Semi - in vivo pull - down assays showing that CBF1 inhibits phyB interaction with PIF5 ( F ) . The total proteins extracted from 4 - d - old 35S : phyB - GFP seedlings grown at 22 ° C in continuous R light served as the bait , and GST - PIF5 fusion proteins served as the prey . Equivalent amounts of phyB - GFP protein extract and GST - PIF5 fusion proteins , and increasing amounts of His - CBF1 were added as indicated before anti - GFP IP assays were performed . The pulled - down proteins were subjected to immunoblotting with antibodies against phyB , GST and His , respectively . Numbers below the anti - GST blots indicate the relative band intensities of co - precipitated GST - PIF5 normalized to those of precipitated phyB - GFP , respectively . The ratio of the first clear band was set to 100 for each blot .

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

Semi-in vivo pull-down assays showing that CBF1 inhibits phyB interaction with PIF5 (F). The total proteins extracted from 4-d-old 35S:phyB-GFP seedlings grown at 22°C in continuous R light served as the bait, and GST-PIF5 fusion proteins served as the prey. Equivalent amounts of phyB-GFP protein extract and GST-PIF5 fusion proteins, and increasing amounts of His-CBF1 were added as indicated before anti-GFP IP assays were performed. The pulled-down proteins were subjected to immunoblotting with antibodies against phyB, GST and His, respectively. Numbers below the anti-GST blots indicate the relative band intensities of co-precipitated GST-PIF5 normalized to those of precipitated phyB-GFP, respectively. The ratio of the first clear band was set to 100 for each blot.


Phenotypes of Col , two cbf1 mutants ( cbf1 - 1 and cbf1 - 2 ) and two CBF1 - OE lines ( CBF1 - myc and CBF1 - flag ) grown under LD conditions at 22 ° C , 17 ° C and 4 ° C , respectively . The seeds of different genotypes were germinated under LD ( 16 - h - light / 8 - h - dark photoperiod ) conditions at 22 ° C for 2 d , respectively , and then grown under LD conditions at 22 ° C , 17 ° C , or 4 ° C , for additional 6 d . Scale bar = 1 mm .

B-EXP_ASSAY O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Phenotypes of Col, two cbf1 mutants (cbf1-1 and cbf1-2) and two CBF1-OE lines (CBF1-myc and CBF1-flag) grown under LD conditions at 22°C, 17°C and 4°C, respectively. The seeds of different genotypes were germinated under LD (16-h-light/8-h-dark photoperiod) conditions at 22°C for 2 d, respectively, and then grown under LD conditions at 22°C, 17°C, or 4°C, for additional 6 d. Scale bar = 1 mm.


Hypocotyl lengths of Col , two cbf1 mutants ( cbf1 - 1 and cbf1 - 2 ) and two CBF1 - OE lines ( CBF1 - myc and CBF1 - flag ) grown under LD conditions at 22 ° C , 17 ° C or 4 ° C . Error bars represent SD from 10 seedlings . * * * P < 0 . 001 ( two - tailed t - test ) for the indicated genotype compared with Col . NS , not significant .

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

Hypocotyl lengths of Col, two cbf1 mutants (cbf1-1 and cbf1-2) and two CBF1-OE lines (CBF1-myc and CBF1-flag) grown under LD conditions at 22°C, 17°C or 4°C. Error bars represent SD from 10 seedlings. ***P < 0.001 (two-tailed t-test) for the indicated genotype compared with Col. NS, not significant.


The ratios of hypocotyl lengths at 17 ° C versus 22 ° C for the indicated seedlings grown under LD conditions . Error bars represent SD from 10 seedlings . * * * P < 0 . 001 ( two - tailed t - test ) for the indicated genotype compared with Col .

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

The ratios of hypocotyl lengths at 17°C versus 22°C for the indicated seedlings grown under LD conditions. Error bars represent SD from 10 seedlings. ***P < 0.001 (two-tailed t-test) for the indicated genotype compared with Col.


qRT - PCR assays showing the CBF1 transcript levels in Col seedlings grown under LD conditions at 22 ° C , 17 ° C and 4 ° C . Col seeds were germinated under LD conditions at 22 ° C for 2 d , and then grown under LD conditions at 22 ° C , 17 ° C or 4 ° C for additional 6 d . The samples were collected at ZT8 , and then subjected to qRT - PCR assays . Error bars represent SD of three technical replicates . Different letters represent significant differences by one - way ANOVA with Duncan ' s post hoc test ( P < 0 . 05 ) .

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

qRT-PCR assays showing the CBF1 transcript levels in Col seedlings grown under LD conditions at 22°C, 17°C and 4°C. Col seeds were germinated under LD conditions at 22°C for 2 d, and then grown under LD conditions at 22°C, 17°C or 4°C for additional 6 d. The samples were collected at ZT8, and then subjected to qRT-PCR assays. Error bars represent SD of three technical replicates. Different letters represent significant differences by one-way ANOVA with Duncan's post hoc test (P < 0.05).


E , F Immunoblots showing the protein levels of PIF4 ( E ) and PIF5 ( F ) in Col , cbf1 - 1 , cbf1 - 2 , and CBF1 - myc seedlings grown under LD conditions at 22 ° C , 17 ° C and 4 ° C . The seeds of different genotypes were germinated under LD conditions at 22 ° C for 2 d , and then grown under LD conditions at 22 ° C , 17 ° C or 4 ° C for additional 6 d . The samples were collected at ZT20 , and then subjected to immunoblotting . Anti - RPN6 was used as a sample loading control . Numbers below the immunoblots indicate the relative band intensities of PIF4 or PIF5 normalized to those of loading control , respectively . The ratio of the first band was set to 100 for each blot .

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

E, F Immunoblots showing the protein levels of PIF4 (E) and PIF5 (F) in Col, cbf1-1, cbf1-2, and CBF1-myc seedlings grown under LD conditions at 22°C, 17°C and 4°C. The seeds of different genotypes were germinated under LD conditions at 22°C for 2 d, and then grown under LD conditions at 22°C, 17°C or 4°C for additional 6 d. The samples were collected at ZT20, and then subjected to immunoblotting. Anti-RPN6 was used as a sample loading control. Numbers below the immunoblots indicate the relative band intensities of PIF4 or PIF5 normalized to those of loading control, respectively. The ratio of the first band was set to 100 for each blot.


A ) Left panel : experimental strategy . Hematopoietic stem cells ( HSPC ) and progenitors were characterized and isolated using specific sets of surface markers . All cells express CD34 . Upon differentiation , progenitors start to express CD38 in contrast to HSPC ( blue ) . Progenitors engaged in the myeloid ( magenta ) and lymphoid ( green ) pathways express respectively CD33 and CD19 markers . Right panel : FACS gating strategy to isolate HSPC ( further referred as CD38 - ( blue box ) , and cells engaged in myeloid and lymphoid differentiation pathways ( referred to as CD33 + [ magenta box ] and CD19 + [ green box ] , respectively ) .

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

A) Left panel: experimental strategy. Hematopoietic stem cells (HSPC) and progenitors were characterized and isolated using specific sets of surface markers. All cells express CD34. Upon differentiation, progenitors start to express CD38 in contrast to HSPC (blue). Progenitors engaged in the myeloid (magenta) and lymphoid (green) pathways express respectively CD33 and CD19 markers. Right panel: FACS gating strategy to isolate HSPC (further referred as CD38- (blue box), and cells engaged in myeloid and lymphoid differentiation pathways (referred to as CD33+ [magenta box] and CD19+ [green box], respectively).


B ) For each population , equatorial Z stacks of two representative nuclei are shown in the left panel ( chromatin is shown in blue ( DAPI staining ) and nuclear envelop in white ( Sun2 staining ) . Scale bar : 5 µm . The corresponding 3D reconstructions are presented in the right panel . Convex and non - convex surfaces are shown in blue and yellow , respectively . C ) Nucleus - invagination ratio is defined as the ratio of the non - convex area over the total nucleus surface area . Nuclei are larger and deformed in CD33 + ( magenta , n = 106 ) than CD38 - and CD19 + cells ( blue [ n = 120 ] and green [ n = 115 ] , respectively ; 3 independent experiments ; Bars correspond to mean ± SD ; * * * * : p < 0 . 0001 , Mann - Whitney test ; n . s : non significant ) .

