B Left panel : illustration of kinetochore configurations in meiotic metaphase I . The kinetochores of sister chromatids cohere to form a single microtubule - binding interface . The inner - outer kinetochore axis connects the pericentric chromatin to the spindle microtubule . CREST ( centromere protein ) and BUBR1 ( budding uninhibited by benzimidazole - related 1 ) served as markers for the inner and outer regions of the kinetochore , respectively . A major satellite DNA ( Maj sat DNA ) probe was used to detect the pericentric chromatin . Right panel : categories of inner kinetochore configurations from A . Right panels : Inner kinetochore configurations in MI were classified as overlapping ( two sister kinetochores which overlap by more than their individual radii , also see yellow arrow in A ) , adjacent ( two sister kinetochores which overlap by less than 100 % of their individual radii , also see magenta arrow in A ) or separated ( two sister kinetochores without overlapping , also see light blue arrow in A ) kinetochores . The scale bar represents 1 µm . C Population of overlapping , adjacent , and separated inner kinetochores shown in B . The results represent the mean ± SD of three independent experiments . n = 3 mice for each genotype . The total numbers of counted cells in Miwi + / - , Miwi - / - and Miwi - / ADH spermatocytes were 88 , 118 and 125 , respectively . The total numbers of counted sister kinetochore pairs in Miwi + / - , Miwi - / - and Miwi - / ADH spermatocytes were 3 , 520 , 4 , 720 and 5 , 000 , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Left panel: illustration of kinetochore configurations in meiotic metaphase I. The kinetochores of sister chromatids cohere to form a single microtubule-binding interface. The inner-outer kinetochore axis connects the pericentric chromatin to the spindle microtubule. CREST (centromere protein) and BUBR1 (budding uninhibited by benzimidazole-related 1) served as markers for the inner and outer regions of the kinetochore, respectively. A major satellite DNA (Maj sat DNA) probe was used to detect the pericentric chromatin. Right panel: categories of inner kinetochore configurations from A. Right panels: Inner kinetochore configurations in MI were classified as overlapping (two sister kinetochores which overlap by more than their individual radii, also see yellow arrow in A), adjacent (two sister kinetochores which overlap by less than 100% of their individual radii, also see magenta arrow in A) or separated (two sister kinetochores without overlapping, also see light blue arrow in A) kinetochores. The scale bar represents 1 µm. C Population of overlapping, adjacent, and separated inner kinetochores shown in B. The results represent the mean ± SD of three independent experiments. n=3 mice for each genotype. The total numbers of counted cells in Miwi+/-, Miwi-/- and Miwi-/ADH spermatocytes were 88, 118 and 125, respectively. The total numbers of counted sister kinetochore pairs in Miwi+/-, Miwi-/- and Miwi-/ADH spermatocytes were 3,520, 4,720 and 5,000, respectively.


B Left panel : illustration of kinetochore configurations in meiotic metaphase I . The kinetochores of sister chromatids cohere to form a single microtubule - binding interface . The inner - outer kinetochore axis connects the pericentric chromatin to the spindle microtubule . CREST ( centromere protein ) and BUBR1 ( budding uninhibited by benzimidazole - related 1 ) served as markers for the inner and outer regions of the kinetochore , respectively . A major satellite DNA ( Maj sat DNA ) probe was used to detect the pericentric chromatin . Right panel : categories of inner kinetochore configurations from A . Right panels : Inner kinetochore configurations in MI were classified as overlapping ( two sister kinetochores which overlap by more than their individual radii , also see yellow arrow in A ) , adjacent ( two sister kinetochores which overlap by less than 100 % of their individual radii , also see magenta arrow in A ) or separated ( two sister kinetochores without overlapping , also see light blue arrow in A ) kinetochores . The scale bar represents 1 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Left panel: illustration of kinetochore configurations in meiotic metaphase I. The kinetochores of sister chromatids cohere to form a single microtubule-binding interface. The inner-outer kinetochore axis connects the pericentric chromatin to the spindle microtubule. CREST (centromere protein) and BUBR1 (budding uninhibited by benzimidazole-related 1) served as markers for the inner and outer regions of the kinetochore, respectively. A major satellite DNA (Maj sat DNA) probe was used to detect the pericentric chromatin. Right panel: categories of inner kinetochore configurations from A. Right panels: Inner kinetochore configurations in MI were classified as overlapping (two sister kinetochores which overlap by more than their individual radii, also see yellow arrow in A), adjacent (two sister kinetochores which overlap by less than 100% of their individual radii, also see magenta arrow in A) or separated (two sister kinetochores without overlapping, also see light blue arrow in A) kinetochores. The scale bar represents 1 µm.


D Combined immunofluorescence and DNA FISH analysis of kinetochore inner and outer plate formation in Miwi heterozygous ( Miwi + / - ) and knockout ( Miwi - / - ) at diakinesis / metaphase I . For slicer activity - deficient mutant spermatocytes ( Miwi - / ADH ) , the images are presented in Fig EV1B . Chromosomes were stained with anti - BUBR1 ( green ) and anti - centromere - protein ( CREST , purple ) antibodies to mark the kinetochore outer and inner plate , respectively . In addition , major satellite DNA regions were further detected using an antisense probe ( red signal ) . Nuclear DNA was stained by DAPI ( blue ) . O - O kinetochore pairs : yellow arrow ; S - S , magenta arrow ; O - S , light blue arrow ; and S - O , light green arrow . Scale bars represent 2 µm . E Magnified images of categories of sister kinetochore configurations from D . One arrow indicates overlapping pair . Double arrows indicate split pair . The scale bar represents 1 µm . F Population of the sister kinetochores shown in E . n = 3 mice for each genotype . The total numbers of counted cells in Miwi + / - , Miwi - / - and Miwi - / ADH spermatocytes were 93 , 89 and 81 , respectively . The total numbers of counted sister kinetochore pairs in Miwi + / - , Miwi - / - and Miwi - / ADH spermatocytes were 3 , 720 , 3 , 560 and 3 , 240 , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Combined immunofluorescence and DNA FISH analysis of kinetochore inner and outer plate formation in Miwi heterozygous (Miwi+/-) and knockout (Miwi-/-) at diakinesis/metaphase I. For slicer activity-deficient mutant spermatocytes (Miwi-/ADH), the images are presented in Fig EV1B. Chromosomes were stained with anti-BUBR1 (green) and anti-centromere-protein (CREST, purple) antibodies to mark the kinetochore outer and inner plate, respectively. In addition, major satellite DNA regions were further detected using an antisense probe (red signal). Nuclear DNA was stained by DAPI (blue). O-O kinetochore pairs: yellow arrow; S-S, magenta arrow; O-S, light blue arrow; and S-O, light green arrow. Scale bars represent 2 µm. E Magnified images of categories of sister kinetochore configurations from D. One arrow indicates overlapping pair. Double arrows indicate split pair. The scale bar represents 1 µm. F Population of the sister kinetochores shown in E. n=3 mice for each genotype. The total numbers of counted cells in Miwi+/-, Miwi-/- and Miwi-/ADH spermatocytes were 93, 89 and 81, respectively. The total numbers of counted sister kinetochore pairs in Miwi+/-, Miwi-/- and Miwi-/ADH spermatocytes were 3,720, 3,560 and 3,240, respectively.


