencing . Error bars represent ± SEM for n = 4 - 10 ( * * P = 0 . 0003 for A , * * P = 0 . 0004 for shControl in B , * * P = 0 . 0002 for shNcoa5 in B versus T0901317 ) .

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A, B RT-qPCR of Abca1 expression from primary BMMs infected with non‐silencing or Ncoa5‐specific shRNAs. Ligand stimulations were performed for 4 h with vehicle control, 1 μM T0901317 alone or together with 6 μg/ml PolyIC (A) or 10 ng/ml LPS (B). Fold changes are shown relative to vehicle‐stimulated shControl. Note only the loss of TLR3‐mediated repression following Ncoa5 silencing. Error bars represent ± SEM for n = 4-10 (**P = 0.0003 for A, **P = 0.0004 for shControl in B, **P = 0.0002 for shNcoa5 in B versus T0901317).


C , D RT - qPCR of Abca1 expression from primary BMMs infected with non ‐ silencing or Ncoa5 ‐ specific shRNAs . Ligand stimulations were performed for 4 h unstimulated , or with 6 μg / ml PolyIC ( C ) or 10 ng / ml LPS ( D ) . Fold changes are shown relative to unstimulated shControl . Error bars represent ± SEM for n = 4 - 6 ( * * P = 0 . 0006 for C , * * P = 0 . 00000001 for shControl in D , * * P = 0 . 000001 for shNcoa5 in D , * P = 0 . 013 versus T0901317 ) .

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C, D RT-qPCR of Abca1 expression from primary BMMs infected with non‐silencing or Ncoa5‐specific shRNAs. Ligand stimulations were performed for 4 h unstimulated, or with 6 μg/ml PolyIC (C) or 10 ng/ml LPS (D). Fold changes are shown relative to unstimulated shControl. Error bars represent ± SEM for n = 4-6 (**P = 0.0006 for C, **P = 0.00000001 for shControl in D, **P = 0.000001 for shNcoa5 in D, *P = 0.013 versus T0901317).


E NCOA5 ChIP assays from primary BMMs stimulated for 3 h with 1 μM T0901317 , 1 μM T0901317 + 6 μg / ml PolyIC , or vehicle control . qPCR was performed for the Abca1 proximal LXRE . Note the increased occupancy only in the presence of both lipid and inflammatory ligands . Error bars represent ± SEM for n = 8 - 12 ( * * P = 0 . 0007 versus T0901317 ) .

O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O

E NCOA5 ChIP assays from primary BMMs stimulated for 3 h with 1 μM T0901317, 1 μM T0901317 + 6 μg/ml PolyIC, or vehicle control. qPCR was performed for the Abca1 proximal LXRE. Note the increased occupancy only in the presence of both lipid and inflammatory ligands. Error bars represent ± SEM for n = 8-12 (**P = 0.0007 versus T0901317).


F NCOA5 ChIP assays from primary BMMs stimulated for 3 h with 6 μg / ml PolyIC or left unstimulated . qPCR was performed for the Abca1 proximal LXRE . Error bars represent ± SEM for n = 5 - 6 .

O B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O

F NCOA5 ChIP assays from primary BMMs stimulated for 3 h with 6 μg/ml PolyIC or left unstimulated. qPCR was performed for the Abca1 proximal LXRE. Error bars represent ± SEM for n = 5-6.


G Cholesterol efflux assays to APOA1 from primary BMMs infected with non ‐ silencing or Ncoa5 ‐ specific shRNAs . Agonist stimulations were performed for 6 h . Error bars represent ± SEM for n = 3 ( * P = 0 . 02 versus T0901317 ) .

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G Cholesterol efflux assays to APOA1 from primary BMMs infected with non‐silencing or Ncoa5‐specific shRNAs. Agonist stimulations were performed for 6 h. Error bars represent ± SEM for n = 3 (*P = 0.02 versus T0901317).


A , B Unmodified / pSer5 RNAPII ( A ) or RNAPII pSer2 ( B ) ChIP assays from primary BMMs stimulated for 4 h with 1 μM T0901317 , 1 μM T0901317 + 6 μg / ml PolyIC , or vehicle control . qPCR was performed for the Abca1 TSS . Error bars represent ± SEM for n = 6 - 9 ( * * P = 0 . 0001 in A , * * P = 0 . 002 in B versus T0901317 ) .

O O O O 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-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O 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-CONTROLLED_VAR O O

A, B Unmodified/pSer5 RNAPII (A) or RNAPII pSer2 (B) ChIP assays from primary BMMs stimulated for 4 h with 1 μM T0901317, 1 μM T0901317 + 6 μg/ml PolyIC, or vehicle control. qPCR was performed for the Abca1 TSS. Error bars represent ± SEM for n = 6-9 (**P = 0.0001 in A, **P = 0.002 in B versus T0901317).


C , D RNAPII ChIP assays as in ( A , B ) but performed from LXR − / − BMMs . Error bars represent ± SEM for n = 5 - 9 ( * * P = 0 . 000000001 , * P = 0 . 02 versus T0901317 ) .

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C, D RNAPII ChIP assays as in (A, B) but performed from LXR−/− BMMs. Error bars represent ± SEM for n = 5-9 (**P = 0.000000001, *P = 0.02 versus T0901317).


E RT - qPCR of Abca1 expression from LXR + / + versus LXR − / − BMMs . Ligand stimulations were performed for 4 h with vehicle control , 1 μM T0901317 , or 1 μM T0901317 together with 6 μg / ml PolyIC . Fold changes are shown relative to vehicle ‐ stimulated LXR + / + . Error bars represent ± SEM for n = 4 ( * * P = 0 . 004 , * P = 0 . 012 versus T0901317 ) .

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E RT-qPCR of Abca1 expression from LXR+/+ versus LXR−/− BMMs. Ligand stimulations were performed for 4 h with vehicle control, 1 μM T0901317, or 1 μM T0901317 together with 6 μg/ml PolyIC. Fold changes are shown relative to vehicle‐stimulated LXR+/+. Error bars represent ± SEM for n = 4 (**P = 0.004, *P = 0.012 versus T0901317).