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

B) For each population, equatorial Z stacks of two representative nuclei are shown in the left panel (chromatin is shown in blue (DAPI staining) and nuclear envelop in white (Sun2 staining). Scale bar: 5 µm. The corresponding 3D reconstructions are presented in the right panel. Convex and non-convex surfaces are shown in blue and yellow, respectively. C) Nucleus-invagination ratio is defined as the ratio of the non-convex area over the total nucleus surface area. Nuclei are larger and deformed in CD33+ (magenta, n=106) than CD38- and CD19+ cells (blue [n=120] and green [n=115], respectively; 3 independent experiments; Bars correspond to mean ± SD; ****: p<0.0001, Mann-Whitney test; n.s: non significant).


A ) For each population ( CD38 - and CD33 + cells ) , Z stacks of two representative cells are shown in the left panel . Microtubules are shown in green , actin in magenta , centrosome in white and chromatin in blue . In the middle panel , inverted image the corresponding actin staining is shown . In the left panel , the corresponding 3D projection of microtubules is presented as inverted image . Scale bar 5 µm .

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

A) For each population (CD38- and CD33+ cells), Z stacks of two representative cells are shown in the left panel. Microtubules are shown in green, actin in magenta, centrosome in white and chromatin in blue. In the middle panel, inverted image the corresponding actin staining is shown. In the left panel, the corresponding 3D projection of microtubules is presented as inverted image. Scale bar 5 µm.


B ) Microtubules are tightly associated with the nucleus . Z stacks of representative CD38 - and CD33 + cells are shown . Microtubules are shown in green , nucleus envelope ( Sun2 immuno - staining ) in magenta and chromatin in blue . Scale bar 5 µm .

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

B) Microtubules are tightly associated with the nucleus. Z stacks of representative CD38- and CD33+ cells are shown. Microtubules are shown in green, nucleus envelope (Sun2 immuno-staining) in magenta and chromatin in blue. Scale bar 5 µm.


C ) As schematized , centrosome - to - nucleus center distance ( d ) over nucleus center - to - nucleus convex envelope ( R ) ratio is calculated to extract relative position of the centrosome to the center . This parameter is significantly lower in myeloid progenitors ( n = 129 ) than in HSPC ( n = 200 ; 3 independent experiments ; * * * * ; p < 0 . 0001 . Mann - Whitney test ) indicating that centrosome becomes internalized within an invaginated region upon differentiation .

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

C) As schematized, centrosome-to-nucleus center distance (d) over nucleus center-to-nucleus convex envelope (R) ratio is calculated to extract relative position of the centrosome to the center. This parameter is significantly lower in myeloid progenitors (n=129) than in HSPC (n=200; 3 independent experiments; ****; p<0.0001. Mann-Whitney test) indicating that centrosome becomes internalized within an invaginated region upon differentiation.


D ) p150 glued associates with the nuclear membrane of HSPC ( CD38 - ) and myeloid progenitors ( CD33 + ) cells . Z stacks of representative cells are shown . Image contrast has been inverted . Stainings show DAPI , Sun2 and p150 glued . Scale bar 5µm .

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

D) p150 glued associates with the nuclear membrane of HSPC (CD38-) and myeloid progenitors (CD33+) cells. Z stacks of representative cells are shown. Image contrast has been inverted. Stainings show DAPI, Sun2 and p150 glued . Scale bar 5µm.


E ) The dynein adaptor BicD2 associates with the nuclear membrane of HSPC ( CD38 - ) and myeloid progenitors ( CD33 + ) cells . Z stacks of representative cells are shown . Image contrast has been inverted . Stainings show DAPI and BicD2 . Scale bar 5µm .

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

E) The dynein adaptor BicD2 associates with the nuclear membrane of HSPC (CD38-) and myeloid progenitors (CD33+) cells. Z stacks of representative cells are shown. Image contrast has been inverted. Stainings show DAPI and BicD2. Scale bar 5µm.


B ) The nucleus is progressively deformed over time in culture . For each time point , equatorial Z stacks of two representative cells are presented . Left panel : inverted image of chromatin ( DAPI ) . Right panel : microtubules are shown in green and chromatin in blue . Scale bar : 5 µm . C ) Nucleus volume ( left panel ) and invagination ratio ( right panel ) increase to attain levels similar to those of freshly isolated CD33 + cells ( 24 h , n = 165 , 4 donors ; 48 h , n = 143 , 3 donors ; and 72h , n = 192 , 4donors ; CD33 + , n = 227 , 5 donors ; Bars correspond to mean ± SD ; n . s : non significant ; * * * : p < 0 . 001 , * * * * : p < 0 . 0001 ; Mann Whitney test ) .

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

B) The nucleus is progressively deformed over time in culture. For each time point, equatorial Z stacks of two representative cells are presented. Left panel: inverted image of chromatin (DAPI). Right panel: microtubules are shown in green and chromatin in blue. Scale bar: 5 µm. C) Nucleus volume (left panel) and invagination ratio (right panel) increase to attain levels similar to those of freshly isolated CD33+ cells (24 h, n=165, 4 donors; 48 h, n=143, 3 donors; and 72h, n=192, 4donors; CD33+, n=227, 5 donors; Bars correspond to mean ± SD; n.s: non significant; ***: p<0.001, ****: p<0.0001; Mann Whitney test).


D ) For each condition ( CTL , untreated cells ; + Bleb , blebbistatin - treated cells ; + Bleb + NZ , blebbistatin and nocodazole - treated cells ; + TX , taxol - treated cells ; and + CilioD , ciliobrevin - D - treated cells ) Z stacks of 2 representative cells are displayed . Microtubules are shown in green , chromatin in blue , centrosome in white and actin in magenta . Asterisks indicate regions where microtubules are not in contact with the nucleus . Scale bar 5 μm .

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

D) For each condition (CTL, untreated cells; +Bleb, blebbistatin-treated cells; +Bleb+NZ, blebbistatin and nocodazole-treated cells; +TX, taxol-treated cells; and +CilioD, ciliobrevin-D-treated cells) Z stacks of 2 representative cells are displayed. Microtubules are shown in green, chromatin in blue, centrosome in white and actin in magenta. Asterisks indicate regions where microtubules are not in contact with the nucleus. Scale bar 5 μm.


F ) Quantifications of nuclear volume ( left panel ) , invagination ratio ( right panel ) in the indicated conditions . Non - treated cells at 48 hours ( n = 137 , 3 donors ) and 72 hours ( n = 143 , 3 donors ) ; and blebbistatin - treated cells ( n = 96 / 3 donors ) , blebbistatin and nocodazole treated cells ( n = 66 , 2 donors ) , taxol - treated cells ( n = 122 , 3 donors ) , and ciliobrevin - D - treated cells ( n = 128 , 3 donors ) . Bars correspond to mean ± SD ; * * * * : p < 0 . 0001 . * * : p < 0 . 01 , n . s : non significant ; Mann - Whitney test .

O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantifications of nuclear volume (left panel), invagination ratio (right panel) in the indicated conditions. Non-treated cells at 48 hours (n=137, 3 donors) and 72 hours (n=143, 3 donors); and blebbistatin-treated cells (n= 96/3 donors), blebbistatin and nocodazole treated cells (n=66, 2 donors), taxol-treated cells (n=122, 3 donors), and ciliobrevin-D-treated cells (n=128, 3 donors). Bars correspond to mean ± SD; ****: p<0.0001. **: p<0.01, n.s: non significant; Mann-Whitney test.


A ) Spatial distribution of lamin A / C in the nuclei of HSPC ( CD38 - ) , myeloid progenitors ( CD33 + ) and lymphoid progenitors ( CD19 + ) . Inverted images of equatorial Z plane of DAPI and lamin A / C are shown . Scale bar : 5 μm . B ) Line scans of lamin A / C signal along nucleus contour of representative cells are presented for each cell population . The corresponding variations in lamin A / C intensity and nucleus curvature are plotted in blue and in red , respectively . Negative curvature values correspond to local nuclear invaginations . Red asterisk underlines local nucleus invaginations . C ) Dispersion of A / C signal along the nucleus periphery is similarly low in CD38 - ( n = 17 , 3 donors ) , CD33 + ( n = 61 , 3 donors ) and CD19 + ( n = 11 , 3 donors ) indicative of an homogenous distribution of the protein along the nuclear membrane . ( Bars correspond to mean ± SD ; n . s : non significant ; Mann Whitney test ) .

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

A) Spatial distribution of lamin A/C in the nuclei of HSPC (CD38-), myeloid progenitors (CD33+) and lymphoid progenitors (CD19+). Inverted images of equatorial Z plane of DAPI and lamin A/C are shown. Scale bar: 5 μm. B) Line scans of lamin A/C signal along nucleus contour of representative cells are presented for each cell population. The corresponding variations in lamin A/C intensity and nucleus curvature are plotted in blue and in red, respectively. Negative curvature values correspond to local nuclear invaginations. Red asterisk underlines local nucleus invaginations. C) Dispersion of A/C signal along the nucleus periphery is similarly low in CD38- (n=17, 3 donors), CD33+ (n=61, 3 donors) and CD19+ (n=11, 3 donors) indicative of an homogenous distribution of the protein along the nuclear membrane. (Bars correspond to mean ± SD; n.s: non significant; Mann Whitney test).


