Concentration - dependent optoDroplet formation exemplified by two neighboring cells that differ in their Cry2 - mCherry - 53BP1 1203 - 1972 W1495A expression level . Cells were imaged at 15 seconds intervals . Quantifications of optoDroplet formation in lowly expressing cells ( mClow ) and highly expressing cells ( mChigh ) from single - cell QIBC analysis of 3 independent experiments are shown with mean ( solid line ) and standard deviation from the mean ( dashed lines ) indicated .

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Concentration-dependent optoDroplet formation exemplified by two neighboring cells that differ in their Cry2-mCherry-53BP1 1203-1972 W1495A expression level. Cells were imaged at 15 seconds intervals. Quantifications of optoDroplet formation in lowly expressing cells (mClow) and highly expressing cells (mChigh) from single-cell QIBC analysis of 3 independent experiments are shown with mean (solid line) and standard deviation from the mean (dashed lines) indicated.


Cry2 - mEGFP - 53BP1 1203 - 1972 W1495A assembles into FokI - induced DNA repair compartments within seconds after light induction . FokI was induced for 1h where indicated before cells were light - activated and imaged at 15 seconds intervals . Red arrowheads mark the FokI - induced lesion with mCherry accumulation .

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Cry2-mEGFP-53BP1 1203-1972 W1495A assembles into FokI-induced DNA repair compartments within seconds after light induction. FokI was induced for 1h where indicated before cells were light-activated and imaged at 15 seconds intervals. Red arrowheads mark the FokI-induced lesion with mCherry accumulation.


Cry2 - mCherry - 53BP1 1203 - 1972 W1495A optoDroplets were induced as indicated and fixed 5 minutes later . Endogenous BRCA1 and p53 were co - stained .

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Cry2-mCherry-53BP1 1203-1972 W1495A optoDroplets were induced as indicated and fixed 5 minutes later. Endogenous BRCA1 and p53 were co-stained.


As in ( A ) , comparing Cry2 - mCherry - 53BP1 constructs with and without the BRCT domains . Quantifications of p53 assembly into 53BP1 optoDroplets from single - cell QIBC analysis of 500 - 600 cells per condition are shown below with mean ( solid line ) and standard deviation from the mean ( dashed lines ) indicated . Red bars indicate the part of 53BP1 that was expressed as Cry2 - mCherry fusion .

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As in (A), comparing Cry2-mCherry-53BP1 constructs with and without the BRCT domains. Quantifications of p53 assembly into 53BP1 optoDroplets from single-cell QIBC analysis of 500-600 cells per condition are shown below with mean (solid line) and standard deviation from the mean (dashed lines) indicated. Red bars indicate the part of 53BP1 that was expressed as Cry2-mCherry fusion.


Western blot of p53 induction upon DNA damage ( 10 Gy , 2h ) with and without 0 . 4 M Sorbitol .

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Western blot of p53 induction upon DNA damage (10 Gy, 2h) with and without 0.4 M Sorbitol.


qPCR of p21 induction upon DNA damage ( 10 Gy , 2h ) with and without 0 . 4 M sorbitol . Mean ± standard deviation is indicated .

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qPCR of p21 induction upon DNA damage (10 Gy, 2h) with and without 0.4 M sorbitol. Mean ± standard deviation is indicated.


Western blot of p21 induction upon DNA damage ( 10 Gy , 2h ) with and without 0 . 4 M sorbitol .

Western blot of p21 induction upon DNA damage (10 Gy, 2h) with and without 0.4 M sorbitol.


QIBC cell cycle analysis based on DNA content and nuclear Cyclin A levels upon DNA damage with and without 0 . 4 M sorbitol . Cells were irradiated with 2 Gy in absence or presence of 0 . 4 M sorbitol . After 1h of recovery , sorbitol - containing medium was exchanged with fresh medium , and cells were allowed to recover for an additional 7h . Percentages in G1 and S / G2 are provided .

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QIBC cell cycle analysis based on DNA content and nuclear Cyclin A levels upon DNA damage with and without 0.4 M sorbitol. Cells were irradiated with 2 Gy in absence or presence of 0.4 M sorbitol. After 1h of recovery, sorbitol-containing medium was exchanged with fresh medium, and cells were allowed to recover for an additional 7h. Percentages in G1 and S/G2 are provided.


( C - J ) All panels show Drosophila 3rd instar larval eye imaginal discs with eyeless - Flippase - induced clones of cells homozygous for EMC3Δ4 labelled by the absence of ubiGFP ( green ) . The red channel shows ELAV , which labels the nuclei of photoreceptor cells . All UAS constructs were expressed under the control of GMR - GAL4 . ( C ) Expression of UAS - Rh1 ( 4C5 , in blue ) and ( D ) Na + K + ATPase ( A5 - C , in blue ) is strongly reduced in EMC3Δ4 homozygous mutant cells . ( E ) Expression of UAS - Xport - A - HA ( anti - HA , in blue ) and ( F ) UAS - fan - HA ( anti - HA , in blue ) is strongly reduced in EMC3Δ4 homozygous mutant cells . ( G ) Expression of UAS - rtv - HA ( anti - HA , in blue ) and ( H ) hid ( anti - hid , in blue ) is increased in EMC3Δ4 homozygous mutant cells . ( I - J ) Normal expression of two TA proteins in EMC3Δ4 homozygous mutant cells . ( I ) UAS - CG8814 - HA ( anti - HA , in blue ) is a TA protein containing a TMD of low hydrophobicity ( J ) UAS - Syx13 - HA is a TA protein with high hydrophobicity TMD . Scale bars represent 10 μm .

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(C-J) All panels show Drosophila 3rd instar larval eye imaginal discs with eyeless-Flippase-induced clones of cells homozygous for EMC3Δ4 labelled by the absence of ubiGFP (green). The red channel shows ELAV, which labels the nuclei of photoreceptor cells. All UAS constructs were expressed under the control of GMR-GAL4. (C) Expression of UAS-Rh1 (4C5, in blue) and (D) Na+K+ATPase (A5-C, in blue) is strongly reduced in EMC3Δ4 homozygous mutant cells. (E) Expression of UAS-Xport-A-HA (anti-HA, in blue) and (F) UAS-fan-HA (anti-HA, in blue) is strongly reduced in EMC3Δ4 homozygous mutant cells. (G) Expression of UAS-rtv-HA (anti-HA, in blue) and (H) hid (anti-hid, in blue) is increased in EMC3Δ4 homozygous mutant cells. (I-J) Normal expression of two TA proteins in EMC3Δ4 homozygous mutant cells. (I) UAS-CG8814-HA (anti-HA, in blue) is a TA protein containing a TMD of low hydrophobicity (J) UAS-Syx13-HA is a TA protein with high hydrophobicity TMD. Scale bars represent 10 μm.


( B - D ) - Immunostaining of 3rd instar larval eye imaginal discs with eyeless - Flippase - induced clones of cells homozygous for EMC3Δ4 , labelled by the absence of ubiGFP ( green ) shows reduced expression of ( B ) UAS - HA - Xport - A ( anti - HA , in blue ) but normal expression of ( C ) UAS - HA - Xport - A4L ( anti - HA , in blue ) or ( D ) UAS - Xport - A4L - HA ( anti - HA , in blue ) in EMC3 mutant homozygous cells . ELAV ( red in B and C ) marks photoreceptor cells and Na + K + ATPase ( red in D ) acts as a positive control for the presence of EMC3Δ4 homozygous mutant clones . The UAS constructs were expressed under the control of GMR - GAL4 . Scale bars represent 10 μm .

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(B-D) - Immunostaining of 3rd instar larval eye imaginal discs with eyeless-Flippase-induced clones of cells homozygous for EMC3Δ4, labelled by the absence of ubiGFP (green) shows reduced expression of (B) UAS-HA-Xport-A (anti-HA, in blue) but normal expression of (C) UAS-HA-Xport-A4L (anti-HA, in blue) or (D) UAS-Xport-A4L-HA (anti-HA, in blue) in EMC3 mutant homozygous cells. ELAV (red in B and C) marks photoreceptor cells and Na+K+ATPase (red in D) acts as a positive control for the presence of EMC3Δ4 homozygous mutant clones. The UAS constructs were expressed under the control of GMR-GAL4. Scale bars represent 10 μm.


( E - F ) Immunostaining of mosaic adult retina with eyeless - Flippase - induced clones of cells homozygous for EMC3e02662 , labelled by the absence of RFP ( red ) , show loss of ( E ) UAS - HA - Xport - A ( anti - HA , in blue ) in EMC3 mutant homozygous cells . ( F ) Expression of UAS - HA - Xport - A4L ( anti - HA , in blue ) is normal in EMC3e02662 homozygous mutant clones . The rhabdomeres are stained for actin ( Phalloidin , in green ) . Scale bars represent 10 μm .

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(E-F) Immunostaining of mosaic adult retina with eyeless-Flippase-induced clones of cells homozygous for EMC3e02662, labelled by the absence of RFP (red), show loss of (E) UAS-HA-Xport-A (anti-HA, in blue) in EMC3 mutant homozygous cells. (F) Expression of UAS-HA-Xport-A4L (anti-HA, in blue) is normal in EMC3e02662 homozygous mutant clones. The rhabdomeres are stained for actin (Phalloidin, in green). Scale bars represent 10 μm.


( B - D ) Histograms of flow cytometry data monitoring the fluorescence protein ratio in the indicated U2OS cell lines for each construct . " EMC5KO + EV " indicates a knockout of EMC5 stably harbouring empty vector , while ' ' EMC5KO + EMC5 " indicates EMC5KO cells rescued by inducible re - expression of a stably integrated EMC5 . ( E - G ) Histograms of flow cytometry data monitoring the fluorescence protein ratio in the indicated U2OS cell lines for each construct . " EMC6KO + EV " indicates a knockout of EMC6 stably harbouring empty vector , while ' ' EMC6KO + EMC6 " indicates EMC6KO cells rescued by re - expression of a stably integrated EMC6 .