D Combined immunofluorescence and DNA FISH analysis of kinetochore inner and outer plate formation in Miwi heterozygous ( Miwi + / - ) and knockout ( Miwi - / - ) at diakinesis / metaphase I . For slicer activity - deficient mutant spermatocytes ( Miwi - / ADH ) , the images are presented in Fig EV1B . Chromosomes were stained with anti - BUBR1 ( green ) and anti - centromere - protein ( CREST , purple ) antibodies to mark the kinetochore outer and inner plate , respectively . In addition , major satellite DNA regions were further detected using an antisense probe ( red signal ) . Nuclear DNA was stained by DAPI ( blue ) . O - O kinetochore pairs : yellow arrow ; S - S , magenta arrow ; O - S , light blue arrow ; and S - O , light green arrow . Scale bars represent 2 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Combined immunofluorescence and DNA FISH analysis of kinetochore inner and outer plate formation in Miwi heterozygous (Miwi+/-) and knockout (Miwi-/-) at diakinesis/metaphase I. For slicer activity-deficient mutant spermatocytes (Miwi-/ADH), the images are presented in Fig EV1B. Chromosomes were stained with anti-BUBR1 (green) and anti-centromere-protein (CREST, purple) antibodies to mark the kinetochore outer and inner plate, respectively. In addition, major satellite DNA regions were further detected using an antisense probe (red signal). Nuclear DNA was stained by DAPI (blue). O-O kinetochore pairs: yellow arrow; S-S, magenta arrow; O-S, light blue arrow; and S-O, light green arrow. Scale bars represent 2 µm.


B RNA pull - down assay using biotinylated major and minor satellite RNA transcripts and whole cell extracts from Miwi heterozygoous ( Miwi + / - ) , knockout ( Miwi - / - ) , slicer activity - deficient mutant ( Miwi - / ADH ) , and Mov10l1 germline knockout ( Mov10l1f / - ; Stra8 - Cre ) testes . Mov10l1f / - ; Stra8 - Cre mice represent a pachytene piRNA - deficient condition . Approximately one repeat of In vitro transcribed major and minor satellite RNAs corresponding to the sequences in A were used as baits . Precipitated proteins were visualized by Western blotting using antibodies against the indicated proteins ( MIWI and MILI ) . The input equals 1 / 10 to 1 / 80 of the protein lysate used in the pull - down . Transcripts corresponding to 300 nt and 162 nt of the 18S rRNA sequence served as controls for the antisense ( As ) and sense ( S ) sequence of major and minor satellite transcripts , respectively . The blot was re - probed with anti - GAPDH antibody serving as a negative control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 RNA pull-down assay using biotinylated major and minor satellite RNA transcripts and whole cell extracts from Miwi heterozygoous (Miwi+/-), knockout (Miwi-/-), slicer activity-deficient mutant (Miwi-/ADH), and Mov10l1 germline knockout (Mov10l1f/-;Stra8-Cre) testes. Mov10l1f/-;Stra8-Cre mice represent a pachytene piRNA-deficient condition. Approximately one repeat of In vitro transcribed major and minor satellite RNAs corresponding to the sequences in A were used as baits. Precipitated proteins were visualized by Western blotting using antibodies against the indicated proteins (MIWI and MILI). The input equals 1/10 to 1/80 of the protein lysate used in the pull-down. Transcripts corresponding to 300 nt and 162 nt of the 18S rRNA sequence served as controls for the antisense (As) and sense (S) sequence of major and minor satellite transcripts, respectively. The blot was re-probed with anti-GAPDH antibody serving as a negative control.


C In vitro RNA processing assay with nuage - nuclear fraction from testicular lysates of Miwi knockout ( - / - ) , heterozygous ( + / - ) , and slicer activity - deficient ( - / ADH ) mutants , and 5 ' - end radioactively labelled RNA targets corresponding to approximately one and two repeats of major and minor satellite sequences ( see panel A for details ) . Transcripts corresponding to 162 , 300 , and 542 nt of the 18S rRNA sequence served as controls . Arrow indicates the cleavage products of In vitro transcribed sense major satellite RNAs after reactions .

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

C In vitro RNA processing assay with nuage-nuclear fraction from testicular lysates of Miwi knockout (-/-), heterozygous (+/-), and slicer activity-deficient (-/ADH) mutants, and 5'-end radioactively labelled RNA targets corresponding to approximately one and two repeats of major and minor satellite sequences (see panel A for details). Transcripts corresponding to 162, 300, and 542 nt of the 18S rRNA sequence served as controls. Arrow indicates the cleavage products of In vitro transcribed sense major satellite RNAs after reactions.


D In vitro RNA processing assay with nuage - nuclear fraction from Mov10l1 heterozygous ( Mov10l1f / + ; Stra8 - Cre ) and Mov10l1 germline knockout ( Mov10l1f / - ; Stra8 - Cre ) testis lysates , and 5 ' - end radioactively labelled RNA targets corresponding to approximately one and two repeats of sense and antisense major satellite sequences . Mov10l1f / - ; Stra8 - Cre mice represent a pachytene piRNA - deficient condition . The arrow indicates the cleavage product of the in vitro transcribed sense major satellite RNA .

