F ) Normalized lactate concentration upon PYCR1 KO . Data represents mean ± SE of three biological experiments . Samples are compared using Kruskal Wallis test followed by Dunnett ' s test for multiple pairwise comparisons . Corrected p values are indicated as follows * p < 0 . 05 . Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances .

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F) Normalized lactate concentration upon PYCR1 KO. Data represents mean ± SE of three biological experiments. Samples are compared using Kruskal Wallis test followed by Dunnett's test for multiple pairwise comparisons. Corrected p values are indicated as follows *p < 0.05. Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances.


A ) Representative pictures of transwell migration and invasion after PYCR1 knockout . Scale bar : 100 μm . B ) Bar plot represents mean ± SD of three biological replicates for transwell migration and invasion . Samples were compared using Kruskal Wallis test followed by Dunnett ' s test for multiple pairwise comparisons . Corrected p - values are reported as follows * * * p < 0 . 001 .

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A) Representative pictures of transwell migration and invasion after PYCR1 knockout. Scale bar :100 μm. B) Bar plot represents mean ± SD of three biological replicates for transwell migration and invasion. Samples were compared using Kruskal Wallis test followed by Dunnett's test for multiple pairwise comparisons. Corrected p-values are reported as follows *** p < 0.001.


C ) Representative images of colony formation in soft agar upon PYCR1 KO . Scale bar : 100 μm .

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C) Representative images of colony formation in soft agar upon PYCR1 KO. Scale bar: 100 μm.


D ) Growth measurements of MCF7 wild type and PYCR1 KO cells over 96 hours . Data represents mean ± SE of three biological experiments . Samples were compared using paired Student ' s t - test .

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D) Growth measurements of MCF7 wild type and PYCR1 KO cells over 96 hours. Data represents mean ± SE of three biological experiments. Samples were compared using paired Student's t-test.


E - H ) Bar plots show percentage of viable cells after treatment with 0 . 75 mM H2O2 ( E ) , 0 . 06 µM paclitaxel ( F ) , 0 . 15 µM doxorubicin ( G ) , 2 . 5 mM cyclophosphamide ( H ) . Data represent mean ± SE of three biological experiments . Samples are compared using Kruskal Wallis test followed by Dunnett ' s test for multiple pairwise comparisons . Corrected p values are indicated as follows * p < 0 . 05 .

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 B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O 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-H) Bar plots show percentage of viable cells after treatment with 0.75 mM H2O2 (E), 0.06 µM paclitaxel (F), 0.15 µM doxorubicin (G), 2.5 mM cyclophosphamide (H). Data represent mean ± SE of three biological experiments. Samples are compared using Kruskal Wallis test followed by Dunnett's test for multiple pairwise comparisons. Corrected p values are indicated as follows *p < 0.05.


I ) Tumor volume measurements for 26 days in MCF7 injected NSG mice . CRISPR control and PYCR1 KO tumors with ( n = 8 ) and without treatment ( n = 5 ) are shown . Data represents mean ± SE . Pax : Paclitaxel , Dox : Doxorubicin . Groups were compared by one way ANOVA followed by Tukey ' s multiple comparisons test for pairwise group comparisons . Corrected p - values are reported as follows * p < 0 . 05 , * * p < 0 . 01 . Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances .

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

I) Tumor volume measurements for 26 days in MCF7 injected NSG mice. CRISPR control and PYCR1 KO tumors with (n=8) and without treatment (n=5) are shown. Data represents mean ± SE. Pax: Paclitaxel, Dox: Doxorubicin. Groups were compared by one way ANOVA followed by Tukey's multiple comparisons test for pairwise group comparisons. Corrected p-values are reported as follows *p < 0.05, ** p < 0.01. Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances.


J ) Bar plot indicates mean ± SE of tumor volume measurements for day 26 . CRISPR control and PYCR1 KO tumors with ( n = 8 ) and without treatment ( n = 5 ) were compared by one way ANOVA followed by Tukey ' s multiple comparisons test for pairwise group comparisons . Corrected p - values are reported as follows * * * p < 0 . 001 . Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances .

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

J) Bar plot indicates mean ± SE of tumor volume measurements for day 26. CRISPR control and PYCR1 KO tumors with (n=8) and without treatment (n=5) were compared by one way ANOVA followed by Tukey's multiple comparisons test for pairwise group comparisons. Corrected p-values are reported as follows *** p < 0.001. Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances.


( A ) HeLa cells expressing HA - Parkin were treated with CCCP or DMSO ( control ) and then immunostained with the indicated antibodies .

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(A) HeLa cells expressing HA-Parkin were treated with CCCP or DMSO (control) and then immunostained with the indicated antibodies.


( B ) HeLa cells stably expressing HA - Parkin or intact SH - SY5Y cells were treated with CCCP or DMSO and subjected to fractionation experiments . I , C , and M indicate input , cytosol - rich supernatant , and mitochondria - rich membrane pellet , respectively .

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(B) HeLa cells stably expressing HA-Parkin or intact SH-SY5Y cells were treated with CCCP or DMSO and subjected to fractionation experiments. I, C, and M indicate input, cytosol-rich supernatant, and mitochondria-rich membrane pellet, respectively.


( D ) Polarized mitochondria stained with MitoTracker red ( red arrowheads ) were not labeled by Parkin . In contrast , damaged mitochondria marked by Parkin ( green arrowheads ) were not stained with MitoTracker red .

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(D) Polarized mitochondria stained with MitoTracker red (red arrowheads) were not labeled by Parkin. In contrast, damaged mitochondria marked by Parkin (green arrowheads) were not stained with MitoTracker red.


( E ) HeLa cells expressing HA - Parkin with various pathogenic mutations were treated with CCCP , followed by immunocytochemistry . ( A , D , and E ) Higher magnification views of the boxed areas are shown in the insets . ( F ) Parkin colocalization with mitochondria was analyzed in > 100 cells per mutation . Example figures indicative of robust colocalization ( counted as 1 ) , weak colocalization ( counted as 0 . 5 ) , and no colocalization ( counted as 0 ) are shown . Error bars represent the mean ± SD values of at least three experiments .

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(E) HeLa cells expressing HA-Parkin with various pathogenic mutations were treated with CCCP, followed by immunocytochemistry. (A, D, and E) Higher magnification views of the boxed areas are shown in the insets. (F) Parkin colocalization with mitochondria was analyzed in >100 cells per mutation. Example figures indicative of robust colocalization (counted as 1), weak colocalization (counted as 0.5), and no colocalization (counted as 0) are shown. Error bars represent the mean ± SD values of at least three experiments.


( A ) HeLa cells expressing wild - type Parkin or E3 - inactivating mutations were treated with CCCP and then immunostained with the indicated antibodies . When E3 - inactivating mutations were introduced into Parkin , the mitochondrial ubiquitylation signal disappeared .

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(A) HeLa cells expressing wild-type Parkin or E3-inactivating mutations were treated with CCCP and then immunostained with the indicated antibodies. When E3-inactivating mutations were introduced into Parkin, the mitochondrial ubiquitylation signal disappeared.


( B and C ) HeLa cells expressing HA - Parkin ( B ) or expressing both Mt - GFP and HA - Parkin ( C ) were treated with CCCP or DMSO ( control ) and then immunostained with the indicated antibodies .

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(B and C) HeLa cells expressing HA-Parkin (B) or expressing both Mt-GFP and HA-Parkin (C) were treated with CCCP or DMSO (control) and then immunostained with the indicated antibodies.


( D ) Localization of GFP - Parkin to the mitochondria after CCCP treatment . ( A - D ) Higher magnification views of the boxed areas are shown in the insets .

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(D) Localization of GFP-Parkin to the mitochondria after CCCP treatment. (A-D) Higher magnification views of the boxed areas are shown in the insets.


( E ) HeLa cell lysates expressing GFP - Parkin were immunoprecipitated by anti - GFP antibody , followed by immunoblotting with the indicated antibodies .

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(E) HeLa cell lysates expressing GFP-Parkin were immunoprecipitated by anti-GFP antibody, followed by immunoblotting with the indicated antibodies.


( F ) Straight immunoblotting of HA - and GFP - Parkin in the absence or presence of CCCP . Note the slower migrating ladders derived from ubiquitylation ( Ub ) in only the GFP - Parkin with CCCP lane .

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(F) Straight immunoblotting of HA- and GFP-Parkin in the absence or presence of CCCP. Note the slower migrating ladders derived from ubiquitylation (Ub) in only the GFP-Parkin with CCCP lane.


( G ) GFP - Parkin - expressing HeLa cells with various pathogenic mutations ( Fig . 1 C ) were treated with CCCP and subjected to immunoblotting . Asterisks show ubiquitylation of GFP - Parkin . Vertical black lines indicate that intervening lanes have been spliced out . IB , immunoblot ; IP , immunoprecipitation .

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(G) GFP-Parkin-expressing HeLa cells with various pathogenic mutations (Fig. 1 C) were treated with CCCP and subjected to immunoblotting. Asterisks show ubiquitylation of GFP-Parkin. Vertical black lines indicate that intervening lanes have been spliced out. IB, immunoblot; IP, immunoprecipitation.