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(B-D) Histograms of flow cytometry data monitoring the fluorescence protein ratio in the indicated U2OS cell lines for each construct. "EMC5KO+EV" indicates a knockout of EMC5 stably harbouring empty vector, while ''EMC5KO+EMC5" indicates EMC5KO cells rescued by inducible re-expression of a stably integrated EMC5. (E-G) Histograms of flow cytometry data monitoring the fluorescence protein ratio in the indicated U2OS cell lines for each construct. "EMC6KO+EV" indicates a knockout of EMC6 stably harbouring empty vector, while ''EMC6KO+EMC6" indicates EMC6KO cells rescued by re-expression of a stably integrated EMC6.


( C - G ) ( C - D , F ) Western - Blot of WT and EMC6KO ( 6KO ) U2OS cell lines transiently transfected with the opsin - tagged constructs probed for HA . When indicated , denatured samples were treated with PNGase to confirm glycosylation . Glycosylated proteins shift upwards in the gel and are indicated by arrows (  ) . ( E , G ) Western - Blot of WT and EMC6KO ( 6KO ) U2OS cell lines transiently transfected with the opsin - tagged constructs probed for Actin and EMC6 .

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(C-G) (C-D, F) Western-Blot of WT and EMC6KO (6KO) U2OS cell lines transiently transfected with the opsin-tagged constructs probed for HA. When indicated, denatured samples were treated with PNGase to confirm glycosylation. Glycosylated proteins shift upwards in the gel and are indicated by arrows (). (E, G) Western-Blot of WT and EMC6KO (6KO) U2OS cell lines transiently transfected with the opsin-tagged constructs probed for Actin and EMC6.


( A - B ) Immunostaining of 3rd instar larval eye imaginal discs with eyeless - Flippase - induced clones of cells homozygous for EMC3Δ4 , labelled by the absence of ubiGFP ( green ) show loss of ( A ) UAS - Xport - A - HA ( anti - HA , in blue ) and UAS - Rh1 ( 4C5 , in red ) . ( B ) Expression of UAS - Xport - A4L ( anti - HA , in blue ) and UAS - Rh1 ( 4C5 , in red ) is observed in EMC3Δ4 homozygous cells . The UAS constructs were expressed under the control of GMR - GAL4 . Scale bars represent 10 μm .

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(A-B) Immunostaining of 3rd instar larval eye imaginal discs with eyeless-Flippase-induced clones of cells homozygous for EMC3Δ4, labelled by the absence of ubiGFP (green) show loss of (A) UAS-Xport-A-HA (anti-HA, in blue) and UAS-Rh1 (4C5, in red). (B) Expression of UAS-Xport-A4L (anti-HA, in blue) and UAS-Rh1 (4C5, in red) is observed in EMC3Δ4 homozygous cells. The UAS constructs were expressed under the control of GMR-GAL4. Scale bars represent 10 μm.


( C - D ) Immunostaining of mosaic adult retinas with eyeless - Flippase - induced clones of cells homozygous for EMC3e02662 , labelled by the absence of RFP ( red ) , show loss of ( C ) TRP ( Mab83F6 , in blue ) when UAS - HA - Xport - A is expressed . ( D ) Expression of TRP ( Mab83F6 , in blue ) is observed in EMC3e02662 homozygous cells , when HA - Xport - A4L is expressed . The rhabdomeres are stained for actin ( Phalloidin , in green ) . The UAS constructs were expressed under the control of Rh1 - GAL4 . Scale bars represent 10 μm .

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(C-D) Immunostaining of mosaic adult retinas with eyeless-Flippase-induced clones of cells homozygous for EMC3e02662, labelled by the absence of RFP (red), show loss of (C) TRP (Mab83F6, in blue) when UAS-HA-Xport-A is expressed. (D) Expression of TRP (Mab83F6, in blue) is observed in EMC3e02662 homozygous cells, when HA-Xport-A4L is expressed. The rhabdomeres are stained for actin (Phalloidin, in green). The UAS constructs were expressed under the control of Rh1-GAL4. Scale bars represent 10 μm.


( E - G ) Western blot of fly heads labelled for V5 , TRP , Rh1 , Xport - A ( Xp - A ) and tubulin . ( E ) Immunoblot of fly heads expressing Rh1Gal4 ( driver ) ( left ) or Rh1 TMD1 - V5 ( right ) in Xport - A heterozygous ( Xport - A1 / TM6B ) or homozygous mutant flies ( Xport - A1 / Xport - A1 ) . Extracts were untreated ( - ) or treated ( + ) with PNGase F enzyme ( lanes 2 , 4 , 6 and 8 ) . For Rh1Gal4 , 4 fly heads were loaded per well , while for Rh1 TMD1 - V5 , 8 fly heads were loaded per well . ( F ) Immunoblot of fly heads expressing Rh1 TMD1 - 3 - V5 in Xport - A heterozygous ( Xport - A1 / TM6B ) or homozygous mutant flies ( Xport - A1 / Xport - A1 ) . Extracts were untreated ( - ) or treated ( + ) with PNGase F enzyme ( lanes 2 and 4 ) . Eight fly heads were loaded per lane . ( G ) Immunoblot of fly heads expressing Rh1 TMD1 - 5 - V5 in Xport - A heterozygote ( Xport - A1 / TM6B ) or homozygous mutant flies ( Xport - A1 / Xport - A1 ) . Extracts were untreated ( - ) or treated ( + ) with either Endo - H ( lanes 2 and 5 ) or PNGase F enzyme ( lanes 3 and 6 ) . Each lane contains approximately 2 , 5 fly heads .

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(E-G) Western blot of fly heads labelled for V5, TRP, Rh1, Xport-A (Xp-A) and tubulin. (E) Immunoblot of fly heads expressing Rh1Gal4 (driver) (left) or Rh1 TMD1-V5 (right) in Xport-A heterozygous (Xport-A1/TM6B) or homozygous mutant flies (Xport-A1/Xport-A1). Extracts were untreated (-) or treated (+) with PNGase F enzyme (lanes 2, 4, 6 and 8). For Rh1Gal4, 4 fly heads were loaded per well, while for Rh1 TMD1-V5, 8 fly heads were loaded per well. (F) Immunoblot of fly heads expressing Rh1 TMD1-3-V5 in Xport-A heterozygous (Xport-A1/TM6B) or homozygous mutant flies (Xport-A1/Xport-A1). Extracts were untreated (-) or treated (+) with PNGase F enzyme (lanes 2 and 4). Eight fly heads were loaded per lane. (G) Immunoblot of fly heads expressing Rh1 TMD1-5-V5 in Xport-A heterozygote (Xport-A1/TM6B) or homozygous mutant flies (Xport-A1/Xport-A1). Extracts were untreated (-) or treated (+) with either Endo-H (lanes 2 and 5) or PNGase F enzyme (lanes 3 and 6). Each lane contains approximately 2,5 fly heads.


( B ) The upper panel showing qRT - PCR analysis of four identified LUAD - associated RBPs ( ADARB1 , CELF2 , QKI and ZFP36 ) mRNA levels in 61 LUAD tissues and matched paracarcinoma tissues . The lower panel showing relative mRNA expression ( T / N ) of the four RBPs in metastatic ( n = 28 ) and non - metastatic ( n = 33 ) LUAD tissues . LUAD tissues were classified into metastatic and non - metastatic tissues as described in Materials and Methods . T , LUAD tissues ; N , paracarcinoma tissues . Y axis represents the log10 transformed fold change of mRNA expression ( T / N ) of four RBPs .

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(B) The upper panel showing qRT-PCR analysis of four identified LUAD-associated RBPs (ADARB1, CELF2, QKI and ZFP36) mRNA levels in 61 LUAD tissues and matched paracarcinoma tissues. The lower panel showing relative mRNA expression (T/N) of the four RBPs in metastatic (n = 28) and non-metastatic (n = 33) LUAD tissues. LUAD tissues were classified into metastatic and non-metastatic tissues as described in Materials and Methods. T, LUAD tissues; N, paracarcinoma tissues. Y axis represents the log10 transformed fold change of mRNA expression (T/N) of four RBPs.


( E - G ) Kaplan - Meier survival curves of LC , LUAD and LUSC patients ( n = 1540 , n = 865 and n = 675 , respectively ) with high or low expression levels of QKI . The log - rank test was used to analyze the difference between two groups .

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

(E-G) Kaplan-Meier survival curves of LC, LUAD and LUSC patients (n=1540, n=865 and n=675, respectively) with high or low expression levels of QKI. The log-rank test was used to analyze the difference between two groups.


( H ) qRT - PCR analysis of endogenous mRNA levels of QKI - 5 , QKI - 6 , and QKI - 7 in human bronchial epithelial cell line BEAS - 2B , LUAD cell lines A549 and H1299 .

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

(H) qRT-PCR analysis of endogenous mRNA levels of QKI-5, QKI-6, and QKI-7 in human bronchial epithelial cell line BEAS-2B, LUAD cell lines A549 and H1299.


( J ) In vitro RNA pull - down assays were performed using biotin - labeled TGFβR1 3 ' UTR segments in A549 and H1299 cells . After pull - down , endogenous QKI - 5 enrichments were detected by western blot analyses . β - actin was used as the protein control .

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

(J) In vitro RNA pull-down assays were performed using biotin-labeled TGFβR1 3' UTR segments in A549 and H1299 cells. After pull-down, endogenous QKI-5 enrichments were detected by western blot analyses. β-actin was used as the protein control.