F RNAPII pSer2 ChIP assays from primary BMMs infected with non ‐ silencing or Ncoa5 ‐ specific shRNAs . Ligand stimulations were performed for 4 h with vehicle control , 1 μM T0901317 , or 1 μM T0901317 + 6 μg / ml PolyIC . qPCR was performed for the Abca1 TSS . Note RNAPII pSer2 returns to baseline occupancy following TLR3 stimulation in shControl but not shNcoa5 BMMs ( * * P = 0 . 004 versus baseline ) . Error bars represent ± SEM for n = 3 from 11 mice .

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F RNAPII pSer2 ChIP assays from primary BMMs infected with non‐silencing or Ncoa5‐specific shRNAs. Ligand stimulations were performed for 4 h with vehicle control, 1 μM T0901317, or 1 μM T0901317 + 6 μg/ml PolyIC. qPCR was performed for the Abca1 TSS. Note RNAPII pSer2 returns to baseline occupancy following TLR3 stimulation in shControl but not shNcoa5 BMMs (**P = 0.004 versus baseline). Error bars represent ± SEM for n = 3 from 11 mice.


( A ) Schematic of experimental setup depicting MECs grown as monolayers on a rigid rBM ( two - dimensional culture model , 2D ; top left ) or within a compliant rBM to generate multicellular spheroid structures with apical - basal polarity ( three - dimensional culture model , 3D ) top right ) . Heatmap of microarray analysis of gene expression in HMT - 3522 S - 1 MECs cultured either as a 2D monolayer or as spheroids ( Bottom ) . Expression of the top 200 differentially expressed genes between the two experimental conditions ( n = 3 independent biological replicates ; R = replicate ) .

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(A) Schematic of experimental setup depicting MECs grown as monolayers on a rigid rBM (two-dimensional culture model, 2D; top left) or within a compliant rBM to generate multicellular spheroid structures with apical-basal polarity (three-dimensional culture model, 3D) top right). Heatmap of microarray analysis of gene expression in HMT-3522 S-1 MECs cultured either as a 2D monolayer or as spheroids (Bottom). Expression of the top 200 differentially expressed genes between the two experimental conditions (n=3 independent biological replicates; R=replicate).


( C ) Samples were stained with antibodies for β4 - integrin or laminin ( green ; yellow arrows ) . The actin and nucleus were counterstained with phalloidin ( red ) and DAPI ( blue ) , respectively . Scale bar , 10 μm .

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(C) Samples were stained with antibodies for β4-integrin or laminin (green; yellow arrows). The actin and nucleus were counterstained with phalloidin (red) and DAPI (blue), respectively. Scale bar, 10 μm.


( D ) HMT - 3522 S - 1 MECs plated as monolayers on a rigid rBM ( 2D ) or as spheroids within rBM ( 3D ) were treated with increasing doses of Paclitaxel ( Pac ) , TRAIL , Doxorubicin ( Doxo ) and irradiation ( IR ) . Percent cell death was quantified by immunofluorescence as percentage of cells stained positive for cleaved caspase - 3 at 48h post - treatment ( mean ± SEM ; n = 3 independent biological replicates ) . * * * * P < 0 . 0001 ( Student ' s t test ) .

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(D) HMT-3522 S-1 MECs plated as monolayers on a rigid rBM (2D) or as spheroids within rBM (3D) were treated with increasing doses of Paclitaxel (Pac), TRAIL, Doxorubicin (Doxo) and irradiation (IR). Percent cell death was quantified by immunofluorescence as percentage of cells stained positive for cleaved caspase-3 at 48h post-treatment (mean ± SEM; n=3 independent biological replicates). ****P < 0.0001 (Student's t test).


( F ) Representative immunofluorescence microscopy images of MCF10A MECs plated in the indicated conditions for 18h and stained with phalloidin to reveal F - actin organization . Cells were plated as single cells on rigid glass coverslips ( 2D glass ; top left ) or fully embedded within rBM ( 3D fully embedded ; bottom left ) . Cell spreading was inhibited by plating cells on either laminin - 111 conjugated , 10 - μm micropatterned glass ( 2D / micropatterned , rigid substrate ; top middle ) or on compliant 75 Pa rBM - laminated polyacrylamide ( PA ) gels ( 2D / PA , soft substrate ; top right ) . The single 2D cells were overlaid with either purified laminin - 111 ( bottom middle ) or rBM ( bottom right ) to create a 3D ECM microenvironment . Images show maximum intensity z - projections of confocal stacks for F - actin phalloidin staining . Scale bar , 10

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative immunofluorescence microscopy images of MCF10A MECs plated in the indicated conditions for 18h and stained with phalloidin to reveal F-actin organization. Cells were plated as single cells on rigid glass coverslips (2D glass; top left) or fully embedded within rBM (3D fully embedded; bottom left). Cell spreading was inhibited by plating cells on either laminin-111 conjugated, 10-μm micropatterned glass (2D/micropatterned, rigid substrate; top middle) or on compliant 75 Pa rBM-laminated polyacrylamide (PA) gels (2D/PA, soft substrate; top right). The single 2D cells were overlaid with either purified laminin-111 (bottom middle) or rBM (bottom right) to create a 3D ECM microenvironment. Images show maximum intensity z-projections of confocal stacks for F-actin phalloidin staining. Scale bar, 10 μ


( G ) Representative immunofluorescence microscopy images of MCF10A MECs stably expressing recombinant mCherry - tagged golgi marker ( mCherry - GalT ; red ; yellow arrows ) ligated with rBM in either 2D or 3D for 18h . The actin cortex and nucleus were counterstained with phalloidin ( green ) and DAPI ( blue ) , respectively . Images show the cross - sectional view of each cell compartment ( dashed lines ; xy plane ) and side view of confocal stacks ( xz plane ) in individual MCF10A MECs . Scale bar , 10 μm . ( H ) Golgi staining was assessed within non - spread MCF10A MECs ligated with rBM in 2D and 3D for 18h and values were plotted as a function of subcellular localization ( 2D , n = 8 ; 3D , n = 9 cells from two independent experiments ) .