D ) Dispersion of lamin B signal along the nucleus periphery is similarly low in CD38 - ( n = 131 , 3 donors ) , and CD19 + ( n = 74 , 3 donors ) indicative of a homogenous distribution of the protein along the nuclear membrane . But is significantly increased in CD33 + ( n = 146 , 3 donors ) . Bars correspond to mean ± SD ; * * * * : p < 0 . 0001 ; Mann Whitney test . E - F - G ) Spatial distribution of lamin B and local measurements of lamin B intensity and curvature of nuclear membrane in CD38 - ( E ) , CD33 + ( F ) and CD19 + ( G ) cells . Inverted images of the equatorial Z plane of DAPI ( left panel ) and lamin B ( right panel ) of a representative nucleus are shown for each population . Scale bar : 5 μm . The variations in lamin B signal intensity and curvature are plotted in blue in red , respectively . Asterisks indicate local invaginations of the nuclear membrane . In the bottom graphs , positive and negative curvatures are shown in grey and red , respectively . The absence of curvature ( curvature = 0 ) is highlighted as a dashed line . In CD33 + cells , negative curvature of the nuclear membrane are associated with low lamin B intensities .

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

D) Dispersion of lamin B signal along the nucleus periphery is similarly low in CD38- (n=131, 3 donors), and CD19+ (n=74, 3 donors) indicative of a homogenous distribution of the protein along the nuclear membrane. But is significantly increased in CD33+ (n=146, 3 donors). Bars correspond to mean ± SD; ****:p<0.0001; Mann Whitney test. E-F-G) Spatial distribution of lamin B and local measurements of lamin B intensity and curvature of nuclear membrane in CD38- (E), CD33+ (F) and CD19+ (G) cells. Inverted images of the equatorial Z plane of DAPI (left panel) and lamin B (right panel) of a representative nucleus are shown for each population. Scale bar: 5 μm. The variations in lamin B signal intensity and curvature are plotted in blue in red, respectively. Asterisks indicate local invaginations of the nuclear membrane. In the bottom graphs, positive and negative curvatures are shown in grey and red, respectively. The absence of curvature (curvature =0) is highlighted as a dashed line. In CD33+ cells, negative curvature of the nuclear membrane are associated with low lamin B intensities.


A ) Spatial distribution of H3K9me3 in CD38 - , CD33 + and CD19 + cells . For each population , two representative nuclei are presented . Inverted images of equatorial Z plane of DAPI ( left panel ) and H3K9me3 ( right panel ) are shown . Scale bar : 5 µm .

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

A) Spatial distribution of H3K9me3 in CD38-, CD33+ and CD19+ cells. For each population, two representative nuclei are presented. Inverted images of equatorial Z plane of DAPI (left panel) and H3K9me3 (right panel) are shown. Scale bar: 5 µm.


B ) Spatial distribution of H3K9me3 at the indicated time points of differentiation of CD38 - cells . For each population , two representative nuclei are presented . Inverted images of equatorial Z plane of DAPI ( left panel ) and H3K9me3 ( right panel ) are shown . Scale bar : 5 µm .

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

B) Spatial distribution of H3K9me3 at the indicated time points of differentiation of CD38- cells. For each population, two representative nuclei are presented. Inverted images of equatorial Z plane of DAPI (left panel) and H3K9me3 (right panel) are shown. Scale bar: 5 µm.


C ) For each cell , the raw DAPI image at the equatorial Z stack is used to extract the nucleus contour and measure H3K9Me3 intensity ( in arbitrary units [ a . u ] ) along the contour ( lower row ) .

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

C) For each cell, the raw DAPI image at the equatorial Z stack is used to extract the nucleus contour and measure H3K9Me3 intensity (in arbitrary units [a.u]) along the contour (lower row).


D ) H3K9me3 loss from the nucleus periphery occurs locally in nuclear invaginations . Line scans of H3K9me3 signal along nucleus contour of 2 representative cells are presented in the upper panel for each time point . Asterisks indicate the local loss of H3K9me3 . The corresponding variations in H3K9Me3 intensity and curvature are plotted in blue in red , respectively . Negative curvature values correspond to local nuclear invaginations .

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

D) H3K9me3 loss from the nucleus periphery occurs locally in nuclear invaginations. Line scans of H3K9me3 signal along nucleus contour of 2 representative cells are presented in the upper panel for each time point. Asterisks indicate the local loss of H3K9me3. The corresponding variations in H3K9Me3 intensity and curvature are plotted in blue in red, respectively. Negative curvature values correspond to local nuclear invaginations.


E ) Local curvature of the nucleus envelope as a function of H3K9Me3 local intensity at the indicated time points of differentiation . Positive and negative curvatures are shown in grey and red , respectively . The absence of curvature is highlighted as a dashed line . During differentiation , negatively curved domains increase in number and size and display low H3K9me3 intensities .

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

E) Local curvature of the nucleus envelope as a function of H3K9Me3 local intensity at the indicated time points of differentiation. Positive and negative curvatures are shown in grey and red, respectively. The absence of curvature is highlighted as a dashed line. During differentiation, negatively curved domains increase in number and size and display low H3K9me3 intensities.


F ) H3K9me3 redistribution during differentiation . H3K9me3 signal dispersion at the nucleus periphery is low in CD38 - ( n = 137 , 3 donors ) indicating that H3K9me3 is homogenously distributed at the nucleus periphery . Upon differentiation , H3K9me3 signal dispersion remains homogenous after 24 hours in culture ( n = 115 , 3 donors ) , but progressively becomes heterogeneous ( 48 h : n = 139 , 3 donors ) to attain a level at 72 h ( n = 148 , 3 donors ) similar to that in freshly collected CD33 + cells ( n = 124 , 3 donors ) , indicative of H3K9me3 redistribution from the nucleus periphery . Bars correspond to mean ± SD ; * * * : p < 0 . 001 ; * * : p < 0 . 01 ; n . s : non significant ; Mann Whitney test .

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

F) H3K9me3 redistribution during differentiation. H3K9me3 signal dispersion at the nucleus periphery is low in CD38- (n=137, 3 donors) indicating that H3K9me3 is homogenously distributed at the nucleus periphery. Upon differentiation, H3K9me3 signal dispersion remains homogenous after 24 hours in culture (n=115, 3 donors), but progressively becomes heterogeneous (48 h: n=139, 3 donors) to attain a level at 72 h (n= 148, 3 donors) similar to that in freshly collected CD33+ cells (n=124, 3 donors), indicative of H3K9me3 redistribution from the nucleus periphery. Bars correspond to mean ± SD; ***:p<0.001; **:p<0.01; n.s: non significant; Mann Whitney test.


G ) Microtubule perturbations impair H3K9me3 redistribution during HSPC differentiation . Microtubule - modifying agents were added to culture medium between 48 and 72 hours . Cells were fixed and analyzed after 72 hours of culture . For each indicated treatment , one representative nucleus is presented . Inverted images of equatorial Z stacks of DAPI ( left panel ) and H3K9me3 ( right panel ) are shown . Scale bar : 5 µm . H ) H3K9me3 signal dispersion significantly increases in non - treated cells between 48h ( n = 139 / 3 donors ) and 72h ( n = 148 / 3 donors ) of culture . At 72h , the dispersion is similar in non - treated and blebbistatin - treated cells ( n = 124 / 3 donors ) . By contrast , H3K9me3 signal dispersion is significantly reduced compared with non - treated cells after treatment with nocodazole and blebbistatin ( n = 55 , 2donors ) , taxol ( n = 109 , 3 donors ) and ciliobrevin - D ( n = 144 , 3donors ) to a level attained by non - treated cells at 48 hours , demonstrating that microtubules affect H3K9me3 redistribution during HSPC differentiation . Bars correspond to mean ± SD ; * * * * : p < 0 . 0001 ; n . s : non significant ; Mann Whitney test .