( A and B ) qRT - PCR and western blot analyses of TGFβR1 mRNA and protein levels in QKI - 5 - overexpressing A549 and H1299 cells . ( C and D ) Expression of TGFβR1 mRNA and protein in QKI - 5 - silenced A549 and H1299 cells .

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

(A and B) qRT-PCR and western blot analyses of TGFβR1 mRNA and protein levels in QKI-5-overexpressing A549 and H1299 cells. (C and D) Expression of TGFβR1 mRNA and protein in QKI-5-silenced A549 and H1299 cells.


( K ) QKI - 5 - overexpressing A549 and H1299 cells were treated and subjected to qRT - PCR assays to detect the mRNA expression of downstream genes of the TGF - β / SMAD signaling , including PAI - 1 , Slug , Snail , E - cadherin , and / or N - cadherin .

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

(K) QKI-5-overexpressing A549 and H1299 cells were treated and subjected to qRT-PCR assays to detect the mRNA expression of downstream genes of the TGF-β/SMAD signaling, including PAI-1, Slug, Snail, E-cadherin, and/or N-cadherin.


( C - F ) Wound - healing migration assays were performed on QKI - 5 - overexpressing A549 and H1299 cells as well as control cells The wound healing was recorded ( C and E ) and quantitatively measured ( D and F ) at least six times .

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

(C-F) Wound-healing migration assays were performed on QKI-5-overexpressing A549 and H1299 cells as well as control cells The wound healing was recorded (C and E) and quantitatively measured (D and F) at least six times.


( G and H ) In the presence or absence of TGF - β1 , QKI - 5 - overexpressing A549 and H1299 cells were allowed to migrate through a polycarbonate membrane in Transwells . After 24 h , migrated cells were stained , photographed and counted in at least four random fields under a light microscope . Representative images ( left ) and the migrated cell numbers ( right ) were presented . ( I and J ) QKI - 5 - overexpressing A549 and H1299 cells were treated and allowed to invade through Matrigel - coated membrane in Transwells . Invasive cells were

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

(G and H) In the presence or absence of TGF-β1, QKI-5-overexpressing A549 and H1299 cells were allowed to migrate through a polycarbonate membrane in Transwells. After 24 h, migrated cells were stained, photographed and counted in at least four random fields under a light microscope. Representative images (left) and the migrated cell numbers (right) were presented. (I and J) QKI-5-overexpressing A549 and H1299 cells were treated and allowed to invade through Matrigel-coated membrane in Transwells. Invasive cells were determined


( E ) H & E staining was performed for the evaluation of lung micrometastases . Representative images showing micrometastases of lung tissues from a pair of mice Blue and red arrowheads indicate lung micrometastases of vector group and QKI - 5 - overexpressing group , respectively . ( F ) Dot plots showing the difference of the micrometastases counts in lung tissues between QKI - 5 - overexpressing group and vector control group ( n = 10 mice per group ) . ( G ) Representative microscopic photographs of H & E staining for liver micrometastases in a pair of mice Blue and red arrowheads indicate liver micrometastases . ( H ) Dot plots showing the difference of the micrometastases counts in liver tissues between QKI - 5 - overexpressing group and control vector group ( n = 10 mice per group ) .

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

(E) H&E staining was performed for the evaluation of lung micrometastases. Representative images showing micrometastases of lung tissues from a pair of mice Blue and red arrowheads indicate lung micrometastases of vector group and QKI-5-overexpressing group, respectively. (F) Dot plots showing the difference of the micrometastases counts in lung tissues between QKI-5-overexpressing group and vector control group (n = 10 mice per group). (G) Representative microscopic photographs of H&E staining for liver micrometastases in a pair of mice Blue and red arrowheads indicate liver micrometastases. (H) Dot plots showing the difference of the micrometastases counts in liver tissues between QKI-5-overexpressing group and control vector group (n = 10 mice per group).


( C - F ) In the presence or absence of TGF - β1 , the migratory and invasive capabilities of above - treated A549 and H1299 cells were evaluated by Transwell assays Migrated and invasive cells were stained and counted under a light microscope .

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

(C-F) In the presence or absence of TGF-β1, the migratory and invasive capabilities of above-treated A549 and H1299 cells were evaluated by Transwell assays Migrated and invasive cells were stained and counted under a light microscope.


( J ) Kaplan - Meier survival curves of LUAD patients ( n = 865 ) with high or low expression levels of TGFβR1 . The log - rank test was used to compare the difference between two groups .

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

(J) Kaplan-Meier survival curves of LUAD patients (n=865) with high or low expression levels of TGFβR1. The log-rank test was used to compare the difference between two groups.


qRT - PCR analyses of mRNA expression of QKI - 5 in A549 cells transfected with siRNAs of TFs , which have potential binding sites in QKI - 5 promoter .

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

qRT-PCR analyses of mRNA expression of QKI-5 in A549 cells transfected with siRNAs of TFs, which have potential binding sites in QKI-5 promoter.


( E ) Western blot analysis of QKI - 5 protein levels in KLF6 - silenced A549 cells .

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

(E) Western blot analysis of QKI-5 protein levels in KLF6-silenced A549 cells.


( F ) An overview of KLF6 - ChIP - seq data ( GSE96355 ) is illustrated using Integrative Genomics Viewer ( IGV ) software . An obvious peak was detected at position - 100 to + 100 in the QKI - 5 promoter .

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

(F) An overview of KLF6-ChIP-seq data (GSE96355) is illustrated using Integrative Genomics Viewer (IGV) software. An obvious peak was detected at position -100 to +100 in the QKI-5 promoter.


qRT - PCR analysis of KLF6 mRNA in 61 LUAD tissues and matched paracarcinoma tissues ( I ) T , LUAD tissues ; N , paracarcinoma tissues . Y axis represents the log10 transformed fold change of mRNA expression of KLF6 . Central dotted line of the violin plot represents median , and upper and lower dotted line represents quartile ( I ) .

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

qRT-PCR analysis of KLF6 mRNA in 61 LUAD tissues and matched paracarcinoma tissues (I) T, LUAD tissues; N, paracarcinoma tissues. Y axis represents the log10 transformed fold change of mRNA expression of KLF6. Central dotted line of the violin plot represents median, and upper and lower dotted line represents quartile (I).


C . HEK293 cells were incubated with 5 µg of purified EVCx43 - or EVCx43 + for 24 h , in the presence or absence of Bafilomycin A1 ( Baf ) in the last 6 h . The levels of LC3 were analyzed by WB and are depicted on graph ( LC3 - II ; n = 4 biological replicates ) . Data information : p - values were derived by Mann - Whitney test . Bars and error bars indicate mean ± SD in all graphs .

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

C. HEK293 cells were incubated with 5 µg of purified EVCx43- or EVCx43+ for 24 h, in the presence or absence of Bafilomycin A1 (Baf) in the last 6 h. The levels of LC3 were analyzed by WB and are depicted on graph (LC3-II; n=4 biological replicates). Data information: p-values were derived by Mann-Whitney test. Bars and error bars indicate mean ± SD in all graphs.


E . HEK293 cells were incubated with EVCx43 - or EVCx43 + for 24 h before WB analysis of ATP2A2 / SERCA2 , a predicted target of miRNAs enriched in Cx43 - containing EV . Graph depicts quantification of ATP2A2 / SERCA2 levels ( n = 4 biological replicates ) . Data information : p - values were derived by Mann - Whitney test . Bars and error bars indicate mean ± SD in all graphs .

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

E. HEK293 cells were incubated with EVCx43- or EVCx43+ for 24 h before WB analysis of ATP2A2/SERCA2, a predicted target of miRNAs enriched in Cx43-containing EV. Graph depicts quantification of ATP2A2/SERCA2 levels (n=4 biological replicates). Data information: p-values were derived by Mann-Whitney test. Bars and error bars indicate mean ± SD in all graphs.


A . Cx43 was immunoprecipitated ( IP ) from HEK293Cx43 + cells , after which interaction with hnRNPA2B1 and hnRNPQ was analyzed by WB .

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

A. Cx43 was immunoprecipitated (IP) from HEK293Cx43+ cells, after which interaction with hnRNPA2B1 and hnRNPQ was analyzed by WB.


B . Co - localization between Cx43 ( green ) and hnRNPA2B1 or hnRNPQ ( red ) in C33a cells was analyzed by confocal microscopy . Scale bars 10 μm . Arrowheads in the inset images highlight co - localization between Cx43 and hnRNPs .

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

B. Co-localization between Cx43 (green) and hnRNPA2B1 or hnRNPQ (red) in C33a cells was analyzed by confocal microscopy. Scale bars 10 μm. Arrowheads in the inset images highlight co-localization between Cx43 and hnRNPs.


A . Cell free EV biogenesis was assessed by relative protection of Cx43 - luciferase . Reactions were incubated at 4ºC or 30ºC in the presence of membranes derived from HEK293Cx43 - transiently transfected with Cx43 - luciferase and cytosol from HEK293Cx43 - cells , except in the negative control . 1 % TX100 was used to disrupt membranes , where indicated ( n = 3 biological replicates ) . Data information : p - values were derived by Mann - Whitney test . Bars and error bars indicate mean ± SD in all graphs .

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

A. Cell free EV biogenesis was assessed by relative protection of Cx43-luciferase. Reactions were incubated at 4ºC or 30ºC in the presence of membranes derived from HEK293Cx43- transiently transfected with Cx43-luciferase and cytosol from HEK293Cx43- cells, except in the negative control. 1% TX100 was used to disrupt membranes, where indicated (n=3 biological replicates). Data information: p-values were derived by Mann-Whitney test. Bars and error bars indicate mean ± SD in all graphs.