( A ) The number of HeLa cells with N - terminal - or C - terminal - tagged PINK1 localized to the mitochondria was counted in > 100 cells .

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

(A) The number of HeLa cells with N-terminal- or C-terminal-tagged PINK1 localized to the mitochondria was counted in >100 cells.


( B and C ) Exogenous nontagged PINK1 ( B ) or endogenous PINK1 ( C ) in HeLa cells was immunostained with the indicated antibodies . ( D ) The number of HeLa cells with endogenous PINK1 localized to the mitochondria was counted as in A . ( A and D ) Error bars represent the mean ± SD values of least three experiments .

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(B and C) Exogenous nontagged PINK1 (B) or endogenous PINK1 (C) in HeLa cells was immunostained with the indicated antibodies. (D) The number of HeLa cells with endogenous PINK1 localized to the mitochondria was counted as in A. (A and D) Error bars represent the mean ± SD values of least three experiments.


( E ) Endogenous PINK1 gradually accumulated after CCCP treatment . The first through the fifth lanes show endogenous PINK1 , and the sixth lane shows overexpressed untagged PINK1 . Note that the asterisk indicates a cross - reacting band because it was not affected by overproduction of untagged PINK1 .

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(E) Endogenous PINK1 gradually accumulated after CCCP treatment. The first through the fifth lanes show endogenous PINK1, and the sixth lane shows overexpressed untagged PINK1. Note that the asterisk indicates a cross-reacting band because it was not affected by overproduction of untagged PINK1.


( F ) Subcellular fractionation of endogenous PINK1 . Intact SH - SY5Y cells were treated with CCCP or DMSO and subjected to fractionation experiments ( same sample as Fig . 1 B ) . I , C , and M indicate input , cytosol - rich supernatant , and the mitochondria - rich membrane pellet , respectively .

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(F) Subcellular fractionation of endogenous PINK1. Intact SH-SY5Y cells were treated with CCCP or DMSO and subjected to fractionation experiments (same sample as Fig. 1 B). I, C, and M indicate input, cytosol-rich supernatant, and the mitochondria-rich membrane pellet, respectively.


( G ) HeLa cells were treated with CCCP and cycloheximide as depicted , followed by immunoblotting with the indicated antibodies . LDH , lactate dehydrogenase . ( F and G ) Asterisks indicate a cross - reacting band .

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(G) HeLa cells were treated with CCCP and cycloheximide as depicted, followed by immunoblotting with the indicated antibodies. LDH, lactate dehydrogenase. (F and G) Asterisks indicate a cross-reacting band.


( H ) N - terminal 34 aa of PINK1 recruited GFP to the mitochondria both in the absence and presence of CCCP . The top panel shows control HeLa cells expressing only GFP . ( B , C , and H ) Higher magnification views of the boxed areas are shown in the insets . Bars , 10 µm .

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(H) N-terminal 34 aa of PINK1 recruited GFP to the mitochondria both in the absence and presence of CCCP. The top panel shows control HeLa cells expressing only GFP. (B, C, and H) Higher magnification views of the boxed areas are shown in the insets. Bars, 10 µm.


( A ) PINK1 knockout ( PINK1 − / − ) or control ( PINK1 + / + ) MEFs were transfected with HA - Parkin , treated with CCCP , and subjected to immunocytochemistry with the indicated antibodies . Higher magnification views of the boxed areas are shown in the insets . ( B ) The number of MEFs with Parkin localized to the mitochondria was counted as in Fig . 3 A .

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

(A) PINK1 knockout (PINK1−/−) or control (PINK1+/+) MEFs were transfected with HA-Parkin, treated with CCCP, and subjected to immunocytochemistry with the indicated antibodies. Higher magnification views of the boxed areas are shown in the insets. (B) The number of MEFs with Parkin localized to the mitochondria was counted as in Fig. 3 A.


( C and D ) Neither activation of Parkin nor mitochondrial degradation was observed in PINK1 − / − MEFs . MEFs stably expressing GFP - Parkin were treated with CCCP for 4 h and subjected to immunoblotting ( C )

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

(C and D) Neither activation of Parkin nor mitochondrial degradation was observed in PINK1−/− MEFs. MEFs stably expressing GFP-Parkin were treated with CCCP for 4 h and subjected to immunoblotting (C)


( C and D ) Neither activation of Parkin nor mitochondrial degradation was observed in PINK1 − / − MEFs . MEFs stably expressing GFP - Parkin were treated with CCCP for 24 h , followed by immunocytochemistry ( D ) . IB , immunoblot ; Ub , ubiquitylation .

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(C and D) Neither activation of Parkin nor mitochondrial degradation was observed in PINK1−/− MEFs. MEFs stably expressing GFP-Parkin were treated with CCCP for 24 h, followed by immunocytochemistry (D). IB, immunoblot; Ub, ubiquitylation.


( E ) The number of MEFs without anti - Tom20 antibody - detectable mitochondria was counted as in Fig . 3 A . In the example figure ( left ) , blue arrowheads indicate cells without anti - Tom20 antibody - detectable mitochondria , and red arrowheads indicate cells harboring anti - Tom20 antibody - detectable mitochondria .

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(E) The number of MEFs without anti-Tom20 antibody-detectable mitochondria was counted as in Fig. 3 A. In the example figure (left), blue arrowheads indicate cells without anti-Tom20 antibody-detectable mitochondria, and red arrowheads indicate cells harboring anti-Tom20 antibody-detectable mitochondria.


( A ) Schematic depiction of pathogenic and deletion mutants of PINK1 used in this study . MTS , mitochondria - targeting sequence ; TMD , transmembrane domain . ( B ) Subcellular localization of Parkin in PINK1 − / − cells complemented by various pathogenic and deletion mutants of PINK1 - Flag . Cells were treated with CCCP . Higher magnification views of the boxed areas are shown in the insets . ( C ) The number of cells with Parkin - positive mitochondria was counted as in Fig . 3 A . Error bars represent the mean ± SD values of least three experiments .

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

(A) Schematic depiction of pathogenic and deletion mutants of PINK1 used in this study. MTS, mitochondria-targeting sequence; TMD, transmembrane domain. (B) Subcellular localization of Parkin in PINK1−/− cells complemented by various pathogenic and deletion mutants of PINK1-Flag. Cells were treated with CCCP. Higher magnification views of the boxed areas are shown in the insets. (C) The number of cells with Parkin-positive mitochondria was counted as in Fig. 3 A. Error bars represent the mean ± SD values of least three experiments.


( D ) PINK1 − / − MEFs complemented by various PINK1 mutants were treated with CCCP and subjected to immunoblotting using anti - Parkin or anti - Flag ( tag of PINK1 ) antibodies . IB , immunoblot ; Ub , ubiquitylation .

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(D) PINK1−/− MEFs complemented by various PINK1 mutants were treated with CCCP and subjected to immunoblotting using anti-Parkin or anti-Flag (tag of PINK1) antibodies. IB, immunoblot; Ub, ubiquitylation.


( A ) Immunoblot analysis with anti - LC3 antibody shows that NO donors reduced LC3 - II levels in rat primary cortical neurons and decreased autophagosome synthesis in bafilomycin A1 - treated rat primary cortical neurons and HeLa cells .

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

(A) Immunoblot analysis with anti-LC3 antibody shows that NO donors reduced LC3-II levels in rat primary cortical neurons and decreased autophagosome synthesis in bafilomycin A1-treated rat primary cortical neurons and HeLa cells.


( B - E ) Confocal microscopy images and analysis of autophagic flux by automated Cellomics microscopy show that NO donors reduced autolysosome numbers in mRFP - GFP - LC3 HeLa cells grown in full medium ( FM ) ( B and D ) or starved with HBSS ( C and E ) . Cells in HBSS had more autolysosomes compared to FM ( C ) . Autolysosome number in untreated cells in FM is set at 100 % .

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

(B-E) Confocal microscopy images and analysis of autophagic flux by automated Cellomics microscopy show that NO donors reduced autolysosome numbers in mRFP-GFP-LC3 HeLa cells grown in full medium (FM) (B and D) or starved with HBSS (C and E). Cells in HBSS had more autolysosomes compared to FM (C). Autolysosome number in untreated cells in FM is set at 100%.


( F ) NO donors increased EGFP - HDQ74 aggregates in a dose - dependent manner in EGFP - HDQ74 - transfected Atg5 + / + , but not in Atg5 − / − , MEFs . Atg5 − / − MEFs had more EGFP - HDQ74 aggregates than Atg5 + / + MEFs .

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(F) NO donors increased EGFP-HDQ74 aggregates in a dose-dependent manner in EGFP-HDQ74-transfected Atg5+/+, but not in Atg5−/−, MEFs. Atg5−/− MEFs had more EGFP-HDQ74 aggregates than Atg5+/+ MEFs.


( G and H ) Confocal microscopy images ( H ) and immunofluorescence analysis with anti - Atg16 antibody ( G ) show that NO donors reduced the percentage of HeLa cells with Atg16 - positive structures ( arrows ) under starvation . Graphical data denote mean ± SEM .