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

D In vitro RNA processing assay with nuage-nuclear fraction from Mov10l1 heterozygous (Mov10l1f/+;Stra8-Cre) and Mov10l1 germline knockout (Mov10l1f/-;Stra8-Cre) testis lysates, and 5'-end radioactively labelled RNA targets corresponding to approximately one and two repeats of sense and antisense major satellite sequences. Mov10l1f/-;Stra8-Cre mice represent a pachytene piRNA-deficient condition. The arrow indicates the cleavage product of the in vitro transcribed sense major satellite RNA.


RNA - immunoprecipitation was performed using anti - MIWI antibody . RNA was extracted from anti - MIWI immunoprecipitates and subjected to Northern blot analysis using probes specific for potential piRNAs ( piR major - a and b ) , as indicated in panel 20 % of input RNA from each group was used as the control . The blot was re - hybridized with piR - A and piR - E complementary probes to demonstrate the efficiency of the RNA - IP . A miR - 16 complementary probe served as a negative control .

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

RNA-immunoprecipitation was performed using anti-MIWI antibody. RNA was extracted from anti-MIWI immunoprecipitates and subjected to Northern blot analysis using probes specific for potential piRNAs (piR major-a and b), as indicated in panel 20% of input RNA from each group was used as the control. The blot was re-hybridized with piR-A and piR-E complementary probes to demonstrate the efficiency of the RNA-IP. A miR-16 complementary probe served as a negative control.


A RNA pull - down was performed using in vitro transcribed biotinylated transcripts and whole cell extracts from Miwi + / - , Miwi - / - , Miwi - / ADH , and Mov10l1f / - ; Stra8 - Cre testes . Mov10l1f / - ; Stra8 - Cre mice represent a pachytene piRNA - deficient condition . In vitro transcribed RNAs corresponding to approximately one repeat of major and minor satellite sequences were used as baits . Precipitated protein was visualized by Western blotting using anti - Dicer antibody .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 RNA pull-down was performed using in vitro transcribed biotinylated transcripts and whole cell extracts from Miwi+/-, Miwi-/-, Miwi-/ADH, and Mov10l1f/-;Stra8-Cre testes. Mov10l1f/-;Stra8-Cre mice represent a pachytene piRNA-deficient condition. In vitro transcribed RNAs corresponding to approximately one repeat of major and minor satellite sequences were used as baits. Precipitated protein was visualized by Western blotting using anti-Dicer antibody.


B Equal amounts of total RNA isolated from spermatocytes of Miwi + / - ; Dicerf / + ; Stra8 - Cre , Miwi - / - ; Dicerf / + ; Stra8 - Cre , Miwi + / - ; Dicerf / - ; Stra8 - Cre and Miwi - / - ; Dicerf / - ; Stra8 - Cre mice , were resolved on a 1 % agarose gel . The gel was subjected to Northern blot analysis using probes specific for the sense and antisense sequences of major and minor satellites . The blot was re - hybridized with a 28S rRNA - complementary probe to demonstrate equal loading ( bottom on the left ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Equal amounts of total RNA isolated from spermatocytes of Miwi+/-;Dicerf/+;Stra8-Cre, Miwi-/-;Dicerf/+;Stra8-Cre, Miwi+/-;Dicerf/-;Stra8-Cre and Miwi-/-;Dicerf/-;Stra8-Cre mice, were resolved on a 1% agarose gel. The gel was subjected to Northern blot analysis using probes specific for the sense and antisense sequences of major and minor satellites. The blot was re-hybridized with a 28S rRNA-complementary probe to demonstrate equal loading (bottom on the left).


C in vitro RNA processing assay with nuage - enriched nuclear fraction from Miwi + / - ; Dicerf / + ; Stra8 - Cre , Miwi - / - ; Dicerf / + ; Stra8 - Cre , and Miwi + / - ; Dicerf / - ; Stra8 - Cre mice , and 5 ' - end radioactively labelled RNA targets corresponding to approximately one and two repeats of sense and antisense major satellite sequences . Arrow indicates the cleavage products of In vitro transcribed sense major satellite RNAs by MIWI - piRNA complex .

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

C in vitro RNA processing assay with nuage-enriched nuclear fraction from Miwi+/-;Dicerf/+;Stra8-Cre, Miwi-/-;Dicerf/+;Stra8-Cre, and Miwi+/-;Dicerf/-;Stra8-Cre mice, and 5'-end radioactively labelled RNA targets corresponding to approximately one and two repeats of sense and antisense major satellite sequences. Arrow indicates the cleavage products of In vitro transcribed sense major satellite RNAs by MIWI-piRNA complex.


D Equal amounts of total RNA isolated from spermatocytes of Miwi + / - ; Dicerf / + ; Stra8 - Cre , Miwi - / - ; Dicerf / + ; Stra8 - Cre , Miwi + / - ; Dicerf / - ; Stra8 - Cre and Miwi - / - ; Dicerf / - ; Stra8 - Cre mice , were digested with RNase III ( RNase III ) or nuclease S1 ( S1 ) , and further resolved on a 1 % agarose gel . The gel was subjected to northern blot analysis using probes specific for the sense ( S ) and antisense ( As ) strands of major ( Maj ) and minor ( Min ) satellite transcripts . The blot was re - hybridized with 18S and 28S - complementary probes to demonstrate equal loading and the same efficiency of RNase III or nuclease S1 digestion .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Equal amounts of total RNA isolated from spermatocytes of Miwi+/-;Dicerf/+;Stra8-Cre, Miwi-/-;Dicerf/+;Stra8-Cre, Miwi+/-;Dicerf/-;Stra8-Cre and Miwi-/-;Dicerf/-;Stra8-Cre mice, were digested with RNase III (RNase III) or nuclease S1 (S1), and further resolved on a 1% agarose gel. The gel was subjected to northern blot analysis using probes specific for the sense (S) and antisense (As) strands of major (Maj) and minor (Min) satellite transcripts. The blot was re-hybridized with 18S and 28S-complementary probes to demonstrate equal loading and the same efficiency of RNase III or nuclease S1 digestion.


