F ) Growth of the cKO - KNL2 cells expressing GFP - fused KNL2 WT or R527A mutant . The upper panel shows examined cell numbers at the indicated time after tet addition ( + tet : KNL2 OFF ) . The cell numbers were normalized to those at 0 h for each line and plotted as relative cell number . The lower panel shows examined untreated cells ( - tet : KNL2 ON ) . Parental cKO - KNL2 chicken DT40 cells ( None ) were also examined .

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F) Growth of the cKO-KNL2 cells expressing GFP-fused KNL2 WT or R527A mutant. The upper panel shows examined cell numbers at the indicated time after tet addition (+tet: KNL2 OFF). The cell numbers were normalized to those at 0 h for each line and plotted as relative cell number. The lower panel shows examined untreated cells (-tet: KNL2 ON). Parental cKO-KNL2 chicken DT40 cells (None) were also examined.


B ) EMSA to examine contribution of the F535 and W536 residues in ggKNL2 to the CENP - A nucleosome binding .

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B) EMSA to examine contribution of the F535 and W536 residues in ggKNL2 to the CENP-A nucleosome binding.


D ) Expression of GFP - KNL2 WT or CENP - A C - terminal tail binding mutants in cKO - KNL2 cells . Expression of full - length KNL2 WT was conditionally turned off by tetracycline ( tet ) addition to the cKO - KNL2 cells ( None ) . GFP - fused KNL2 WT , F535A , W536A or F535A / W536A was stably expressed in the cKO - KNL2 cells . These cells were cultured in the presence or absence of tet ( + tet : KNL2 OFF or − tet : KNL2 ON ) for 48 h . α - Tubulin ( Tub ) was probed as a loading control .

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D) Expression of GFP-KNL2 WT or CENP-A C-terminal tail binding mutants in cKO-KNL2 cells. Expression of full-length KNL2 WT was conditionally turned off by tetracycline (tet) addition to the cKO-KNL2 cells (None). GFP-fused KNL2 WT, F535A, W536A or F535A/W536A was stably expressed in the cKO-KNL2 cells. These cells were cultured in the presence or absence of tet (+tet: KNL2 OFF or −tet: KNL2 ON) for 48 h. α-Tubulin (Tub) was probed as a loading control.


E ) Localization analyses of GFP - fused KNL2 WT and F535A , W536A or F535A / W536A mutant ( green ) . CENP - T was stained as a kinetochore marker ( red ) , and DNA was stained using DAPI ( blue ) . Magnified views are shown in insets . Scale bar indicates 10 μm .

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E) Localization analyses of GFP-fused KNL2 WT and F535A, W536A or F535A/W536A mutant (green). CENP-T was stained as a kinetochore marker (red), and DNA was stained using DAPI (blue). Magnified views are shown in insets. Scale bar indicates 10 μm.


F ) Growth of the cKO - KNL2 cells expressing GFP - fused KNL2 WT or each CENP - A C - terminal tail binding mutant . The upper panel shows examined cell numbers at the indicated time after tet addition ( + tet : KNL2 OFF ) . The cell numbers were normalized to those at 0 h for each line and plotted as relative cell number . The lower panel shows examined untreated cells ( - tet : KNL2 ON ) . Parental cKO - KNL2 chicken DT40 cells ( None ) were also examined .

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F) Growth of the cKO-KNL2 cells expressing GFP-fused KNL2 WT or each CENP-A C-terminal tail binding mutant. The upper panel shows examined cell numbers at the indicated time after tet addition (+tet: KNL2 OFF). The cell numbers were normalized to those at 0 h for each line and plotted as relative cell number. The lower panel shows examined untreated cells (-tet: KNL2 ON). Parental cKO-KNL2 chicken DT40 cells (None) were also examined.


A ) Immunostaining with anti - Mis18α antibody in AID - based KNL2 knockout cells expressing GFP fused KNL2 wild type or each KNL2 mutant in the presence of IAA for 15 h . Clear colocalization of KNL2 signals with Mis18α was observed ( Insets ) . DNA was stained with DAPI . Scale bar indicates 10 µm .

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A) Immunostaining with anti-Mis18α antibody in AID-based KNL2 knockout cells expressing GFP fused KNL2 wild type or each KNL2 mutant in the presence of IAA for 15 h. Clear colocalization of KNL2 signals with Mis18α was observed (Insets). DNA was stained with DAPI. Scale bar indicates 10 µm.


B ) AID - based KNL2 knockout cells expressing GFP fused each KNL2 mutant and SNAP - tagged CENP - A were used . After addition of IAA , SNAP - CENP - A signals were visualized by TMR - Star staining . CENP - T was stained as a kinetochore marker . Representative images were presented . Mitotic centromere localization of each KNL2 mutant was shown as a merged image ( Insets ) : SNAP - CENP - A ( red ) and each KNL2 mutant ( green ) . DNA was stained with DAPI . Scale bar indicates 10 µm .

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B) AID-based KNL2 knockout cells expressing GFP fused each KNL2 mutant and SNAP-tagged CENP-A were used. After addition of IAA, SNAP-CENP-A signals were visualized by TMR-Star staining. CENP-T was stained as a kinetochore marker. Representative images were presented. Mitotic centromere localization of each KNL2 mutant was shown as a merged image (Insets): SNAP-CENP-A (red) and each KNL2 mutant (green). DNA was stained with DAPI. Scale bar indicates 10 µm.


A ) KNL2 ( red ) localization in CENP - C conditional knockout chicken DT40 cells ( cKO - CENP - C ) with or without CENP - C expression . DNA was stained with DAPI ( blue ) . CENP - T was stained as a kinetochore marker ( light blue ) . Scale bar indicates 10 μm .

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A) KNL2 (red) localization in CENP-C conditional knockout chicken DT40 cells (cKO-CENP-C) with or without CENP-C expression. DNA was stained with DAPI (blue). CENP-T was stained as a kinetochore marker (light blue). Scale bar indicates 10 μm.


B ) KNL2 intensities were evaluated in six cells before and after CENP - C knock out . KNL2 signal intensities on the mitotic centromeres ( 14 - 90 centromeres ) in each cell were plotted with the average and the standard deviation . The ratio of KNL2 intensities between cells before and after CENP - C knock out was estimated . KNL2 intensities were reduced by ~ 55 % , when CENP - C was knocked out .

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B) KNL2 intensities were evaluated in six cells before and after CENP-C knock out. KNL2 signal intensities on the mitotic centromeres (14-90 centromeres) in each cell were plotted with the average and the standard deviation. The ratio of KNL2 intensities between cells before and after CENP-C knock out was estimated. KNL2 intensities were reduced by ~55%, when CENP-C was knocked out.


C ) Competitive pull - down assays for the CENP - A nucleosome binding of ggKNL2 and ggCENP - C . A complex of GST - KNL2456 - 560 with the CENP - A nucleosome ( CA - nuc ) was incubated in the absence or presence of MBP - CENP - C619 - 690 ( right panel ) or phosphorylated MBP - CENP - C619 - 690 ( middle panel ) and pulled down by GST affinity . The concentration of CENP - C619 - 690 was increased from 0 μM to 1 μM ( lanes 3 - 7 ) . The ratio of band intensities of pull - downed histones ( * ) to GST - KNL2456 - 560 in each lane ( lanes 3 - 7 ) was estimated and presented in the bar graph ( left panel ) . Data are mean with standard deviations calculated from the technical replicates , n = 3 .

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C) Competitive pull-down assays for the CENP-A nucleosome binding of ggKNL2 and ggCENP-C. A complex of GST-KNL2456-560 with the CENP-A nucleosome (CA-nuc) was incubated in the absence or presence of MBP-CENP-C619-690 (right panel) or phosphorylated MBP-CENP-C619-690 (middle panel) and pulled down by GST affinity. The concentration of CENP-C619-690 was increased from 0 μM to 1 μM (lanes 3-7). The ratio of band intensities of pull-downed histones (*) to GST-KNL2456-560 in each lane (lanes 3-7) was estimated and presented in the bar graph (left panel). Data are mean with standard deviations calculated from the technical replicates, n=3.


Immunohistochemistry of NSMCE2 on mouse testis . The inset illustrates the accumulation of NSMCE2 on the XY body . Scale bar , 50 μm .

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Immunohistochemistry of NSMCE2 on mouse testis. The inset illustrates the accumulation of NSMCE2 on the XY body. Scale bar, 50 μm.


Immunofluorescence ( IF ) of NSMCE2 and SCP3 on isolated mouse spermatocytes . Threads of NSMCE2 can be detected on regions of weak SCP3 signal , indicative of asynapsis ( arrowheads ) . Scale bar , 2 . 5 μm .

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Immunofluorescence (IF) of NSMCE2 and SCP3 on isolated mouse spermatocytes. Threads of NSMCE2 can be detected on regions of weak SCP3 signal, indicative of asynapsis (arrowheads). Scale bar, 2.5 μm.


Colocalization of NSMCE2 and BRCA1 at the XY body of mouse spermatocytes .

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Colocalization of NSMCE2 and BRCA1 at the XY body of mouse spermatocytes.


Representative image of NSMCE2 and BRCA1 foci in control and MMS ‐ treated MEFs . Scale bar , 2 . 5 μm .

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Representative image of NSMCE2 and BRCA1 foci in control and MMS‐treated MEFs. Scale bar, 2.5 μm.


Quantification of NSMCE2 and BRCA1 foci in wild ‐ type MEFs exposed to IR ( 10 Gy ; 1 ‐ h recovery ) , MMS ( 1 mM for 1 h ; 24 ‐ h recovery ) , and PARP inhibitor ( 1 μM for 1 h ; 24 ‐ h recovery ) . Data indicate mean values and are representative of three independent experiments . Error bars indicate SD .