B . Crude membranes and cytosol from broken HEK293Cx43 + or HEK293Cx43 - cells were mixed in an ATP regenerating system , supplemented with synthetic miR - 133b or miR - 509 - 3p , as indicated . Graph depicts the indicated miRNAs levels , assessed by RT - qPCR and represented as percent protected after RNAse I digestion of unincorporated miRNAs ( n = 7 - 8 biological replicates ) . Data information : p - values were derived by Mann - Whitney test . Bars and error bars indicate mean ± SD in all graphs .

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

B. Crude membranes and cytosol from broken HEK293Cx43+ or HEK293Cx43- cells were mixed in an ATP regenerating system, supplemented with synthetic miR-133b or miR-509-3p, as indicated. Graph depicts the indicated miRNAs levels, assessed by RT-qPCR and represented as percent protected after RNAse I digestion of unincorporated miRNAs (n=7-8 biological replicates). Data information: p-values were derived by Mann-Whitney test. Bars and error bars indicate mean ± SD in all graphs.


C . Co - immunoprecipitation of endogenous miR - 133b and miR - 509 - 3p following UV crosslinking immunoprecipitation ( CLIP ) of Cx43 ( n = 3 - 5 biological replicates ) . Data information : p - values were derived by Mann - Whitney test . Bars and error bars indicate mean ± SD in all graphs .

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

C. Co-immunoprecipitation of endogenous miR-133b and miR-509-3p following UV crosslinking immunoprecipitation (CLIP) of Cx43 (n=3-5 biological replicates). Data information: p-values were derived by Mann-Whitney test. Bars and error bars indicate mean ± SD in all graphs.


D . WB analysis for Cx43 in samples derived by miRNAs pulldowns performed with whole lysates of HEK293Cx43 + cells and biotinylated miR - 133b and miR - 509 - 3p ( n = 5 biological replicates ) . Data information : p - values were derived by Mann - Whitney test . Bars and error bars indicate mean ± SD in all graphs .

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

D. WB analysis for Cx43 in samples derived by miRNAs pulldowns performed with whole lysates of HEK293Cx43+ cells and biotinylated miR-133b and miR-509-3p (n=5 biological replicates). Data information: p-values were derived by Mann-Whitney test. Bars and error bars indicate mean ± SD in all graphs.


EMSA was performed using either Cy3 - labeled miR - 133b ( E , and Cx43 produced by in vitro translation inserted into lipid nanodiscs , For competition EMSA , an excess of unlabeled miR - 133b was used ( competitor ) . Empty nanodiscs were used to control unspecific binding .

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

EMSA was performed using either Cy3-labeled miR-133b (E , and Cx43 produced by in vitro translation inserted into lipid nanodiscs, For competition EMSA, an excess of unlabeled miR-133b was used (competitor). Empty nanodiscs were used to control unspecific binding.


EMSA was performed using either Cy3 - labeled miR - 133b F ) and Cx43 produced by in vitro translation inserted into lipid nanodiscs , in the presence or absence of recombinant GST - hnRNPA2B1 or GST - hnRNPQ , as indicated .

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

EMSA was performed using either Cy3-labeled miR-133b F) and Cx43 produced by in vitro translation inserted into lipid nanodiscs, in the presence or absence of recombinant GST-hnRNPA2B1 or GST-hnRNPQ, as indicated.


EMSA was performed using either Cy3 - labeled miR - 509 - 3p ( G and Cx43 produced by in vitro translation inserted into lipid nanodiscs , For competition EMSA , an excess of unlabeled miR - 133b or miR - 509 - 3p was used ( competitor ) . Empty nanodiscs were used to control unspecific binding .

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

EMSA was performed using either Cy3-labeled miR-509-3p (G and Cx43 produced by in vitro translation inserted into lipid nanodiscs, For competition EMSA, an excess of unlabeled miR-133b or miR-509-3p was used (competitor). Empty nanodiscs were used to control unspecific binding.


EMSA was performed using either Cy3 - labeled miR - 509 - 3p H ) and Cx43 produced by in vitro translation inserted into lipid nanodiscs , in the presence or absence of recombinant GST - hnRNPA2B1 or GST - hnRNPQ , as indicated .

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

EMSA was performed using either Cy3-labeled miR-509-3p H) and Cx43 produced by in vitro translation inserted into lipid nanodiscs, in the presence or absence of recombinant GST-hnRNPA2B1 or GST-hnRNPQ, as indicated.


I . EMSA was performed using either Cy3 - labeled wild type or mutant miR - 199a - 3p , and Cx43 - enriched membranes prepared from C33a parental cells ( Cx43 + ) . Cx43 - membranes were used to control unspecific binding .

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

I. EMSA was performed using either Cy3-labeled wild type or mutant miR-199a-3p, and Cx43-enriched membranes prepared from C33a parental cells (Cx43+). Cx43- membranes were used to control unspecific binding.


Representative maximum projection images of microscopy - based miRNA - protein interaction using beads coated with GST - tagged NT / CT - Cx43 , hnRNPA2B1 or hnRNPQ , as indicated , following the addition of Cy3 - labelled miR - 133b or miR - 509 - 3p . Graph depicts quantification of total fluorescence / bead , as a measure of protein - miRNAs binding . Scale bars 50 μm ( n = 2 biological replicates ; average of 23 beads analyzed / condition ) . Data information : p - values were derived by Kruskal - Wallis ( Dunn ' s post - hoc ) test . Bars and error bars indicate mean ± SD in all graphs .

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

Representative maximum projection images of microscopy-based miRNA-protein interaction using beads coated with GST-tagged NT/CT-Cx43, hnRNPA2B1 or hnRNPQ, as indicated, following the addition of Cy3-labelled miR-133b or miR-509-3p. Graph depicts quantification of total fluorescence/bead, as a measure of protein-miRNAs binding. Scale bars 50 μm (n=2 biological replicates; average of 23 beads analyzed/condition). Data information: p-values were derived by Kruskal-Wallis (Dunn's post-hoc) test. Bars and error bars indicate mean ± SD in all graphs.


A . Equal amounts of EVCx43 + and EVCx43 - labelled with SYTO ® RNASelect ™ green fluorescent stain were incubated with HEK293Cx43 + cells for 30 min , at 37ºC . Scale bars 10 μm ( n = 5 biological replicates ) . p - values were derived by Mann - Whitney test .

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

A. Equal amounts of EVCx43+ and EVCx43- labelled with SYTO® RNASelect™ green fluorescent stain were incubated with HEK293Cx43+ cells for 30 min, at 37ºC. Scale bars 10 μm (n=5 biological replicates). p-values were derived by Mann-Whitney test.


A Left , kymograph from sequential epifluorescence ( 20 × ) images showing cytosolic Ca2 + oscillations in speed ‐ fluctuating immature DC after slow motility phases ( upper and lower panel ) and a change in direction ( lower panel ) . DCs were loaded with the Ca2 + dye Oregon GreenBAPTA 1 ‐ AM , introduced in micro ‐ channels , and imaged every 10 s . Right , sequential epifluorescence ( 20 × ) images ( 1 image / 20 s is shown ) . Instantaneous velocities and intracellular calcium oscillations were measured as described in Materials and Methods . Scale bar : 10 μm .

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

A Left, kymograph from sequential epifluorescence (20×) images showing cytosolic Ca2+ oscillations in speed‐fluctuating immature DC after slow motility phases (upper and lower panel) and a change in direction (lower panel). DCs were loaded with the Ca2+ dye Oregon GreenBAPTA 1‐AM, introduced in micro‐channels, and imaged every 10 s. Right, sequential epifluorescence (20×) images (1 image/20 s is shown). Instantaneous velocities and intracellular calcium oscillations were measured as described in Materials and Methods. Scale bar: 10 μm.


B , C Mean cell velocity of immature DCs ( n > 125 cells from two independent experiments ) ( B ) or CD8 + T lymphocytes ( n > 40 cells from one experiment ) ( C ) migrating in micro ‐ channels with or without BAPTA . Boxes illustrate 10 - 90 percentiles of values , and whiskers represent the range of values . P ‐ values were calculated using a Kruskal - Wallis test .

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 B-GENEPROD 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 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

B, C Mean cell velocity of immature DCs (n > 125 cells from two independent experiments) (B) or CD8+T lymphocytes (n > 40 cells from one experiment) (C) migrating in micro‐channels with or without BAPTA. Boxes illustrate 10-90 percentiles of values, and whiskers represent the range of values. P‐values were calculated using a Kruskal-Wallis test.


D Mean intracellular calcium concentration in speed ‐ fluctuating ( ΔV / V0 > 0 . 3 ; ΔV / V0 relative speed variation divided by the global median velocity ) and non ‐ speed ‐ fluctuating ( ΔV / V0 0 . 3 ) immature DCs ( n = 187 cells from more than three independent experiments ) . P ‐ values were calculated using a Mann - Whitney test .

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

D Mean intracellular calcium concentration in speed‐fluctuating (ΔV/V0 > 0.3; ΔV/V0 relative speed variation divided by the global median velocity) and non‐speed‐fluctuating (ΔV/V0 0.3) immature DCs (n = 187 cells from more than three independent experiments). P‐values were calculated using a Mann-Whitney test.


E , F Velocity fluctuations ( ΔV / V0 ) ( E ) or mean Ca2 + activities ( F ) displayed by DCs migrating in micro ‐ channels in the presence of 100 ng / ml CCL3 . P ‐ values were calculated using a Mann - Whitney test , with respect to non ‐ treated DCs ( NT ) .