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

G) Microtubule perturbations impair H3K9me3 redistribution during HSPC differentiation. Microtubule-modifying agents were added to culture medium between 48 and 72 hours. Cells were fixed and analyzed after 72 hours of culture. For each indicated treatment, one representative nucleus is presented. Inverted images of equatorial Z stacks of DAPI (left panel) and H3K9me3 (right panel) are shown. Scale bar: 5 µm. H) H3K9me3 signal dispersion significantly increases in non-treated cells between 48h (n=139/3 donors) and 72h (n=148/3 donors) of culture. At 72h, the dispersion is similar in non-treated and blebbistatin-treated cells (n=124/ 3 donors). By contrast, H3K9me3 signal dispersion is significantly reduced compared with non-treated cells after treatment with nocodazole and blebbistatin (n=55, 2donors), taxol (n= 109, 3 donors) and ciliobrevin-D (n=144, 3donors) to a level attained by non-treated cells at 48 hours, demonstrating that microtubules affect H3K9me3 redistribution during HSPC differentiation. Bars correspond to mean ± SD; ****:p<0.0001; n.s: non significant; Mann Whitney test.


A ) Microtubules perturbations impact the trancriptome of differentiating cells . Volcano - plot representation of down - regulated ( blue ) and up - regulated genes ( red ) in non - treated / control cells relative to ciliobrevin - D treated cells . Each circle represents the fold change ( FC ) of the quantity of mRNA and the significance of the change based on three independent replicates . Expressed genes with a false discovery rate ( FDR ) lower than 0 . 05 are shown in grey , whereas genes with a FDR greater than 0 . 05 are shown in blue or red . Genes significantly mis - regulated i . e . with an absolute value of FC higher than 1 . 8 are shown as full disks . Names of genes of interest are indicated within the graph . Genes that are in the signature of the myeloid - differentiation pathway are shown in magenta . Genes that are the signature of the megakaryocyte - differentiation pathway are shown in green . B ) Heatmap representation and hierarchical clustering of gene expression changes between non - treated and ciliobrevin - D - treated cells collected from 3 donors in each condition . The color code corresponds to a log2 scale of the differential expression levels .

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

A) Microtubules perturbations impact the trancriptome of differentiating cells. Volcano-plot representation of down-regulated (blue) and up-regulated genes (red) in non-treated/control cells relative to ciliobrevin-D treated cells. Each circle represents the fold change (FC) of the quantity of mRNA and the significance of the change based on three independent replicates. Expressed genes with a false discovery rate (FDR) lower than 0.05 are shown in grey, whereas genes with a FDR greater than 0.05 are shown in blue or red. Genes significantly mis-regulated i.e. with an absolute value of FC higher than 1.8 are shown as full disks. Names of genes of interest are indicated within the graph. Genes that are in the signature of the myeloid-differentiation pathway are shown in magenta. Genes that are the signature of the megakaryocyte-differentiation pathway are shown in green. B) Heatmap representation and hierarchical clustering of gene expression changes between non-treated and ciliobrevin-D-treated cells collected from 3 donors in each condition. The color code corresponds to a log2 scale of the differential expression levels.


E ) In vitro tumor T cell co - culture competition assay in IFNGR1 - KO D10 cells containing sgRNAs targeting TSC1 , TSC2 or both . Statistics was done by Kruskal - Wallis test with Dunn ' s post hoc test . Error bars indicate SD of four biological replicates with different T cell donors ( n = 4 ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

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

E) In vitro tumor T cell co-culture competition assay in IFNGR1-KO D10 cells containing sgRNAs targeting TSC1, TSC2 or both. Statistics was done by Kruskal-Wallis test with Dunn's post hoc test. Error bars indicate SD of four biological replicates with different T cell donors (n = 4). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.


F ) In vitro tumor T cell co - culture competition assay in multiple melanoma cell lines containing indicated sgRNAs . Statistics was done by Kruskal - Wallis test with Dunn ' s post hoc test . Error bars indicate SD of six biological replicates with different cell lines ( n = 6 ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

O O O O O B-CELL_TYPE I-CELL_TYPE B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) In vitro tumor T cell co-culture competition assay in multiple melanoma cell lines containing indicated sgRNAs. Statistics was done by Kruskal-Wallis test with Dunn's post hoc test. Error bars indicate SD of six biological replicates with different cell lines (n = 6). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.


I ) In vitro T cell co - culture competition assay of sgCtrl and sgTsc2 - expressing lung cancer cell lines . Statistics was done by Mann - Whitney test . Error bars indicate SD of five biological replicates with different T cell donors ( n = 5 ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

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

I) In vitro T cell co-culture competition assay of sgCtrl and sgTsc2-expressing lung cancer cell lines. Statistics was done by Mann-Whitney test. Error bars indicate SD of five biological replicates with different T cell donors (n = 5). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.


B ) Tumor growth upon adoptive cell transfer with Ctrl ( n = 9 ) or MART - 1 ( n = 8 ) T cells . Data points represent average tumor volume , and error bars represent SEM .

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

B) Tumor growth upon adoptive cell transfer with Ctrl (n = 9) or MART-1 (n = 8) T cells. Data points represent average tumor volume, and error bars represent SEM.


D ) Quantification of the in vivo competition assay ( output ) at end point by flow cytometry analysis . Error bars indicate SD . Statistical analysis was performed by Mann - Whitney test . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 . For this specific experiment , mice that deceased before the analysis ( n = 1 , Ctrl T cell group ) , with failed ACT injection ( n = 1 , MART - 1 T cell group ) or were completely tumor - free after ACT ( n = 1 , MART - 1 T cell group ) were excluded from the analysis .

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

D) Quantification of the in vivo competition assay (output) at end point by flow cytometry analysis. Error bars indicate SD. Statistical analysis was performed by Mann-Whitney test. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. For this specific experiment, mice that deceased before the analysis (n=1, Ctrl T cell group), with failed ACT injection (n=1, MART-1 T cell group) or were completely tumor-free after ACT (n=1, MART-1 T cell group) were excluded from the analysis.


A ) Western blot analysis of baseline mTOR signaling in sgCtrl - or sgTsc2 - expressing melanoma and lung cancer cell lines . Representative plot of 2 - 6 independent experiments . All blots shown in this panel were run in parallel .

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

A) Western blot analysis of baseline mTOR signaling in sgCtrl- or sgTsc2-expressing melanoma and lung cancer cell lines. Representative plot of 2-6 independent experiments. All blots shown in this panel were run in parallel.


E ) Western blot analysis of sgCtrl - or sgTSC2 - expressing D10 melanoma cells upon MART - 1 T cell challenge for indicated time . Representative of three biological replicates with different T cell donors ( n = 3 ) .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-CELL_LINE B-DISEASE O O B-GENEPROD I-GENEPROD I-GENEPROD B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O

E) Western blot analysis of sgCtrl- or sgTSC2-expressing D10 melanoma cells upon MART-1 T cell challenge for indicated time. Representative of three biological replicates with different T cell donors (n = 3).


F ) In vitro competition assay of sgCtrl - and sgTsc2 - expressing D10 cells co - cultured with MART - 1 T cells in the presence of necroptosis ( Nec1 - s , 20uM ) or apoptosis inhibitors ( Q - VD - Oph , 50uM ) , normalized to inhibitor plus Ctrl T cell - treated groups , showing the net MART - 1 T cell effect . Statistical analysis was performed by one - way ANOVA with Holm - Sidak ' s multiple comparisons test . Error bars indicate SD of three biological replicates with different T cell donors ( n = 3 ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

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

F) In vitro competition assay of sgCtrl- and sgTsc2-expressing D10 cells co-cultured with MART-1 T cells in the presence of necroptosis (Nec1-s, 20uM) or apoptosis inhibitors (Q-VD-Oph, 50uM), normalized to inhibitor plus Ctrl T cell-treated groups, showing the net MART-1 T cell effect. Statistical analysis was performed by one-way ANOVA with Holm-Sidak's multiple comparisons test. Error bars indicate SD of three biological replicates with different T cell donors (n = 3). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.


A ) Cell viability analysis by CellTiter - Blue assay of sgCtrl - or sgTSC2 - expressing D10 melanoma and A549 lung cancer cells treated with TRAIL at indicated concentrations for three days . Statistical analysis was performed by two - tailed unpaired Student t test . For D10 , error bars represent SD from three independent experiments ( n = 3 ) . For A549 , error bars represent SD of pooled data from two independent experiments with three technical replicates each ( n = 2 ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-CELL_LINE B-DISEASE O B-CELL_LINE B-DISEASE I-DISEASE O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cell viability analysis by CellTiter-Blue assay of sgCtrl- or sgTSC2-expressing D10 melanoma and A549 lung cancer cells treated with TRAIL at indicated concentrations for three days. Statistical analysis was performed by two-tailed unpaired Student t test. For D10, error bars represent SD from three independent experiments (n = 3). For A549, error bars represent SD of pooled data from two independent experiments with three technical replicates each (n = 2). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.