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(G and H) Confocal microscopy images (H) and immunofluorescence analysis with anti-Atg16 antibody (G) show that NO donors reduced the percentage of HeLa cells with Atg16-positive structures (arrows) under starvation. Graphical data denote mean ± SEM.


( A ) NO donors reduced JNK1 phosphorylation in HeLa cells , as analyzed by immunoprecipitation with anti - JNK1 agarose - conjugated beads and immunoblotting with anti - phospho - JNK antibody .

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(A) NO donors reduced JNK1 phosphorylation in HeLa cells, as analyzed by immunoprecipitation with anti-JNK1 agarose-conjugated beads and immunoblotting with anti-phospho-JNK antibody.


( B ) NO donors reduced Bcl - 2 phosphorylation in HeLa cells , as analyzed by immunoblotting with anti - phospho - Bcl - 2 antibody .

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(B) NO donors reduced Bcl-2 phosphorylation in HeLa cells, as analyzed by immunoblotting with anti-phospho-Bcl-2 antibody.


C ) Immunoblot analysis with anti - phospho - Bcl - 2 antibody shows that DETA NONOate could not reduce phospho - Bcl - 2 in Flag - CA JNK1 - transfected HeLa cells , where phospho - Bcl - 2 was higher compared to mock - transfected cells .

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

C) Immunoblot analysis with anti-phospho-Bcl-2 antibody shows that DETA NONOate could not reduce phospho-Bcl-2 in Flag-CA JNK1-transfected HeLa cells, where phospho-Bcl-2 was higher compared to mock-transfected cells.


( D and E ) Immunoprecipitation with anti - Flag M2 affinity agarose gel and immunoblotting with anti - Myc antibody shows that NO donors increased the interaction of Flag - Beclin 1 with WT Myc - Bcl - 2 ( D ) , but not with AAA Myc - Bcl - 2 ( E ) , in HeLa cells transfected with WT Myc - Bcl - 2 ( D ) or AAA Myc - Bcl - 2 ( E ) along with either empty Flag or Flag - Beclin 1 . Asterisk denotes IgG band ( E ) .

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

(D and E) Immunoprecipitation with anti-Flag M2 affinity agarose gel and immunoblotting with anti-Myc antibody shows that NO donors increased the interaction of Flag-Beclin 1 with WT Myc-Bcl-2 (D), but not with AAA Myc-Bcl-2 (E), in HeLa cells transfected with WT Myc-Bcl-2 (D) or AAA Myc-Bcl-2 (E) along with either empty Flag or Flag-Beclin 1. Asterisk denotes IgG band (E).


( F ) Immunoprecipitation with anti - Flag M2 affinity agarose gel and immunoblotting with anti - Vps34 antibody shows that NO donors decreased Flag - Beclin 1 - hVps34 interaction in HeLa cells transfected with hVps34 along with either empty Flag or Flag - Beclin 1 .

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

(F) Immunoprecipitation with anti-Flag M2 affinity agarose gel and immunoblotting with anti-Vps34 antibody shows that NO donors decreased Flag-Beclin 1-hVps34 interaction in HeLa cells transfected with hVps34 along with either empty Flag or Flag-Beclin 1.


( G ) Immunoprecipitation with anti - Flag M2 affinity agarose gel and immunoblotting with anti - Vps34 antibody shows that AAA Myc - Bcl - 2 decreased Flag - Beclin 1 - hVps34 interaction in the presence ( right panel ) or absence ( left panel ) of DETA NONOate in HeLa cells transfected with hVps34 along with either empty Flag or Flag - Beclin 1 , and with pcDNA3 . 1 or AAA Myc - Bcl - 2 . Graphical data denote mean ± SEM .

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

(G) Immunoprecipitation with anti-Flag M2 affinity agarose gel and immunoblotting with anti-Vps34 antibody shows that AAA Myc-Bcl-2 decreased Flag-Beclin 1-hVps34 interaction in the presence (right panel) or absence (left panel) of DETA NONOate in HeLa cells transfected with hVps34 along with either empty Flag or Flag-Beclin 1, and with pcDNA3.1 or AAA Myc-Bcl-2. Graphical data denote mean ± SEM.


( A ) Immunoblot analysis with anti - LC3 antibody shows that DETA NONOate reduced autophagosome synthesis in bafilomycin A1 - treated WT Bcl - 2 and AAA Bcl - 2 MEFs .

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

(A) Immunoblot analysis with anti-LC3 antibody shows that DETA NONOate reduced autophagosome synthesis in bafilomycin A1-treated WT Bcl-2 and AAA Bcl-2 MEFs.


( B and C ) NO donors decreased EGFP - LC3 vesicles in EGFP - LC3 - transfected WT Bcl - 2 and AAA Bcl - 2 MEFs ( C ) . Images were acquired by a confocal microscope ( B ) . WT Bcl - 2 and AAA Bcl - 2 MEFs were analyzed separately .

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

(B and C) NO donors decreased EGFP-LC3 vesicles in EGFP-LC3-transfected WT Bcl-2 and AAA Bcl-2 MEFs (C). Images were acquired by a confocal microscope (B). WT Bcl-2 and AAA Bcl-2 MEFs were analyzed separately.


( D ) NO donors increased EGFP - HDQ74 aggregates in EGFP - HDQ74 - transfected WT Bcl - 2 and AAA Bcl - 2 MEFs . WT Bcl - 2 and AAA Bcl - 2 MEFs were analyzed separately .

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

(D) NO donors increased EGFP-HDQ74 aggregates in EGFP-HDQ74-transfected WT Bcl-2 and AAA Bcl-2 MEFs. WT Bcl-2 and AAA Bcl-2 MEFs were analyzed separately.


( E ) Immunoblot analysis with anti - LC3 antibody shows that DETA NONOate reduced autophagosome synthesis in bafilomycin A1 - treated WT Jnk , Jnk1 − / − , and Jnk2 − / − MEFs .

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

(E) Immunoblot analysis with anti-LC3 antibody shows that DETA NONOate reduced autophagosome synthesis in bafilomycin A1-treated WT Jnk, Jnk1−/−, and Jnk2−/− MEFs.


( F ) Immunoblot analyses with anti - phospho - S6K and anti - phospho - S6 antibodies show that NO donors activated mTORC1 in HeLa cells .

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

(F) Immunoblot analyses with anti-phospho-S6K and anti-phospho-S6 antibodies show that NO donors activated mTORC1 in HeLa cells.


( G ) Confocal microscope images of immunofluorescence with anti - phospho - S6 antibody show that DETA NONOate increased S6 phosphorylation ; arrowhead shows a cell where this effect was not observed .

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

(G) Confocal microscope images of immunofluorescence with anti-phospho-S6 antibody show that DETA NONOate increased S6 phosphorylation; arrowhead shows a cell where this effect was not observed.


( H ) Immunoblot analyses with anti - phospho - S6K and anti - phospho - S6 antibodies show that NO donors activated mTORC1 in WT Bcl - 2 and AAA Bcl - 2 MEFs .

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

(H) Immunoblot analyses with anti-phospho-S6K and anti-phospho-S6 antibodies show that NO donors activated mTORC1 in WT Bcl-2 and AAA Bcl-2 MEFs.


( I ) Confocal microscope images of immunofluorescence with anti - phospho - mTOR and anti - LAMP1 antibodies in HeLa cells show that DETA NONOate increased phospho - mTOR but did not alter its distribution with lysosomes . Graphical data denote mean ± SEM .

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

(I) Confocal microscope images of immunofluorescence with anti-phospho-mTOR and anti-LAMP1 antibodies in HeLa cells show that DETA NONOate increased phospho-mTOR but did not alter its distribution with lysosomes. Graphical data denote mean ± SEM.


( A ) Immunoblot analyses with anti - phospho - S6K and anti - phospho - S6 antibodies show that NO donors activated mTORC1 in Tsc2 + / + MEFs , but not in Tsc2 − / − , MEFs .

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

(A) Immunoblot analyses with anti-phospho-S6K and anti-phospho-S6 antibodies show that NO donors activated mTORC1 in Tsc2+/+ MEFs, but not in Tsc2−/−, MEFs.


( B ) NO donors reduced phosphorylation of IKKα / β and AMPKα in MEFs , as analyzed by immunoblotting with anti - phospho - IKKα / β and anti - phospho - AMPKα antibodies , respectively .

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

(B) NO donors reduced phosphorylation of IKKα/β and AMPKα in MEFs, as analyzed by immunoblotting with anti-phospho-IKKα/β and anti-phospho-AMPKα antibodies, respectively.


( C ) Immunoblot analyses with anti - phospho - S6K and anti - phospho - S6 antibodies show that NO donors activated mTORC1 in Ikkβ + / + MEFs , but not in Ikkβ − / − MEFs .

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

(C) Immunoblot analyses with anti-phospho-S6K and anti-phospho-S6 antibodies show that NO donors activated mTORC1 in Ikkβ+/+ MEFs, but not in Ikkβ−/− MEFs.


( D ) Immunoblot analysis with anti - LC3 antibody shows that DETA NONOate reduced autophagosome synthesis in bafilomycin A1 - treated Tsc2 + / + and Tsc2 − / − MEFs .