E Sequential in vitro RNA processing assay using 5 ' - end radioactively labelled RNA targets corresponding to approximately one repeat of sense and antisense major satellite sequences . The nuage - nuclear fraction from Miwi + / - testes is indicated as N . Total cellular extracts ( T ) from Miwi + / - testes were subjected to immunodepletion using anti - Dicer antibodies ( α - Dicer ) , with IgG ( IgG ) as a negative control . The incubation time ( Incub . t ) was 0 . 5 and 1 hour . The arrow indicates the product of MIWI - mediated cleavage of the in vitro transcribed sense major satellite RNA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Sequential in vitro RNA processing assay using 5'-end radioactively labelled RNA targets corresponding to approximately one repeat of sense and antisense major satellite sequences. The nuage-nuclear fraction from Miwi+/- testes is indicated as N. Total cellular extracts (T) from Miwi+/- testes were subjected to immunodepletion using anti-Dicer antibodies (α-Dicer), with IgG (IgG ) as a negative control. The incubation time (Incub. t) was 0.5 and 1 hour. The arrow indicates the product of MIWI-mediated cleavage of the in vitro transcribed sense major satellite RNA.


E Sequential in vitro RNA processing assay using 5 ' - end radioactively labelled RNA targets corresponding to approximately one repeat of sense and antisense major satellite sequences . The nuage - nuclear fraction from Miwi + / - testes is indicated as N . Total cellular extracts ( T ) from Miwi + / - testes were subjected to immunodepletion using anti - Dicer antibodies ( α - Dicer ) , with IgG (

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Sequential in vitro RNA processing assay using 5'-end radioactively labelled RNA targets corresponding to approximately one repeat of sense and antisense major satellite sequences. The nuage-nuclear fraction from Miwi+/- testes is indicated as N. Total cellular extracts (T) from Miwi+/- testes were subjected to immunodepletion using anti-Dicer antibodies (α-Dicer), with IgG (IgG


A Frequency of overlapping , adjacent , and separated inner kinetochores . Spermatocytes from Miwi + / - ; Dicerf / + ; Stra8 - Cre , Miwi - / - ; Dicerf / + ; Stra8 - Cre , Miwi + / - ; Dicerf / - ; Stra8 - Cre , and Miwi - / - ; Dicerf / - ; Stra8 - Cre mice were used to examine sister kinetochore configuration at meiosis I as shown in Fig 3A and 3B . n = 3 mice for each genotype . The total numbers of counted cells in the four types of spermatocytes were 101 , 117 , 129 and 115 , respectively . The total numbers of counted sister kinetochore pairs in the four types of spermatocytes were 4 , 040 , 4 , 680 , 5 , 160 and 4 , 600 , respectively . B Frequency of the different sister kinetochore configurations . The four types of spermatocytes in A were used to examine sister kinetochore configurations in meiosis I metaphase with O - O , S - S , O - S , or S - O as defined in the text . n = 3 mice for each genotype . The total numbers of counted cells in the four types of spermatocytes were 134 , 140 , 140 and 130 , respectively . The total numbers of counted sister kinetochore pairs in the four types of spermatocytes were 5 , 360 , 5 , 600 , 5 , 600 and 5 , 200 , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Frequency of overlapping, adjacent, and separated inner kinetochores. Spermatocytes from Miwi+/-;Dicerf/+;Stra8-Cre, Miwi-/-;Dicerf/+;Stra8-Cre, Miwi+/-;Dicerf/-;Stra8-Cre, and Miwi-/-;Dicerf/-;Stra8-Cre mice were used to examine sister kinetochore configuration at meiosis I as shown in Fig 3A and 3B. n=3 mice for each genotype. The total numbers of counted cells in the four types of spermatocytes were 101, 117, 129 and 115, respectively. The total numbers of counted sister kinetochore pairs in the four types of spermatocytes were 4,040, 4,680, 5,160 and 4,600, respectively. B Frequency of the different sister kinetochore configurations. The four types of spermatocytes in A were used to examine sister kinetochore configurations in meiosis I metaphase with O-O, S-S, O-S, or S-O as defined in the text. n=3 mice for each genotype. The total numbers of counted cells in the four types of spermatocytes were 134, 140, 140 and 130, respectively. The total numbers of counted sister kinetochore pairs in the four types of spermatocytes were 5,360, 5,600, 5,600 and 5,200, respectively.


A Frequency of overlapping , adjacent , and separated inner kinetochores . Spermatocytes from Miwi + / - ; Dicerf / + ; Stra8 - Cre , Miwi - / - ; Dicerf / + ; Stra8 - Cre , Miwi + / - ; Dicerf / - ; Stra8 - Cre , and Miwi - / - ; Dicerf / - ; Stra8 - Cre mice were used to examine sister kinetochore configuration at meiosis I as shown in Fig 3A and 3B . n = 3 mice for each genotype . The total numbers of counted cells in the four types of spermatocytes were 101 , 117 , 129 and 115 , respectively . The total numbers of counted sister kinetochore pairs in the four types of spermatocytes were 4 , 040 , 4 , 680 , 5 , 160 and 4 , 600 , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Frequency of overlapping, adjacent, and separated inner kinetochores. Spermatocytes from Miwi+/-;Dicerf/+;Stra8-Cre, Miwi-/-;Dicerf/+;Stra8-Cre, Miwi+/-;Dicerf/-;Stra8-Cre, and Miwi-/-;Dicerf/-;Stra8-Cre mice were used to examine sister kinetochore configuration at meiosis I as shown in Fig 3A and 3B. n=3 mice for each genotype. The total numbers of counted cells in the four types of spermatocytes were 101, 117, 129 and 115, respectively. The total numbers of counted sister kinetochore pairs in the four types of spermatocytes were 4,040, 4,680, 5,160 and 4,600, respectively.


C Frequency of abnormal arrangement in the meiotic metaphase I plate . The four types of spermatocytes in A and B were used to perform immunofluorescence as shown in Fig 1B and C . n = 3 mice for each genotype . The total numbers of counted metaphase I plates in the four types of spermatocytes were 221 , 181 , 421 and 240 , respectively . D Frequency of meiosis I metaphase cells with chromosomal abnormalities . The four types of spermatocytes in A - C were used to perform a metaphase spread as shown in Fig 1H and 1I . n = 3 mice for each genotype . The total numbers of counted MI cells in the four types of spermatocytes were 89 , 73 , 132 and 104 , respectively . E Frequency of meiosis II metaphase cells with chromosomal abnormalities . The four types of spermatocytes in A - D were used to perform a metaphase spread as shown in Fig 1K and L . n = 3 mice for each genotype . The total numbers of counted MII cells in the four types of spermatocytes were 74 , 87 , 85 and 94 , respectively .