C ) Flow cytometry analysis of TRAIL - R2 surface expression level in multiple sgTSC2 - expressing tumor cell lines compared to their sgCtrl - expressing controls . Statistical analysis was performed by two - way ANOVA with Sidak ' s multiple comparisons test . Error bars represent SD of three biological replicates ( n = 3 ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

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

C) Flow cytometry analysis of TRAIL-R2 surface expression level in multiple sgTSC2- expressing tumor cell lines compared to their sgCtrl-expressing controls. Statistical analysis was performed by two-way ANOVA with Sidak's multiple comparisons test. Error bars represent SD of three biological replicates (n = 3). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.


D ) In vitro competition assay performed by mixing control and tumor cells with ablation for either TSC2 , TRAIL receptors ( TNFRSF10A and TNFRSF10B ) , or both in a co - culture with MART - 1 T cells . Statistical analysis was performed with a one - way ANOVA , followed by a Tukey post - hoc test . Error bars indicate SD of four biological replicates with different T cell donors . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

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

D) In vitro competition assay performed by mixing control and tumor cells with ablation for either TSC2, TRAIL receptors (TNFRSF10A and TNFRSF10B), or both in a co-culture with MART-1 T cells. Statistical analysis was performed with a one-way ANOVA, followed by a Tukey post-hoc test. Error bars indicate SD of four biological replicates with different T cell donors. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.


B ) TSC2 expression : TRAIL signaling expression ratio for predicting ICB treatment responses in pre - treatment patient samples Combo , anti - PD - 1 plus anti - CTLA - 4 combination treatment . Statistical significance was calculated with Student t test for normally distributed data , or with Mann - Whitney test for non - normally distributed data . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 . Partial response and complete response ( PRCR ) are defined as responder ( R ) ; stable disease ( SD ) and progressive disease ( PD ) are defined as non - responder ( NR ) .

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

B) TSC2 expression: TRAIL signaling expression ratio for predicting ICB treatment responses in pre-treatment patient samples Combo, anti-PD-1 plus anti-CTLA-4 combination treatment. Statistical significance was calculated with Student t test for normally distributed data, or with Mann-Whitney test for non-normally distributed data. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Partial response and complete response (PRCR) are defined as responder (R); stable disease (SD) and progressive disease (PD) are defined as non-responder (NR).


C ) TSC2 immune challenge signature expression in cohorts of patients after ICB treatment . Early during treatment ( EDT ) , anti - PD - 1 + anti - CTLA - 4 , and anti - PD - 1 alone treatment cohorts . Statistical significance was calculated with Student t test for normally distributed data , or with Mann - Whitney test for non - normally distributed data . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 . Partial response and complete response ( PRCR ) are defined as responder ( R ) ; stable disease ( SD ) and progressive disease ( PD ) are defined as non - responder ( NR ) .

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

C) TSC2 immune challenge signature expression in cohorts of patients after ICB treatment. Early during treatment (EDT), anti-PD-1 + anti-CTLA-4, and anti-PD-1 alone treatment cohorts. Statistical significance was calculated with Student t test for normally distributed data, or with Mann-Whitney test for non-normally distributed data. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Partial response and complete response (PRCR) are defined as responder (R); stable disease (SD) and progressive disease (PD) are defined as non-responder (NR).


( A ) RAW 264 . 7 macrophages infected with heat killed ( top panels ) or live ( bottom panels ) S . aureus USA300 ( in red ) were imaged by DIC time - lapse microscopy at 6 h post - infection . The images on the left are of time zero in the image sequence and show the presence of S . aureus . The images on the right are of the same cells and the presence of membrane ruffles and dynamic filopodia are denoted by the yellow arrow heads . Candidate pinosomes are denoted by yellow arrows .

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

(A) RAW 264.7 macrophages infected with heat killed (top panels) or live (bottom panels) S. aureus USA300 (in red) were imaged by DIC time-lapse microscopy at 6 h post-infection. The images on the left are of time zero in the image sequence and show the presence of S. aureus. The images on the right are of the same cells and the presence of membrane ruffles and dynamic filopodia are denoted by the yellow arrow heads. Candidate pinosomes are denoted by yellow arrows.


( B ) RAW 264 . 7 macrophages expressing the lipid biosensor for PI ( 4 , 5 ) P2 , PH - Plcδ - RFP ( in red ) , were left uninfected or were infected with live GFP - expressing S . aureus USA300 ( in green ) for 8 h prior to fixation . The macrophage plasmalemma and extracellular bacteria are indicated by Cy5 - conjugated wheat germ agglutinin ( WGA , in blue ) . As a control , to demonstrate displacement of PH - Plcδ - RFP from the macrophage membrane , transfected cells were treated with 10 µM ionomycin in the presence of 1 mM CaCl2 for 20 min prior to fixation . These images are representative of three independent experiments .

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

(B) RAW 264.7 macrophages expressing the lipid biosensor for PI(4,5)P2, PH-Plcδ-RFP (in red), were left uninfected or were infected with live GFP-expressing S. aureus USA300 (in green) for 8 h prior to fixation. The macrophage plasmalemma and extracellular bacteria are indicated by Cy5-conjugated wheat germ agglutinin (WGA, in blue). As a control, to demonstrate displacement of PH-Plcδ-RFP from the macrophage membrane, transfected cells were treated with 10 µM ionomycin in the presence of 1 mM CaCl2 for 20 min prior to fixation. These images are representative of three independent experiments.


( C , D ) Internalization of FITC - Dextran by uninfected ( top row ) and S . aureus ( in blue ) - infected ( bottom row ) RAW 264 . 7 macrophages at 10 h post - infection . These images are representative of three independent experiments . The quantitation of FITC - dextran uptake by either uninfected or infected RAW 264 . 7 and primary bone marrow derived macrophages is shown in ( D ) . For each experiment , macrophages were treated with 1 mM concanamycin A for 30 min prior to image acquisition . Shown is the mean fluorescence intensity ( MFI ) in arbitrary units ( a . u . ) ± standard deviation where each symbol represents the measurement of a single cell . The data derive from three independent experiments and n . s . indicates the data are not significant as determined by an unpaired two - tailed t - test ( p > 0 . 05 ) .

O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE B-CELL_TYPE O O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O B-CELL_TYPE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C, D) Internalization of FITC-Dextran by uninfected (top row) and S. aureus (in blue)-infected (bottom row) RAW 264.7 macrophages at 10 h post-infection. These images are representative of three independent experiments. The quantitation of FITC-dextran uptake by either uninfected or infected RAW 264.7 and primary bone marrow derived macrophages is shown in (D). For each experiment, macrophages were treated with 1 mM concanamycin A for 30 min prior to image acquisition. Shown is the mean fluorescence intensity (MFI) in arbitrary units (a.u.) ± standard deviation where each symbol represents the measurement of a single cell. The data derive from three independent experiments and n.s. indicates the data are not significant as determined by an unpaired two-tailed t-test (p>0.05).


( E ) Potential for co - localization of FITC - dextran ( in green ) with phagocytosed S . aureus expressing mCherry ( in red ) . The micrographs depict representative macrophages infected with S . aureus at 10 h post - infection and the white arrows point to mCherry positive bacteria that are co - localizing with dextran . Macrophages were treated with 1 μM concanamycin A prior to imaging live .

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

(E) Potential for co-localization of FITC-dextran (in green) with phagocytosed S. aureus expressing mCherry (in red). The micrographs depict representative macrophages infected with S. aureus at 10 h post-infection and the white arrows point to mCherry positive bacteria that are co-localizing with dextran. Macrophages were treated with 1 μM concanamycin A prior to imaging live.


( A ) Internalization and processing of DQ ™ Green BSA by uninfected and S . aureus ( in blue ) infected RAW 264 . 7 macrophages at 10 h post - infection . These images are representative of three independent experiments . ( B ) Quantitation of DQ BSA green fluorescence displayed by either uninfected or S . aureus ( in blue ) infected RAW 264 . 7 . Shown is the mean fluorescence intensity ( MFI ) in arbitrary units ( a . u ) ± standard deviation where each symbol represents the measurement of a single cell . The data derive from three independent experiments and n . s . indicates the data are not significant as determined by an unpaired two - tailed t - test ( p > 0 . 05 ) .

O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE B-CELL_TYPE O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Internalization and processing of DQ™ Green BSA by uninfected and S. aureus (in blue) infected RAW 264.7 macrophages at 10 h post-infection. These images are representative of three independent experiments. (B) Quantitation of DQ BSA green fluorescence displayed by either uninfected or S. aureus (in blue) infected RAW 264.7. Shown is the mean fluorescence intensity (MFI) in arbitrary units (a.u) ± standard deviation where each symbol represents the measurement of a single cell. The data derive from three independent experiments and n.s. indicates the data are not significant as determined by an unpaired two-tailed t-test (p>0.05).