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

(D) Immunoblot analysis with anti-LC3 antibody shows that DETA NONOate reduced autophagosome synthesis in bafilomycin A1-treated Tsc2+/+ and Tsc2−/− MEFs.


( E ) DETA NONOate decreased EGFP - LC3 vesicles in Tsc2 + / + and Tsc2 − / − MEFs transfected with EGFP - LC3 , along with either pcDNA3 . 1 or AAA Myc - Bcl - 2 . Although it further reduced EGFP - LC3 vesicles in Tsc2 + / + MEFs expressing AAA Myc - Bcl - 2 compared to mock - transfected Tsc2 + / + MEFs , this effect was not seen in Tsc2 − / − MEFs . Tsc2 + / + and Tsc2 − / − MEFs were analyzed separately . Graphical data denote mean ± SEM .

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

(E) DETA NONOate decreased EGFP-LC3 vesicles in Tsc2+/+ and Tsc2−/−MEFs transfected with EGFP-LC3, along with either pcDNA3.1 or AAA Myc-Bcl-2. Although it further reduced EGFP-LC3 vesicles in Tsc2+/+MEFs expressing AAA Myc-Bcl-2 compared to mock-transfected Tsc2+/+MEFs, this effect was not seen in Tsc2−/−MEFs. Tsc2+/+ and Tsc2−/−MEFs were analyzed separately. Graphical data denote mean ± SEM.


( A - D ) Immunoblot analyses with anti - LC3 , along with anti - nNOS , anti - iNOS , and anti - eNOS antibodies , show decreased LC3 - II levels in stable HEK293 cell lines overexpressing nNOS ( A and D ) , iNOS - GFP ( B and D ) , or eNOS ( C and D ) compared to HEK293 control cells in the presence or absence of bafilomycin A1 .

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

(A-D) Immunoblot analyses with anti-LC3, along with anti-nNOS, anti-iNOS, and anti-eNOS antibodies, show decreased LC3-II levels in stable HEK293 cell lines overexpressing nNOS (A and D), iNOS-GFP (B and D), or eNOS (C and D) compared to HEK293 control cells in the presence or absence of bafilomycin A1.


( E and F ) mCherry - LC3 vesicles were reduced in mCherry - LC3 - transfected stable HEK293 NOS cell lines compared to HEK293 control cells cultured in HBSS , an effect that was restored in the NOS cells by L - NAME ( F ) . Images were acquired by a confocal microscope ( E ) .

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

(E and F) mCherry-LC3 vesicles were reduced in mCherry-LC3-transfected stable HEK293 NOS cell lines compared to HEK293 control cells cultured in HBSS, an effect that was restored in the NOS cells by L-NAME (F). Images were acquired by a confocal microscope (E).


( G and H ) Confocal microscopy images ( G ) and immunofluorescence analysis with anti - Atg16 antibody show that stable HEK293 NOS cell lines had fewer Atg16 - positive structures under starvation , compared to HEK293 control cells ( H ) . Graphical data denote mean ± SEM .

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

(G and H) Confocal microscopy images (G) and immunofluorescence analysis with anti-Atg16 antibody show that stable HEK293 NOS cell lines had fewer Atg16-positive structures under starvation, compared to HEK293 control cells (H). Graphical data denote mean ± SEM.


( A - F ) Immunoblot analyses with anti - nNOS , anti - iNOS , anti - eNOS , anti - phospho - JNK1 , anti - phospho - Bcl - 2 , anti - phospho - S6K , and anti - phospho - S6 antibodies show that stable HEK293 cell lines overexpressing nNOS ( A and D ) , iNOS - GFP ( B and E ) , or eNOS ( C and F ) inhibited both the JNK1 - Bcl - 2 and mTORC1 pathways compared to the HEK293 control cells under basal ( FM ) ( A - C ) or starvation ( HBSS ) ( D - F ) conditions . Graphical data denote mean ± SEM .

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

(A-F) Immunoblot analyses with anti-nNOS, anti-iNOS, anti-eNOS, anti-phospho-JNK1, anti-phospho-Bcl-2, anti-phospho-S6K, and anti-phospho-S6 antibodies show that stable HEK293 cell lines overexpressing nNOS (A and D), iNOS-GFP (B and E), or eNOS (C and F) inhibited both the JNK1-Bcl-2 and mTORC1 pathways compared to the HEK293 control cells under basal (FM) (A-C) or starvation (HBSS) (D-F) conditions. Graphical data denote mean ± SEM.


( A ) Immunoblot analyses with anti - LC3 antibody show that rapamycin ( 8 hr ) and L - NAME ( 24 hr ) increased autophagosome synthesis in bafilomycin A1 - treated mouse primary cortical neurons and HeLa cells .

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

(A) Immunoblot analyses with anti-LC3 antibody show that rapamycin (8 hr) and L-NAME (24 hr) increased autophagosome synthesis in bafilomycin A1-treated mouse primary cortical neurons and HeLa cells.


( B and C ) Confocal microscopy images ( B ) and analysis of autophagic flux by automated Cellomics microscope ( C ) show that L - NAME increased autolysosomes in mRFP - GFP - LC3 HeLa cells .

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

(B and C) Confocal microscopy images (B) and analysis of autophagic flux by automated Cellomics microscope (C) show that L-NAME increased autolysosomes in mRFP-GFP-LC3 HeLa cells.


( D ) L - NAME reduced EGFP - HDQ74 aggregates in EGFP - HDQ74 - transfected Atg5 + / + , but not in Atg5 − / − , MEFs .

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

(D) L-NAME reduced EGFP-HDQ74 aggregates in EGFP-HDQ74-transfected Atg5+/+, but not in Atg5−/−, MEFs.


( E ) Drosophila expressing mutant huntingtin exon 1 ( Q120 ) shows a significant decrease in neurodegeneration ( p < 0 . 001 , paired t test ) upon L - NAME treatment compared to DMSO .

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(E) Drosophila expressing mutant huntingtin exon 1 (Q120) shows a significant decrease in neurodegeneration (p < 0.001, paired t test) upon L-NAME treatment compared to DMSO.


( F ) Expression of an endogenous negative regulator of NOS activity ( NOS4 ) significantly attenuates neurodegeneration ( p < 0 . 05 , paired t test ) in Drosophila expressing mutant huntingtin exon 1 ( Q120 ) .

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

(F) Expression of an endogenous negative regulator of NOS activity (NOS4) significantly attenuates neurodegeneration (p < 0.05, paired t test) in Drosophila expressing mutant huntingtin exon 1 (Q120).


( G - I ) Images from the retina of transgenic HD zebrafish show mutant huntingtin aggregates ( arrows ) ( G ) . Treatment with rapamycin or L - NAME reduced the number of aggregates ( H ) . L - NAME did not reduce aggregates in the presence of NH4Cl , which increased aggregate count ( I ) .

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

(G-I) Images from the retina of transgenic HD zebrafish show mutant huntingtin aggregates (arrows) (G). Treatment with rapamycin or L-NAME reduced the number of aggregates (H). L-NAME did not reduce aggregates in the presence of NH4Cl, which increased aggregate count (I).


IFM images of KIF13A - YFP transfected control and Rab - knockdown HeLa cells . TN : average tubule number ( mean ± SEM , n = 3 ) . B , C Graphs represent the measurement of KIF13A - positive TN ( B ) and TL ( C ) in HeLa cells of Fig 1A ( mean ± SEM ) . n = 3 . nc : total number of cells . nt : total number of tubules . arrowheads and arrows point to the KIF13A - / Rab22A - positive tubular REs and E / SEs respectively . Scale bars : 10 μm .

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

IFM images of KIF13A-YFP transfected control and Rab-knockdown HeLa cells. TN: average tubule number (mean±SEM, n=3). B, C Graphs represent the measurement of KIF13A-positive TN (B) and TL (C) in HeLa cells of Fig 1A (mean± SEM). n=3. nc: total number of cells. nt: total number of tubules.arrowheads and arrows point to the KIF13A-/Rab22A-positive tubular REs and E/SEs respectively. Scale bars: 10 μm.


IFM images of KIF13A - YFP and mCherry - Rab7A / 11A / 22A transfected HeLa cells arrowheads and arrows point to the KIF13A - / Rab22A - positive tubular REs and E / SEs respectively . Scale bars : 10

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

IFM images of KIF13A-YFP and mCherry-Rab7A/11A/22A transfected HeLa cells arrowheads and arrows point to the KIF13A-/Rab22A-positive tubular REs and E/SEs respectively. Scale bars: 10 μm


Live cell imaging of GFP / mCherry - Rab22A with respect to mCherry - Rab11A or GFP - Rab7A in HeLa cells . Magnified view of insets ( at 0 , 20 , 40 seconds ) are shown separately ) , arrowheads and arrows point to the KIF13A - / Rab22A - positive tubular REs and E / SEs respectively . Scale bars : 10

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

Live cell imaging of GFP/mCherry-Rab22A with respect to mCherry-Rab11A or GFP-Rab7A in HeLa cells. Magnified view of insets (at 0, 20, 40 seconds) are shown separately ), arrowheads and arrows point to the KIF13A-/Rab22A-positive tubular REs and E/SEs respectively. Scale bars: 10 μm


Pull - down of different His - KIF13A domains using HeLa cell lysate and then probed with indicated Rab proteins . The bead bound His - KIF13A in each pull - down was shown as coomassie gel . * , non - specific bands .