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Quantification of NSMCE2 and BRCA1 foci in wild‐type MEFs exposed to IR (10 Gy; 1‐h recovery), MMS (1 mM for 1 h; 24‐h recovery), and PARP inhibitor (1 μM for 1 h; 24‐h recovery). Data indicate mean values and are representative of three independent experiments. Error bars indicate SD.


Representative image of IR ‐ ( 10 Gy ; 1 ‐ h recovery ) and MMS ‐ ( 1 mM for 1 ‐ h ; 24 ‐ h recovery ) induced NSMCE2 and γH2AX foci in MEFs . Scale bar , 2 . 5 μm .

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Representative image of IR‐ (10 Gy; 1‐h recovery) and MMS‐ (1 mM for 1‐h; 24‐h recovery) induced NSMCE2 and γH2AX foci in MEFs. Scale bar, 2.5 μm.


Brightfield microscopy of 2 . 5 dpc embryos obtained from crosses of Nsmce2GT / + mice . The right panel is of the same 2 embryos shown on the left panel , 36 h after being cultured in vitro . The arrow indicates embryos that showed asymmetric cell sizes when isolated , which degenerated upon culture . These embryos were identified as Nsmce2GT / GT through genotyping .

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Brightfield microscopy of 2.5 dpc embryos obtained from crosses of Nsmce2GT/+ mice. The right panel is of the same 2 embryos shown on the left panel, 36 h after being cultured in vitro. The arrow indicates embryos that showed asymmetric cell sizes when isolated, which degenerated upon culture. These embryos were identified as Nsmce2GT/GT through genotyping.


Examples of the three genotypes found at Mendelian frequencies ( see Appendix Table S1 ) on 2 . 5 dpc embryos .

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Examples of the three genotypes found at Mendelian frequencies (see Appendix Table S1) on 2.5 dpc embryos.


Examples of the type of nuclei found on Nsmce2GT / GT and Nsmce2 + ( + / + or + / GT ) 2 . 5 dpc embryos , 12 h after in vitro culture . Mutant embryos were identified due to the absence of NSMCE2 signal ( green ) . DAPI was used to stain DNA ( blue ) . White arrows indicate examples of cells with condensed chromosomes or irregular shapes , which are hardly seen in wild type embryos .

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Examples of the type of nuclei found on Nsmce2GT/GT and Nsmce2+ (+/+ or +/GT) 2.5 dpc embryos, 12 h after in vitro culture. Mutant embryos were identified due to the absence of NSMCE2 signal (green). DAPI was used to stain DNA (blue). White arrows indicate examples of cells with condensed chromosomes or irregular shapes, which are hardly seen in wild type embryos.


Immunoprecipitation of SMC5 ( and IgG , as a control ) from a 48 ‐ h culture of B lymphocytes of wt and Nsmce2SD / SD animals . NSMCE2 and SMC6 levels on the immunoprecipitates were analyzed by WB . The image of the Ponceau staining is shown as a loading control .

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Immunoprecipitation of SMC5 (and IgG, as a control) from a 48‐h culture of B lymphocytes of wt and Nsmce2SD/SD animals. NSMCE2 and SMC6 levels on the immunoprecipitates were analyzed by WB. The image of the Ponceau staining is shown as a loading control.


IF of NSMCE2 foci in MMS ‐ treated Nsmce2 + / + and Nsmce2SD / SD MEFs . Scale bar , 2 . 5 μm .

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IF of NSMCE2 foci in MMS‐treated Nsmce2+/+ and Nsmce2SD/SD MEFs. Scale bar, 2.5 μm.


Representative picture of 6 ‐ month ‐ old Nsmce2 + / + and Nsmce2SD / SD littermates .

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Representative picture of 6‐month‐old Nsmce2+/+ and Nsmce2SD/SD littermates.


Kaplan - Meyer survival curves of Nsmce2 + / + and Nsmce2SD / SD mice . The P ‐ value was calculated with the Mantel - Cox long ‐ rank test .

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Kaplan-Meyer survival curves of Nsmce2+/+ and Nsmce2SD/SD mice. The P‐value was calculated with the Mantel-Cox long‐rank test.


A Kaplan - Meyer survival curves of Nsmce2 + / + and Nsmce2 + / GT mice . The P ‐ value was calculated with the Mantel - Cox long ‐ rank test . * * * P 0 . 001 .

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A Kaplan-Meyer survival curves of Nsmce2+/+ and Nsmce2+/GT mice. The P‐value was calculated with the Mantel-Cox long‐rank test. ***P 0.001.


B Tumor incidence found on Nsmce2 + / + and Nsmce2 + / GT mice at the time of death ( i . e . moribund mice ) .

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B Tumor incidence found on Nsmce2+/+ and Nsmce2+/GT mice at the time of death (i.e. moribund mice).


C NSMCE2 levels evaluated by Western blot in 2 independent pairs of Nsmce2 + / + and Nsmce2 + / GT MEFs . β ‐ actin was used as a loading control . Numbers shown bellow indicate the relative amounts of NSMCE2 in each sample .

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C NSMCE2 levels evaluated by Western blot in 2 independent pairs of Nsmce2+/+ and Nsmce2+/GT MEFs. β‐actin was used as a loading control. Numbers shown bellow indicate the relative amounts of NSMCE2 in each sample.


D Sister chromatid exchange ( SCE ) events found per metaphase on Nsmce2 + / + and Nsmce2 + / GT MEFs . * * * P 0 . 001 .

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D Sister chromatid exchange (SCE) events found per metaphase on Nsmce2+/+ and Nsmce2+/GT MEFs. ***P 0.001.


E , F Relative percentages of micronuclei ( E ) and polynucleated cells ( F ) on cultures of Nsmce2 + / + and Nsmce2 + / GT MEFs . Data are representative of three independent experiments . Error bars indicate SD . * * * P 0 . 001 .

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E, F Relative percentages of micronuclei (E) and polynucleated cells (F) on cultures of Nsmce2+/+ and Nsmce2+/GT MEFs. Data are representative of three independent experiments. Error bars indicate SD. ***P 0.001.


NSMCE2 levels assessed by WB in UQ . CreERT2 transgenic Nsmce2 + / + and Nsmce2lox / lox littermate MEFs , 24 h after exposure to 4 ‐ OHT . Tubulin levels are shown as a loading control .

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NSMCE2 levels assessed by WB in UQ.CreERT2 transgenic Nsmce2+/+ and Nsmce2lox/lox littermate MEFs, 24 h after exposure to 4‐OHT. Tubulin levels are shown as a loading control.


Clonogenic assay on immortalized UQ . CreERT2 / Nsmce2lox / lox MEFs . The picture shows the colonies detected after 10 days of growth in the presence or absence of 4 ‐ OHT , upon staining with crystal violet .

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Clonogenic assay on immortalized UQ.CreERT2/Nsmce2lox/lox MEFs. The picture shows the colonies detected after 10 days of growth in the presence or absence of 4‐OHT, upon staining with crystal violet.


NSMCE2 and BRCA1 foci in MMS ‐ treated ( 1 mM for 1 h ; 24 ‐ h recovery ) UQ . CreERT2 / Nsmce2lox / lox MEFs , 48 h after being treated ( or not ) with 4 ‐ OHT . Scale bar , 5 μm .

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NSMCE2 and BRCA1 foci in MMS‐treated (1 mM for 1 h; 24‐h recovery) UQ.CreERT2/Nsmce2lox/lox MEFs, 48 h after being treated (or not) with 4‐OHT. Scale bar, 5 μm.


SCE events per metaphase on UQ . CreERT2 / Nsmce2lox / lox MEFs grown in the presence of 4 ‐ OHT for 72 h . * * * P 0 . 001 .

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SCE events per metaphase on UQ.CreERT2/Nsmce2lox/lox MEFs grown in the presence of 4‐OHT for 72 h. ***P 0.001.


Example of SCE events ( arrows ) found on Nsmce2 ‐ deleted MEFs .

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Example of SCE events (arrows) found on Nsmce2‐deleted MEFs.


SCE events at telomeres on UQ . CreERT2 / Nsmce2lox / lox MEFs grown in the presence of 4 ‐ OHT for 72 h , as detected by CO ‐ FISH ( see ) . Data are representative of three independent experiments . Error bars indicate SD . * P 0 . 05 .

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SCE events at telomeres on UQ.CreERT2/Nsmce2lox/lox MEFs grown in the presence of 4‐OHT for 72 h, as detected by CO‐FISH (see ). Data are representative of three independent experiments. Error bars indicate SD. *P 0.05.


Telomere lengths of UQ . CreERT2 Nsmce2lox / lox MEFs treated or not with 4 ‐ OHT for 72 h .

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Telomere lengths of UQ.CreERT2 Nsmce2lox/lox MEFs treated or not with 4‐OHT for 72 h.


Example of the intensity of telomere signals ( yellow ) used for ( H ) .

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Example of the intensity of telomere signals (yellow) used for (H).


Percentage of cells with micronuclei found on cultures of UQ . CreERT2 / Nsmce2lox / lox MEFs treated or not with 4 ‐ OHT for 72 h . Data are representative of three independent experiments . Error bars indicate SD . * * * P 0 . 001 .

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Percentage of cells with micronuclei found on cultures of UQ.CreERT2/Nsmce2lox/lox MEFs treated or not with 4‐OHT for 72 h. Data are representative of three independent experiments. Error bars indicate SD. ***P 0.001.