( C ) Fluorescence - based proliferation assays were performed in conjunction with the probe DQ ™ Green BSA . Processed DQ ™ Green BSA could be found co - localizing with intracellular mCherry - expressing S . aureus ( in red ) and with intracellular S . aureus has replicated ( i . e . is proliferation dye negative ) at 10h post - infection as depicted here . Data information : Scale bars equal 10 μm .

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

(C) Fluorescence-based proliferation assays were performed in conjunction with the probe DQ™ Green BSA. Processed DQ™ Green BSA could be found co-localizing with intracellular mCherry-expressing S. aureus (in red) and with intracellular S. aureus has replicated (i.e. is proliferation dye negative) at 10h post-infection as depicted here. Data information: Scale bars equal 10 μm.


( A ) Uptake of Alexa ® Fluor647 - conjugated transferrin by RAW 264 . 7 macrophages in the presence and absence of PIK - III and Dynasore . The fluorescence micrographs show transferrin ( in blue ) and the macrophage plasmalemma demarcated by TMR - conjugated wheat germ agglutinin ( in red ) . The dashed line indicates the region of the cell for which the line scan analysis , shown graphically on the right , was done . These micrographs are representative images obtained from three independent experiments . Line scan analysis was performed to indicate the distribution of fluorescent transferrin ( blue line ) relative to the plasmalemma ( red line ) for the DMSO and Dynasore treated cells presented in the fluorescence micrographs . Scale bars equal 10 μm .

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

(A) Uptake of Alexa® Fluor647-conjugated transferrin by RAW 264.7 macrophages in the presence and absence of PIK-III and Dynasore. The fluorescence micrographs show transferrin (in blue) and the macrophage plasmalemma demarcated by TMR-conjugated wheat germ agglutinin (in red). The dashed line indicates the region of the cell for which the line scan analysis, shown graphically on the right, was done. These micrographs are representative images obtained from three independent experiments. Line scan analysis was performed to indicate the distribution of fluorescent transferrin (blue line) relative to the plasmalemma (red line) for the DMSO and Dynasore treated cells presented in the fluorescence micrographs. Scale bars equal 10 μm.


( B ) RAW macrophages were pre - treated with vehicle ( DMSO ) or the vps34 inhibitor PIK - III prior to the addition of 10 kDa FITC - dextran ( 100 µg / mL ) for 2 hrs . Prior to imaging live , macrophages were also stained with Cy5 - conjugated WGA . The representative micrographs depict inhibited dextran uptake indicating PIK - III treatment can perturb fluid phase uptake . Scale bars equal 10 µm .

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

(B) RAW macrophages were pre-treated with vehicle (DMSO) or the vps34 inhibitor PIK-III prior to the addition of 10 kDa FITC-dextran (100 µg/mL) for 2 hrs. Prior to imaging live, macrophages were also stained with Cy5-conjugated WGA. The representative micrographs depict inhibited dextran uptake indicating PIK-III treatment can perturb fluid phase uptake. Scale bars equal 10 µm.


( C ) The intensity of dextran fluorescence was quantified for RAW cells pre - treated with vehicle , PIK - III ( 10 µM ) , Dynasore ( 100 µM ) or PIK - III and Dynasore ( 10 and 100 µM ) respectively . The data shown are the mean fluorescence intensity ± standard deviation for each experiment calculated from at least 30 cells from two independent experiments . * indicates p < 0 . 05 , as determined by an ordinary one - way ANOVA with a Bonferroni multiple comparisons test .

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

(C) The intensity of dextran fluorescence was quantified for RAW cells pre-treated with vehicle, PIK-III (10 µM), Dynasore (100 µM) or PIK-III and Dynasore (10 and 100 µM) respectively. The data shown are the mean fluorescence intensity ± standard deviation for each experiment calculated from at least 30 cells from two independent experiments. * indicates p<0.05, as determined by an ordinary one-way ANOVA with a Bonferroni multiple comparisons test.


( A ) The effects of prolonged exposure to gentamicin ( 300 µg / mL ) or vancomycin ( 300 µg / mL ) on the survival of phagocytosed S . aureus USA300 in RAW macrophages is shown . Depicted above the graph is a timeline detailing the treatment regime . The data are presented as the mean fold change in CFU / mL + / - standard deviation for 15 biological replicates of S . aureus , represented by each symbol , from five independent experiments . For each replicate the fold change was calculated by dividing the bacterial count at 10 h post - infection by the counts at 1 . 5 h post - infection . The dashed line indicates a fold change value equal to 1 ( i . e . no change in bacterial burden ) . * * p < 0 . 01 and statistical significance was determined by a one - way ANOVA with a Dunnett ' s post - hoc test .

O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O B-CELL_LINE B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The effects of prolonged exposure to gentamicin (300 µg/mL) or vancomycin (300 µg/mL) on the survival of phagocytosed S. aureus USA300 in RAW macrophages is shown. Depicted above the graph is a timeline detailing the treatment regime. The data are presented as the mean fold change in CFU/mL +/- standard deviation for 15 biological replicates of S. aureus, represented by each symbol, from five independent experiments. For each replicate the fold change was calculated by dividing the bacterial count at 10 h post-infection by the counts at 1.5 h post-infection. The dashed line indicates a fold change value equal to 1 (i.e. no change in bacterial burden). **p<0.01 and statistical significance was determined by a one-way ANOVA with a Dunnett's post-hoc test.


( B ) The ability to protect S . aureus USA300 from intracellular killing by gentamicin is shown . Here , experiments were performed as in ( A ) except during the initial gentamicin treatment ( 100 μg / mL for 1h ) to kill extracellular bacteria macrophages were also treated with either DMSO or PIK - III / Dynasore to inhibit fluid phase uptake . At 1 . 5 h post - infection cells were washed and treated with gentamicin ( 300 µg / mL ) in the presence of either DMSO ( vehicle ) or PIK - III + Dynasore which were maintained throughout the experiment . The data are presented as the mean number of surviving bacteria in CFU / mL at 10 h post - infection + / - standard deviation . These data derive from a total of 15 biological replicates of S . aureus from at least three independent experiments . * * * p < 0 . 001 and statistical significance was determined by an unpaired two - tailed t - test with a Welch ' s correction .

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

(B) The ability to protect S. aureus USA300 from intracellular killing by gentamicin is shown. Here, experiments were performed as in (A) except during the initial gentamicin treatment (100 μg/mL for 1h) to kill extracellular bacteria macrophages were also treated with either DMSO or PIK-III/Dynasore to inhibit fluid phase uptake. At 1.5 h post-infection cells were washed and treated with gentamicin (300 µg/mL) in the presence of either DMSO (vehicle) or PIK-III + Dynasore which were maintained throughout the experiment. The data are presented as the mean number of surviving bacteria in CFU/mL at 10 h post-infection +/- standard deviation. These data derive from a total of 15 biological replicates of S. aureus from at least three independent experiments. *** p<0.001 and statistical significance was determined by an unpaired two-tailed t-test with a Welch's correction.


( C ) The effect of PIK - III / Dynasore treatment on the ability of S . aureus USA300 to replicate within RAW macrophages is shown . After gentamicin treatment at 1 . 5 h , RAW 264 . 7 macrophages having phagocytosed S . aureus were lysed and plated to determine the number of gentamicin protected bacteria . Alternatively , infected samples treated with either DMSO as a vehicle control or with PIK - III / Dynasore which was added at 1 . 5h post - infection after gentamicin . At 10 h post - infection macrophages exposed to DMSO or inhibitors , that were maintained throughout the experiment , were lysed and plated to determine the bacterial burden . The data shown are the mean fold change in CFU / mL at 10 h where the data are normalized to the bacterial burden obtained at 1 . 5 h . Each symbol represents a biological replicate ( n = 8 ) of S . aureus and derive from three independent experiments . The data presented are the mean ± standard deviation . * indicates p < 0 . 05 as determine by an unpaired two - tailed t - test with a Welch ' s correction .