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Pull-down of different His-KIF13A domains using HeLa cell lysate and then probed with indicated Rab proteins. The bead bound His-KIF13A in each pull-down was shown as coomassie gel. *, non-specific bands.


IFM images of KIF13A - YFP and mCherry - Rab22AWT / Q64L / S19N cotransfected HeLa cells . Arrowheads and arrows point to the KIF13A - positive tubular REs and E / SEs respectively . The colocalization coefficient ( r , in mean ± SEM ) between two markers was indicated separately . Scale bars : 10 μm Graphs represent the measurement of KIF13A - positive TN ( B ) and TL ( C ) in HeLa cells of Fig 2A . n = 3 . nc : total number of cells . nt : total number of

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IFM images of KIF13A-YFP and mCherry-Rab22AWT/Q64L/S19N cotransfected HeLa cells. Arrowheads and arrows point to the KIF13A-positive tubular REs and E/SEs respectively. The colocalization coefficient (r, in mean±SEM) between two markers was indicated separately. Scale bars: 10 μm Graphs represent the measurement of KIF13A-positive TN (B) and TL (C) in HeLa cells of Fig 2A. n=3. nc: total number of cells. nt: total number of tubules


Subcellular fractionation of HeLa cells to probe the localization of Rab22A ( red box ) with respect to other organelle specific proteins . * , non - specific bands .

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Subcellular fractionation of HeLa cells to probe the localization of Rab22A (red box) with respect to other organelle specific proteins. *, non-specific bands.


IFM images of KIF13A - YFP transfected control and Rab22A - , BLOC - 1Mu - , BLOC - 2HPS6 - knockdown HeLa cells . Cells were stained for AP - 1 ( γ ) or internalized with Tf - Alexa fluor 594 arrowheads and arrows point to the KIF13A - / Rab22A - positive tubular REs and E / SEs respectively . Scale bars : 10

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IFM images of KIF13A-YFP transfected control and Rab22A-, BLOC-1Mu-, BLOC-2HPS6- knockdown HeLa cells. Cells were stained for AP-1 (γ) or internalized with Tf-Alexa fluor 594 arrowheads and arrows point to the KIF13A-/Rab22A-positive tubular REs and E/SEs respectively. Scale bars: 10 μm


Graphs represent the measurement of KIF13A - positive TN ( B ) and TL ( C ) in HeLa cells of Fig 3A . n = 3 . nc : total number of cells . nt : total number of

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Graphs represent the measurement of KIF13A-positive TN (B) and TL (C) in HeLa cells of Fig 3A. n=3. nc: total number of cells. nt: total number of tubules


Immunoblotting analysis of proteins in control and Rab22A - , BLOC - 1 - , BLOC - 2 - depleted HeLa cells . * , non - specific bands . Protein band intensities were quantified and indicated on the gels .

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Immunoblotting analysis of proteins in control and Rab22A-, BLOC-1-, BLOC-2-depleted HeLa cells. *, non-specific bands. Protein band intensities were quantified and indicated on the gels.


IFM images of mCherry - Rab22A and KIF13A - YFP cotransfected control and BLOC - 1Mu - , BLOC - 2HPS6 - knockdown HeLa cells ) , arrowheads and arrows point to the KIF13A - / Rab22A - positive tubular REs and E / SEs respectively . Scale bars : 10

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IFM images of mCherry-Rab22A and KIF13A-YFP cotransfected control and BLOC-1Mu-, BLOC-2HPS6- knockdown HeLa cells ), arrowheads and arrows point to the KIF13A-/Rab22A-positive tubular REs and E/SEs respectively. Scale bars: 10 μm


BF and IFM images of control and Rab22A - depleted melanocytes . The colocalization coefficient ( r , in mean ± SEM ) between the two markers indicated separately . Nuclei are stained with Hoechst33258 ) , arrows indicate the melanocyte pigmentation and arrowheads point to the localization of TYRP1 / GFP - Rab22A . Scale bars : 10

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BF and IFM images of control and Rab22A-depleted melanocytes. The colocalization coefficient (r, in mean±SEM) between the two markers indicated separately. Nuclei are stained with Hoechst33258 ), arrows indicate the melanocyte pigmentation and arrowheads point to the localization of TYRP1/GFP-Rab22A. Scale bars: 10 μm


Graph represents the quantified melanin content in control sh , Rab22A sh and BLOC - 1 - melanocytes . n = 3 . The fold - change in melanin content ( mean ± SEM ) indicated

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Graph represents the quantified melanin content in control sh, Rab22A sh and BLOC-1- melanocytes. n=3. The fold-change in melanin content (mean±SEM) indicated separately


Immunoblotting analysis of proteins in Control sh and Rab22A sh melanocytes ) , protein band intensities were quantified and indicated on the

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Immunoblotting analysis of proteins in Control sh and Rab22A sh melanocytes ), protein band intensities were quantified and indicated on the gels


EM of control and Rab22A - knockdown MNT - 1 melanocytes . Arrowheads indicate PMEL fibrils . Scale bars : 1 μm . I , II , III , IV : stages of melanosomes . M : mitochondria . Magnified view of insets are shown separately and they emphasize the stage I melanosomes in both conditions . Note : the presence of hybrid stage I / II melanosomes in Rab22A - knockdown cells . Insets ( b ) - ( d ) and ( g ) - ( h ) are from different cells of respective condition . Scale bars : 200 nm .

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EM of control and Rab22A-knockdown MNT-1 melanocytes. Arrowheads indicate PMEL fibrils. Scale bars: 1 μm. I, II, III, IV: stages of melanosomes. M: mitochondria. Magnified view of insets are shown separately and they emphasize the stage I melanosomes in both conditions. Note: the presence of hybrid stage I/II melanosomes in Rab22A-knockdown cells. Insets (b)-(d) and (g)-(h) are from different cells of respective condition. Scale bars: 200 nm.


BF and IFM images of GFP - Rab22A transfected BLOC - 1 - ( Mu ) melanocytes ) , arrows indicate the melanocyte pigmentation and arrowheads point to the localization of TYRP1 / GFP - Rab22A . Scale bars : 10

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BF and IFM images of GFP-Rab22A transfected BLOC-1-(Mu) melanocytes ), arrows indicate the melanocyte pigmentation and arrowheads point to the localization of TYRP1/GFP-Rab22A. Scale bars: 10 μm


Immunoblotting analysis of proteins in BLOC - 1 - and BLOC - 1R melanocytes protein band intensities were quantified and indicated on the

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Immunoblotting analysis of proteins in BLOC-1- and BLOC-1R melanocytes protein band intensities were quantified and indicated on the gels


Subcellular membrane fractionation of control and Rab22A - , BLOC - 1 - , BLOC - 2 - knockdown HeLa cells and probed the fractions for pallidin , dysbindin and muted . Red colored box emphasizes BLOC - 1 membrane association in the respective cell types .

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Subcellular membrane fractionation of control and Rab22A-, BLOC-1-, BLOC-2-knockdown HeLa cells and probed the fractions for pallidin, dysbindin and muted. Red colored box emphasizes BLOC-1 membrane association in the respective cell types.


Cytosol - membrane fractionation of homogenates from HeLa cells ( B as indicated . Protein band intensities were quantified and indicated the percentage membrane association on the

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Cytosol-membrane fractionation of homogenates from HeLa cells (B as indicated. Protein band intensities were quantified and indicated the percentage membrane association on the gels


Cytosol - membrane fractionation of homogenates fro melanocyte as indicated . Protein band intensities were quantified and indicated the percentage membrane association on the

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Cytosol-membrane fractionation of homogenates fro melanocyte as indicated. Protein band intensities were quantified and indicated the percentage membrane association on the gels


Pull - down of His - Rab22AWT using HeL lysate the bead bound His - Rab22A / His - KIF13A domains were shown as coomassie stained gel

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

Pull-down of His-Rab22AWT using HeL lysate the bead bound His-Rab22A/His-KIF13A domains were shown as coomassie stained gel separatel


Pull - down of His - Rab22AWT usin melanocyte ( E ) lysate . In E , the beads were preloaded with GTPγS or GDP the bead bound His - Rab22A / His - KIF13A domains were shown as coomassie stained gel

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Pull-down of His-Rab22AWT usin melanocyte (E) lysate. In E, the beads were preloaded with GTPγS or GDP the bead bound His-Rab22A/His-KIF13A domains were shown as coomassie stained gel separatel


Pull - down of different His - KIF13A domains using HeLa cell lysate the bead bound His - Rab22A / His - KIF13A domains were shown as coomassie stained gel

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

Pull-down of different His-KIF13A domains using HeLa cell lysate the bead bound His-Rab22A/His-KIF13A domains were shown as coomassie stained gel separatel


C . Biotinylation of the centrosome and microtubules by V5 - BirA * - AURKA in different cell cycle stages . After 18 h biotin incubation , HEK293T : : V5 - BirA * - AURKA were fixed and stained for the fusion protein with V5 antibody , biotinylated proteins with the fluorescent streptavidin and the centrosome with gamma - tubulin antibody . DNA was stained with DAPI . Scale bar , 5 μm .