Intrauterine dwarfism observed upon NSMCE2 deletion on 17 . 5 dpc embryos . Pregnant females were treated at 14 . 5 dpc for 3 days with intraperitoneal injections of 4 ‐ OHT . The image represents the difference in size observed between Nsmce2 + / + and Nsmce2lox / lox embryos carrying the UQ . CreERT2 transgene ( Nsmce2 + / + and Nsmce2lox / lox , from now on ) .

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Intrauterine dwarfism observed upon NSMCE2 deletion on 17.5 dpc embryos. Pregnant females were treated at 14.5 dpc for 3 days with intraperitoneal injections of 4‐OHT. The image represents the difference in size observed between Nsmce2+/+ and Nsmce2lox/lox embryos carrying the UQ.CreERT2 transgene (Nsmce2+/+ and Nsmce2lox/lox, from now on).


Representative picture of Nsmce2 + / + and Nsmce2lox / lox animals fed for 40 weeks with a 4 ‐ OHT ‐ containing diet ( see also Movies EV1 and EV2 ) .

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Representative picture of Nsmce2+/+ and Nsmce2lox/lox animals fed for 40 weeks with a 4‐OHT‐containing diet (see also Movies EV1 and EV2).


Images illustrating the presence of altered pigmentation problems that arise on Nsmce2lox / lox mice .

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Images illustrating the presence of altered pigmentation problems that arise on Nsmce2lox/lox mice.


Percentage of fat against total body mass on Nsmce2 + / + and Nsmce2lox / lox animals . * * * P 0 . 001 .

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Percentage of fat against total body mass on Nsmce2+/+ and Nsmce2lox/lox animals. ***P 0.001.


White bloodcell counts ( WBC ) from Nsmce2 + / + and Nsmce2lox / lox animals . * * * P 0 . 001 .

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White bloodcell counts (WBC) from Nsmce2+/+ and Nsmce2lox/lox animals. ***P 0.001.


Percentage of γH2AX ‐ positive micronuclei found on the intestinal epithelia of Nsmce2 + / + and Nsmce2lox / lox animals ( see Appendix Fig S11 ) . Data are representative of three independent experiments . Error bars indicate SD . * P 0 . 05 .

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Percentage of γH2AX‐positive micronuclei found on the intestinal epithelia of Nsmce2+/+ and Nsmce2lox/lox animals (see Appendix Fig S11). Data are representative of three independent experiments. Error bars indicate SD. *P 0.05.


A WB illustrating the depletion of NSMCE2 in B cells isolated from 2 ‐ month ‐ old CD19 + / Cre animals of the indicated genotypes , 48 h after being stimulated in vitro with lipopolysaccharide ( LPS ) . Tubulin was used as a loading control .

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A WB illustrating the depletion of NSMCE2 in B cells isolated from 2‐month‐old CD19+/Cre animals of the indicated genotypes, 48 h after being stimulated in vitro with lipopolysaccharide (LPS). Tubulin was used as a loading control.


B Representative FACS analysis from the B ‐ cell cultures mentioned in ( A ) . DNA content was measured with propidium iodide ( PI ) . Note the presence of cells with > 4n DNA content in NSMCE2 ‐ deleted cells .

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

B Representative FACS analysis from the B‐cell cultures mentioned in (A). DNA content was measured with propidium iodide (PI). Note the presence of cells with > 4n DNA content in NSMCE2‐deleted cells.


C , D Frequency of origin firing ( C ) and fork rate ( D ) were analyzed by DNA fiber analyses ( see ) from fibers prepared from B ‐ cell cultures explained in ( A ) . No significant differences were observed between Nsmce2 + / + and Nsmce2lox / loxB cells . Data are representative of two independent experiments . Error bars indicate SD . Between 200 and 300 tracks were measured to estimate fork rate and 300 - 500 for the calculation of origin firing frequencies .

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

C, D Frequency of origin firing (C) and fork rate (D) were analyzed by DNA fiber analyses (see ) from fibers prepared from B‐cell cultures explained in (A). No significant differences were observed between Nsmce2+/+ and Nsmce2lox/loxB cells. Data are representative of two independent experiments. Error bars indicate SD. Between 200 and 300 tracks were measured to estimate fork rate and 300-500 for the calculation of origin firing frequencies.


E Representative images of the DNA fibers used for the analysis explained in ( C , D ) . CldU ( red ) and IdU ( green ) channels are shown .

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E Representative images of the DNA fibers used for the analysis explained in (C, D). CldU (red) and IdU (green) channels are shown.


Representative pictures of the spleens from 2 ‐ month ‐ old CD19 + / Cre animals of the indicated genotypes .

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Representative pictures of the spleens from 2‐month‐old CD19+/Cre animals of the indicated genotypes.


Numbers of B and T cells from 2 ‐ month ‐ old CD19 + / Cre animals of the indicated genotypes evaluated by flow cytometry ( B220 : B ‐ cell marker ; CD3 : T ‐ cell marker ) .

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Numbers of B and T cells from 2‐month‐old CD19+/Cre animals of the indicated genotypes evaluated by flow cytometry (B220: B‐cell marker; CD3: T‐cell marker).


SCE events per metaphase on B cells obtained from CD19 + / Cre animals of the indicated genotypes , 72 h after being stimulated in vitro with LPS . * * P 0 . 01 , * * * P 0 . 001 .

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SCE events per metaphase on B cells obtained from CD19+/Cre animals of the indicated genotypes, 72 h after being stimulated in vitro with LPS. **P 0.01, ***P 0.001.


Analysis of forward ( FSC ) and side ( SSC ) scatter parameters of B cells obtained from CD19 + / Cre animals of the indicated genotypes , 48 h after being stimulated in vitro with LPS . The emergence of bigger cells is noticeable on cells lacking both BLM and NSMCE2 .

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Analysis of forward (FSC) and side (SSC) scatter parameters of B cells obtained from CD19+/Cre animals of the indicated genotypes, 48 h after being stimulated in vitro with LPS. The emergence of bigger cells is noticeable on cells lacking both BLM and NSMCE2.


Representative images of B ‐ cell cultures of the indicated genotypes 48 h after being stimulated in vitro with LPS . Insets provide a magnified view of an individual cell ( arrowhead ) for better comparison of the observed differences in cell size . Scale bar , 100 μm .

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Representative images of B‐cell cultures of the indicated genotypes 48 h after being stimulated in vitro with LPS. Insets provide a magnified view of an individual cell (arrowhead) for better comparison of the observed differences in cell size. Scale bar, 100 μm.


Examples of the types of nuclei found on Blm and Nsmce2 double ‐ mutant B cells ( compared with wild ‐ type B cells ) , in cells obtained from ( E ) . DAPI was used to stain DNA . Whereas wt nuclei are regular in size , double ‐ mutant cells invariably presented enlarged , multilobulated , and irregular nuclei . Scale bar , 2 . 5 μm .

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

Examples of the types of nuclei found on Blm and Nsmce2 double‐mutant B cells (compared with wild‐type B cells), in cells obtained from (E). DAPI was used to stain DNA. Whereas wt nuclei are regular in size, double‐mutant cells invariably presented enlarged, multilobulated, and irregular nuclei. Scale bar, 2.5 μm.


B Western blot analyses after induced expression of FLAG - tagged UPF3A in WT HEK293 and HeLa cells ( n = 1 ) . Tubulin serves as control . The asterisk indicates unspecific bands .

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B Western blot analyses after induced expression of FLAG-tagged UPF3A in WT HEK293 and HeLa cells (n=1). Tubulin serves as control. The asterisk indicates unspecific bands.


C Fraction of expressed genes ( genes with non - zero counts in DESeq2 ) were calculated which exhibit individual or combinations of differential gene expression ( DGE ) , differential transcript usage ( DTU ) and / or alternative splicing ( AS ) events in HEK293 and HeLa WT cells overexpressing UPF3A using the respective computational analysis ( cutoffs are indicated ) . AS and DTU events were collapsed on the gene level . For DGE , p - values were calculated by DESeq2 using a two - sided Wald test and corrected for multiple testing using the Benjamini - Hochberg method . For DTU , p - values were calculated by IsoformSwitchAnalyzeR ( ISAR ) using a DEXSeq - based test and corrected for multiple testing using the Benjamini - Hochberg method . For AS , p - values were calculated by LeafCutter using an asymptotic Chi - squared distribution and corrected for multiple testing using the Benjamini - Hochberg method .

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 B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-CELL_LINE O B-CELL_LINE 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 O O O O O O O O O O O O O O O O O O O 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 Fraction of expressed genes (genes with non-zero counts in DESeq2) were calculated which exhibit individual or combinations of differential gene expression (DGE), differential transcript usage (DTU) and/or alternative splicing (AS) events in HEK293 and HeLa WT cells overexpressing UPF3A using the respective computational analysis (cutoffs are indicated). AS and DTU events were collapsed on the gene level. For DGE, p-values were calculated by DESeq2 using a two-sided Wald test and corrected for multiple testing using the Benjamini-Hochberg method. For DTU, p-values were calculated by IsoformSwitchAnalyzeR (ISAR) using a DEXSeq-based test and corrected for multiple testing using the Benjamini-Hochberg method. For AS, p-values were calculated by LeafCutter using an asymptotic Chi-squared distribution and corrected for multiple testing using the Benjamini-Hochberg method.


D Volcano plot showing the differential gene expression analyses from the RNA - Seq dataset of HEK293 and HeLa WT cells overexpressing UPF3A . The log2 fold change is plotted against the - log10 adjusted p - value ( adj . p - value ) . P - values were calculated by DESeq2 using a two - sided Wald test and corrected for multiple testing using the Benjamini - Hochberg method . OE = overexpression .