O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Representative immunofluorescence microscopy images of MCF10A MECs stably expressing recombinant mCherry-tagged golgi marker (mCherry-GalT; red; yellow arrows) ligated with rBM in either 2D or 3D for 18h. The actin cortex and nucleus were counterstained with phalloidin (green) and DAPI (blue), respectively. Images show the cross-sectional view of each cell compartment (dashed lines; xy plane) and side view of confocal stacks (xz plane) in individual MCF10A MECs. Scale bar, 10 μm. (H) Golgi staining was assessed within non-spread MCF10A MECs ligated with rBM in 2D and 3D for 18h and values were plotted as a function of subcellular localization (2D, n=8; 3D, n=9 cells from two independent experiments).


( I ) Heatmap of RNA - seq experiment from MCF10A MECs ligated with rBM in 2D and 3D 12h post - plating . Data show the expression of the top 200 genes that are differentially expressed between the 2D and 3D rBM conditions . ( n = 3 independent biological replicates ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(I) Heatmap of RNA-seq experiment from MCF10A MECs ligated with rBM in 2D and 3D 12h post-plating. Data show the expression of the top 200 genes that are differentially expressed between the 2D and 3D rBM conditions. (n=3 independent biological replicates).


( A ) Bar graph showing qPCR quantification of SEC61B level in MCF10A MECs ligated with rBM in 2D and 3D 12h post - plating ( mean ± SEM ; n = 3 independent biological replicates ) . Statistical analysis by one - way ANOVA followed by Uncorrected Fisher ' s LSD , * * P = 0 . 0022 .

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(A) Bar graph showing qPCR quantification of SEC61B level in MCF10A MECs ligated with rBM in 2D and 3D 12h post-plating (mean ± SEM; n=3 independent biological replicates). Statistical analysis by one-way ANOVA followed by Uncorrected Fisher's LSD, **P = 0.0022.


( B ) MCF10A MECs expressing VSVGts045 - EGFP were ligated to rBM in 2D or 3D and incubated at 40 ° C for 16h to trap VSVGts045 - EGFP protein in the ER . Cells were shifted to 32 ° C for 2h to release VSVGts045 - EGFP into the secretory pathway . Representative fluorescence microscopy images of MECs expressing VSVGts045 - EGFP 2h post incubation at 32 ° C . Scale bar ( whole cell ) , 10 μm ; Scale bar ( magnified ) , 2 μm . ( C ) Scatter plot of the secretory trafficking efficiency of VSVGts045 - EGFP based on the fluorescence of VSVGts045 at the plasma membrane versus total fluorescence measured in cells from Panel B ( mean ± SEM ; 2D , n = 45 ; 3D , n = 49 cells from three independent experiments ) . * * * * P < 0 . 0001 ( Student ' s t test ) .

O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) MCF10A MECs expressing VSVGts045-EGFP were ligated to rBM in 2D or 3D and incubated at 40°C for 16h to trap VSVGts045-EGFP protein in the ER. Cells were shifted to 32°C for 2h to release VSVGts045-EGFP into the secretory pathway. Representative fluorescence microscopy images of MECs expressing VSVGts045-EGFP 2h post incubation at 32°C. Scale bar (whole cell), 10 μm; Scale bar (magnified), 2 μm. (C) Scatter plot of the secretory trafficking efficiency of VSVGts045-EGFP based on the fluorescence of VSVGts045 at the plasma membrane versus total fluorescence measured in cells from Panel B (mean ± SEM; 2D, n=45; 3D, n=49 cells from three independent experiments). ****P < 0.0001 (Student's t test).


( D ) Representative line graph showing the changes in cytosolic [ Ca2 + ] levels of MCF10A MECs under different treatment . Fura - 2 - loaded MECs ligated to 2D ( blue ) or 3D rBM ( red ) were pre - incubated with EGTA to chelate extracellular Ca2 + in the bath solution , challenged with 2μM thapsigargin to induce ER Ca2 + release , and replenished with 4 mM Ca2 + in bath solution at the indicated time . F340 / F380 values for each cell were quantified over the course of imaging ( mean ± SD ; 2D , n = 7 ; 3D , n = 7 cells from one experiment ) . ( E ) Quantification of the amplitude of the Ca2 + response induced by thapsigargin ( ∆ F = F - F0 ) , where F0 is the basal fluorescence before thapsigargin treatment ( mean ± SEM ( 2D , n = 20 ; 3D , n = 21 cells from three independent experiments ) . * * * P = 0 . 0007 ( Student ' s t test ) .

O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Representative line graph showing the changes in cytosolic [Ca2+] levels of MCF10A MECs under different treatment. Fura-2-loaded MECs ligated to 2D (blue) or 3D rBM (red) were pre-incubated with EGTA to chelate extracellular Ca2+ in the bath solution, challenged with 2μM thapsigargin to induce ER Ca2+ release, and replenished with 4 mM Ca2+ in bath solution at the indicated time. F340/F380 values for each cell were quantified over the course of imaging (mean ± SD; 2D, n=7; 3D, n=7 cells from one experiment). (E) Quantification of the amplitude of the Ca2+ response induced by thapsigargin (∆F=F-F0), where F0 is the basal fluorescence before thapsigargin treatment (mean ± SEM (2D, n=20; 3D, n=21 cells from three independent experiments). ***P = 0.0007 (Student's t test).


( F ) Bar graph showing qPCR of the relative level of ATF3 mRNA in MCF10A MECs ligated with rBM in 2D and 3D 12h post - plating ( mean ± SEM ; n = 3 independent biological replicates ) . Statistical analysis by one - way ANOVA followed by Uncorrected Fisher ' s LSD , * * P = 0 . 0037 .

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(F) Bar graph showing qPCR of the relative level of ATF3 mRNA in MCF10A MECs ligated with rBM in 2D and 3D 12h post-plating (mean ± SEM; n=3 independent biological replicates). Statistical analysis by one-way ANOVA followed by Uncorrected Fisher's LSD, **P = 0.0037.