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C. Biotinylation of the centrosome and microtubules by V5-BirA*-AURKA in different cell cycle stages. After 18 h biotin incubation, HEK293T::V5-BirA*-AURKA were fixed and stained for the fusion protein with V5 antibody, biotinylated proteins with the fluorescent streptavidin and the centrosome with gamma-tubulin antibody. DNA was stained with DAPI. Scale bar, 5 μm.


D . Control and biotin - treated HEK293T cells stably expressing V5 - BirA * and V5 - BirA * - AURKA were lysed and biotinylated proteins were precipitated by streptavidin beads . The initial sample ( initial ) and captured biotinylated proteins ( pulldown ) were run on an SDS gel and immunoblotted with the fluorescent streptavidin and antibodies against V5 and vinculin ( loading control ) .

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D. Control and biotin-treated HEK293T cells stably expressing V5-BirA* and V5-BirA*-AURKA were lysed and biotinylated proteins were precipitated by streptavidin beads. The initial sample (initial) and captured biotinylated proteins (pulldown) were run on an SDS gel and immunoblotted with the fluorescent streptavidin and antibodies against V5 and vinculin (loading control).


C . AURKA interacts with the centriolar satellite proteins PCM1 , CEP131 , CEP72 , CSPP1 , KIAA0586 / Talpid3 and CP110 . HEK293T cells were transiently transfected with FLAG - BirA * or FLAG - BirA * - AURKA . Following 18 h biotin incubation , cells were lysed , and biotinylated proteins were precipitated by streptavidin beads . The initial sample and immunoprecipitated biotinylated proteins were run on a gel and immunoblotted with fluorescent streptavidin and antibodies against FLAG , CEP72 , CEP131 , PCM1 , CEP63 , CSPP1 , KIAA0586 / Talpid3 , CP110 , BBS4 and SSX2IP .

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

C. AURKA interacts with the centriolar satellite proteins PCM1, CEP131, CEP72, CSPP1, KIAA0586/Talpid3 and CP110. HEK293T cells were transiently transfected with FLAG-BirA* or FLAG-BirA*-AURKA. Following 18 h biotin incubation, cells were lysed, and biotinylated proteins were precipitated by streptavidin beads. The initial sample and immunoprecipitated biotinylated proteins were run on a gel and immunoblotted with fluorescent streptavidin and antibodies against FLAG, CEP72, CEP131, PCM1, CEP63, CSPP1, KIAA0586/Talpid3, CP110, BBS4 and SSX2IP.


E . Endogenous interaction of PCM1 and AURKA . Lysates from synchronous and serum - starved HEK293T were immunoprecipitated with AURKA antibody and rabbit IgG control . The initial sample and immunoprecipitated proteins were run on a gel and immunoblotted with antibodies against PCM1 and AURKA .

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

E. Endogenous interaction of PCM1 and AURKA. Lysates from synchronous and serum-starved HEK293T were immunoprecipitated with AURKA antibody and rabbit IgG control. The initial sample and immunoprecipitated proteins were run on a gel and immunoblotted with antibodies against PCM1 and AURKA.


F . AURKA co - localize with PCM1 at the peripheral satellite clusters upon chemical dimerization of satellites with Kif5b motor domain . U2OS cells were co - transfected with GFP - PCM1 - FKBP and HA - Kif5b ( 1 - 269 a . a . ) - FRB and treated with 500 nM rapamycin for 1 hour followed by fixation at 6 hours . Cells that were not treated with rapamycin were processed in parallel as a control . Fixed cells were stained with antibodies against GFP , AURKA and gamma - tubulin . DNA was stained with DAPI . Cell edges are outlined . Scale bar , 10 μm . G . Quantification of pericentrosomal levels of AURKA and gamma tubulin for F . n > 25 cells per experiment . Data represent mean value from two experiments per condition ± SEM ( * * * * p < 0 . 0001 ; * * p < 0 . 01 ; Unpaired Student ' s t - test . ) .

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

F. AURKA co-localize with PCM1 at the peripheral satellite clusters upon chemical dimerization of satellites with Kif5b motor domain. U2OS cells were co-transfected with GFP-PCM1-FKBP and HA-Kif5b(1-269 a.a.)-FRB and treated with 500 nM rapamycin for 1 hour followed by fixation at 6 hours. Cells that were not treated with rapamycin were processed in parallel as a control. Fixed cells were stained with antibodies against GFP, AURKA and gamma-tubulin. DNA was stained with DAPI. Cell edges are outlined. Scale bar, 10 μm. G. Quantification of pericentrosomal levels of AURKA and gamma tubulin for F. n>25 cells per experiment. Data represent mean value from two experiments per condition ± SEM (****p < 0.0001; **p < 0.01; Unpaired Student's t-test.).


A , B . Representative images and quantification of basal body levels of ( A ) AURKA and ( B ) phospho - AURKA ( p - AURKA ) in control and PCM1 - depleted cells . RPE1 cells were transfected with control or PCM1 siRNA # 1 or siRNA # 2 for 48 h . Following 24 h serum starvation , cells were fixed and stained with the indicated antibodies . Centrosomal AURKA and p - AURKA fluorescence intensities were measured from maximum projections , and average means of the levels in control cells were normalized to 1 . n > 500 cells per experiment . Data for ( A ) AURKA and ( B ) p - AURKA represent mean value from three experiments per condition ± SEM ( * * * * p < 0 . 0001 , Unpaired Student ' s t - test . ) Scale bar , 10

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

A, B. Representative images and quantification of basal body levels of (A) AURKA and (B) phospho-AURKA (p-AURKA) in control and PCM1-depleted cells. RPE1 cells were transfected with control or PCM1 siRNA #1 or siRNA #2 for 48 h. Following 24 h serum starvation, cells were fixed and stained with the indicated antibodies. Centrosomal AURKA and p-AURKA fluorescence intensities were measured from maximum projections, and average means of the levels in control cells were normalized to 1. n>500 cells per experiment. Data for (A) AURKA and (B) p-AURKA represent mean value from three experiments per condition ± SEM (****p < 0.0001, Unpaired Student's t-test.) Scale bar, 10 μm


D . Cycloheximide chase experiment for quantification of AURKA half - life . Cells were transfected with control or PCM1 siRNA # 1 for 48 h , then treated with 200 nM cycloheximide along with serum starvation for indicated time points . AURKA intensities were quantified by immunoblotting for AURKA and vinculin ( loading control ) and AURKA levels were normalized to vinculin levels . Data represent mean value from three experiments per condition ± SEM ( * * p < 0 . 01 , Unpaired Student ' s t - test )

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

D. Cycloheximide chase experiment for quantification of AURKA half-life. Cells were transfected with control or PCM1 siRNA#1 for 48 h, then treated with 200 nM cycloheximide along with serum starvation for indicated time points. AURKA intensities were quantified by immunoblotting for AURKA and vinculin (loading control) and AURKA levels were normalized to vinculin levels. Data represent mean value from three experiments per condition ± SEM (**p < 0.01, Unpaired Student's t-test)


A . Representative immunofluorescence images of cilium assembly experiments in control and PCM1 - depleted RPE1 cells treated with DMSO or MLN8237 . Cells were transfected with control or PCM1 siRNA # 1 for 48 h and treated with DMSO ( vehicle control ) or 0 . 5 μM MLN8237 in serum starvation medium for 24 h . Cells were fixed and immunostained for the primary cilium with acetylated tubulin antibody ( Acet - tub ) and the centrosome with gamma - tubulin antibody . DNA was stained with DAPI . Scale bar , 10 μm . B . Quantification of ciliogenesis efficiency for A . n > 100 cells per experiment . Data represent mean value from three experiments per condition ± SEM ( * * * p < 0 . 001 ; * * p < 0 . 01 ; ns , non - significant , Unpaired Student ' s t - test . ) . C . Quantification of cilium length for A . n > 100 cells per experiment . Data represent mean value from three experiments per condition ± SEM ( * * * * p < 0 . 0001 ; ns , non - significant , Unpaired Student ' s t - test . ) .

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

A. Representative immunofluorescence images of cilium assembly experiments in control and PCM1-depleted RPE1 cells treated with DMSO or MLN8237. Cells were transfected with control or PCM1 siRNA#1 for 48 h and treated with DMSO (vehicle control) or 0.5 μM MLN8237 in serum starvation medium for 24 h. Cells were fixed and immunostained for the primary cilium with acetylated tubulin antibody (Acet-tub) and the centrosome with gamma-tubulin antibody. DNA was stained with DAPI. Scale bar, 10 μm. B. Quantification of ciliogenesis efficiency for A. n>100 cells per experiment. Data represent mean value from three experiments per condition ± SEM (***p < 0.001; **p < 0.01; ns, non-significant, Unpaired Student's t-test.). C. Quantification of cilium length for A. n>100 cells per experiment. Data represent mean value from three experiments per condition ± SEM (****p < 0.0001; ns, non-significant, Unpaired Student's t-test.).