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D Volcano plot showing the differential gene expression analyses from the RNA-Seq dataset of HEK293 and HeLa WT cells overexpressing UPF3A. The log2 fold change is plotted against the -log10 adjusted p-value (adj. p-value). P-values were calculated by DESeq2 using a two-sided Wald test and corrected for multiple testing using the Benjamini-Hochberg method. OE = overexpression.


E Read coverage of SMG5 from WT HEK293 and HeLa RNA - seq data with or without induced UPF3A overexpression shown as Integrative Genomics Viewer ( IGV ) snapshot . Differential gene expression ( from DESeq2 ) is indicated as log2 fold change ( log2FC ) on the right . Schematic representation of the protein coding transcript below .

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

E Read coverage of SMG5 from WT HEK293 and HeLa RNA-seq data with or without induced UPF3A overexpression shown as Integrative Genomics Viewer (IGV) snapshot. Differential gene expression (from DESeq2) is indicated as log2 fold change (log2FC) on the right. Schematic representation of the protein coding transcript below.


A Western blot analysis of WT and UPF3A KO cells ( clones 4 , 14 and 20 ) with the indicated siRNA treatments ( n = 1 ) . UPF3A and UPF3B protein levels were detected , Tubulin serves as control . The asterisk indicates unspecific bands .

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A Western blot analysis of WT and UPF3A KO cells (clones 4, 14 and 20) with the indicated siRNA treatments (n=1). UPF3A and UPF3B protein levels were detected, Tubulin serves as control. The asterisk indicates unspecific bands.


B Quantitative RT - PCR of the indicated cell lines treated with the indicated siRNAs for 2 or 6 days . For RSRC2 and SRSF2 the ratio of NMD isoform to canonical isoform was calculated . ZFAS1 expression was normalized to C1orf43 reference . Data points and means are plotted as log2 fold change ( log2FC ) ( n = 3 for RSRC2 and SRSF2 , n = 4 for ZFAS1 ) .

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B Quantitative RT-PCR of the indicated cell lines treated with the indicated siRNAs for 2 or 6 days. For RSRC2 and SRSF2 the ratio of NMD isoform to canonical isoform was calculated. ZFAS1 expression was normalized to C1orf43 reference. Data points and means are plotted as log2 fold change (log2FC) (n=3 for RSRC2 and SRSF2, n=4 for ZFAS1).


C Fraction of expressed genes ( genes with non - zero counts in DESeq2 ) were calculated which exhibit individual or combinations of differential gene expression ( DGE ) , differential transcript usage ( DTU ) and / or alternative splicing ( AS ) events in the indicated conditions using the respective computational analysis ( cutoffs are indicated ) . AS and DTU events were collapsed on the gene level . For DGE , p - values were calculated by DESeq2 using a two - sided Wald test and corrected for multiple testing using the Benjamini - Hochberg method . For DTU , p - values were calculated by IsoformSwitchAnalyzeR using a DEXSeq - based test and corrected for multiple testing using the Benjamini - Hochberg method . For AS , p - values were calculated by LeafCutter using an asymptotic Chi - squared distribution and corrected for multiple testing using the Benjamini - Hochberg method .

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 B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Fraction of expressed genes (genes with non-zero counts in DESeq2) were calculated which exhibit individual or combinations of differential gene expression (DGE), differential transcript usage (DTU) and/or alternative splicing (AS) events in the indicated conditions using the respective computational analysis (cutoffs are indicated). AS and DTU events were collapsed on the gene level. For DGE, p-values were calculated by DESeq2 using a two-sided Wald test and corrected for multiple testing using the Benjamini-Hochberg method. For DTU, p-values were calculated by IsoformSwitchAnalyzeR using a DEXSeq-based test and corrected for multiple testing using the Benjamini-Hochberg method. For AS, p-values were calculated by LeafCutter using an asymptotic Chi-squared distribution and corrected for multiple testing using the Benjamini-Hochberg method.


D , E Volcano plots showing the differential gene expression analyses from the indicated RNA - Seq datasets ( D UPF3A KO clone 14 , E UPF3A KO clone 20 ) . The log2 fold change is plotted against the - log10 adjusted p - value ( adj . p - value ) . P - values were calculated by DESeq2 using a two - sided Wald test and corrected for multiple testing using the Benjamini - Hochberg

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

D,E Volcano plots showing the differential gene expression analyses from the indicated RNA-Seq datasets (D UPF3A KO clone 14, E UPF3A KO clone 20). The log2 fold change is plotted against the -log10 adjusted p-value (adj. p-value). P-values were calculated by DESeq2 using a two-sided Wald test and corrected for multiple testing using the Benjamini-Hochberg method


A Western blot analysis of WT and UPF3B KO cells ( clones 90 and 91 ) combined with the indicated knockdowns ( n = 1 ) . UPF3A and UPF3B ( AK - 141 ) protein levels were detected , Tubulin serves as control . Protein levels of UPF3A were quantified , normalized to tubulin expression , and shown as datapoints and mean . Fold - changes of relevant conditions are shown . The asterisk indicates unspecific bands .

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A Western blot analysis of WT and UPF3B KO cells (clones 90 and 91) combined with the indicated knockdowns (n=1). UPF3A and UPF3B (AK-141) protein levels were detected, Tubulin serves as control. Protein levels of UPF3A were quantified, normalized to tubulin expression, and shown as datapoints and mean. Fold-changes of relevant conditions are shown. The asterisk indicates unspecific bands.


B Quantitative RT - PCR of the indicated cell lines with the indicated knockdowns . For RSRC2 and SRSF2 the ratio of NMD isoform to canonical isoform was calculated . Data points and means are plotted as log2 fold change ( log2FC , n = 3 ) .

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B Quantitative RT-PCR of the indicated cell lines with the indicated knockdowns. For RSRC2 and SRSF2 the ratio of NMD isoform to canonical isoform was calculated. Data points and means are plotted as log2 fold change (log2FC, n=3).


D , E Volcano plots showing the differential transcript usage ( via IsoformSwitchAnalyzeR ) in various RNA - Seq data . Isoforms containing GENCODE ( release 33 ) annotated PTC ( red , TRUE ) , regular stop codons ( blue , FALSE ) or having no annotated open reading frame ( grey , NA ) are indicated . The change in isoform fraction ( dIF ) is plotted against the - log10 adjusted p - value ( adj . p - value ) . Density plots show the distribution of filtered isoforms in respect to the dIF , cutoffs were | dIF | > 0 . 1 and adj . p - value < 0 . 05 . P - values were calculated by IsoformSwitchAnalyzeR using a DEXSeq - based test and corrected for multiple testing using the Benjamini - Hochberg method .

O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Volcano plots showing the differential transcript usage (via IsoformSwitchAnalyzeR) in various RNA-Seq data. Isoforms containing GENCODE (release 33) annotated PTC (red, TRUE), regular stop codons (blue, FALSE) or having no annotated open reading frame (grey, NA) are indicated. The change in isoform fraction (dIF) is plotted against the -log10 adjusted p-value (adj. p-value). Density plots show the distribution of filtered isoforms in respect to the dIF, cutoffs were |dIF| > 0.1 and adj. p-value < 0.05. P-values were calculated by IsoformSwitchAnalyzeR using a DEXSeq-based test and corrected for multiple testing using the Benjamini-Hochberg method.


A Western blot analysis of WT , UPF3B KO and UPF3A - UPF3B double KO cells ( clones 1 and 2 ) ( n = 1 ) . UPF3A and UPF3B protein levels were detected , Tubulin serves as control . The asterisk indicates unspecific bands .

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A Western blot analysis of WT, UPF3B KO and UPF3A-UPF3B double KO cells (clones 1 and 2) (n=1). UPF3A and UPF3B protein levels were detected, Tubulin serves as control. The asterisk indicates unspecific bands.


C Quantitative RT - PCR of the indicated samples with the indicated KDs . For RSRC2 and SRSF2 the ratio of NMD isoform to canonical isoform was calculated . ZFAS1 expression was normalized to C1orf43 reference . Data points and means are plotted as log2 fold change ( log2FC , n = 3 ) .

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C Quantitative RT-PCR of the indicated samples with the indicated KDs. For RSRC2 and SRSF2 the ratio of NMD isoform to canonical isoform was calculated. ZFAS1 expression was normalized to C1orf43 reference. Data points and means are plotted as log2 fold change (log2FC, n=3).


E Northern blot analysis of globin reporter and xrFrag . Ethidium bromide stained 28S and 18S rRNAs are shown as controls . Quantification results are shown as data points and mean ( n = 3 ) .

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E Northern blot analysis of globin reporter and xrFrag. Ethidium bromide stained 28S and 18S rRNAs are shown as controls. Quantification results are shown as data points and mean (n=3).