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

E, F Velocity fluctuations (ΔV/V0) (E) or mean Ca2+ activities (F) displayed by DCs migrating in micro‐channels in the presence of 100 ng/ml CCL3. P‐values were calculated using a Mann-Whitney test, with respect to non‐treated DCs (NT).


G Mean Ca2 + activities during phases of fast ( > 50 % of max velocity ) and slow ( 50 % of max velocity ) motility in speed ‐ fluctuating DCs ( Supplementary Fig S2F ) ( n = 64 cells , from three independent experiments ) . Activities were normalized to the mean Ca2 + activity shown by each cell during the entire movie . The P ‐ value was calculated using a Mann - Whitney test .

O O B-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 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 B-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

G Mean Ca2+ activities during phases of fast (> 50% of max velocity) and slow ( 50% of max velocity) motility in speed‐fluctuating DCs (Supplementary Fig S2F) (n = 64 cells, from three independent experiments). Activities were normalized to the mean Ca2+ activity shown by each cell during the entire movie. The P‐value was calculated using a Mann-Whitney test.


A Relative gene expression of IP3R1 ( blue ) , IP3R2 ( red ) , and IP3R3 ( green ) in IP3R ‐ silenced DCs . DCs were infected with two lentiviruses encoding for different shRNAs for IP3R isoforms . ShScramble ‐ infected DCs were used as a control . Gene expression was determined by a quantitative PCR . It was calculated with respect to GAPDH expression and normalized to the levels observed in the shScramble . The median plus standard deviation of three independent experiments are shown .

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

A Relative gene expression of IP3R1 (blue), IP3R2 (red), and IP3R3 (green) in IP3R‐silenced DCs. DCs were infected with two lentiviruses encoding for different shRNAs for IP3R isoforms. ShScramble‐infected DCs were used as a control. Gene expression was determined by a quantitative PCR. It was calculated with respect to GAPDH expression and normalized to the levels observed in the shScramble. The median plus standard deviation of three independent experiments are shown.


B Immunoblot for IP3R type 1 and IP3R type 3 in IP3R ‐ silenced DCs . Actin was used as a control . IP3R3 appears as two bands as previously described ( 22 ) .

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

B Immunoblot for IP3R type 1 and IP3R type 3 in IP3R‐silenced DCs. Actin was used as a control. IP3R3 appears as two bands as previously described (22).


C Immunoblot for IP3R type 1 ( top ) and IP3R type 3 ( bottom ) in DCs transduced with lentivirus encoding for different IP3R shRNAs . Actin was used as a control .

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C Immunoblot for IP3R type 1 (top) and IP3R type 3 (bottom) in DCs transduced with lentivirus encoding for different IP3R shRNAs. Actin was used as a control.


D Relative IP3R2 expression in ShScramble ‐ infected DCs ( gray ) , shIP3R1 ( blue ) , and IP3R3 ( green ) ‐ silenced DCs . The experiment was performed as described in ( A ) .

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D Relative IP3R2 expression in ShScramble‐infected DCs (gray), shIP3R1 (blue), and IP3R3 (green)‐silenced DCs. The experiment was performed as described in (A).


F Transmigration assay of shScramble ‐ , IP3R ( 1 , 3 ) A ( blue ) ‐ , IP3R ( 2 , 3 ) B ( red ) ‐ , and IP3R ( 1 , 3 ) C ( green ) ‐ expressing DCs . Cells were loaded in the upper chamber of a 5 ‐ μm pore collagen ‐ coated transwell assay , recovered from the upper and lower chambers after 16 h , and counted . The median from three independent experiments is shown .

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F Transmigration assay of shScramble‐, IP3R(1,3)A (blue)‐, IP3R(2,3)B (red)‐, and IP3R(1,3)C (green)‐expressing DCs. Cells were loaded in the upper chamber of a 5‐μm pore collagen‐coated transwell assay, recovered from the upper and lower chambers after 16 h, and counted. The median from three independent experiments is shown.


G Quantification of the mean cell velocity of shIP3R ( 1 , 3 ) A ( blue ) ‐ , shIP3R ( 2 , 3 ) B ( red ) ‐ , and shIP3R ( 1 , 3 ) C ( green ) ‐ silenced immature DCs migrating in micro ‐ channels . shScramble ‐ infected DCs were used as a control ( gray ) ( n > 100 cells from three independent experiments for shIP3R ( 1 , 3 ) A and shIP3R ( 1 , 3 ) C and two independent experiments for shIP3R ( 2 , 3 ) B ) . Boxes illustrate 10 - 90 percentiles of values , and whiskers represent the range of values . P ‐ values were calculated using a Kruskal - Wallis test .

O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O 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 Quantification of the mean cell velocity of shIP3R(1,3)A (blue)‐, shIP3R(2,3)B (red)‐, and shIP3R(1,3)C (green)‐silenced immature DCs migrating in micro‐channels. shScramble‐infected DCs were used as a control (gray) (n > 100 cells from three independent experiments for shIP3R(1,3)A and shIP3R(1,3)C and two independent experiments for shIP3R(2,3)B). Boxes illustrate 10-90 percentiles of values, and whiskers represent the range of values. P‐values were calculated using a Kruskal-Wallis test.


H Quantification of the mean cell velocity of shIP3R ( 1 , 3 ) C ( green ) ‐ silenced immature DCs migrating in micro ‐ channels in the presence of 2 mM BAPTA . shScramble ‐ infected DCs were used as a control ( gray ) ( n > 70 cells from two independent experiments ) . P ‐ values were calculated using a Kruskal - Wallis test .

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H Quantification of the mean cell velocity of shIP3R(1,3)C (green)‐silenced immature DCs migrating in micro‐channels in the presence of 2 mM BAPTA. shScramble‐infected DCs were used as a control (gray) (n > 70 cells from two independent experiments). P‐values were calculated using a Kruskal-Wallis test.


Analysis of shScramble ( gray ) ‐ , shIP3R ( 1 , 3 ) A ( blue ) ‐ , shIP3R ( 2 , 3 ) B ( red ) ‐ , and shIP3R ( 1 , 3 ) C ( green ) ‐ expressing DCs migrating in micro ‐ channels ( n > 100 cells per condition from 3 independent experiments for shIP3R ( 1 , 3 ) A and shIP3R ( 1 , 3 ) C and two independent experiments for shIP3R ( 2 , 3 ) B ) . A Velocity fluctuations ( ΔV / V0 ) of immature DCs migrating in micro ‐ channels . Boxes illustrate 10 - 90 percentiles of values , and whiskers represent the range of values . P ‐ values were calculated using a Kruskal - Wallis test .

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

Analysis of shScramble (gray)‐, shIP3R(1,3)A (blue)‐, shIP3R(2,3)B (red)‐, and shIP3R(1,3)C (green)‐expressing DCs migrating in micro‐channels (n > 100 cells per condition from 3 independent experiments for shIP3R(1,3)A and shIP3R(1,3)C and two independent experiments for shIP3R(2,3)B).A Velocity fluctuations (ΔV/V0) of immature DCs migrating in micro‐channels. Boxes illustrate 10-90 percentiles of values, and whiskers represent the range of values. P‐values were calculated using a Kruskal-Wallis test.


Analysis of shScramble ( gray ) ‐ , shIP3R ( 1 , 3 ) A ( blue ) ‐ , shIP3R ( 2 , 3 ) B ( red ) ‐ , and shIP3R ( 1 , 3 ) C ( green ) ‐ expressing DCs migrating in micro ‐ channels ( n > 100 cells per condition from 3 independent experiments for shIP3R ( 1 , 3 ) A and shIP3R ( 1 , 3 ) C and two independent experiments for shIP3R ( 2 , 3 ) B ) . B Dot plot showing velocity fluctuations and migration speeds of shScramble ( gray ) ‐ and shIP3R ( 1 , 3 ) C ( green ) ‐ expressing DCs .

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

Analysis of shScramble (gray)‐, shIP3R(1,3)A (blue)‐, shIP3R(2,3)B (red)‐, and shIP3R(1,3)C (green)‐expressing DCs migrating in micro‐channels (n > 100 cells per condition from 3 independent experiments for shIP3R(1,3)A and shIP3R(1,3)C and two independent experiments for shIP3R(2,3)B).B Dot plot showing velocity fluctuations and migration speeds of shScramble (gray)‐ and shIP3R(1,3)C (green)‐expressing DCs.


Analysis of shScramble ( gray ) ‐ , shIP3R ( 1 , 3 ) A ( blue ) ‐ , shIP3R ( 2 , 3 ) B ( red ) ‐ , and shIP3R ( 1 , 3 ) C ( green ) ‐ expressing DCs migrating in micro ‐ channels ( n > 100 cells per condition from 3 independent experiments for shIP3R ( 1 , 3 ) A and shIP3R ( 1 , 3 ) C and two independent experiments for shIP3R ( 2 , 3 ) B ) . C Inverted sequential epifluorescence images ( 20 × ) of a representative control DC ( left ) and an immature shIP3R ( 1 , 3 ) C ‐ silenced DC ( right ) migrating in micro ‐ channels . DCs were differentiated from LifeAct ‐ GFP transgenic mice and imaged every 2 . 2 min .

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

Analysis of shScramble (gray)‐, shIP3R(1,3)A (blue)‐, shIP3R(2,3)B (red)‐, and shIP3R(1,3)C (green)‐expressing DCs migrating in micro‐channels (n > 100 cells per condition from 3 independent experiments for shIP3R(1,3)A and shIP3R(1,3)C and two independent experiments for shIP3R(2,3)B).C Inverted sequential epifluorescence images (20×) of a representative control DC (left) and an immature shIP3R(1,3)C‐silenced DC (right) migrating in micro‐channels. DCs were differentiated from LifeAct‐GFP transgenic mice and imaged every 2.2 min.