D . Representative immunofluorescence images of cilium assembly experiments in control and PCM1 - depleted RPE1 cells treated with DMSO or tubacin . Cells were transfected with control or PCM1 siRNA # 1 for 48 h and treated with DMSO or 2 μM tubacin in serum starvation medium for 24 h . Cells were fixed and immunostained for the primary cilium with Arl13b and acetylated tubulin antibody ( Acet - tub ) and the centrosome with gamma - tubulin antibody . DNA was stained with DAPI . Scale bar , 10 μm E . Quantification of ciliogenesis efficiency for D . n > 100 cells per experiment . Data represent mean value from three experiments per condition ± SEM ( * p < 0 . 05 ; ns , non - significant , Unpaired Student ' s t - test ) .

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

D. Representative immunofluorescence images of cilium assembly experiments in control and PCM1-depleted RPE1 cells treated with DMSO or tubacin. Cells were transfected with control or PCM1 siRNA#1 for 48 h and treated with DMSO or 2 μM tubacin in serum starvation medium for 24 h. Cells were fixed and immunostained for the primary cilium with Arl13b and acetylated tubulin antibody (Acet-tub) and the centrosome with gamma-tubulin antibody. DNA was stained with DAPI. Scale bar, 10 μm E. Quantification of ciliogenesis efficiency for D. n>100 cells per experiment. Data represent mean value from three experiments per condition ± SEM (*p < 0.05; ns, non-significant, Unpaired Student's t-test).


F . Representative immunofluorescence images and quantification of IFT88 levels at the centrosome and primary cilium . RPE1 cells were transfected with control or PCM1 siRNA # 1 for 48 h , serum starved for 24 h and treated with DMSO or 0 . 5 μM MLN8237 . Cells were fixed and immunostained with antibodies against IFT88 , acetylated tubulin and gamma tubulin . DNA was stained with DAPI . Scale bar , 10 μm . G . Quantification of IFT88 centrosomal intensity and ciliary concentration for F . IFT88 fluorescence intensities at the centrosome and axoneme were measured from maximum projections , and average means of the levels in control cells were normalized to 1 . Ciliary concentration was calculated by dividing ciliary fluorescence intensity to the cilium length . n > 100 cells per experiment . Data represent mean value from three experiments per condition ± SEM ( * * * p < 0 . 001 ; * * * * p < 0 . 0001 , Unpaired Student ' s t - test )

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

F. Representative immunofluorescence images and quantification of IFT88 levels at the centrosome and primary cilium. RPE1 cells were transfected with control or PCM1 siRNA#1 for 48 h, serum starved for 24 h and treated with DMSO or 0.5 μM MLN8237. Cells were fixed and immunostained with antibodies against IFT88, acetylated tubulin and gamma tubulin. DNA was stained with DAPI. Scale bar, 10 μm. G. Quantification of IFT88 centrosomal intensity and ciliary concentration for F. IFT88 fluorescence intensities at the centrosome and axoneme were measured from maximum projections, and average means of the levels in control cells were normalized to 1. Ciliary concentration was calculated by dividing ciliary fluorescence intensity to the cilium length. n>100 cells per experiment. Data represent mean value from three experiments per condition ± SEM (***p < 0.001; ****p < 0.0001, Unpaired Student's t-test)


H , I . Quantification of cilium disassembly and centrosomal AURKA levels after serum stimulation . RPE1 cells were transfected with control or PCM1 siRNA # 1 for 48 h , serum starved for 24 h and treated with DMSO or 0 . 5 μM MLN8237 in serum stimulation medium for 2 h and 24 h . Cells were fixed and immunostained with antibodies against acetylated tubulin and gamma tubulin , and percentage of ciliated cells were quantified . x - axis indicates the hours after serum stimulation . ( H ) n > 100 cells per experiment . Data represent mean value from three experiments per condition ± SEM . ( I ) n > 95 cells per experiment . Data represent mean value from three experiments per condition ± SEM ( * * * p < 0 . 001 ; * * * * p < 0 . 0001 , Unpaired Student ' s t - test ) .

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

H, I. Quantification of cilium disassembly and centrosomal AURKA levels after serum stimulation. RPE1 cells were transfected with control or PCM1 siRNA#1 for 48 h, serum starved for 24 h and treated with DMSO or 0.5 μM MLN8237 in serum stimulation medium for 2 h and 24 h. Cells were fixed and immunostained with antibodies against acetylated tubulin and gamma tubulin, and percentage of ciliated cells were quantified. x-axis indicates the hours after serum stimulation. (H) n>100 cells per experiment. Data represent mean value from three experiments per condition ± SEM. (I) n>95 cells per experiment. Data represent mean value from three experiments per condition ± SEM (***p < 0.001; ****p < 0.0001, Unpaired Student's t-test).


( A , B ) C57BL / 6 mice were left untreated ( NT ) or were intraperitoneally treated with indicated amounts of AG for 3 d . Lung sections stained with Haematoxylin and eosin ( H & E ) ( A ) and quantification of lung lesion burden from H & E - stained sections ( B ) . Data information : Data are means + SD of indicated numbers of mice from 1 of n = 3 independent experiments with similar results and each symbol represents 1 mouse . Data are representative of n = 3 independent experiments . One - way ANOVA followed by Dunnett ' s post hoc test were used for statistical analysis , respectively . ns , not significant ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 . Scale bar , 200 μm .

O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-TISSUE O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-TISSUE O 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 O O O O O B-ORGANISM O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) C57BL/6 mice were left untreated (NT) or were intraperitoneally treated with indicated amounts of AG for 3 d. Lung sections stained with Haematoxylin and eosin(H&E) (A) and quantification of lung lesion burden from H&E-stained sections (B). Data information: Data are means + SD of indicated numbers of mice from 1 of n=3 independent experiments with similar results and each symbol represents 1 mouse. Data are representative of n=3 independent experiments. One-way ANOVA followed by Dunnett's post hoc test were used for statistical analysis, respectively. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Scale bar, 200 μm.


( D - G ) H & E staining of lung sections from mice 4 weeks after intranasal infection with M . bovis BCG ( D and E ) or Mtb H37Rv ( F and G ) in the absence or presence of intranasally administrated AG aptamers ( 1 μg ) once at a 1 week - interval . Quantification of lung inflamed regions shown in ( E and G ) . Data information : Data in are means + SD of indicated numbers of mice from 1 of n = 3 independent experiments with similar results and each symbol represents 1 mouse . Data are representative of n = 3 independent experiments . One - way ANOVA followed by Dunnett ' s post hoc test and Student ' s t test were used for statistical analysis , respectively . ns , not significant ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 . Scale bar , 200 μm .

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

(D-G) H&E staining of lung sections from mice 4 weeks after intranasal infection with M. bovis BCG (D and E) or Mtb H37Rv (F and G) in the absence or presence of intranasally administrated AG aptamers (1 μg) once at a 1 week-interval. Quantification of lung inflamed regions shown in (E and G). Data information: Data in are means + SD of indicated numbers of mice from 1 of n=3 independent experiments with similar results and each symbol represents 1 mouse. Data are representative of n=3 independent experiments. One-way ANOVA followed by Dunnett's post hoc test and Student's t test were used for statistical analysis, respectively. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Scale bar, 200 μm.


( B ) Quantitative polymerase chain reaction ( qPCR ) analysis of Mmps including Mmp2 , Mmp9 , Mmp10 , Mmp12 and Mmp13 mRNA from mouse peritoneal macrophages stimulated with AG ( 1 μg / ml ) for indicated times . ( C ) Immunoblots of cell supernatants to analyze secreted MMP2 , MMP9 , MMP10 , MMP12 and MMP13 by mouse peritoneal macrophages stimulated with AG ( 1 μg / mL ) for indicated times ; GADPH of cell lysates served as a loading control . Data information : Data are means + SD averaged from 3 independent experiments performed with technical triplicates and each symbol represents the mean of technical triplicates . Two - way ANOVA followed by Tukey ' s post hoc test was used for statistical analysis . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 .

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

(B) Quantitative polymerase chain reaction (qPCR) analysis of Mmps including Mmp2, Mmp9, Mmp10, Mmp12 and Mmp13 mRNA from mouse peritoneal macrophages stimulated with AG (1 μg/ml) for indicated times. (C) Immunoblots of cell supernatants to analyze secreted MMP2, MMP9, MMP10, MMP12 and MMP13 by mouse peritoneal macrophages stimulated with AG (1 μg/mL) for indicated times; GADPH of cell lysates served as a loading control. Data information: Data are means + SD averaged from 3 independent experiments performed with technical triplicates and each symbol represents the mean of technical triplicates. Two-way ANOVA followed by Tukey's post hoc test was used for statistical analysis. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.


( H ) qPCR analysis of Mmps including Mmp9 , Mmp10 , Mmp12 , and Mmp13 from mouse peritoneal macrophages infected with H37Rv for 24h ( MOI = 5 ) in absence or presence of AG aptamers ( 1 μg / ml ) . ( I ) Immunoblots of cell supernatants to analyze secreted MMP9 , MMP10 , MMP12 and MMP13 by mouse peritoneal macrophages infected with H37Rv for indicated times ( MOI = 5 ) in the absence or presence of AG aptamers ( 0 . 5 μg / ml ) ; GADPH of cell lysates served as the loading control . Data information : Data are means + SD averaged from 3 independent experiments performed with technical triplicates and each symbol represents the mean of technical triplicates . Two - way ANOVA followed by Tukey ' s post hoc test was used for statistical analysis . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 .