O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O B-CELL_LINE B-CELL_TYPE O O O O B-SMALL_MOLECULE O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE B-CELL_TYPE O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-CELL_TYPE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) The effect of PIK-III/Dynasore treatment on the ability of S. aureus USA300 to replicate within RAW macrophages is shown. After gentamicin treatment at 1.5 h, RAW 264.7 macrophages having phagocytosed S. aureus were lysed and plated to determine the number of gentamicin protected bacteria. Alternatively, infected samples treated with either DMSO as a vehicle control or with PIK-III/Dynasore which was added at 1.5h post-infection after gentamicin. At 10 h post-infection macrophages exposed to DMSO or inhibitors, that were maintained throughout the experiment, were lysed and plated to determine the bacterial burden. The data shown are the mean fold change in CFU/mL at 10 h where the data are normalized to the bacterial burden obtained at 1.5 h. Each symbol represents a biological replicate (n=8) of S. aureus and derive from three independent experiments. The data presented are the mean ± standard deviation. * indicates p<0.05 as determine by an unpaired two-tailed t-test with a Welch's correction.


( D ) The effect of PIK - III + Dynasore or DMSO on the growth of S . aureus USA300 in RPMI with 5 % ( v / v ) FBS is shown . The data are the mean OD600nm + / - standard deviation of four biological replicates of S . aureus for each condition and are representative of three independent experiments .

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

(D) The effect of PIK-III + Dynasore or DMSO on the growth of S. aureus USA300 in RPMI with 5% (v/v) FBS is shown. The data are the mean OD600nm +/- standard deviation of four biological replicates of S. aureus for each condition and are representative of three independent experiments.


( E ) Analysis of the effects of PIK - III / Dynasore treatment on S . aureus replication using a fluorescence based bacterial proliferation assay is shown . The micrographs depict S . aureus USA300 expressing GFP that were co - labelled with proliferation dye . The top row of micrographs depict vehicle treated macrophages where GFP - positive , yet proliferation dye - negative bacteria were present . The bottom row depicts inhibitor macrophages treated macrophages where the bacteria fail to replicate and remain proliferation dye positive . The macrophage plasmalemma and extracellular bacteria are demarcated by TMR - WGA ( in red ) . Images were acquired at 10 h post - infection and are representative of three independent experiments . Scale bars equal 10 μm .

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

(E) Analysis of the effects of PIK-III/Dynasore treatment on S. aureus replication using a fluorescence based bacterial proliferation assay is shown. The micrographs depict S. aureus USA300 expressing GFP that were co-labelled with proliferation dye. The top row of micrographs depict vehicle treated macrophages where GFP-positive, yet proliferation dye-negative bacteria were present. The bottom row depicts inhibitor macrophages treated macrophages where the bacteria fail to replicate and remain proliferation dye positive. The macrophage plasmalemma and extracellular bacteria are demarcated by TMR-WGA (in red). Images were acquired at 10 h post-infection and are representative of three independent experiments. Scale bars equal 10 μm.


( F ) The normalized fraction of macrophages containing replicating bacteria is shown . The graph represents the mean ± standard deviation of the number of macrophages that contain replicating bacteria where the data are normalized to the vehicle control condition . Each symbol represents a biological replicate ( n = 6 ) of S . aureus and derive from three independent experiments . * * * * p < 0 . 0001 and statistical significance was determined by an unpaired two - tailed t - test using a Welch ' s correction .

O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) The normalized fraction of macrophages containing replicating bacteria is shown. The graph represents the mean ± standard deviation of the number of macrophages that contain replicating bacteria where the data are normalized to the vehicle control condition. Each symbol represents a biological replicate (n=6) of S. aureus and derive from three independent experiments. ****p<0.0001 and statistical significance was determined by an unpaired two-tailed t-test using a Welch's correction.


( A ) Growth of wild - type S . aureus USA300 or a purK mutant . The data are the mean ± standard deviation of the end point optical density measured at 18 h at 600nm for each strain in RPMI . Additives are indicated on the x axis and were either 5 % ( v / v ) FBS , 50 μM IMP , 10 μM PIK - III and 100 μM Dynasore or an equivalent volume of DMSO . Each symbol represents a biological replicate of S . aureus and the data derive from three independent experiments . Statistical significance was determined by a one - way ANOVA with a Holm - Sidak ' s multiple comparison test . n . s . indicates not significant , * * * indicates p < 0 . 0001 .

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

(A) Growth of wild-type S. aureus USA300 or a purK mutant. The data are the mean ± standard deviation of the end point optical density measured at 18 h at 600nm for each strain in RPMI. Additives are indicated on the x axis and were either 5% (v/v) FBS, 50 μM IMP, 10 μM PIK-III and 100 μM Dynasore or an equivalent volume of DMSO. Each symbol represents a biological replicate of S. aureus and the data derive from three independent experiments. Statistical significance was determined by a one-way ANOVA with a Holm-Sidak's multiple comparison test. n.s. indicates not significant, *** indicates p<0.0001.


( B ) The graph depicts the fold change in bacterial burden for RAW 264 . 7 macrophages infected with purK S . aureus . Macrophages were left untreated or were exposed to 50 μM IMP or 50 μM IMP after pretreatment with PIK - III and Dynasore which were maintained throughout the infection . The data are the mean ± standard deviation of the burden at 10 h post - infection normalized to the burden at 1 . 5h post - infection prior to the addition of IMP . Each symbol represents a single biological replicate of S . aureus and derive from three independent experiments . Significance was determined by one - way ANOVA with a Turkeys multiple comparison test . * p < 0 . 05 , * * p < 0 . 01 .

O O O O O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE B-CELL_TYPE O O B-GENEPROD B-ORGANISM I-ORGANISM I-ORGANISM O B-CELL_TYPE O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) The graph depicts the fold change in bacterial burden for RAW 264.7 macrophages infected with purK S. aureus. Macrophages were left untreated or were exposed to 50 μM IMP or 50 μM IMP after pretreatment with PIK-III and Dynasore which were maintained throughout the infection. The data are the mean ± standard deviation of the burden at 10 h post-infection normalized to the burden at 1.5h post-infection prior to the addition of IMP. Each symbol represents a single biological replicate of S. aureus and derive from three independent experiments. Significance was determined by one-way ANOVA with a Turkeys multiple comparison test. *p<0.05, **p<0.01.


( C , D ) The ability of purK S . aureus to grow in an IMP - dependent manner within primary bone marrow - derived macrophages is shown . ( C ) Fluorescence micrographs depicting purK replication analyzed by fluorescence proliferation assay where non - replicating bacteria are both GFP and proliferation dye positive . In contrast , replicating bacteria ( white arrows ) are only GFP positive . The top row depicts purK infected BMDMs without IMP whereas the bottom row depicts macrophages carrying purK S . aureus in the presence of 50 μM IMP . Images were acquired at 12 h post - infection and are representative of three independent experiments . Scale bars equal 10 μm . ( D ) The fraction of macrophages containing replicating bacteria was determined for purK infected BMDMs . Each data point represents a biological replicate ( n = 8 ) and derived from three independent experiments . The data shown are the mean ± standard deviation where the data were normalized to the no IMP condition . * * indicates p < 0 . 01 as determined by an unpaired two - tailed t - test with a Wilcoxon matched pairs test .

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

(C, D) The ability of purK S. aureus to grow in an IMP-dependent manner within primary bone marrow-derived macrophages is shown. (C) Fluorescence micrographs depicting purK replication analyzed by fluorescence proliferation assay where non-replicating bacteria are both GFP and proliferation dye positive. In contrast, replicating bacteria (white arrows) are only GFP positive. The top row depicts purK infected BMDMs without IMP whereas the bottom row depicts macrophages carrying purK S. aureus in the presence of 50 μM IMP. Images were acquired at 12 h post-infection and are representative of three independent experiments. Scale bars equal 10 μm. (D) The fraction of macrophages containing replicating bacteria was determined for purK infected BMDMs. Each data point represents a biological replicate (n=8) and derived from three independent experiments. The data shown are the mean ± standard deviation where the data were normalized to the no IMP condition. ** indicates p<0.01 as determined by an unpaired two-tailed t-test with a Wilcoxon matched pairs test.


B Fatty acid composition of B . subtilis WT cells grown in PF medium , and Δbkd grown with MB , IB or depleted for precursor for 90 min ( IBPF ) . For detailed analyses , see Appendix Fig S1B .

O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O

B Fatty acid composition of B. subtilis WT cells grown in PF medium, and Δbkd grown with MB, IB or depleted for precursor for 90 min (IBPF). For detailed analyses, see Appendix Fig S1B.


E Fatty acid composition of E . coli WT and fabA ( Ts ) cells grown at 30 ° C and shifted to different temperatures for 120 min . For detailed analyses , see Appendix Fig S2B and C .

O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-ORGANISM I-ORGANISM I-ORGANISM O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E Fatty acid composition of E. coli WT and fabA(Ts) cells grown at 30°C and shifted to different temperatures for 120 min. For detailed analyses, see Appendix Fig S2B and C.