Analysis of shScramble ( gray ) ‐ , shIP3R ( 1 , 3 ) A ( blue ) ‐ , shIP3R ( 2 , 3 ) B ( red ) ‐ , and shIP3R ( 1 , 3 ) C ( green ) ‐ expressing DCs migrating in micro ‐ channels ( n > 100 cells per condition from 3 independent experiments for shIP3R ( 1 , 3 ) A and shIP3R ( 1 , 3 ) C and two independent experiments for shIP3R ( 2 , 3 ) B ) . D Percentage of DCs changing their direction of locomotion while migrating in micro ‐ channels . P ‐ values were calculated with a paired test with respect to the shScramble condition .

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

Analysis of shScramble (gray)‐, shIP3R(1,3)A (blue)‐, shIP3R(2,3)B (red)‐, and shIP3R(1,3)C (green)‐expressing DCs migrating in micro‐channels (n > 100 cells per condition from 3 independent experiments for shIP3R(1,3)A and shIP3R(1,3)C and two independent experiments for shIP3R(2,3)B).D Percentage of DCs changing their direction of locomotion while migrating in micro‐channels. P‐values were calculated with a paired test with respect to the shScramble condition.


Analysis of shScramble ( gray ) ‐ , shIP3R ( 1 , 3 ) A ( blue ) ‐ , shIP3R ( 2 , 3 ) B ( red ) ‐ , and shIP3R ( 1 , 3 ) C ( green ) ‐ expressing DCs migrating in micro ‐ channels ( n > 100 cells per condition from 3 independent experiments for shIP3R ( 1 , 3 ) A and shIP3R ( 1 , 3 ) C and two independent experiments for shIP3R ( 2 , 3 ) B ) . E Dot plot showing the frequency of changes in direction of immature DCs while migrating in micro ‐ channels . Bars show the mean plus the standard deviation . A Kruskal - Wallis test was applied for statistical analysis .

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

Analysis of shScramble (gray)‐, shIP3R(1,3)A (blue)‐, shIP3R(2,3)B (red)‐, and shIP3R(1,3)C (green)‐expressing DCs migrating in micro‐channels (n > 100 cells per condition from 3 independent experiments for shIP3R(1,3)A and shIP3R(1,3)C and two independent experiments for shIP3R(2,3)B).E Dot plot showing the frequency of changes in direction of immature DCs while migrating in micro‐channels. Bars show the mean plus the standard deviation. A Kruskal-Wallis test was applied for statistical analysis.


Analysis of shScramble ( gray , n = 2 , 400 cells ) ‐ , shIP3R ( 1 , 3 ) A ( blue , n = 1 , 611 cells ) ‐ , and shIP3R ( 1 , 3 ) C ( green , n = 1 , 454 cells ) ‐ expressing DCs migrating in the device described in Supplementary Fig S3I , stained with Hoechst and imaged every 2 min during 24 h at 10 × magnification . Values were obtained from three independent experiments . Cell trajectories were obtained by tracking their nucleus . A Graph showing the DC mean instantaneous speed . P ‐ values were calculated with a t ‐ test analysis . B Mean instantaneous speed distribution of migrating DCs .

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

Analysis of shScramble (gray, n = 2,400 cells)‐, shIP3R(1,3)A (blue, n = 1,611 cells)‐, and shIP3R(1,3)C (green, n = 1,454 cells)‐expressing DCs migrating in the device described in Supplementary Fig S3I, stained with Hoechst and imaged every 2 min during 24 h at 10× magnification. Values were obtained from three independent experiments. Cell trajectories were obtained by tracking their nucleus.A Graph showing the DC mean instantaneous speed. P‐values were calculated with a t‐test analysis.B Mean instantaneous speed distribution of migrating DCs.


Analysis of shScramble ( gray , n = 2 , 400 cells ) ‐ , shIP3R ( 1 , 3 ) A ( blue , n = 1 , 611 cells ) ‐ , and shIP3R ( 1 , 3 ) C ( green , n = 1 , 454 cells ) ‐ expressing DCs migrating in the device described in Supplementary Fig S3I , stained with Hoechst and imaged every 2 min during 24 h at 10 × magnification . Values were obtained from three independent experiments . Cell trajectories were obtained by tracking their nucleus . C Representation of individual DC trajectories .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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-SUBCELLULAR O O O O O O O O

Analysis of shScramble (gray, n = 2,400 cells)‐, shIP3R(1,3)A (blue, n = 1,611 cells)‐, and shIP3R(1,3)C (green, n = 1,454 cells)‐expressing DCs migrating in the device described in Supplementary Fig S3I, stained with Hoechst and imaged every 2 min during 24 h at 10× magnification. Values were obtained from three independent experiments. Cell trajectories were obtained by tracking their nucleus.C Representation of individual DC trajectories.


Analysis of shScramble ( gray , n = 2 , 400 cells ) ‐ , shIP3R ( 1 , 3 ) A ( blue , n = 1 , 611 cells ) ‐ , and shIP3R ( 1 , 3 ) C ( green , n = 1 , 454 cells ) ‐ expressing DCs migrating in the device described in Supplementary Fig S3I , stained with Hoechst and imaged every 2 min during 24 h at 10 × magnification . Values were obtained from three independent experiments . Cell trajectories were obtained by tracking their nucleus . D Path persistence ( calculated as the ratio between the effective length and the total length of the cell trajectory ) of migrating DCs . P ‐ values were determined with a t ‐ test analysis .

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

Analysis of shScramble (gray, n = 2,400 cells)‐, shIP3R(1,3)A (blue, n = 1,611 cells)‐, and shIP3R(1,3)C (green, n = 1,454 cells)‐expressing DCs migrating in the device described in Supplementary Fig S3I, stained with Hoechst and imaged every 2 min during 24 h at 10× magnification. Values were obtained from three independent experiments. Cell trajectories were obtained by tracking their nucleus.D Path persistence (calculated as the ratio between the effective length and the total length of the cell trajectory) of migrating DCs. P‐values were determined with a t‐test analysis.


Analysis of shScramble ( gray , n = 2 , 400 cells ) ‐ , shIP3R ( 1 , 3 ) A ( blue , n = 1 , 611 cells ) ‐ , and shIP3R ( 1 , 3 ) C ( green , n = 1 , 454 cells ) ‐ expressing DCs migrating in the device described in Supplementary Fig S3I , stained with Hoechst and imaged every 2 min during 24 h at 10 × magnification . Values were obtained from three independent experiments . Cell trajectories were obtained by tracking their nucleus . E Bars showing the proportion of the effective length displacement ( L ) of DCs . A Fisher ' s exact test ( P ‐ value 2 . 2 × 10 − 16 ) was used for statistics .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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-SUBCELLULAR 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

Analysis of shScramble (gray, n = 2,400 cells)‐, shIP3R(1,3)A (blue, n = 1,611 cells)‐, and shIP3R(1,3)C (green, n = 1,454 cells)‐expressing DCs migrating in the device described in Supplementary Fig S3I, stained with Hoechst and imaged every 2 min during 24 h at 10× magnification. Values were obtained from three independent experiments. Cell trajectories were obtained by tracking their nucleus.E Bars showing the proportion of the effective length displacement (L) of DCs. A Fisher's exact test (P‐value 2.2 × 10−16) was used for statistics.


A Immunoblot showing the intracellular levels of phosphorylated MLC in DCs transduced with the shScramble or shRNAs for IP3Rs ( shIP3R ( 1 , 3 ) A ′ , shIP3R ( 2 , 3 ) B ′ , and shIP3R ( 1 , 3 ) C ′ ) . Actin was used as a control .

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A Immunoblot showing the intracellular levels of phosphorylated MLC in DCs transduced with the shScramble or shRNAs for IP3Rs (shIP3R(1,3)A′, shIP3R(2,3)B′, and shIP3R(1,3)C′). Actin was used as a control.


B Inverted sequential epifluorescence images ( 20 × ) showing myosin IIA ‐ GFP localization of a representative shScramble ( left ) ‐ and shIP3R ( 1 , 3 ) C ‐ expressing DC ( right ) migrating in micro ‐ channels . Myosin IIA ‐ GFP knock ‐ in DCs were imaged every 1 min ( 1 out of 2 images is shown ) . Asterisks highlight changes of direction during locomotion .

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B Inverted sequential epifluorescence images (20×) showing myosin IIA‐GFP localization of a representative shScramble (left)‐ and shIP3R(1,3)C‐expressing DC (right) migrating in micro‐channels. Myosin IIA‐GFP knock‐in DCs were imaged every 1 min (1 out of 2 images is shown). Asterisks highlight changes of direction during locomotion.


C , D Myosin IIA ‐ GFP localization ( red ) versus velocity fluctuations ( blue , ΔV / V0 ) in a representative shScramble ‐ transduced DC ( C ) or shIP3R ( 1 , 3 ) C ‐ silenced ( D ) migrating in micro ‐ channels . DCs were recorded as in ( B ) . E Quantification of the interval between curves ( highlighted in D ) obtained for myosin IIA positioning and velocity from shScramble ‐ and shIP3R ( 1 , 3 ) C ‐ expressing DCs . Cells were recorded as in ( B ) ( n = 20 cells from two experiments ) . The Mann - Whitney test was applied for statistical analysis .

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

C, D Myosin IIA‐GFP localization (red) versus velocity fluctuations (blue, ΔV/V0) in a representative shScramble‐transduced DC (C) or shIP3R(1,3)C‐silenced (D) migrating in micro‐channels. DCs were recorded as in (B).E Quantification of the interval between curves (highlighted in D) obtained for myosin IIA positioning and velocity from shScramble‐ and shIP3R(1,3)C‐expressing DCs. Cells were recorded as in (B) (n = 20 cells from two experiments). The Mann-Whitney test was applied for statistical analysis.