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

(H) qPCR analysis of Mmps including Mmp9, Mmp10, Mmp12, and Mmp13 from mouse peritoneal macrophages infected with H37Rv for 24h (MOI=5) in absence or presence of AG aptamers (1 μg/ml). (I) Immunoblots of cell supernatants to analyze secreted MMP9, MMP10, MMP12 and MMP13 by mouse peritoneal macrophages infected with H37Rv for indicated times (MOI=5) in the absence or presence of AG aptamers (0.5 μg/ml); GADPH of cell lysates served as the loading control. Data information: Data are means + SD averaged from 3 independent experiments performed with technical triplicates and each symbol represents the mean of technical triplicates. Two-way ANOVA followed by Tukey's post hoc test was used for statistical analysis. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.


( G ) Immunoblots of cell supernatants to analyze secreted MMP9 , MMP10 , MMP12 , and MMP13 by mouse peritoneal macrophages infected with H37Rv for indicated times ( MOI = 5 ) in absence or presence of inhibitor targeting ERK ( PD98059 ) at the concentration of 10 μM ; GADPH of cell lysates served as the loading control .

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

(G) Immunoblots of cell supernatants to analyze secreted MMP9, MMP10, MMP12, and MMP13 by mouse peritoneal macrophages infected with H37Rv for indicated times (MOI=5) in absence or presence of inhibitor targeting ERK (PD98059) at the concentration of 10 μM; GADPH of cell lysates served as the loading control.


( A , B ) C57BL / 6 mice were left untreated ( NT ) or were intraperitoneally treated with indicated amounts of AG for 3 d in the absence or presence of the MMP inhibitor marimastat ( 10 mg / kg ) given intraperitoneally prior to AG stimulation . Lung sections stained with H & E ( A ) and quantification of lung lesion burden from H & E stained sections ( B ) . Data information : Data in ( A ) are representative of n = 3 independent experiments . Data in ( B ) are means + SD of indicated numbers of mice from one of n = 3 independent experiments and each symbol represents data from 1 mouse . One - way ANOVA followed by Dunnett ' s post hoc test ( B ) was used for statistical analysis . ns , not significant ; * * * * , p < 0 . 0001 . Scale bar , 200 μm .

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

(A, B) C57BL/6 mice were left untreated (NT) or were intraperitoneally treated with indicated amounts of AG for 3 d in the absence or presence of the MMP inhibitor marimastat (10 mg/kg) given intraperitoneally prior to AG stimulation. Lung sections stained with H&E (A) and quantification of lung lesion burden from H&E stained sections (B). Data information: Data in (A) are representative of n=3 independent experiments. Data in (B) are means + SD of indicated numbers of mice from one of n=3 independent experiments and each symbol represents data from 1 mouse. One-way ANOVA followed by Dunnett's post hoc test (B) was used for statistical analysis. ns, not significant; ****, p<0.0001. Scale bar, 200 μm.


( A ) Surface plasmon resonance ( SPR ) assay of the direct interaction of AG with galectin - 9 . Curve fittings to a 1 : 1 Langmuir binding model calculated with TraceDrawer are shown as smooth black lines .

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

(A) Surface plasmon resonance (SPR) assay of the direct interaction of AG with galectin-9. Curve fittings to a 1:1 Langmuir binding model calculated with TraceDrawer are shown as smooth black lines.


( C ) SPR assay of the interaction of AG with CRD2 of galectin - 9 . Curve fittings to a 1 : 1 Langmuir binding model calculated with TraceDrawer are shown as smooth black lines .

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

(C) SPR assay of the interaction of AG with CRD2 of galectin-9. Curve fittings to a 1:1 Langmuir binding model calculated with TraceDrawer are shown as smooth black lines.


( I ) Immunoblot of lysates of shCtrl and shGalectin - 9 THP - 1 cells stimulated with AG ( 1 μg / ml ) for indicated times . Data are representative of n = 3 independent experiments .

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

(I) Immunoblot of lysates of shCtrl and shGalectin-9 THP-1 cells stimulated with AG (1 μg/ml) for indicated times. Data are representative of n=3 independent experiments.


( K ) qRT - PCR detection of MMP transcripts including Mmp9 , Mmp10 , and Mmp12 in shCtrl and shGalectin - 9 THP - 1 cells stimulated with AG ( 1 μg / ml ) for 24 h in absence or presence of ERK inhibitor PD98059 ( 10 μM ) . Data information : Data are means + SD averaged from 3 independent experiments performed with technical triplicates and each symbol represents the mean of technical triplicates . Two - way ANOVA followed by Dunnett ' s post hoc test were used for statistical analysis . ns , not significant ; * , p < 0 . 05 ; * * * , p < 0 . 001 * * * * , p < 0 . 0001 .

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

(K) qRT-PCR detection of MMP transcripts including Mmp9, Mmp10, and Mmp12 in shCtrl and shGalectin-9 THP-1 cells stimulated with AG (1 μg/ml) for 24 h in absence or presence of ERK inhibitor PD98059 (10 μM). Data information: Data are means + SD averaged from 3 independent experiments performed with technical triplicates and each symbol represents the mean of technical triplicates. Two-way ANOVA followed by Dunnett's post hoc test were used for statistical analysis. ns, not significant; *, p<0.05; ***, p<0.001 ****, p<0.0001.


( D ) Confocal microscopy of mouse peritoneal macrophages left untreated ( NC ) ( upper row ) or stimulated with AG ( 1 μg / mL ) for 2 h ( middle row ) or infected with H37Rv for 3 h ( MOI = 5 ) ( bottom row ) , staining with anti - Galectin - 9 and anti - TAK1 antibody . DAPI , nuclei , blue . Scale bar , 5 μm . Data in the right graph show mean + SD of n = 12 fields from three independent experiments . The symbols indicate the colocalization ratio of at least 10 cells in each field .

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

(D) Confocal microscopy of mouse peritoneal macrophages left untreated (NC) (upper row) or stimulated with AG (1 μg/mL) for 2 h (middle row) or infected with H37Rv for 3 h (MOI=5) (bottom row), staining with anti-Galectin-9 and anti-TAK1 antibody. DAPI, nuclei, blue. Scale bar, 5 μm. Data in the right graph show mean + SD of n=12 fields from three independent experiments. The symbols indicate the colocalization ratio of at least 10 cells in each field.


( F ) Immunoblots of cell lysates of peritoneal macrophages isolated from WT or Galectin - 9 KO mice stimulated with AG ( 1 μg / ml ) in absence or presence of TAK1 inhibitor 5Z - 7 - OZ ( 1 μM ) for indicated times . Data are representative of n = 3 independent experiments .

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

(F) Immunoblots of cell lysates of peritoneal macrophages isolated from WT or Galectin-9 KO mice stimulated with AG (1 μg/ml) in absence or presence of TAK1 inhibitor 5Z-7-OZ (1 μM) for indicated times. Data are representative of n=3 independent experiments.


( B , WT or Galectin - 9 KO mice were intraperitoneally treated with AG for 3 d in the absence or presence of the MMP inhibitor marimastat ( 10 mg / kg ) given intraperitoneally prior to AG stimulation . Lung sections stained with H & E Data information : Data are representative of n = 3 independent experiments .

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

(B, WT or Galectin-9 KO mice were intraperitoneally treated with AG for 3 d in the absence or presence of the MMP inhibitor marimastat (10 mg/kg) given intraperitoneally prior to AG stimulation. Lung sections stained with H&E Data information: Data are representative of n=3 independent experiments.


( D , WT or Galectin - 9 KO mice were intranasally infected with H37Rv for 4 weeks in absence or presence of intranasally administrated AG aptamers ( 1 μg ) once at a 1 week - interval . Lung sections stained with H & E Data information : Data are representative of n = 3 independent experiments .

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

(D, WT or Galectin-9 KO mice were intranasally infected with H37Rv for 4 weeks in absence or presence of intranasally administrated AG aptamers (1 μg) once at a 1 week-interval. Lung sections stained with H&E Data information: Data are representative of n=3 independent experiments.


Recovery of infectious recombinant MHV - GFP . Cell culture supernatants containing viruses produced after virus rescue from two MHV - GFP YAC clones ( Clone1 , Clone 2 ) were used to infect 17Cl - 1 cells . At 48 hours post - infection , infected cells were visualised for GFP expression ( left panels ) and by bright field microscopy ( right panels ) . Mock represents 17Cl - 1 cells inoculated with supernatant from BHK - MHV - N cells electroporated without viral RNAs .

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

Recovery of infectious recombinant MHV-GFP. Cell culture supernatants containing viruses produced after virus rescue from two MHV-GFP YAC clones (Clone1, Clone 2) were used to infect 17Cl-1 cells. At 48 hours post-infection, infected cells were visualised for GFP expression (left panels) and by bright field microscopy (right panels). Mock represents 17Cl-1 cells inoculated with supernatant from BHK-MHV-N cells electroporated without viral RNAs.