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

C Frequency of abnormal arrangement in the meiotic metaphase I plate. The four types of spermatocytes in A and B were used to perform immunofluorescence as shown in Fig 1B and C. n=3 mice for each genotype. The total numbers of counted metaphase I plates in the four types of spermatocytes were 221, 181, 421 and 240, respectively. D Frequency of meiosis I metaphase cells with chromosomal abnormalities. The four types of spermatocytes in A-C were used to perform a metaphase spread as shown in Fig 1H and 1I. n=3 mice for each genotype. The total numbers of counted MI cells in the four types of spermatocytes were 89, 73, 132 and 104, respectively. E Frequency of meiosis II metaphase cells with chromosomal abnormalities. The four types of spermatocytes in A-D were used to perform a metaphase spread as shown in Fig 1K and L. n=3 mice for each genotype. The total numbers of counted MII cells in the four types of spermatocytes were 74, 87, 85 and 94, respectively.


C Frequency of abnormal arrangement in the meiotic metaphase I plate . The four types of spermatocytes in A and B were used to perform immunofluorescence as shown in Fig 1B and C . n = 3 mice for each genotype . The total numbers of counted metaphase I plates in the four types of spermatocytes were 221 , 181 , 421 and 240 , respectively .

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

C Frequency of abnormal arrangement in the meiotic metaphase I plate. The four types of spermatocytes in A and B were used to perform immunofluorescence as shown in Fig 1B and C. n=3 mice for each genotype. The total numbers of counted metaphase I plates in the four types of spermatocytes were 221, 181, 421 and 240, respectively.


A - F ' Upper panels ( A - F ) show morphology of c4da neurons of the indicated genotypes at the third instar larval stage , lower panels ( A ' - F ' ) show c4da neuron morphology at 18 h after puparium formation ( APF ) . Neurons were labeled by CD8 : : GFP expression under ppk - GAL4 or by tdtomato expression in MARCM clones . ( A , A ' ) C4da neurons expressing control Orco dsRNA . Scale bars are 100 μm in A ( for larval images ) and 50 μm in A ' ( for pupal images ) . ( B , B ' ) C4da neurons expressing PP2A - 29B dsRNA . ( C , C ' ) Homozygous PP2A - 29BGE16781 mutant c4da neuron MARCM clones . ( D , D ' ) Homozygous PP2A - 29BGE16781 mutant c4da neuron MARCM clones expressing UAS - HAPP2A - 29B . ( E , E ' ) C4da neurons expressing dominant - negative Mts . ( F , F ' ) Homozygous mtss5286 mutant c4da neuron MARCM clones . G Penetrance of pruning defects at 18 h APF in A ' - E ' . * * P < 0 . 005 , * * * P < 0 . 0005 , Fisher ' s exact test . Number of neurons for each genotype are given in the figure , the number of animals is given below in parentheses . H Severity of pruning defects at 18 h APF in A ' - E ' as assessed by number of primary and secondary dendrites attached to soma at 18 h APF . Data are mean + / - s . d . , * * P < 0 . 005 , * * * P < 0 . 0005 , Wilcoxon ' s test . Numbers of neurons ( animals ) for each genotype are given in the figure .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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' Upper panels (A - F) show morphology of c4da neurons of the indicated genotypes at the third instar larval stage, lower panels (A' - F') show c4da neuron morphology at 18 h after puparium formation (APF). Neurons were labeled by CD8::GFP expression under ppk-GAL4 or by tdtomato expression in MARCM clones. (A, A') C4da neurons expressing control Orco dsRNA. Scale bars are 100 μm in A (for larval images) and 50 μm in A' (for pupal images). (B, B') C4da neurons expressing PP2A-29B dsRNA. (C, C') Homozygous PP2A-29BGE16781 mutant c4da neuron MARCM clones. (D, D') Homozygous PP2A-29BGE16781 mutant c4da neuron MARCM clones expressing UAS-HAPP2A-29B. (E, E') C4da neurons expressing dominant-negative Mts. (F, F') Homozygous mtss5286 mutant c4da neuron MARCM clones. G Penetrance of pruning defects at 18 h APF in A' - E'. ** P<0.005, *** P<0.0005, Fisher's exact test. Number of neurons for each genotype are given in the figure, the number of animals is given below in parentheses. H Severity of pruning defects at 18 h APF in A' - E' as assessed by number of primary and secondary dendrites attached to soma at 18 h APF. Data are mean +/- s. d., ** P<0.005, *** P<0.0005, Wilcoxon's test. Numbers of neurons (animals) for each genotype are given in the figure.


A - J The indicated Sox14 or Mical constructs were tested for their ability to suppress pruning defects induced by PP2A - 29B dsRNA expression . C4da neurons were labeled by CD8 : : GFP expression under ppk - GAL4 , and dendrite pruning defects were assessed at 18 h APF . Panels A - E show effects of Sox14 / Mical pathway manipulations alone , panels F - J show neurons coexpressing PP2A - 29B dsRNA . The scale bar in A is 50 μm . ( A , F ) C4da neurons ( co - ) expressing lacZ . ( B , G ) C4da neurons ( co - ) overexpressing Sox14 . ( C , H ) C4da neurons ( co - ) overexpressing Mical . ( D , I ) C4da neurons ( co - ) expressing Mical ∆ redox . ( E , J ) C4da neurons ( co - ) expressing Mical ∆ CH . K Penetrance of pruning defects in panels A - J . * P < 0 . 05 , * * * P < 0 . 0005 , Fisher ' s exact test . Numbers of neurons ( animals ) for each genotype are given in the figure the numbers of animals are given below in parentheses . L Severity of pruning defects in panels A - J as assessed by number of primary and secondary dendrites attached to soma at 18 h APF . Data are mean + / - s . d . , * P < 0 . 05 , * * * P < 0 . 0005 , Wilcoxon ' s test . Numbers of neurons ( animals ) for each genotype are given in the figure .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-J The indicated Sox14 or Mical constructs were tested for their ability to suppress pruning defects induced by PP2A-29B dsRNA expression. C4da neurons were labeled by CD8::GFP expression under ppk-GAL4, and dendrite pruning defects were assessed at 18 h APF. Panels A - E show effects of Sox14/Mical pathway manipulations alone, panels F - J show neurons coexpressing PP2A-29B dsRNA. The scale bar in A is 50 μm. (A, F) C4da neurons (co-)expressing lacZ. (B, G) C4da neurons (co-)overexpressing Sox14. (C, H) C4da neurons (co-)overexpressing Mical. (D, I) C4da neurons (co-)expressing Mical∆redox. (E, J) C4da neurons (co-)expressing Mical∆CH. K Penetrance of pruning defects in panels A - J. * P<0.05, *** P<0.0005, Fisher's exact test. Numbers of neurons (animals) for each genotype are given in the figure the numbers of animals are given below in parentheses. L Severity of pruning defects in panels A - J as assessed by number of primary and secondary dendrites attached to soma at 18 h APF. Data are mean +/- s. d., * P<0.05, *** P<0.0005, Wilcoxon's test. Numbers of neurons (animals) for each genotype are given in the figure.