( G ) Representative immunoblots of phosphorylated EIF2a ( pEIF2a ) , total EIF2a and alpha - tubulin in cell lysate from MCF10A MECs ligated to a rBM in 2D or 3D and corresponding quantification data ( mean ± SEM ; n = 6 independent biological replicates ) . Statistical analysis by Student ' s t test , * * P = 0 . 0049 .

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(G) Representative immunoblots of phosphorylated EIF2a (pEIF2a), total EIF2a and alpha-tubulin in cell lysate from MCF10A MECs ligated to a rBM in 2D or 3D and corresponding quantification data (mean ± SEM; n=6 independent biological replicates). Statistical analysis by Student's t test, **P = 0.0049.


( H ) Bar graph of qPCR data measuring the relative levels of CRACR2B mRNA in MCF10A MECs ligated with rBM in 2D and 3D 12h post - plating ( mean ± SEM ; n = 4 independent biological replicates ) . * P = 0 . 0371 ( Student ' s t test ) .

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(H) Bar graph of qPCR data measuring the relative levels of CRACR2B mRNA in MCF10A MECs ligated with rBM in 2D and 3D 12h post-plating (mean ± SEM; n=4 independent biological replicates). *P = 0.0371 (Student's t test).


( I ) Representative fluorescence microscopy images of the ER - plasma membrane contact site reporter GFP - MAPPER in MCF10A MECs ligated with rBM in 2D or 3D 18h post plating . Scale bar ( whole cell ) , 10 μm ; Scale bar ( magnified ) , 1 μm . ( J ) Graph of the levels of GFP - MAPPER at the plasma membrane relative total cellular GFP - MAPPER fluorescence in MCF10A MECs ligated with rBM in 2D or 3D in Panel I ( mean ± SEM ; 2D , n = 52 ; 3D , n = 57 cells from three independent experiments ) . * * P = 0 . 0032 ( Student ' s t test ) .

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(I) Representative fluorescence microscopy images of the ER-plasma membrane contact site reporter GFP-MAPPER in MCF10A MECs ligated with rBM in 2D or 3D 18h post plating. Scale bar (whole cell), 10 μm; Scale bar (magnified), 1 μm. (J) Graph of the levels of GFP-MAPPER at the plasma membrane relative total cellular GFP-MAPPER fluorescence in MCF10A MECs ligated with rBM in 2D or 3D in Panel I (mean ± SEM; 2D, n=52; 3D, n=57 cells from three independent experiments). **P = 0.0032 (Student's t test).


( A ) Representative immunofluorescence microscopy images of MCF10A MECs ligated with either a rBM in 2D or 3D for 18h and stained with antibody targeting filamin ( green ) . F - actin was counterstained with phalloidin ( red ) . Images show maximum intensity z - projections of xy confocal stacks ( left ) and middle focal plane of confocal stacks from MECs ( right ) . Yellow arrows indicate filamin aggregates in individual MECs ligated to rBM in 2D . Yellow blunt - end lines highlight the extent of filamin distribution away from the cell edge . Scale bar , 10 μm .

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(A) Representative immunofluorescence microscopy images of MCF10A MECs ligated with either a rBM in 2D or 3D for 18h and stained with antibody targeting filamin (green). F-actin was counterstained with phalloidin (red). Images show maximum intensity z-projections of xy confocal stacks (left) and middle focal plane of confocal stacks from MECs (right). Yellow arrows indicate filamin aggregates in individual MECs ligated to rBM in 2D. Yellow blunt-end lines highlight the extent of filamin distribution away from the cell edge. Scale bar, 10 μm.


( B ) The expression level of filamin in MCF10A MECs ligated to rBM in 2D and 3D for 18h was assessed via immunoblot and quantified relative to actin loading control ( mean ± SEM ; n = 5 independent biological replicates ) . * * * P = 0 . 0008 ( Student ' s t test ) .

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(B) The expression level of filamin in MCF10A MECs ligated to rBM in 2D and 3D for 18h was assessed via immunoblot and quantified relative to actin loading control (mean ± SEM; n=5 independent biological replicates). ***P = 0.0008 (Student's t test).


( C ) Representative fluorescence microscopy images of MCF10A MECs co - expressing the ER - plasma membrane contact site reporter GFP - MAPPER and shRNA targeting filamin ( FLMN ) or luciferase ( Luc ) ligated to rBM in 2D for 18h . Scale bar ( whole cell ) , 10 μm ; Scale bar ( magnified ) , 2 μm .

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(C) Representative fluorescence microscopy images of MCF10A MECs co-expressing the ER-plasma membrane contact site reporter GFP-MAPPER and shRNA targeting filamin (FLMN) or luciferase (Luc) ligated to rBM in 2D for 18h. Scale bar (whole cell), 10 μm; Scale bar (magnified), 2 μm.


( D ) Quantification of the levels of GFP - MAPPER at the plasma membrane versus total cellular fluorescence in MCF10A MECs stably expressing an shRNA targeting filamin ( FLMN ) or luciferase ( Luc ) ligated to rBM in 2D ( mean ± SEM ; 2D + shLuc , n = 38 ; 2D + shFLMN , n = 39 cells from three independent experiments ) . * * P < 0 . 0021 ( Student ' s t test ) .

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(D) Quantification of the levels of GFP-MAPPER at the plasma membrane versus total cellular fluorescence in MCF10A MECs stably expressing an shRNA targeting filamin (FLMN) or luciferase (Luc) ligated to rBM in 2D (mean ± SEM; 2D + shLuc, n=38; 2D + shFLMN , n=39 cells from three independent experiments). **P < 0.0021 (Student's t test).


( E ) Representative immunoblots of filamin , phosphorylated EIF2a ( pEIF2a ) , total EIF2a and alpha - tubulin in cell lysate from MCF10A MECs that express shRNA targeting filamin ( shFLMN ) or luciferase ( shLuc ) and ligated to a rBM in 2D or 3D for 18h and corresponding quantification data ( mean ± SEM ; n = 4 independent biological replicates ) . Statistical analysis by one - way ANOVA followed by Uncorrected Fisher ' s LSD . 2D - shLuc versus 3D - shLuc , * * * P = 0 . 0003 . 2D - shLuc versus 2D - shFLMN , * * * P = 0 . 0004 .