A Immunoblot showing the intracellular levels of phosphorylated MLC in DCs treated overnight with increasing amounts of the MLCK inhibitor , ML7 .

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A Immunoblot showing the intracellular levels of phosphorylated MLC in DCs treated overnight with increasing amounts of the MLCK inhibitor, ML7.


B , C Analysis of immature DCs migrating in micro ‐ channels in the presence of ML7 or DMSO as a control ( n > 170 cells from two independent experiments ) . Velocity fluctuations ( ΔV / V0 ) of immature DCs migrating in micro ‐ channels ( B ) . Whiskers show 10 - 90 percentiles plus the median . P ‐ values were calculated using a Kruskal - Wallis test . Frequency of changes ( C ) in direction of DCs migrating in micro ‐ channels . Mean plus SD are shown . A Kruskal - Wallis test was applied for statistical analysis .

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

B, C Analysis of immature DCs migrating in micro‐channels in the presence of ML7 or DMSO as a control (n > 170 cells from two independent experiments). Velocity fluctuations (ΔV/V0) of immature DCs migrating in micro‐channels (B). Whiskers show 10-90 percentiles plus the median. P‐values were calculated using a Kruskal-Wallis test. Frequency of changes (C) in direction of DCs migrating in micro‐channels. Mean plus SD are shown. A Kruskal-Wallis test was applied for statistical analysis.


D , E Analysis of shScramble ( gray ) ‐ and shIP3R ( 1 , 3 ) C ( green ) ‐ expressing DCs migrating in micro ‐ channels in the presence of 20 μM ML7 ( n > 70 cells per condition from 3 independent experiments ) . Velocity fluctuations ( ΔV / V0 ) of immature DCs migrating in micro ‐ channels ( D ) . Boxes illustrate 10 - 90 percentiles of values , and whiskers represent the range of values . P ‐ values were calculated using a Kruskal - Wallis test . Frequency of changes ( E ) in direction observed in DCs migrating in micro ‐ channels . Means plus SD are shown . A Kruskal - Wallis test was applied for statistical analysis .

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

D, E Analysis of shScramble (gray)‐ and shIP3R(1,3)C (green)‐expressing DCs migrating in micro‐channels in the presence of 20 μM ML7 (n > 70 cells per condition from 3 independent experiments). Velocity fluctuations (ΔV/V0) of immature DCs migrating in micro‐channels (D). Boxes illustrate 10-90 percentiles of values, and whiskers represent the range of values. P‐values were calculated using a Kruskal-Wallis test. Frequency of changes (E) in direction observed in DCs migrating in micro‐channels. Means plus SD are shown. A Kruskal-Wallis test was applied for statistical analysis.


Cumulative distribution function of differential ribosome occupancy as detected by ribosome profiling in miRNA _ KO mutants versus WT . Of 759 predicted miRNA target genes , 728 ( 96 % ) were detectable in the ribosome profiling data and appear in the plot . Data information : In A , student ' s t - test was applied to assess the significance for the increase in ribosome occupancy of the 728 / 759 miRNA targets ( p - value < 1e51 for every mutant ) .

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

Cumulative distribution function of differential ribosome occupancy as detected by ribosome profiling in miRNA_KO mutants versus WT. Of 759 predicted miRNA target genes, 728 (96%) were detectable in the ribosome profiling data and appear in the plot. Data information: In A, student's t-test was applied to assess the significance for the increase in ribosome occupancy of the 728/759 miRNA targets (p-value<1e51 for every mutant).


Top : Immunoblot analysis of CIC in WT , Drosha _ KO , Dicer _ KO and Ago2 & 1 _ KO mESCs . TUBULIN was used as a loading control . Blot is a representative image of three biological replicates . Bottom : Bar graph showing quantification of CIC intensity , normalized to TUBULIN and relative to the WT sample in three biological replicates . Data information : Bar graph in Fig 2F shows mean intensity of CIC signal ± SD normalized to TUBULIN . Values are relative to WT , which was set to 1 . P - values were calculated using a student ' s t - test comparing each value to the WT . * p - value < 0 . 05 .

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

Top: Immunoblot analysis of CIC in WT, Drosha_KO, Dicer_KO and Ago2&1_KO mESCs. TUBULIN was used as a loading control. Blot is a representative image of three biological replicates. Bottom: Bar graph showing quantification of CIC intensity, normalized to TUBULIN and relative to the WT sample in three biological replicates. Data information: Bar graph in Fig 2F shows mean intensity of CIC signal ± SD normalized to TUBULIN. Values are relative to WT, which was set to 1. P-values were calculated using a student's t-test comparing each value to the WT. *p-value<0.05.


MA plot of the DGE analysis in miR - 290 - 295 _ KO mESCs . Data information : In A , significant genes in MA plot were determined using an adjusted p - value of 0 . 1 .

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MA plot of the DGE analysis in miR-290-295_KO mESCs. Data information: In A, significant genes in MA plot were determined using an adjusted p-value of 0.1.


F , G . Integrated data for Tfap4 gene shows multiple lines of evidence for its regulation by miR - 290 - 295 . In addition to the data shown and described in Fig 1D , E , a second AGO2 - binding dataset , based on cross - linking immunoprecipitation and sequencing ( CLIP - seq ) , has been integrated . Response elements for miR - 290 - 295 cluster members are denoted in red and labeled .

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

F, G. Integrated data for Tfap4 gene shows multiple lines of evidence for its regulation by miR-290-295. In addition to the data shown and described in Fig 1D, E, a second AGO2-binding dataset, based on cross-linking immunoprecipitation and sequencing (CLIP-seq), has been integrated. Response elements for miR-290-295 cluster members are denoted in red and labeled.


Top : Immunoblot analysis of TFAP4 in WT , Drosha _ KO , Dicer _ KO , Ago2 & 1 _ KO and miR - 290 - 295 _ KO mESCs . LAMINB1 was used as a loading control . Blot is a representative image of three biological replicates . Bottom : Bar graph showing quantification of CIC intensity , normalized to LAMINB1 and relative to the WT sample in three biological replicates . Data information : In H bar graphs show mean intensity of TFAP4 signal ± SD normalized to LAMINB1 Values are relative to WT or the negative control mimic , which was set to 1 . P - values were calculated using a student ' s t - test comparing each value to the WT . * p - value < 0 . 05 , * * p - value < 0 . 01 , * * * * p - value < 0 . 0001 .

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

Top: Immunoblot analysis of TFAP4 in WT, Drosha_KO, Dicer_KO, Ago2&1_KO and miR-290-295_KO mESCs. LAMINB1 was used as a loading control. Blot is a representative image of three biological replicates. Bottom: Bar graph showing quantification of CIC intensity, normalized to LAMINB1 and relative to the WT sample in three biological replicates. Data information: In H bar graphs show mean intensity of TFAP4 signal ± SD normalized to LAMINB1 Values are relative to WT or the negative control mimic, which was set to 1. P-values were calculated using a student's t-test comparing each value to the WT. *p-value<0.05, **p-value<0.01, ****p-value<0.0001.


Top : Immunoblot analysis of TFAP4 after transfection of miRNA mimics ( miR - 291a - 5p , miR - 291a - 3p and miR - 291a - 5p + miR - 291a - 3p combined ) in miR - 290 - 295 _ KO mESCs . Immunoblots were stained with Coomassie blue dye as a loading control . Blot is a representative image of three biological replicates . Bottom : Bar graph showing quantification of TFAP4 intensity , normalized to Coomassie and relative to the WT sample in three biological replicates . Data information : In I , bar graphs show mean intensity of TFAP4 signal ± SD normalized to Coomassie . Values are relative to WT or the negative control mimic , which was set to 1 . P - values were calculated using a student ' s t - test comparing each value to the WT . * p - value < 0 . 05 , * * p - value < 0 . 01 , * * * * p - value < 0 . 0001 .

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

Top: Immunoblot analysis of TFAP4 after transfection of miRNA mimics (miR-291a-5p, miR-291a-3p and miR-291a-5p+miR-291a-3p combined) in miR-290-295_KO mESCs. Immunoblots were stained with Coomassie blue dye as a loading control. Blot is a representative image of three biological replicates. Bottom: Bar graph showing quantification of TFAP4 intensity, normalized to Coomassie and relative to the WT sample in three biological replicates. Data information: In I, bar graphs show mean intensity of TFAP4 signal ± SD normalized to Coomassie. Values are relative to WT or the negative control mimic, which was set to 1. P-values were calculated using a student's t-test comparing each value to the WT. *p-value<0.05, **p-value<0.01, ****p-value<0.0001.


Top : Mature sequences of 5p and 3p miRNAs of the miR - 290 - 295 family and the mature sequence of the human miR - 302c - 3p . Colors indicate identical seed sequences . Bottom : AGO2 CLIP - seq shows a peak at the miR - 302c - 3p binding site on human TFAP4 .

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Top: Mature sequences of 5p and 3p miRNAs of the miR-290-295 family and the mature sequence of the human miR-302c-3p. Colors indicate identical seed sequences. Bottom: AGO2 CLIP-seq shows a peak at the miR-302c-3p binding site on human TFAP4.


Immunoblot validation of siPOOL - mediated knock down of Tfap4 . TFAP4 levels were compared between untreated WT versus miR - 290 - 295 _ KO cells treated with a negative control and a Tfap4 - targeted siPOOL . Immunoblots were stained with Coomassie blue dye as a loading control . Blot is a representative image of two biological replicates .

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Immunoblot validation of siPOOL-mediated knock down of Tfap4. TFAP4 levels were compared between untreated WT versus miR-290-295_KO cells treated with a negative control and a Tfap4-targeted siPOOL. Immunoblots were stained with Coomassie blue dye as a loading control. Blot is a representative image of two biological replicates.