C4da neurons were labeled by CD8 : : GFP expression under ppk - GAL4 , and expression of Sox14 was assessed by immunofluorescence . A - C Sox14 stainings at 2 h APF . ( A ) Control c4da neurons . ( B ) C4da neurons expressing PP2A - 29B dsRNA . ( C ) C4da neuron expressing dominant - negative Mts . D Quantification of Sox14 expression in ( A - C ) . C4da neuron Sox14 expression was normalized to Sox14 expression in neighboring ddaE c1da neurons ( identified by characteristic cell body shape ) . Data are mean + / - s . d . . The numbers of neurons ( animals ) for each genotype are given in each graph ( data were not derived from independent experiments ) . n . s . , not significant , * * * P < 0 . 0005 , Wilcoxon ' s test .

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

C4da neurons were labeled by CD8::GFP expression under ppk-GAL4, and expression of Sox14 was assessed by immunofluorescence. A - C Sox14 stainings at 2 h APF. (A) Control c4da neurons. (B) C4da neurons expressing PP2A-29B dsRNA. (C) C4da neuron expressing dominant-negative Mts. D Quantification of Sox14 expression in (A - C). C4da neuron Sox14 expression was normalized to Sox14 expression in neighboring ddaE c1da neurons (identified by characteristic cell body shape). Data are mean +/- s. d.. The numbers of neurons (animals) for each genotype are given in each graph (data were not derived from independent experiments). n. s., not significant, *** P<0.0005, Wilcoxon's test.


C4da neurons were labeled by CD8 : : GFP expression under ppk - GAL4 , and expression of Mical was assessed by immunofluorescence . E - H Mical stainings at the indicated time points . ( E ) Mical expression in control c4da neurons at 2 h APF . ( F ) Mical expression in c4da neurons expressing dominant - negative Mts at 2 h APF . ( G ) Mical expression in c4da neurons expressing PP2A - 29B dsRNA at 2 h APF . ( H ) Mical expression in c4da neurons expressing Orco dsRNA at 0 h APF . ( I ) Mical expression in c4da neurons expressing PP2A - 29B dsRNA at 0 h APF . J Quantification of Mical expression in ( E - I ) . C4da neuron Mical expression was normalized to Mical expression in neighboring ddaE c1da neurons ( marked by asterisks ) . Data are mean + / - s . d . . The numbers of neurons ( animals ) for each genotype are given in each graph ( data were not derived from independent experiments ) . n . s . , not significant , * * P < 0 . 005 , * * * P < 0 . 0005 , Wilcoxon ' s test . Data information : Scale bars in A and E are 10 μm .

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

C4da neurons were labeled by CD8::GFP expression under ppk-GAL4, and expression of Mical was assessed by immunofluorescence. E - H Mical stainings at the indicated time points. (E) Mical expression in control c4da neurons at 2 h APF. (F) Mical expression in c4da neurons expressing dominant-negative Mts at 2 h APF. (G) Mical expression in c4da neurons expressing PP2A-29B dsRNA at 2 h APF. (H) Mical expression in c4da neurons expressing Orco dsRNA at 0 h APF. (I) Mical expression in c4da neurons expressing PP2A-29B dsRNA at 0 h APF. J Quantification of Mical expression in (E - I). C4da neuron Mical expression was normalized to Mical expression in neighboring ddaE c1da neurons (marked by asterisks). Data are mean +/- s. d.. The numbers of neurons (animals) for each genotype are given in each graph (data were not derived from independent experiments). n. s., not significant, ** P<0.005, *** P<0.0005, Wilcoxon's test. Data information: Scale bars in A and E are 10 μm.


A - G The indicated UAS - tsr constructs were tested for their ability to suppress pruning defects induced by PP2A - 29B dsRNA expression . Suppression tests were performed as in Figure 2 . Panels A - C show effects of the expression of wild type or phosphomutant tsr variants on dendrite pruning , panels D - G show neurons coexpressing PP2A - 29B dsRNA . ( A , E ) C4da neuron ( co - ) overexpressing wild type tsr . ( B , F ) C4da neuron ( co - ) overexpressing tsr S3A . ( C , G ) C4da neuron ( co - ) overexpressing tsr S3E . The arrow in panel G denotes a characteristic varicosity at the tip of an unpruned dendrite . ( D ) C4da neuron co - expressing PP2A - 29B dsRNA and lacZ as a UAS titration control . H Penetrance of pruning defects in panels A - G . * P < 0 . 05 , Fisher ' s exact test . Numbers of neurons ( animals ) for each genotype are given in the figure . I Severity of pruning defects in panels A - G as assessed by number of primary and secondary dendrites attached to soma at 18 h APF . Data are mean + / - s . d . , * P < 0 . 05 , Wilcoxon ' s test . Numbers of neurons ( animals ) for each genotype are given in the figure .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - G The indicated UAS-tsr constructs were tested for their ability to suppress pruning defects induced by PP2A-29B dsRNA expression. Suppression tests were performed as in Figure 2. Panels A - C show effects of the expression of wild type or phosphomutant tsr variants on dendrite pruning, panels D - G show neurons coexpressing PP2A-29B dsRNA. (A, E) C4da neuron (co-)overexpressing wild type tsr. (B, F) C4da neuron (co-)overexpressing tsr S3A. (C, G) C4da neuron (co-)overexpressing tsr S3E. The arrow in panel G denotes a characteristic varicosity at the tip of an unpruned dendrite. (D) C4da neuron co-expressing PP2A-29B dsRNA and lacZ as a UAS titration control. H Penetrance of pruning defects in panels A - G. * P<0.05, Fisher's exact test. Numbers of neurons (animals) for each genotype are given in the figure. I Severity of pruning defects in panels A - G as assessed by number of primary and secondary dendrites attached to soma at 18 h APF. Data are mean +/- s. d., * P<0.05, Wilcoxon's test. Numbers of neurons (animals) for each genotype are given in the figure.