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(E) Representative immunoblots of filamin, phosphorylated EIF2a (pEIF2a), total EIF2a and alpha-tubulin in cell lysate from MCF10A MECs that express shRNA targeting filamin (shFLMN) or luciferase (shLuc) and ligated to a rBM in 2D or 3D for 18h and corresponding quantification data (mean ± SEM; n=4 independent biological replicates). Statistical analysis by one-way ANOVA followed by Uncorrected Fisher's LSD. 2D-shLuc versus 3D-shLuc, ***P = 0.0003. 2D-shLuc versus 2D-shFLMN, ***P = 0.0004.


( F ) Bar graph of the relative levels of ATF3 mRNA as measured by qPCR in MCF10A MECs expressing shRNA targeting filamin ( shFLMN ) or luciferase ( shLuc ) and ligated with rBM in 2D or 3D for 18h ( mean ± SEM ; n = 4 independent biological replicates ) . Statistical analysis by one - way ANOVA followed by Uncorrected Fisher ' s LSD . 2D - shLuc versus 3D - shLuc , * * * P = 0 . 0005 . 2D - shLuc versus 2D - shFLMN , * P = 0 . 0359 .

O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Bar graph of the relative levels of ATF3 mRNA as measured by qPCR in MCF10A MECs expressing shRNA targeting filamin (shFLMN) or luciferase (shLuc) and ligated with rBM in 2D or 3D for 18h (mean ± SEM; n=4 independent biological replicates). Statistical analysis by one-way ANOVA followed by Uncorrected Fisher's LSD. 2D-shLuc versus 3D-shLuc, ***P = 0.0005. 2D-shLuc versus 2D-shFLMN, *P = 0.0359.


( G ) Representative immunoblots of filamin , phosphorylated EIF2a ( pEIF2a ) , total EIF2a and alpha - tubulin in cell lysate from MCF10A MECs ligated to rBM in 3D and treated with ethanol ( mock ) or doxycycline ( FLMN ) for 18h to induce filamin expression and corresponding quantification data ( mean ± SEM ; n = 5 independent biological replicates ) . * * P = 0 . 0018 ( Student ' s t test ) .

O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O 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-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Representative immunoblots of filamin, phosphorylated EIF2a (pEIF2a), total EIF2a and alpha-tubulin in cell lysate from MCF10A MECs ligated to rBM in 3D and treated with ethanol (mock) or doxycycline (FLMN) for 18h to induce filamin expression and corresponding quantification data (mean ± SEM; n=5 independent biological replicates). **P = 0.0018 (Student's t test).


( A ) Schematic showing the principles behind Atomic Force Microscopy ( AFM ) ( left ) . A cantilever at the end of the microscope probe is deflected when it is in contact with the cell surface . Cell cortex - mediated resistance to indentation alters the path of the laser beam focused on the cantilever that is then reflected onto a photodetector to enable measurement of cellular cortical actin tension . AFM was used to measure the cortical actin tension in MCF10A MECs ligated to a laminin - 111 substrate in 2D or 3D ( right ) and treated with blebbistatin ( Bleb ) to reduce cortical actin tension , induced to overexpress filamin expression ( FLMN ) , or activated ROCK ( ROCK ) to increase cortical actin tension . MECs were indented using a 2 - µm beaded tip on the AFM cantilever and the Hertz model was used to fit each indentation curve to extract the Young ' s modulus of the cell cortex ( mean ± SEM ; 2D , n = 123 ; 3D , n = 184 ; 3D + FLMN , n = 83 ; 2D + Bleb , n = 66 ; 3D + Bleb , n = 65 ; 3D + ROCK , n = 212 ; n = AFM indentation from > 30 cells from three independent experiments ) . Statistical analysis by one - way ANOVA followed by Tukey ' s multiple comparisons test . 2D versus 3D , * * * * P < 0 . 0001 ; 3D versus 3D + FLMN , * * * * P < 0 . 0001 ; 3D versus 3D + ROCK , * * * * P < 0 . 0001 ; 2D versus 2D + Bleb , * * * P = 0 . 0004 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O

(A) Schematic showing the principles behind Atomic Force Microscopy (AFM) (left). A cantilever at the end of the microscope probe is deflected when it is in contact with the cell surface. Cell cortex-mediated resistance to indentation alters the path of the laser beam focused on the cantilever that is then reflected onto a photodetector to enable measurement of cellular cortical actin tension. AFM was used to measure the cortical actin tension in MCF10A MECs ligated to a laminin-111 substrate in 2D or 3D (right) and treated with blebbistatin (Bleb) to reduce cortical actin tension, induced to overexpress filamin expression (FLMN), or activated ROCK (ROCK) to increase cortical actin tension. MECs were indented using a 2-µm beaded tip on the AFM cantilever and the Hertz model was used to fit each indentation curve to extract the Young's modulus of the cell cortex (mean ± SEM; 2D, n=123; 3D, n=184; 3D+FLMN, n=83; 2D+Bleb, n=66; 3D + Bleb, n= 65; 3D+ROCK, n=212; n=AFM indentation from >30 cells from three independent experiments). Statistical analysis by one-way ANOVA followed by Tukey's multiple comparisons test. 2D versus 3D, ****P < 0.0001; 3D versus 3D + FLMN, ****P < 0.0001; 3D versus 3D + ROCK, ****P < 0.0001; 2D versus 2D + Bleb, ***P=0.0004.