A - C Volcano plots of label free mass spectrometry - based analysis of the interaction partners of UPF2 in WT cells treated with control siRNAs and the UPF3B KO clone 90 and dKO clone 1 both treated with siRNAs targeting UPF3B ( n = 4 biologically independent samples ) . ( A ) FLAG - UPF2 in WT against FLAG - GST control in WT cells , ( B ) UPF2 in 3B KO cells against FLAG control in WT cells , ( C ) UPF2 in dKO cells against FLAG control in WT cells . Points labeled in purple indicate NMD factors ; points labeled in turquoise indicate EJC components . Cut offs : Log2 fold change ≥

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

A-C Volcano plots of label free mass spectrometry-based analysis of the interaction partners of UPF2 in WT cells treated with control siRNAs and the UPF3B KO clone 90 and dKO clone 1 both treated with siRNAs targeting UPF3B (n = 4 biologically independent samples). (A) FLAG-UPF2 in WT against FLAG-GST control in WT cells, (B) UPF2 in 3B KO cells against FLAG control in WT cells, (C) UPF2 in dKO cells against FLAG control in WT cells. Points labeled in purple indicate NMD factors; points labeled in turquoise indicate EJC components. Cut offs: Log2 fold change ≥ 0


E Representative Western blot after FLAG co - immunoprecipitation ( IP ) of FLAG - tagged GST ( control ) or UPF3B WT and single mutant constructs in UPF3B KO cells ( n = 3 ) .

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

E Representative Western blot after FLAG co-immunoprecipitation (IP) of FLAG-tagged GST (control) or UPF3B WT and single mutant constructs in UPF3B KO cells (n = 3).


F Western blot analysis of WT and UPF3B KO clone 90 with Luciferase and UPF3A KDs respectively . Monitored expression of the FLAG - tagged UPF3B rescue construct shown in ( D ) . Rescue construct protein levels were detected with anti - FLAG and anti - UPF3B ( AK - 141 ) antibodies . Tubulin serves as control ( n = 1 ) .

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

F Western blot analysis of WT and UPF3B KO clone 90 with Luciferase and UPF3A KDs respectively. Monitored expression of the FLAG-tagged UPF3B rescue construct shown in (D). Rescue construct protein levels were detected with anti-FLAG and anti-UPF3B (AK-141) antibodies. Tubulin serves as control (n=1).


G Quantitative RT - PCR of the samples from ( F ) . For RSRC2 and SRSF2 the ratio of NMD isoform to canonical isoform was calculated . ZFAS1 expression was normalized to C1orf43 reference . Data points and means are plotted as log2 fold change ( log2FC , n = 3 ) .

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

G Quantitative RT-PCR of the samples from (F). For RSRC2 and SRSF2 the ratio of NMD isoform to canonical isoform was calculated. ZFAS1 expression was normalized to C1orf43 reference. Data points and means are plotted as log2 fold change (log2FC, n=3).


B Western blot analysis of WT and UPF3B KO clone 90 with Luciferase and UPF3A KDs respectively . Monitored expression of the FLAG - tagged UPF3A and UPF3B rescue construct shown in ( A ) . Rescue construct protein levels were detected with anti - FLAG , anti - UPF3A and anti - UPF3B ( AK - 141 ) antibodies . Tubulin serves as control ( n = 1 ) . The asterisk indicates unspecific bands .

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

B Western blot analysis of WT and UPF3B KO clone 90 with Luciferase and UPF3A KDs respectively. Monitored expression of the FLAG-tagged UPF3A and UPF3B rescue construct shown in (A). Rescue construct protein levels were detected with anti-FLAG, anti-UPF3A and anti-UPF3B (AK-141) antibodies. Tubulin serves as control (n=1). The asterisk indicates unspecific bands.


C Quantitative RT - PCR of the samples from ( B ) . For RSRC2 and SRSF2 the ratio of NMD isoform to canonical isoform was calculated . Data points and means are plotted as log2 fold change ( log2FC ) ( n = 3 ) .

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

C Quantitative RT-PCR of the samples from (B). For RSRC2 and SRSF2 the ratio of NMD isoform to canonical isoform was calculated. Data points and means are plotted as log2 fold change (log2FC) (n=3).


B Representative adhesion frequency ( Pa ) vs . contact duration ( tc ) curves forNKG2D expressing NK cells ( n ≥ 3 ) in contact with RBCs ( n ≥ 3 ) coated with a ligand ( MICA in red , MICB in orange , ULBP1 in green , or ULBP3 in blue ) at different contact durations , fitted by non - linear in - situ binding kinetics model Site densities of NKG2D ( mr ) and its ligands ( ml ) are indicated . Data information : Error bars are mean ± SEM for at least three independent biological experiments where * P < 0 . 05 , * * P < 0 . 01 ( two - tailed unpaired t - test ) .

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

B Representative adhesion frequency (Pa) vs. contact duration (tc) curves forNKG2D expressing NK cells (n ≥ 3) in contact with RBCs (n ≥ 3) coated with a ligand (MICA in red, MICB in orange, ULBP1 in green, or ULBP3 in blue) at different contact durations, fitted by non-linear in-situ binding kinetics model Site densities of NKG2D (mr) and its ligands (ml) are indicated. Data information: Error bars are mean ± SEM for at least three independent biological experiments where *P < 0.05, **P < 0.01 (two-tailed unpaired t-test).


In - situ force - free affinities ( C ) , on - rates ( D ) of NKG2D binding with indicated ligands from mammalian cells . The in - situ force - free kinetics were obtained from fittings with in - situ binding kinetics modeling in ( B ) . Data information : Every dot represents one independent binding experiment . Error bars are mean ± SEM for at least three independent biological experiments where * P < 0 . 05 , * * P < 0 . 01 ( two - tailed unpaired t - test ) .

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

In-situ force-free affinities (C), on-rates (D) of NKG2D binding with indicated ligands from mammalian cells. The in-situ force-free kinetics were obtained from fittings with in-situ binding kinetics modeling in (B). Data information: Every dot represents one independent binding experiment. Error bars are mean ± SEM for at least three independent biological experiments where *P < 0.05, **P < 0.01 (two-tailed unpaired t-test).


In - situ force - free off - rates ( E ) of NKG2D binding with indicated ligands from mammalian cells . The in - situ force - free kinetics were obtained from fittings with in - situ binding kinetics modeling in ( B ) . Data information : Every dot represents one independent binding experiment . Error bars are mean ± SEM for at least three independent biological experiments where * P < 0 . 05 , * * P < 0 . 01 ( two - tailed unpaired t - test ) .

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

In-situ force-free off-rates (E) of NKG2D binding with indicated ligands from mammalian cells. The in-situ force-free kinetics were obtained from fittings with in-situ binding kinetics modeling in (B). Data information: Every dot represents one independent binding experiment. Error bars are mean ± SEM for at least three independent biological experiments where *P < 0.05, **P < 0.01 (two-tailed unpaired t-test).


F Detection range comparison in affinity measurement of NKG2D and indicated ligands between in - situ and in - solution assay . Bars in different colors are the ratios of the affinities of indicated ligands divided by that of ULBP1 . Data information : In - solution affinities of proteins purified from E . coli In - solution affinities of proteins purified from mammalian cells were measured by

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

F Detection range comparison in affinity measurement of NKG2D and indicated ligands between in-situ and in-solution assay. Bars in different colors are the ratios of the affinities of indicated ligands divided by that of ULBP1. Data information: In-solution affinities of proteins purified from E. coli In-solution affinities of proteins purified from mammalian cells were measured by BLI


IFN - γ release assay and IFN - γ production ( B ) by human peripheral NK cells stimulated with plate - coated NKG2D ligands ( MICA in red , MICB in green and ULBP3 in blue ) assessed by Cytometric Bead Array , of which are their 50 % effective concentration ( EC50 ) ( C ) . Data information : The IFN - γ release assay in ( B ) was one representative experiment of three total independent experiments . Data points in ( B ) and bars in ( C ) represent mean values . Error bars in ( B ) and ( C ) represent mean ± SEM . * * * P < 0 . 001 , * * * * P < 0 . 0001 ( two - tailed unpaired t - test ) .

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

IFN-γ release assay and IFN-γ production (B) by human peripheral NK cells stimulated with plate-coated NKG2D ligands (MICA in red, MICB in green and ULBP3 in blue) assessed by Cytometric Bead Array, of which are their 50% effective concentration (EC50) (C). Data information: The IFN-γ release assay in (B) was one representative experiment of three total independent experiments. Data points in (B) and bars in (C) represent mean values. Error bars in (B) and (C) represent mean ± SEM. ***P < 0.001, **** P <0.0001 (two-tailed unpaired t-test).


D - F Plots of reciprocals of EC50 vs . the effective in - situ affinities ( AcKa ) ( D ) , the effective in - situ on - rate ( AcKon ) ( E ) and in - situ off - rate ( koff ) ( F ) .

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D-F Plots of reciprocals of EC50 vs. the effective in-situ affinities (AcKa) (D), the effective in-situ on-rate (AcKon) (E) and in-situ off-rate (koff) (F).


C Verification of the binding specificity of NKG2D with different ligands . Data information : Every dot represents the adhesion frequency of one cell - bead pair and the experimental data came from at least two to three repeated trials . Horizontal lines represent mean values . Error bars represent mean ± SEM .

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

C Verification of the binding specificity of NKG2D with different ligands. Data information: Every dot represents the adhesion frequency of one cell-bead pair and the experimental data came from at least two to three repeated trials. Horizontal lines represent mean values. Error bars represent mean ± SEM.


E , F Force - dependent bond lifetimes of NKG2D and various ligands at 5 pN , 10 pN and 15 pN ( E ) and full force spectrum ( F ) . Data information : Bond lifetimes in ( E ) and ( F ) ( in which n = 1505 for MICA , n = 1852 for MICB , n = 531 for ULBP1 , n = 1241 for ULBP3 ) of all ligands with NKG2D came from at least 19 NK cell - bead pairs of at least four independent biological experiments . bars in ( E ) , and data points in ( F ) represent mean values . Error bars represent mean ± SEM .