A - G The indicated UAS - tsr constructs were tested for their ability to suppress pruning defects induced by PP2A - 29B dsRNA expression . Suppression tests were performed as in Figure 2 . Panels A - C show effects of the expression of wild type or phosphomutant tsr variants on dendrite pruning , panels D - G show neurons coexpressing PP2A - 29B dsRNA . ( A , E ) C4da neuron ( co - ) overexpressing wild type tsr . ( B , F ) C4da neuron ( co - ) overexpressing tsr S3A . ( C , G ) C4da neuron ( co - ) overexpressing tsr S3E . The arrow in panel G denotes a characteristic varicosity at the tip of an unpruned dendrite . ( D ) C4da neuron co - expressing PP2A - 29B dsRNA and lacZ as a UAS titration control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - G The indicated UAS-tsr constructs were tested for their ability to suppress pruning defects induced by PP2A-29B dsRNA expression. Suppression tests were performed as in Figure 2. Panels A - C show effects of the expression of wild type or phosphomutant tsr variants on dendrite pruning, panels D - G show neurons coexpressing PP2A-29B dsRNA. (A, E) C4da neuron (co-)overexpressing wild type tsr. (B, F) C4da neuron (co-)overexpressing tsr S3A. (C, G) C4da neuron (co-)overexpressing tsr S3E. The arrow in panel G denotes a characteristic varicosity at the tip of an unpruned dendrite. (D) C4da neuron co-expressing PP2A-29B dsRNA and lacZ as a UAS titration control.


J - L GFP - tagged Drosophila cofilin ( GFP : : tsr ) was expressed in c4da neurons under ppk - GAL4 , and its localization was assessed at the indicated developmental stages by live imaging . Panels J - L show GFP : : tsr localization in c4da neurons at the third instar stage , panels J ' - L ' show GFP : : tsr localization in c4da neurons at 6 h APF . ( J , J ' ) Control c4da neurons expressing Orco dsRNA . ( K , K ' ) C4da neurons expressing Mical dsRNA . ( L , L ' ) C4da neurons expressing PP2A - 29B dsRNA . M Quantification of GFP : : tsr punctae in samples J - L ' . Data are mean + / - s . d . , the numbers of neurons ( animals ) analyzed for each genotype are given in the figure ( data were not derived from independent experiments ) . * * P < 0 . 005 , * * * P < 0 . 0005 , n . s . , not significant , Wilcoxon ' s test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - L GFP-tagged Drosophila cofilin (GFP::tsr) was expressed in c4da neurons under ppk-GAL4, and its localization was assessed at the indicated developmental stages by live imaging. Panels J - L show GFP::tsr localization in c4da neurons at the third instar stage, panels J' - L' show GFP::tsr localization in c4da neurons at 6 h APF. (J, J') Control c4da neurons expressing Orco dsRNA. (K, K') C4da neurons expressing Mical dsRNA. (L, L') C4da neurons expressing PP2A-29B dsRNA. M Quantification of GFP::tsr punctae in samples J - L'. Data are mean +/- s. d., the numbers of neurons (animals) analyzed for each genotype are given in the figure (data were not derived from independent experiments). ** P<0.005, *** P<0.0005, n. s., not significant, Wilcoxon's test.


A - H Genetic interactions between PP2A - 29B and actin regulators during dendrite pruning . The indicated manipulations of actin regulators were tested for their ability to suppress pruning defects induced by PP2A - 29B dsRNA expression . Suppression tests were performed as in Figure 2 . ( A ) C4da neuron coexpressing PP2A - 29B dsRNA and tdtomato as suppression control . ( B ) C4da neuron coexpressing PP2A - 29B dsRNA and GFP : : tsr . ( C ) C4da neuron coexpressing PP2A - 29B dsRNA and RacN17 . ( D ) C4da neuron coexpressing PP2A - 29B dsRNA and RhoN19 . ( E ) C4da neuron expressing PP2A - 29B dsRNA in ena210 / + background . ( F ) C4da neuron coexpressing PP2A - 29B dsRNA and Orco dsRNA as suppression control . ( G ) C4da neuron coexpressing PP2A - 29B dsRNA and Chicadee dsRNA . ( H ) C4da neuron coexpressing PP2A - 29B dsRNA and moesin dsRNA . I Penetrance of pruning defects in panels E - L . n . s . , not significant , Fisher ' s exact test . Numbers of neurons ( animals ) for each genotype are given in the figure . J Severity of pruning defects in panels E - L as assessed by number of primary and secondary dendrites attached to soma at 18 h APF . Data are mean + / - s . d . , * P < 0 . 05 , * * P < 0 . 005 , * * * P < 0 . 0005 , Wilcoxon ' s test . Numbers of neurons ( animals ) for each genotype are given in the figure .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - H Genetic interactions between PP2A-29B and actin regulators during dendrite pruning. The indicated manipulations of actin regulators were tested for their ability to suppress pruning defects induced by PP2A-29B dsRNA expression. Suppression tests were performed as in Figure 2. (A) C4da neuron coexpressing PP2A-29B dsRNA and tdtomato as suppression control. (B) C4da neuron coexpressing PP2A-29B dsRNA and GFP::tsr. (C) C4da neuron coexpressing PP2A-29B dsRNA and RacN17. (D) C4da neuron coexpressing PP2A-29B dsRNA and RhoN19. (E) C4da neuron expressing PP2A-29B dsRNA in ena210/+ background. (F) C4da neuron coexpressing PP2A-29B dsRNA and Orco dsRNA as suppression control. (G) C4da neuron coexpressing PP2A-29B dsRNA and Chicadee dsRNA. (H) C4da neuron coexpressing PP2A-29B dsRNA and moesin dsRNA. I Penetrance of pruning defects in panels E - L. n. s., not significant, Fisher's exact test. Numbers of neurons (animals) for each genotype are given in the figure. J Severity of pruning defects in panels E - L as assessed by number of primary and secondary dendrites attached to soma at 18 h APF. Data are mean +/- s. d., * P<0.05, ** P<0.005, *** P<0.0005, Wilcoxon's test. Numbers of neurons (animals) for each genotype are given in the figure.