( B ) Schematic of the principles behind traction force microscopy ( left ) . MECs are plated on compliant polyacrylamide gels containing 100 nm fluorescent beads ( close to the cell - polyacrylamide gel interface ) . Traction stresses are calculated based on the bead displacement induced by substrate deformation and relaxation . Quantification of the traction stresses in individual MCF10A MECs ligated with rBM in 2D and 3D and treated in the absence and presence of blebbistatin ( Bleb ) for 18h ( right ; mean ± SEM ; 2D , n = 139 ; 3D , n = 124 ; 2D + Bleb , n = 42 ; 3D + Bleb , n = 43 cells from three independent experiments ) . The background bead displacement was measured from gel areas that lacked ligated cells ( n = 8 fields from one experiment ) . Statistical analysis by one - way ANOVA followed by Tukey ' s multiple comparisons test . * * * * P < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O B-CONTROLLED_VAR 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-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Schematic of the principles behind traction force microscopy (left). MECs are plated on compliant polyacrylamide gels containing 100 nm fluorescent beads (close to the cell-polyacrylamide gel interface). Traction stresses are calculated based on the bead displacement induced by substrate deformation and relaxation. Quantification of the traction stresses in individual MCF10A MECs ligated with rBM in 2D and 3D and treated in the absence and presence of blebbistatin (Bleb) for 18h (right; mean ± SEM; 2D, n=139; 3D, n=124; 2D+Bleb, n=42; 3D + Bleb, n= 43 cells from three independent experiments). The background bead displacement was measured from gel areas that lacked ligated cells (n=8 fields from one experiment). Statistical analysis by one-way ANOVA followed by Tukey's multiple comparisons test. ****P < 0.0001.


( C ) Schematic of the principle of active microrheology ( left ) . MCF10A MECs endocytosed 0 . 5 μm polystyrene particles , which were trapped and oscillated using laser optical tweezers to measure the cytoplasmic modulus . The cytoplasmic modulus was measured for MECs ligated to a rBM in 2D or 3D and treated in the absence or presence of blebbistatin ( Bleb ) to reduce cortical actin tension ( right ) ; individual modulus values were calculated based on the slope in the linear range of the normalized force - displacement curve ( mean ± SEM ; 2D , n = 66 ; 3D , n = 69 ; 2D + Bleb , n = 46 ; 3D + Bleb , n = 60 cells from three independent experiments ) . Statistical analysis by one - way ANOVA followed by Uncorrected Fisher ' s LSD . 2D versus 3D , * * P = 0 . 0028 . 2D versus 2D + Bleb , * P = 0 . 0182 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O

(C) Schematic of the principle of active microrheology (left). MCF10A MECs endocytosed 0.5 μm polystyrene particles, which were trapped and oscillated using laser optical tweezers to measure the cytoplasmic modulus. The cytoplasmic modulus was measured for MECs ligated to a rBM in 2D or 3D and treated in the absence or presence of blebbistatin (Bleb) to reduce cortical actin tension (right); individual modulus values were calculated based on the slope in the linear range of the normalized force-displacement curve (mean ± SEM; 2D, n= 66; 3D, n=69; 2D+Bleb, n=46; 3D+Bleb, n=60 cells from three independent experiments). Statistical analysis by one-way ANOVA followed by Uncorrected Fisher's LSD. 2D versus 3D, **P = 0.0028. 2D versus 2D + Bleb, *P = 0.0182.


( D ) Schematic depicting strategy used to measure cortical tension using laser ablation . ( Top ) Cells with high cortical tension exhibit plasma membrane blebbing when cortical actin is severed by a pulsed laser , whereas cells with lower cortical tension do not . ( Bottom left ) Bar graph of the laser ablation response of MCF10A MECs ligated to a rBM in 2D or 3D ( mean ± SD ; 2D , n = 35 ; 3D , n = 40 cells from three independent experiments ) . ( Bottom right ) Bar graph showing the laser ablation response of MCF10A MECs ligated to a rBM in 2D or 3D and treated in the absence or presence of blebbistatin ( 2D + Bleb and 3D + Bleb ) or expressing constitutively active ROCK ( 3D + ROCK ) ( mean ; 2D , n = 14 ; 3D , n = 15 ; 2D + Bleb , n = 17 ; 3D + Bleb , n = 14 ; 3D + ROCK , n = 10 cells from one experiment ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O

(D) Schematic depicting strategy used to measure cortical tension using laser ablation. (Top) Cells with high cortical tension exhibit plasma membrane blebbing when cortical actin is severed by a pulsed laser, whereas cells with lower cortical tension do not. (Bottom left) Bar graph of the laser ablation response of MCF10A MECs ligated to a rBM in 2D or 3D (mean ± SD; 2D, n= 35; 3D, n=40 cells from three independent experiments). (Bottom right) Bar graph showing the laser ablation response of MCF10A MECs ligated to a rBM in 2D or 3D and treated in the absence or presence of blebbistatin (2D+Bleb and 3D+Bleb) or expressing constitutively active ROCK (3D+ROCK) (mean; 2D, n= 14; 3D, n=15; 2D+Bleb, n=17; 3D+Bleb, n=14; 3D+ ROCK, n=10 cells from one experiment).


( E ) Representative fluorescence and brightfield images of bleb formation induced by laser ablation in MECs stably expressing LifeAct - RFP . Arrowhead : the site of laser ablation . Scale bar , 10 μm .

O O O O O O O O O O O O O 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) Representative fluorescence and brightfield images of bleb formation induced by laser ablation in MECs stably expressing LifeAct-RFP. Arrowhead: the site of laser ablation. Scale bar, 10 μm.


( A ) Representative line graph showing the changes in cytosolic [ Ca2 + ] levels of MCF10A MECs in response to treatment . Fura - 2 - loaded MECs ligated with rBM in 2D were treated with DMSO ( 2D ; blue ) or with blebbistatin ( 2D + Bleb ; red ) and preincubated with EGTA to chelate extracellular Ca2 + in the bath solution , challenged with 2 μM thapsigargin to induce ER Ca2 + release , and replenished with 4 mM Ca2 + in bath solution at the indicated times . F340 / F380 values for each cell were quantified over the course of imaging and plotted ( mean ± SD ; 2D , n = 7 ; 2D + Bleb , n = 7 cells from one experiment ) . ( B ) Quantification of the amplitude of the Ca2 + response ( ∆ F = F - F0 ) induced by thapsigargin , where F0 is the basal fluorescence before thapsigargin treatment . The data shown indicated mean ± SEM ( 2D , n = 13 ; 2D + Bleb , n = 12 cells from two independent experiments ) . * * * * P < 0 . 0001 ( Student ' s t test ) .