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

E, F Force-dependent bond lifetimes of NKG2D and various ligands at 5 pN, 10 pN and 15 pN (E) and full force spectrum (F). Data information: Bond lifetimes in (E) and (F) (in which n = 1505 for MICA, n = 1852 for MICB, n = 531 for ULBP1, n = 1241 for ULBP3) of all ligands with NKG2D came from at least 19 NK cell-bead pairs of at least four independent biological experiments. bars in (E), and data points in (F) represent mean values. Error bars represent mean ± SEM.


C The force vs . extension trajectories for the simulations are shown in ( A ) and ( B ) . Occurrence of the sudden extension changes are indicated in the shaded area and time points correspond with the snapshots in ( A ) and ( B ) are marked .

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C The force vs. extension trajectories for the simulations are shown in (A) and (B). Occurrence of the sudden extension changes are indicated in the shaded area and time points correspond with the snapshots in (A) and (B) are marked.


F Distance vs . time curves for force - induced H - bond formation between indicated residues within NKG2D - MICA binding interfaces . The dashed red lines represent H - bonds whose distances are less than 3 . 5 Å .

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

F Distance vs. time curves for force-induced H-bond formation between indicated residues within NKG2D-MICA binding interfaces. The dashed red lines represent H-bonds whose distances are less than 3.5 Å.


A , B Abolishments of force - induced binding residues by 3A mutations ( MICA 3A , n = 526 ) and TAT mutations ( MICA TAT , n = 580 ) impairs NKG2D ' s catch bond with MICA WT ( n = 1505 ) ( A ) and IFN - γ release ( B ) . Data information : Bond lifetimes in ( A ) of MICA WT and mutants with NKG2D came from at least 21 pairs of cells and beads of at least three repeated experiments . IFN - γ release of NK cells in ( B ) was one representative experiment of three total independent experiments . Data points represent mean values . Error bars represent mean ± SEM for biological triplicate experiments . * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ( two - tailed unpaired t - test ) .

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

A, B Abolishments of force-induced binding residues by 3A mutations (MICA 3A, n = 526) and TAT mutations (MICA TAT, n= 580) impairs NKG2D's catch bond with MICA WT (n = 1505) (A) and IFN-γ release (B). Data information: Bond lifetimes in (A) of MICA WT and mutants with NKG2D came from at least 21 pairs of cells and beads of at least three repeated experiments. IFN-γ release of NK cells in (B) was one representative experiment of three total independent experiments. Data points represent mean values. Error bars represent mean ± SEM for biological triplicate experiments. **P < 0.01, ***P < 0.001, **** P <0.0001 (two-tailed unpaired t-test).


50 % effective concentration ( EC50 ) of NK cells stimulated by indicated MICA mutants Data information : horizontal lines represent mean values . Error bars represent mean ± SEM for biological triplicate experiments . * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ( two - tailed unpaired t - test ) .

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50% effective concentration (EC50) of NK cells stimulated by indicated MICA mutants Data information: horizontal lines represent mean values. Error bars represent mean ± SEM for biological triplicate experiments. **P < 0.01, ***P < 0.001, **** P <0.0001 (two-tailed unpaired t-test).


IFN - γ release of NK cells stimulated by indicated MICA mutants at a concentration of 11nM ( D ) and 22nM ( E ) . Data information : bars in ( E ) , and data point in ( F ) represent mean values . Error bars represent mean ± SEM for biological triplicate experiments . * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ( two - tailed unpaired t - test ) .

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IFN-γ release of NK cells stimulated by indicated MICA mutants at a concentration of 11nM (D) and 22nM (E). Data information: bars in (E), and data point in (F) represent mean values. Error bars represent mean ± SEM for biological triplicate experiments. **P < 0.01, ***P < 0.001, **** P <0.0001 (two-tailed unpaired t-test).


A - C Plots and Pearson correlation analysis of reciprocals of EC50 vs . the force - dependent in - situ koff at 5 pN ( A ) , 10 pN ( B ) 15pN ( C ) .

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A-C Plots and Pearson correlation analysis of reciprocals of EC50 vs. the force-dependent in-situ koff at 5 pN (A), 10 pN (B) 15pN (C).


I Detection range comparison in force - dependent affinity of NKG2D and indicated ligands under difference force . Bars in different colors are the ratios of the affinities of indicated ligands divided by that of ULBP1 .

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I Detection range comparison in force-dependent affinity of NKG2D and indicated ligands under difference force. Bars in different colors are the ratios of the affinities of indicated ligands divided by that of ULBP1.


D Contour plots showed probabilities of output signals at 300s produced by continuously variable initial on - rates and force - dependent off - rates .

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D Contour plots showed probabilities of output signals at 300s produced by continuously variable initial on-rates and force-dependent off-rates.


( A ) Assessment of vacuole ( arrows ) formation in neutrophils after stimulation with PMA ( II ) and rapamycin ( III ) , compared with control cells ( I : treated with PBS and serum ) ( magnification 1000 × ) .

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(A) Assessment of vacuole (arrows) formation in neutrophils after stimulation with PMA (II) and rapamycin (III), compared with control cells (I: treated with PBS and serum) (magnification 1000×).


( B ) ( I ) Presence of late autophagosomes as observed with MDC staining in cells treated with PMA and rapamycin ( RAP ) compared with control cells ( PBS ) and cells treated with PMA and 3 ‐ MA ( magnification 400 and 1000 × ) . ( II ) Percentage of PMN with acidified vacuoles as detected by MDC staining . Data are representative of six independent experiments and are presented as mean ± SD ; Wilcoxon matched ‐ pairs test , * p < 0 . 05 : PMA ( bar 2 : 86 . 16 ± 6 . 49 % ) , rapamycin ( bar 3 : 65 . 83 ± 8 . 47 % ) treated cells , cells treated with PMA and 3 ‐ MA ( bar 4 : 9 . 17 ± 0 . 75 % ) and cells treated with rapamycin and 3 ‐ MA ( bar 5 : 8 . 5 ± 1 . 87 % ) compared with control conditions ( bar 1 : 15 . 67 ± 3 . 50 % ) . The percentage of cells with acidified autophagosomes in PMN treated with PMA and 3 ‐ MA ( bar 4 ) or rapamycin and 3 ‐ MA ( bar 5 ) was significantly lower ( p < 0 . 05 ) compared with cells treated with PMA ( bar 2 ) and rapamycin ( bar 3 ) , respectively .

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

(B) (I) Presence of late autophagosomes as observed with MDC staining in cells treated with PMA and rapamycin (RAP) compared with control cells (PBS) and cells treated with PMA and 3‐MA (magnification 400 and 1000×). (II) Percentage of PMN with acidified vacuoles as detected by MDC staining. Data are representative of six independent experiments and are presented as mean±SD; Wilcoxon matched‐pairs test, *p<0.05: PMA (bar 2: 86.16±6.49%), rapamycin (bar 3: 65.83±8.47%) treated cells, cells treated with PMA and 3‐MA (bar 4: 9.17±0.75%) and cells treated with rapamycin and 3‐MA (bar 5: 8.5±1.87%) compared with control conditions (bar 1: 15.67±3.50%). The percentage of cells with acidified autophagosomes in PMN treated with PMA and 3‐MA (bar 4) or rapamycin and 3‐MA (bar 5) was significantly lower (p<0.05) compared with cells treated with PMA (bar 2) and rapamycin (bar 3), respectively.


( C ) Analysis of LC3B ‐ I to LC3B ‐ II conversion in cells treated with PMA ( lane IV ) and rapamycin ( lane II ) , as observed by LC3B immunoblotting . Effect of 3 ‐ MA in both PMA ( lane V ) and rapamycin ( lane I ) ‐ treated cells . Lane III : control cells , and lane VI : cells treated only with 3 ‐ MA . One representative out of six independent experiments is shown .

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

(C) Analysis of LC3B‐I to LC3B‐II conversion in cells treated with PMA (lane IV) and rapamycin (lane II), as observed by LC3B immunoblotting. Effect of 3‐MA in both PMA (lane V) and rapamycin (lane I)‐treated cells. Lane III: control cells, and lane VI: cells treated only with 3‐MA. One representative out of six independent experiments is shown.


( D ) Impact of PI ‐ 3K inhibition using LY294002 on LC3B conversion after PMA challenge ( lane III ) , ( lane I : control cells , lane II : PMA , lane IV : LY294002 only ) . One representative out of four independent experiments is shown .

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

(D) Impact of PI‐3K inhibition using LY294002 on LC3B conversion after PMA challenge (lane III), (lane I: control cells, lane II: PMA, lane IV: LY294002 only). One representative out of four independent experiments is shown.


( E ) Immunofluorescence confocal microscopy staining of LC3B . ( I ) Control cells treated with PBS and serum , ( II ) PMA ‐ treated cells . 1 . DAPI : blue , 2 . LC3B : red and 3 . merge . ( III ) Percentage of PMN with LC3B aggregates . Control conditions ( bar 1 : 17 . 7 ± 2 . 4 % ) , 3 ‐ MA ( bar 2 : 3 ± 1 . 01 % ) , PMA ( bar 3 : 69 ± 2 . 1 % ) , PMA and 3 ‐ MA ( bar 4 : 4 ± 1 . 9 % ) , rapamycin ( bar 5 : 56 ± 1 . 6 % ) . Data are representative of five independent experiments and presented as mean ± SD ; Student ' s t ‐ test , * p < 0 . 01 compared to the control ( bar 1 ) or the indicated comparision ( bar 3 vs 4 ) .