A DPH anisotropy of B . subtilis WT or Δbkd cells supplemented either with MB , IB or depleted for precursor ( IBPF ) for the times indicated . High DPH anisotropy indicates low membrane fluidity . B DPH anisotropy of E . coli WT cells grown steady state at 30 ° C , 37 ° C or shifted from 30 ° C to 37 ° C followed by immediate measurement . In addition , DPH anisotropy of fabA ( Ts ) cells grown steady state at 30 ° C or shifted from 30 ° C to 37 ° C followed by measurement at the times indicated .

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

A DPH anisotropy of B. subtilis WT or Δbkd cells supplemented either with MB, IB or depleted for precursor (IBPF) for the times indicated. High DPH anisotropy indicates low membrane fluidity. B DPH anisotropy of E. coli WT cells grown steady state at 30°C, 37°C or shifted from 30°C to 37°C followed by immediate measurement. In addition, DPH anisotropy of fabA(Ts) cells grown steady state at 30°C or shifted from 30°C to 37°C followed by measurement at the times indicated. D


A Images of B . subtilis WT and Δbkd cells co - labelled with the membrane potential - sensitive dye DISC3 ( 5 ) and the membrane permeability indicator Sytox Green . Membrane properties were assessed for Δbkd cells grown in the presence of MB , IB or washed precursor - free ( IBPF ) for the times indicated . As controls , WT cells were measured in the presence of depolarising antimicrobial peptide gramicidin ABC ( gABC ) or pore - forming lantibiotic Nisin . For cross - correlation between membrane depolarisation and membrane permeabilisation , see Appendix Fig S3A - C . B Quantification of DISC3 ( 5 ) fluorescence for cells ( n = 100 - 142 ) depicted in panel A . Median represented by red line . C Images of E . coli WT and fabA ( Ts ) cells co - labelled with the same indicator dyes as in panel A . Membrane properties were assessed for fabA ( Ts ) at 30 ° C and upon transfer to non - permissive 37 ° C for the times indicated . As controls , WT cells were incubated with the pore - forming antibiotic Polymyxin B ( PolyB ) . For cross - correlation between membrane depolarisation and membrane permeabilisation , see Appendix Fig S3D and E . The integrity of the diffusion barrier function was additionally studied via ONPG permeability in a ΔlacY background ( see Fig EV1 ) . D Quantification of DISC3 ( 5 ) fluorescence for cells ( n = 76 - 141 ) depicted in panel C . Median represented by red line .

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

A Images of B. subtilis WT and Δbkd cells co-labelled with the membrane potential-sensitive dye DISC3(5) and the membrane permeability indicator Sytox Green. Membrane properties were assessed for Δbkd cells grown in the presence of MB, IB or washed precursor-free (IBPF) for the times indicated. As controls, WT cells were measured in the presence of depolarising antimicrobial peptide gramicidin ABC (gABC) or pore-forming lantibiotic Nisin. For cross-correlation between membrane depolarisation and membrane permeabilisation, see Appendix Fig S3A-C. B Quantification of DISC3(5) fluorescence for cells (n=100-142) depicted in panel A. Median represented by red line. C Images of E. coli WT and fabA(Ts) cells co-labelled with the same indicator dyes as in panel A. Membrane properties were assessed for fabA(Ts) at 30°C and upon transfer to non-permissive 37°C for the times indicated. As controls, WT cells were incubated with the pore-forming antibiotic Polymyxin B (PolyB). For cross-correlation between membrane depolarisation and membrane permeabilisation, see Appendix Fig S3D and E. The integrity of the diffusion barrier function was additionally studied via ONPG permeability in a ΔlacY background (see Fig EV1). D Quantification of DISC3(5) fluorescence for cells (n=76-141) depicted in panel C. Median represented by red line. Dat


A Images of B . subtilis Δbkd cells stained with membrane dye FM 5 - 95 and expressing GFP fusions of DNA - binding protein HBsu ( top ) , cell division protein FtsZ ( middle ) or cell elongation protein MreB ( bottom ) . Cells were grown with IB or depleted for precursors for 90 min ( IBPF ) . For further examples and additional time points , see Appendix Fig S4A . B Images of E . coli fabA ( Ts ) cells stained with FM 5 - 95 for the outer membrane and with DNA - intercalating dye DAPI ( top ) , or expressing GFP sandwich ( SW ) fusions to the cell division protein FtsZ ( middle ) and the cell elongation protein MreB ( bottom ) , respectively . Depicted are cells grown at 30 ° C or with a temperature shift to 37 ° C for 120 min . For a more detailed view on the influence of low membrane fluidity on membrane dissociation of RNase E as well as on the cell division machinery , see Figs EV2 , EV3 , EV4 , and S5 . For further examples and additional time points , see Appendix Fig S4B .

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

A Images of B. subtilis Δbkd cells stained with membrane dye FM 5-95 and expressing GFP fusions of DNA-binding protein HBsu (top), cell division protein FtsZ (middle) or cell elongation protein MreB (bottom). Cells were grown with IB or depleted for precursors for 90 min (IBPF). For further examples and additional time points, see Appendix Fig S4A. B Images of E. coli fabA(Ts) cells stained with FM 5-95 for the outer membrane and with DNA-intercalating dye DAPI (top), or expressing GFP sandwich (SW) fusions to the cell division protein FtsZ (middle) and the cell elongation protein MreB (bottom), respectively. Depicted are cells grown at 30°C or with a temperature shift to 37°C for 120 min. For a more detailed view on the influence of low membrane fluidity on membrane dissociation of RNase E as well as on the cell division machinery, see Figs EV2, EV3, EV4, and S5. For further examples and additional time points, see Appendix Fig S4B. D


B Images of B . subtilis Δbkd cells grown with IB or without precursor ( IBPF ) . Cells were stained with membrane fluidity - sensitive dye Laurdan and imaged at 460 nm , 520 nm and as the corresponding colour - coded Laurdan GP map . C Images of E . coli fabA ( Ts ) cells grown at 30 ° C or shifted to non - permissive 37 ° C for 120 min . Cells were stained and imaged as described in panel B . For a more pronounced view on domain formation associated with local differences in membrane fluidity , see Appendix Fig S7 , showing E . coli fabA ( Ts ) grown in LB instead of M9 minimal medium with glucose / casamino acids . D Images of B . subtilis Δbkd cells grown and stained with Laurdan ( imaged at 460 nm ) as in panel B , but additionally expressing WALP23 - mCherry . For corresponding fluorescence intensity correlation between images , see Appendix Fig S8A . Co - localisation of membrane dye FM 5 - 95 and transmembrane peptide WALP23 is shown in Appendix Figs S9 and S8C . E Images of E . coli fabA ( Ts ) cells grown and stained as in panel C , but additionally expressing WALP23 - mScarlet - I . For corresponding fluorescence intensity correlation between images , see Appendix Fig S8B .

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

B Images of B. subtilis Δbkd cells grown with IB or without precursor (IBPF). Cells were stained with membrane fluidity-sensitive dye Laurdan and imaged at 460 nm, 520 nm and as the corresponding colour-coded Laurdan GP map. C Images of E. coli fabA(Ts) cells grown at 30°C or shifted to non-permissive 37°C for 120 min. Cells were stained and imaged as described in panel B. For a more pronounced view on domain formation associated with local differences in membrane fluidity, see Appendix Fig S7, showing E. coli fabA(Ts) grown in LB instead of M9 minimal medium with glucose/casamino acids. D Images of B. subtilis Δbkd cells grown and stained with Laurdan (imaged at 460 nm) as in panel B, but additionally expressing WALP23-mCherry. For corresponding fluorescence intensity correlation between images, see Appendix Fig S8A. Co-localisation of membrane dye FM 5-95 and transmembrane peptide WALP23 is shown in Appendix Figs S9 and S8C. E Images of E. coli fabA(Ts) cells grown and stained as in panel C, but additionally expressing WALP23-mScarlet-I. For corresponding fluorescence intensity correlation between images, see Appendix Fig S8B. Dat


A Time lapse images of E . coli fabA ( Ts ) cells expressing FOF1 a - mNG . For corresponding controls , see Appendix Fig S10 . Cells were grown at 30 ° C and shifted from 30 ° C to non - permissive 37 ° C . See Movie EV2 for full time lapse .

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

A Time lapse images of E. coli fabA(Ts) cells expressing FOF1 a-mNG. For corresponding controls, see Appendix Fig S10. Cells were grown at 30°C and shifted from 30°C to non-permissive 37°C. See Movie EV2 for full time lapse.