O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O 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-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Representative line graph showing the changes in cytosolic [Ca2+] levels of MCF10A MECs in response to treatment. Fura-2-loaded MECs ligated with rBM in 2D were treated with DMSO (2D; blue) or with blebbistatin (2D+Bleb; red) and preincubated with EGTA to chelate extracellular Ca2+ in the bath solution, challenged with 2 μM thapsigargin to induce ER Ca2+ release, and replenished with 4 mM Ca2+ in bath solution at the indicated times. F340/F380 values for each cell were quantified over the course of imaging and plotted (mean ± SD; 2D, n=7; 2D + Bleb, n=7 cells from one experiment). (B) Quantification of the amplitude of the Ca2+ response (∆F=F-F0) induced by thapsigargin, where F0 is the basal fluorescence before thapsigargin treatment. The data shown indicated mean ± SEM (2D, n=13; 2D + Bleb, n=12 cells from two independent experiments). ****P < 0.0001 (Student's t test).


( C ) Representative immunoblots of phosphorylated EIF2a ( pEIF2a ) , total EIF2a and alpha - tubulin in cell lysate from MCF10A MECs ligated to a rBM in 2D or 3D and treated in the absence or presence of blebbistatin ( Bleb ) . Corresponding quantification data are shown at bottom ( mean ± SEM ; n = 4 independent biological replicates ) . Statistical analysis by one - way ANOVA followed by Uncorrected Fisher ' s LSD . 2D versus 3D , * * P = 0 . 0022 . 2D versus 2D + Bleb , * P = 0 . 0248 .

O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O 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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O

(C) Representative immunoblots of phosphorylated EIF2a (pEIF2a), total EIF2a and alpha-tubulin in cell lysate from MCF10A MECs ligated to a rBM in 2D or 3D and treated in the absence or presence of blebbistatin (Bleb). Corresponding quantification data are shown at bottom (mean ± SEM; n=4 independent biological replicates). Statistical analysis by one-way ANOVA followed by Uncorrected Fisher's LSD. 2D versus 3D, **P = 0.0022. 2D versus 2D + Bleb, *P = 0.0248.


( D ) Bar graph of qPCR data measuring the relative levels of ATF3 mRNA in MECs ligated with rBM in 2D and treated in the absence or presence of blebbistatin ( Bleb ) ( mean ± SEM ; n = 4 independent biological replicates ) . * P = 0 . 0158 ( Student ' s t test ) .

O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Bar graph of qPCR data measuring the relative levels of ATF3 mRNA in MECs ligated with rBM in 2D and treated in the absence or presence of blebbistatin (Bleb) (mean ± SEM; n=4 independent biological replicates). *P = 0.0158 (Student's t test).


( E ) Graph of the levels of GFP - MAPPER at the plasma membrane relative total cellular GFP - MAPPER fluorescence in MCF10A MECs ligated with rBM in 2D and treated in the absence or presence of blebbistatin ( Bleb ) ( mean ± SEM ; 2D , n = 39 ; 2D + Bleb , n = 39 cells from three independent experiments ) . * * * P = 0 . 0002 ( Student ' s t test ) .

O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O 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) Graph of the levels of GFP-MAPPER at the plasma membrane relative total cellular GFP-MAPPER fluorescence in MCF10A MECs ligated with rBM in 2D and treated in the absence or presence of blebbistatin (Bleb) (mean ± SEM; 2D, n=39; 2D + Bleb, n=39 cells from three independent experiments). ***P = 0.0002 (Student's t test).


( G ) Scatter plot of the number of proximity ligation assay ( PLA ) puncta within individual cells expressing myc - PERK that were ligated to rBM in 2D or 3D , treated in the absence or presence of blebbistatin ( Bleb ) for 25h and stained with PLA antibody probes specific for endogenous filamin and myc - PERK . Representative images of PLA staining from these experiments can be found in Panel H . Background PLA signal was measured from cells stained in the absence of primary antibodies ( mean ± SEM ; 2D , n = 47 ; 3D , n = 32 cells ; 2D + Bleb , n = 39 ; background , n = 14 cells from three independent experiments ) . Statistical analysis by one - way ANOVA followed by Uncorrected Fisher ' s LSD . * * * * P < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Scatter plot of the number of proximity ligation assay (PLA) puncta within individual cells expressing myc-PERK that were ligated to rBM in 2D or 3D, treated in the absence or presence of blebbistatin (Bleb) for 25h and stained with PLA antibody probes specific for endogenous filamin and myc-PERK. Representative images of PLA staining from these experiments can be found in Panel H. Background PLA signal was measured from cells stained in the absence of primary antibodies (mean ± SEM; 2D, n=47; 3D, n=32 cells; 2D + Bleb, n=39; background, n=14 cells from three independent experiments). Statistical analysis by one-way ANOVA followed by Uncorrected Fisher's LSD. ****P < 0.0001.


( H ) Representative fluorescence microscopy images of cells expressing myc - PERK that were ligated to rBM in 2D or 3D , treated in the absence or presence of blebbistatin ( Bleb ) for 25h and stained with PLA antibody probes specific for endogenous filamin and myc - PERK ( red puncta ) . F - actin was stained with

O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O

(H) Representative fluorescence microscopy images of cells expressing myc-PERK that were ligated to rBM in 2D or 3D, treated in the absence or presence of blebbistatin (Bleb) for 25h and stained with PLA antibody probes specific for endogenous filamin and myc-PERK (red puncta). F-actin was stained with phalloidin


( I ) Representative line graph showing the changes in cytosolic [ Ca2 + ] levels of MCF10A MECs in response to treatment . Fura - 2 - loaded MCF10A MECs ligated with rBM in 2D and expressing filamin repeats 21 - 23 ( 2D + FLMNIg21 - Ig23 ) or vector control ( 2D ) were pre - incubated with EGTA to chelate extracellular Ca2 + in the bath solution , challenged with 2 μM thapsigargin to induce ER Ca2 + release , and replenished with 4 mM Ca2 + in bath solution at the indicated time . F340 / F380 values of individual cells were quantified over the course of imaging ( mean ± SD ( 2D , n = 8 ; 2D + FLMNIg21 - Ig23 , n = 7 cells from one experiment ) . ( J ) Quantification of the amplitude of the Ca2 + response ( ∆ F = F - F0 ) induced by thapsigargin , where F0 is the basal fluorescence before thapsigargin treatment . The data shown indicated mean ± SEM ( 2D , n = 21 ; 2D + FLMNIg21 - Ig23 , n = 19 cells from three independent experiments ) . * * * * P < 0 . 0001 ( Student ' s t test ) .