Scatterplot of DEG in miR - 290 - 295 _ KO control versus siPOOL - Tfap4 . SiPOOL experiments were performed in miR - 290 - 295 _ KO cells and DE was assessed relative to a negative control siPOOL transfection . Only genes that are statistically significantly differentially expressed in miR - 290 - 295 _ KO are shown . Genes predicted to be targeted by miR - 290 - 295 are marked in blue . Genes are defined as rescued ( orange ) if the log2FoldChange - ratio between miR - 290 - 295 _ KO control and siPOOL - Tfap4 is in the range [ - 2 , - 1 / 2 ] . Tfap4 is marked by a red dot .

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

Scatterplot of DEG in miR-290-295_KO control versus siPOOL-Tfap4. SiPOOL experiments were performed in miR-290-295_KO cells and DE was assessed relative to a negative control siPOOL transfection. Only genes that are statistically significantly differentially expressed in miR-290-295_KO are shown. Genes predicted to be targeted by miR-290-295 are marked in blue. Genes are defined as rescued (orange) if the log2FoldChange-ratio between miR-290-295_KO control and siPOOL-Tfap4 is in the range [-2, -1/2]. Tfap4 is marked by a red dot.


Representative images of NANOG ( red ) and OCT4 ( green ) immunofluorescence staining in WT , Drosha _ KO and Drosha & Tfap4 _ KO mESCs . Scale bar = 20 μm . Quantification of NANOG fluorescence signal intensity in WT , Drosha _ KO and Drosha & Tfap4 _ KO mESCs show no significant rescue of NANOG intensity in Drosha & Tfap4 _ KO Data information : In E , box plots show nuclear intensity of NANOG in ~ 300 cells per genotype . P - values were generated using ordinary one - way ANOVA . * * * * p - value < 0 . 0001 . In panel E , the box plot ' s central band represents the median relative intensity for each genotype . The boxes represent the interquartile range and the whiskers represent the maximum and minimum relative intensity values .

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

Representative images of NANOG (red) and OCT4 (green) immunofluorescence staining in WT, Drosha_KO and Drosha & Tfap4_KO mESCs. Scale bar = 20 μm. Quantification of NANOG fluorescence signal intensity in WT, Drosha_KO and Drosha & Tfap4_KO mESCs show no significant rescue of NANOG intensity in Drosha & Tfap4_KO Data information: In E, box plots show nuclear intensity of NANOG in ~300 cells per genotype. P-values were generated using ordinary one-way ANOVA. ****p-value<0.0001. In panel E, the box plot's central band represents the median relative intensity for each genotype. The boxes represent the interquartile range and the whiskers represent the maximum and minimum relative intensity values.


( D ) Two - color dSTORM imaging of RIM1 / 2 and gephyrin in cryosections . From left to right : rendered images of RIM1 / 2 and gephyrin clusters showing the aligned SSDs ( arrows ) ; gephyrin SSDs segmented by watershed outlined with different colors ; RIM1 / 2 SSDs outlined with different colors ; binary SSDs of gephyrin and RIM1 / 2 . Inhibitory synapses were identified by the gephyrin clusters in the epifluorescence images . Scale bar : 200 nm . RIM1 / 2 was labelled with Alexa 647 , gephyrin ( mAb7a ) with Cy3B in these experiments .

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

(D) Two-color dSTORM imaging of RIM1/2 and gephyrin in cryosections. From left to right: rendered images of RIM1/2 and gephyrin clusters showing the aligned SSDs (arrows); gephyrin SSDs segmented by watershed outlined with different colors; RIM1/2 SSDs outlined with different colors; binary SSDs of gephyrin and RIM1/2. Inhibitory synapses were identified by the gephyrin clusters in the epifluorescence images. Scale bar: 200 nm. RIM1/2 was labelled with Alexa 647, gephyrin (mAb7a) with Cy3B in these experiments.


( A - C ) Alignment of SSDs of pre - synaptic RIM1 / 2 and SSDs of post - synaptic gephyrin , GlyRs , and GABAARs , respectively . ( A1 - C1 ) Distances were measured between the intensity peaks of paired SSDs in rendered dual - color dSTORM images ( yellow line , values given in mean ± SD ) . RIM1 / 2 was labelled with Alexa 647 and gephyrin ( mAb7a ) with Cy3B in ( A1 ) . RIM1 / 2 was labelled with Cy3B , while GlyRs or GABAARs were labelled with Alexa 647 in ( B1 ) and ( C1 ) . ( A2 - C2 ) Trans - synaptic alignment of SSDs of gephyrin , GlyRs , GABAARs and RIM1 / 2 . SSDs were segmented by H - watershed . Data are plotted as mean ± SD . Number of synapses : n = 48 ( A1 , A2 ) from three independent experiments ( no treatment ) , n = 30 ( B1 , B2 ) from two experiments with TTX treatment , n = 16 ( C1 , C2 ) from two experiments with TTX . Only synapses with side view profiles were included . All images are adjusted to the same scale . Scale bar : 200 nm .

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

(A-C) Alignment of SSDs of pre-synaptic RIM1/2 and SSDs of post-synaptic gephyrin, GlyRs, and GABAARs, respectively. (A1-C1) Distances were measured between the intensity peaks of paired SSDs in rendered dual-color dSTORM images (yellow line, values given in mean ± SD). RIM1/2 was labelled with Alexa 647 and gephyrin (mAb7a) with Cy3B in (A1). RIM1/2 was labelled with Cy3B, while GlyRs or GABAARs were labelled with Alexa 647 in (B1) and (C1). (A2-C2) Trans-synaptic alignment of SSDs of gephyrin, GlyRs, GABAARs and RIM1/2. SSDs were segmented by H-watershed. Data are plotted as mean ± SD. Number of synapses: n = 48 (A1, A2) from three independent experiments (no treatment), n = 30 (B1, B2) from two experiments with TTX treatment, n = 16 (C1, C2) from two experiments with TTX. Only synapses with side view profiles were included. All images are adjusted to the same scale. Scale bar: 200 nm.


( A - B ) Representative dSTORM images of GlyRs and GABAARs at synapses in side view ( A ) or en face view ( B ) . The top row of images shows the pointillist representation of the detections , the second row the rendered images and the bottom row the corresponding segmented SSDs . GlyRs were labelled with Alexa 647 and GABAARs with Cy3B in these experiments . Scale bar : 200 nm .

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

(A-B) Representative dSTORM images of GlyRs and GABAARs at synapses in side view (A) or en face view (B). The top row of images shows the pointillist representation of the detections, the second row the rendered images and the bottom row the corresponding segmented SSDs. GlyRs were labelled with Alexa 647 and GABAARs with Cy3B in these experiments. Scale bar: 200 nm.


( A ) Triple immuno - labelling of gephyrin , GlyRs and GABAARs at mixed inhibitory synapses ( arrow ) imaged with conventional fluorescence microscopy . Scale bar : 5 µm .

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

(A) Triple immuno-labelling of gephyrin, GlyRs and GABAARs at mixed inhibitory synapses (arrow) imaged with conventional fluorescence microscopy. Scale bar: 5 µm.


Characterization of the nanoscale organization of GABAARs and GlyRs at mixed inhibitory synapses . ( A ) Synaptic receptor cluster rendered from detections of both GABAARs ( labelled with Alexa 647 ) and GlyRs ( labelled with Cy3B ) . ( B ) Detections of GABAARs ( red dots ) and GlyRs ( blue dots ) overlaid with binary synaptic mask ( white ) .

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

Characterization of the nanoscale organization of GABAARs and GlyRs at mixed inhibitory synapses. (A) Synaptic receptor cluster rendered from detections of both GABAARs (labelled with Alexa 647) and GlyRs (labelled with Cy3B). (B) Detections of GABAARs (red dots) and GlyRs (blue dots) overlaid with binary synaptic mask (white).


Sub - synaptic characterization of GABAAR and GlyR domains . ( F ) Watershed segmentation of sub - synaptic domains ( SSDs ) . Each SSD is shown in different colors . ( G ) Detections of GABAARs ( red dots ) and GlyRs ( blue dots ) overlaid with SSDs ( white ) .

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

Sub-synaptic characterization of GABAAR and GlyR domains. (F) Watershed segmentation of sub-synaptic domains (SSDs). Each SSD is shown in different colors. (G) Detections of GABAARs (red dots) and GlyRs (blue dots) overlaid with SSDs (white).


( A ) The number of receptor SSDs , segmented from the combined receptor clusters of GABAARs and GlyRs , was decreased after 4 - AP treatment ( MW test , p < 0 . 0001 ) . ( B ) This was mostly due to the loss of GABAAR SSDs that were segmented from the rendered images of GABAAR detections only ( MW , * * * * p < 0 . 0001 ) . ( C ) The number of GlyR SSDs ( based on GlyR detections ) were only marginally reduced ( MW , * * p < 0 . 01 ) . Data information : Number of synapses : n = 452 in TTX , n = 583 in 4 - AP condition , from three independent experiments . Mean ± SEM .

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

(A) The number of receptor SSDs, segmented from the combined receptor clusters of GABAARs and GlyRs, was decreased after 4-AP treatment (MW test, p < 0.0001). (B) This was mostly due to the loss of GABAAR SSDs that were segmented from the rendered images of GABAAR detections only (MW, ****p < 0.0001). (C) The number of GlyR SSDs (based on GlyR detections) were only marginally reduced (MW, **p < 0.01). Data information: Number of synapses: n = 452 in TTX, n = 583 in 4-AP condition, from three independent experiments. Mean ± SEM.