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

(E) Immunofluorescence confocal microscopy staining of LC3B. (I) Control cells treated with PBS and serum, (II) PMA‐treated cells. 1. DAPI: blue, 2. LC3B: red and 3. merge. (III) Percentage of PMN with LC3B aggregates. Control conditions (bar 1: 17.7±2.4%), 3‐MA (bar 2: 3±1.01%), PMA (bar 3: 69±2.1%), PMA and 3‐MA (bar 4: 4±1.9%), rapamycin (bar 5: 56±1.6%). Data are representative of five independent experiments and presented as mean±SD; Student's t‐test, *p<0.01 compared to the control (bar 1) or the indicated comparision (bar 3 vs 4).


ATG3 and LC3B gene expression pattern in neutrophils after stimulation with PMA or rapamycin . ATG3 mRNA expression analysis ( fold ) after PMA ( 2 . 44 ± 0 . 78 ) and rapamycin ( RAP : 1 . 81 ± 0 . 41 ) stimulation . LC3B gene alterations ( PMA : 4 . 98 ± 0 . 59 ) and rapamycin ( RAP : 1 . 7 ± 0 . 55 ) . Effect of 3 ‐ MA pre ‐ treatment , for both stimuli , was also assessed ( PMA + 3 ‐ MA : ATG3 : 1 . 36 ± 0 . 64 , LC3B : 1 . 5 ± 0 . 49 and rapamycin + 3 ‐ MA : ATG3 : 1 . 28 ± 0 . 46 , LC3B : 1 . 24 ± 0 . 57 ) . No other significant alteration in ATG genes mRNA levels was detected . Representative data of ATG7 mRNA expression analysis after treatment with PMA ( 1 . 21 ± 0 . 42 ) and rapamycin ( 0 . 94 ± 0 . 64 ) . Data are representative of six independent experiments and presented as mean relative expression ± SD . Wilcoxon matched ‐ pairs test , * p < 0 . 05 and n . s . = not significant compared with control conditions ( dot line ) as indicated in the figure . Relative expression ( folds ) of each gene is derived from substitution of DCT values ( normalized by GAPDH DCT value ) in 2DDCT equation 36 .

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

ATG3 and LC3B gene expression pattern in neutrophils after stimulation with PMA or rapamycin. ATG3 mRNA expression analysis (fold) after PMA (2.44±0.78) and rapamycin (RAP: 1.81±0.41) stimulation. LC3B gene alterations (PMA: 4.98±0.59) and rapamycin (RAP: 1.7±0.55). Effect of 3‐MA pre‐treatment, for both stimuli, was also assessed (PMA+3‐MA: ATG3: 1.36±0.64, LC3B: 1.5±0.49 and rapamycin+3‐MA: ATG3: 1.28±0.46, LC3B: 1.24±0.57). No other significant alteration in ATG genes mRNA levels was detected. Representative data of ATG7 mRNA expression analysis after treatment with PMA (1.21±0.42) and rapamycin (0.94±0.64). Data are representative of six independent experiments and presented as mean relative expression±SD. Wilcoxon matched‐pairs test, *p<0.05 and n.s.=not significant compared with control conditions (dot line) as indicated in the figure. Relative expression (folds) of each gene is derived from substitution of DCT values (normalized by GAPDH DCT value) in 2DDCT equation 36.


( A ) ( I ) MDC staining in neutrophils treated with LPS , LTA , peptidoglycan ( PGN ) , loxorubin ( LOX ) and R848 ( 400 × ) . ( II ) Percentage of PMN with acidified vacuoles as detected by MDC staining . LPS ( 50 ng / mL ) ( bar 2 : 69 . 67 ± 8 . 21 % ) , LTA ( bar 3 : 75 . 17 ± 9 . 80 % ) , peptidoglycan ( bar 4 : 77 ± 6 . 96 % ) , loxorubin ( bar 5 : 66 . 67 ± 5 . 61 % ) , R848 ( bar 6 : 69 . 5 ± 7 . 45 % ) , poly I : C ( bar 7 : 16 . 33 ± 3 . 14 % ) and flagellin ( bar 8 : 19 . 83 ± 5 . 16 % ) − treated cells compared with control conditions ( bar 1 : 14 . 5 ± 4 . 97 % ) . Data are representative of six independent experiments and presented as mean ± SD ; Wilcoxon matched − pairs test , * p < 0 . 05 , n . s . = not significant .

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

(A) (I) MDC staining in neutrophils treated with LPS, LTA, peptidoglycan (PGN), loxorubin (LOX) and R848 (400×). (II) Percentage of PMN with acidified vacuoles as detected by MDC staining. LPS (50 ng/mL) (bar 2: 69.67±8.21%), LTA (bar 3: 75.17±9.80%), peptidoglycan (bar 4: 77±6.96%), loxorubin (bar 5: 66.67±5.61%), R848 (bar 6: 69.5±7.45%), poly I:C (bar 7: 16.33±3.14%) and flagellin (bar 8: 19.83±5.16%)−treated cells compared with control conditions (bar 1: 14.5±4.97%). Data are representative of six independent experiments and presented as mean±SD; Wilcoxon matched−pairs test, *p<0.05, n.s.=not significant.


( B ) LC3B lipidation status of cells treated with LPS ( lane I ) , LTA ( lane II ) , peptidoglycan ( lane III ) , loxoribin ( lane IV ) and R848 ( lane V ) , detected by immunoblotting . One representative out of six independent experiments is shown .

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

(B) LC3B lipidation status of cells treated with LPS (lane I), LTA (lane II), peptidoglycan (lane III), loxoribin (lane IV) and R848 (lane V), detected by immunoblotting. One representative out of six independent experiments is shown.


( C ) LC3B − II / LC3B - I ratio as obtained from integrated optical density measurements . LTA ( bar 2 : 1 . 75 ± 0 . 84 ) , peptidoglycan ( bar 3 : 1 . 82 ± 1 . 17 ) , LPS ( bar 4 : 1 . 9 ± 1 . 7 ) , loxorubin ( bar 5 : 1 . 32 ± 0 . 49 ) and R848 ( bar 6 : 1 . 4 ± 0 . 77 ) compared with control neutrophils ( bar 1 : 0 . 76 ± 0 . 30 ) . Data are representative of six independent experiments and are presented as mean ± SD . Wilcoxon matched − pairs test , * p < 0 . 05 .

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

(C) LC3B−II/LC3B-I ratio as obtained from integrated optical density measurements. LTA (bar 2: 1.75±0.84), peptidoglycan (bar 3: 1.82±1.17), LPS (bar 4: 1.9±1.7), loxorubin (bar 5: 1.32±0.49) and R848 (bar 6: 1.4±0.77) compared with control neutrophils (bar 1: 0.76±0.30). Data are representative of six independent experiments and are presented as mean±SD. Wilcoxon matched−pairs test, *p<0.05.


( D ) Analysis of LC3B gene expression levels in neutrophils treated with TLR agonists . LPS ( bar 1 : 2 . 27 ± 0 . 43 ) , peptidoglycan ( bar 2 : 2 . 07 ± 0 . 42 ) , LTA ( bar 3 : 2 . 1 ± 0 . 49 ) and R848 ( bar 4 : 1 . 89 ± 0 . 35 ) compared with medium − treated control cells . Dotted line represents LC3B mRNA levels of medium - treated control cells . Data are representative of six independent experiments and are presented as mean relative expression ± SD . Wilcoxon matched − pairs test , * p < 0 . 05 .

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

(D) Analysis of LC3B gene expression levels in neutrophils treated with TLR agonists. LPS (bar 1: 2.27±0.43), peptidoglycan (bar 2: 2.07±0.42), LTA (bar 3: 2.1±0.49) and R848 (bar 4: 1.89±0.35) compared with medium−treated control cells. Dotted line represents LC3B mRNA levels of medium-treated control cells. Data are representative of six independent experiments and are presented as mean relative expression±SD. Wilcoxon matched−pairs test, *p<0.05.


( E ) Assessment of ATG gene expression levels in PMN after LPS stimulation ( 50 ng / mL ) . ATG3 ( bar 1 : 2 . 94 ± 0 . 52 ) , ATG4 ( bar 2 : 1 . 72 ± 0 . 42 ) , ATG5 ( bar 3 : 1 . 84 ± 0 . 34 ) , ATG6 ( bar 4 : 1 . 8 ± 0 . 3 ) , ATG7 ( bar 5 : 3 . 13 ± 0 . 64 ) and LC3B ( bar 6 : 2 . 27 ± 0 . 43 ) compared with control cells . Dotted line represents mRNA levels of medium - treated cells . Data are representative of six independent experiments and are presented as mean relative expression ± SD . Wilcoxon matched − pairs test , * p < 0 . 05 .

O O O O O O O O O O B-CELL_TYPE O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Assessment of ATG gene expression levels in PMN after LPS stimulation (50 ng/mL). ATG3 (bar 1: 2.94±0.52), ATG4 (bar 2: 1.72±0.42), ATG5 (bar 3: 1.84±0.34), ATG6 (bar 4: 1.8±0.3), ATG7 (bar 5: 3.13±0.64) and LC3B (bar 6: 2.27±0.43) compared with control cells. Dotted line represents mRNA levels of medium-treated cells. Data are representative of six independent experiments and are presented as mean relative expression±SD. Wilcoxon matched−pairs test, *p<0.05.


( A ) ( I ) Formation of acidified autophagosomes after stimulation with 10 ng / mL of IL ‐ 1β , compared with medium ‐ treated control cells . ( II ) Percentage of PMN with acidified vacuoles as detected by MDC staining . IL ‐ 1β ‐ treated cells ( bar 2 : 65 . 17 ± 10 . 23 % ) compared with control conditions ( bar 1 : 15 . 83 ± 6 . 08 % ) . Data are representative of six independent experiments and presented as mean ± SD ; Wilcoxon matched ‐ pairs test , * p < 0 . 05 .