O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR 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-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(I) Representative line graph showing the changes in cytosolic [Ca2+] levels of MCF10A MECs in response to treatment. Fura-2-loaded MCF10A MECs ligated with rBM in 2D and expressing filamin repeats 21-23 (2D+FLMNIg21-Ig23) or vector control (2D) were pre-incubated with EGTA to chelate extracellular Ca2+ in the bath solution, challenged with 2 μM thapsigargin to induce ER Ca2+ release, and replenished with 4 mM Ca2+ in bath solution at the indicated time. F340/F380 values of individual cells were quantified over the course of imaging (mean ± SD (2D, n=8; 2D + FLMNIg21-Ig23, n=7 cells from one experiment). (J) Quantification of the amplitude of the Ca2+ response (∆F=F-F0) induced by thapsigargin, where F0 is the basal fluorescence before thapsigargin treatment. The data shown indicated mean ± SEM (2D, n=21; 2D + FLMNIg21-Ig23, n=19 cells from three independent experiments). ****P < 0.0001 (Student's t test).


( K ) Bar graph of qPCR data measuring the relative levels of ATF3 mRNA in MCF10A MECs expressing luciferase control or filamin repeats 21 - 23 ( FLMNIg21 - Ig23 ) ligated with rBM in 2D for 18h ( mean ± SEM ; n = 3 independent biological replicates ) . * P = 0 . 032 ( Student ' s t test ) .

O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(K) Bar graph of qPCR data measuring the relative levels of ATF3 mRNA in MCF10A MECs expressing luciferase control or filamin repeats 21-23 (FLMNIg21-Ig23) ligated with rBM in 2D for 18h (mean ± SEM; n=3 independent biological replicates). *P = 0.032 (Student's t test).


( L ) Representative immunoblots of phosphorylated EIF2a ( pEIF2a ) , total EIF2a and alpha - tubulin in cell lysate from MCF10A MECs expressing luciferase ( control ) or filamin repeats 21 - 23 ( FLMNIg21 - Ig23 - myc ) that were ligated with rBM in 2D . Corresponding quantification data are shown at bottom ( mean ± SEM ; n = 5 independent biological replicates ) . * P = 0 . 0112 ( Student ' s t test ) .

O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(L) Representative immunoblots of phosphorylated EIF2a (pEIF2a), total EIF2a and alpha-tubulin in cell lysate from MCF10A MECs expressing luciferase (control) or filamin repeats 21-23 (FLMNIg21-Ig23-myc) that were ligated with rBM in 2D. Corresponding quantification data are shown at bottom (mean ± SEM; n=5 independent biological replicates). *P = 0.0112 (Student's t test).


( A ) Bar graph of qPCR data measuring the levels of SEC61B mRNA in MECs ligated with rBM in 2D and treated in the absence and presence of blebbistatin ( Bleb ) ( mean ± SEM ; n = 3 independent biological replicates ) . * * P = 0 . 0046 ( Student ' s t test ) .

O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Bar graph of qPCR data measuring the levels of SEC61B mRNA in MECs ligated with rBM in 2D and treated in the absence and presence of blebbistatin (Bleb) (mean ± SEM; n=3 independent biological replicates). **P = 0.0046 (Student's t test).


( B ) Change in the Helmholtz free energy of membrane protrusion assembly ( ∆ F ) as a function of cell membrane excess area ( A / Ap ) , which is the variable conjugate to the cortical actin tension .

O O O O O O O O O O O O O O O O O O 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) Change in the Helmholtz free energy of membrane protrusion assembly (∆F) as a function of cell membrane excess area (A/Ap), which is the variable conjugate to the cortical actin tension.


( D ) Representative time - lapse confocal microscopy images of MECs stably expressing LifeAct - RFP that were ligated to a rBM in 2D ( 2D ) or 3D ( 3D ) . Time in seconds ( s ) is indicated in each inset ; scale bar , 10 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Representative time-lapse confocal microscopy images of MECs stably expressing LifeAct-RFP that were ligated to a rBM in 2D (2D) or 3D (3D). Time in seconds (s) is indicated in each inset; scale bar, 10 μm.


( E ) Bar graph of protrusion length measurements in MECs ligated to rBM in 2D , 3D , or in 2D treated with blebbistatin ( 2D + Bleb ) ( mean ± SEM ; 2D , n = 139 ; 3D , n = 106 ; 2D + Bleb , n = 88 protrusions from three independent experiments ) . Statistical analysis by one - way ANOVA followed by Uncorrected Fisher ' s LSD . * * * * P < 0 . 0001 . ( F ) Bar graph of protrusion residence time measurements in MECs ligated to rBM in 2D ( 2D ) , 3D ( 3D ) , or in 2D treated with blebbistatin ( 2D + Bleb ) ( mean ± SEM ; 2D , n = 30 ; 3D , n = 30 ; 2D + Bleb , n = 38 protrusions from three independent experiments ) . Statistical analysis by one - way ANOVA followed by Uncorrected Fisher ' s LSD . * * * * P < 0 . 0001 .

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-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O 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) Bar graph of protrusion length measurements in MECs ligated to rBM in 2D, 3D, or in 2D treated with blebbistatin (2D + Bleb) (mean ± SEM; 2D, n= 139; 3D, n=106; 2D + Bleb, n=88 protrusions from three independent experiments). Statistical analysis by one-way ANOVA followed by Uncorrected Fisher's LSD. ****P < 0.0001. (F) Bar graph of protrusion residence time measurements in MECs ligated to rBM in 2D (2D), 3D (3D), or in 2D treated with blebbistatin (2D+ Bleb) (mean ± SEM; 2D, n= 30; 3D, n=30; 2D + Bleb, n=38 protrusions from three independent experiments). Statistical analysis by one-way ANOVA followed by Uncorrected Fisher's LSD. ****P < 0.0001.