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

(A) (I) Formation of acidified autophagosomes after stimulation with 10 ng/mL of IL‐1β, compared with medium‐treated control cells. (II) Percentage of PMN with acidified vacuoles as detected by MDC staining. IL‐1β‐treated cells (bar 2: 65.17±10.23%) compared with control conditions (bar 1: 15.83±6.08%). Data are representative of six independent experiments and presented as mean±SD; Wilcoxon matched‐pairs test, *p<0.05.


( B ) LC3B conversion in neutrophils treated with 10 ng / mL ( lane II ) and 100 ng / mL ( lane III ) of IL ‐ 1β . ( lane I : medium ‐ treated control cells ) . One representative out of five independent experiments is shown .

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

(B) LC3B conversion in neutrophils treated with 10 ng/mL (lane II) and 100 ng/mL (lane III) of IL‐1β. (lane I: medium‐treated control cells). One representative out of five independent experiments is shown.


( A ) Effect of 3 ‐ MA on neutrophil phagocytic capacity of opsonized E . coli as observed by flow cytometry . Percentage of phagocytosing neutrophils after 3 ‐ MA pretreatment ( III ) compared with cells incubated with opsonized bacteria ( II ) . Data are representative of six independent experiments and are presented as mean ± SD , ( I : control unstimulated group ) , ( IV ) MFI representation ( bar 1 : control : 1 ± 1 . 9 , bar 2 : E . coli : 387 . 7 ± 33 . 3 , bar 3 : E . coli + 3 ‐ MA : 25 . 13 ± 18 , Wilcoxon matched ‐ pairs test , * p < 0 . 05 ; bar 2 compared with bar 1 and bar 3 compared to bar 2 ) .

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

(A) Effect of 3‐MA on neutrophil phagocytic capacity of opsonized E. coli as observed by flow cytometry. Percentage of phagocytosing neutrophils after 3‐MA pretreatment (III) compared with cells incubated with opsonized bacteria (II). Data are representative of six independent experiments and are presented as mean±SD, (I: control unstimulated group), (IV) MFI representation (bar 1: control: 1±1.9, bar 2: E. coli: 387.7±33.3, bar 3: E. coli+3‐MA: 25.13±18, Wilcoxon matched‐pairs test, *p<0.05; bar 2 compared with bar 1 and bar 3 compared to bar 2).


( B ) ( I ) Presence of acidified structures in E . coli ‐ phagocytosing neutrophils and medium ‐ treated control cells . ( II ) Percentage of PMN with acidified vacuoles as detected by MDC staining . E . coli ‐ phagocytosing cells ( bar 2 : 79 . 33 ± 7 . 15 % ) compared with control conditions ( bar 1 : 14 . 17 ± 4 . 79 % ) . Data are representative of six independent experiments and presented as mean ± SD ; Wilcoxon matched ‐ pairs test , * p < 0 . 05 . (

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

(B) (I) Presence of acidified structures in E. coli‐phagocytosing neutrophils and medium‐treated control cells. (II) Percentage of PMN with acidified vacuoles as detected by MDC staining. E. coli‐phagocytosing cells (bar 2: 79.33±7.15%) compared with control conditions (bar 1: 14.17±4.79%). Data are representative of six independent experiments and presented as mean±SD; Wilcoxon matched‐pairs test, *p<0.05. (


( C ) LC3B conversion analysis in E . coli ‐ phagocytosing neutrophils ( lane II ) , control neutrophils ( lane I ) and E . coli ‐ phagocytosing neutrophils after 3 ‐ MA pretreatment ( lane III ) . One representative out of six independent experiments is shown .

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

(C) LC3B conversion analysis in E. coli‐phagocytosing neutrophils (lane II), control neutrophils (lane I) and E. coli‐phagocytosing neutrophils after 3‐MA pretreatment (lane III). One representative out of six independent experiments is shown.


( D ) Effect of 3 ‐ MA administration to E . coli ‐ phagocytosing neutrophils 15 min after the initiation of phagocytosis ( lane III ) as detected by LC3B conversion analysis . Lane II represents E . coli ‐ phagocytosing neutrophils and lane I control neutrophils . One representative out of four independent experiments is shown .

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

(D) Effect of 3‐MA administration to E. coli‐phagocytosing neutrophils 15 min after the initiation of phagocytosis (lane III) as detected by LC3B conversion analysis. Lane II represents E. coli‐phagocytosing neutrophils and lane I control neutrophils. One representative out of four independent experiments is shown.


( E ) ( I ) Percentage of neutrophils with LC3B punctuated structures . E . coli ( bar 1 : 84 ± 2 . 2 % ) compared with E . coli + 3 ‐ MA ( bar 2 : 4 ± 1 . 95 % ) . Data are representative of six independent experiments and presented as mean ± SD . Wilcoxon matched ‐ pairs test , * p < 0 . 05 . ( II ) Formation of LC3B puncta in phagocytosing neutrophils and ( III ) in cells treated with 3 ‐ MA before incubation with opsonized E . coli , as observed by immunofluorescence confocal microscopy ( 1 . LC3B : red ; 2 . FITC conjugated E . coli : green ; 3 . DAPI : blue ; and 4 . merge ) . Solid line arrows point the colocalization of LC3B puncta with E . coli bacteria . Dashed line arrows point LC3B aggregates that are not colocalized with E . coli .

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

(E) (I) Percentage of neutrophils with LC3B punctuated structures. E. coli (bar 1: 84±2.2%) compared with E. coli+3‐MA (bar 2: 4±1.95%). Data are representative of six independent experiments and presented as mean±SD. Wilcoxon matched‐pairs test, *p<0.05. (II) Formation of LC3B puncta in phagocytosing neutrophils and (III) in cells treated with 3‐MA before incubation with opsonized E. coli, as observed by immunofluorescence confocal microscopy (1. LC3B: red; 2. FITC conjugated E. coli: green; 3. DAPI: blue; and 4. merge). Solid line arrows point the colocalization of LC3B puncta with E. coli bacteria. Dashed line arrows point LC3B aggregates that are not colocalized with E. coli.


Alterations in ATG gene expression after phagocytosis of opsonized E . coli . ATG3 ( 2 . 05 ± 0 . 27 ) , ATG4 ( 2 . 20 ± 0 . 27 ) , ATG5 ( 2 . 89 ± 0 . 42 ) , ATG6 ( 2 . 04 ± 0 . 40 ) , ATG7 ( 1 . 98 ± 0 . 30 ) and LC3B ( 2 . 46 ± 0 . 39 ) gene expression ( fold ) in E . coli ‐ phagocytosing neutrophils compared with control cells . Dotted line represents mRNA expression levels of control cells . Data are representative of six independent experiments and are presented as mean relative expression ± SD Wilcoxon matched ‐ pairs test , * p < 0 . 05 .

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

Alterations in ATG gene expression after phagocytosis of opsonized E. coli. ATG3 (2.05±0.27), ATG4 (2.20±0.27), ATG5 (2.89±0.42), ATG6 (2.04±0.40), ATG7 (1.98±0.30) and LC3B (2.46±0.39) gene expression (fold) in E. coli‐phagocytosing neutrophils compared with control cells. Dotted line represents mRNA expression levels of control cells. Data are representative of six independent experiments and are presented as mean relative expression±SD Wilcoxon matched‐pairs test, *p<0.05.


( A ) LC3B conversion status after pretreatment with the ROS scavenger BHA prior to PMA or E . coli stimulation ( lane I : PMA , lane II : PMA + BHA , lane III : E . coli , lane IV : E . coli + BHA , lane V : control cells ) . One representative out of four independent experiments is shown .

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

(A) LC3B conversion status after pretreatment with the ROS scavenger BHA prior to PMA or E. coli stimulation (lane I: PMA, lane II: PMA+BHA, lane III: E. coli, lane IV: E. coli+BHA, lane V: control cells). One representative out of four independent experiments is shown.


( B ) LC3B conversion status after pretreatment with the NADPH oxidase inhibitor DPI in E . coli − phagocytosing neutrophils ( lane I : PBS , lane II : E . coli , lane III : E . coli + DPI ) . One representative out of four independent experiments is shown .

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

(B) LC3B conversion status after pretreatment with the NADPH oxidase inhibitor DPI in E. coli−phagocytosing neutrophils (lane I: PBS, lane II: E. coli, lane III: E. coli+DPI). One representative out of four independent experiments is shown.


( C ) ( I ) Effect of NADPH ‐ oxidase inhibition with DPI in the formation of acidified autophagosomes in E . coli ‐ phagocytosing neutrophils . ( II ) Percentage of PMN with acidified vacuoles as detected by MDC staining . E . coli − phagocytosing PMN ( 77 . 17 ± 8 . 33 % ) and PMN treated with DPI before incubation with opsonized E . coli ( 21 . 67 ± 4 . 37 % ) . Data are representative of six independent experiments and presented as mean ± SD ; Wilcoxon matched ‐ pairs test , * p < 0 .

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

(C) (I) Effect of NADPH‐oxidase inhibition with DPI in the formation of acidified autophagosomes in E. coli‐phagocytosing neutrophils. (II) Percentage of PMN with acidified vacuoles as detected by MDC staining. E. coli−phagocytosing PMN (77.17±8.33%) and PMN treated with DPI before incubation with opsonized E. coli (21.67±4.37%). Data are representative of six independent experiments and presented as mean±SD; Wilcoxon matched‐pairs test, *p<0.05