A . Immunohistochemical analysis of SHARPIN expression in the human mammary gland . Cross - section of a mammary duct ( upper panel ) and magnification of the marked area ( lower panel ) . SHARPIN - positive luminal ( grey arrow ) and stromal cells ( red arrow ) , and the approximate position of the basal lamina ( dashed red line ) are indicated . Scale bar represents 50 μm .

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

A. Immunohistochemical analysis of SHARPIN expression in the human mammary gland. Cross-section of a mammary duct (upper panel) and magnification of the marked area (lower panel). SHARPIN-positive luminal (grey arrow) and stromal cells (red arrow), and the approximate position of the basal lamina (dashed red line) are indicated. Scale bar represents 50 μm.


B . Western blot analysis of SHARPIN protein expression in isolated primary mammary epithelial cells ( MECs ) and mammary stromal fibroblasts ( MSFs ) . CDH1 and vimentin were used as markers of epithelial and stromal cell lineages , respectively . GAPDH served as a control for protein loading .

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B. Western blot analysis of SHARPIN protein expression in isolated primary mammary epithelial cells (MECs) and mammary stromal fibroblasts (MSFs). CDH1 and vimentin were used as markers of epithelial and stromal cell lineages, respectively. GAPDH served as a control for protein loading.


C . FACS - based isolation of mouse mammary gland basal epithelial cells ( LinnegCD24intICAM - 1hi ) , mature luminal epithelial cells ( LinnegCD24hiICAM - 1neg ) , luminal progenitor cells ( LinnegCD24hiICAM - 1int ) , and stromal cells ( LinnegCD24neg ) .

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C. FACS-based isolation of mouse mammary gland basal epithelial cells (LinnegCD24intICAM-1hi), mature luminal epithelial cells (LinnegCD24hiICAM-1neg), luminal progenitor cells (LinnegCD24hiICAM-1int), and stromal cells (LinnegCD24neg).


D . Quantitative PCR analysis of SHARPIN mRNA expression in cell populations isolated in ( C ) ( mean ± SEM , n = 3 - 5 ) .

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D. Quantitative PCR analysis of SHARPIN mRNA expression in cell populations isolated in (C) (mean ± SEM, n = 3 - 5).


A - G . Mammary ductal outgrowth in 3 - 7 weeks old wt or Sharpincpdm female mice . A . Representative carmine - alum stained mammary gland whole mounts . Arrow indicates the inguinal lymph node . Scale bars represent 2 mm . B . Quantification of mammary ductal outgrowth area ( n = 4 - 15 glands ) . C . Quantification of the number of ductal branch points per mammary gland ( n = 7 - 9 glands , 7 weeks old mice ) .

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A-G. Mammary ductal outgrowth in 3-7 weeks old wt or Sharpincpdm female mice. A. Representative carmine-alum stained mammary gland whole mounts. Arrow indicates the inguinal lymph node. Scale bars represent 2 mm. B. Quantification of mammary ductal outgrowth area (n = 4 - 15 glands). C. Quantification of the number of ductal branch points per mammary gland (n = 7 - 9 glands, 7 weeks old mice).


A - G . Mammary ductal outgrowth in 3 - 7 weeks old wt or Sharpincpdm female mice . D . Cryosections from 7 weeks old wt or Sharpincpdm mouse mammary glands stained with HE ( upper panel ) or immunolabelled with the indicated antibodies . Scale bars represent 20

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A-G. Mammary ductal outgrowth in 3-7 weeks old wt or Sharpincpdm female mice. D. Cryosections from 7 weeks old wt or Sharpincpdm mouse mammary glands stained with HE (upper panel) or immunolabelled with the indicated antibodies. Scale bars represent 20 μm


A - G . Mammary ductal outgrowth in 3 - 7 weeks old wt or Sharpincpdm female mice . E . Representative carmine - alum stained images highlighting terminal end buds ( TEBs ) in 7 weeks old wt and Sharpincpdm mouse mammary glands and F . quantification of the number of TEBs per gland ( n = 9 - 10 glands ) . Scale bar represents 500 μm .

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A-G. Mammary ductal outgrowth in 3-7 weeks old wt or Sharpincpdm female mice. E. Representative carmine-alum stained images highlighting terminal end buds (TEBs) in 7 weeks old wt and Sharpincpdm mouse mammary glands and F. quantification of the number of TEBs per gland (n = 9 - 10 glands). Scale bar represents 500 μm.


A - G . Mammary ductal outgrowth in 3 - 7 weeks old wt or Sharpincpdm female mice . G . TEBs in paraffin sections from wt or Sharpincpdm mouse mammary glands stained with HE ( upper panel ) or immunolabelled with the indicated antibodies ( lower panel ) . Scale bars represent 50 µm . Mean ± SEM .

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A-G. Mammary ductal outgrowth in 3-7 weeks old wt or Sharpincpdm female mice. G. TEBs in paraffin sections from wt or Sharpincpdm mouse mammary glands stained with HE (upper panel) or immunolabelled with the indicated antibodies (lower panel). Scale bars represent 50 µm. Mean ± SEM.


A - C . Monitoring of mammary gland development following transplantation of wt or Sharpincpdm mouse mammary epithelium in the mammary fat pads ( epithelium - free ) of virgin wt mice . A . Representative carmine - alum stained images of mouse mammary glands generated from wt or Sharpincpdm epithelial tissue transplants . B . Quantification of transplant growth take - on - rate ( n = 17 ) . C . Quantification of fat pad filling rate of grown transplants ( n = 7 - 10 ) in wt hosts .

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A-C. Monitoring of mammary gland development following transplantation of wt or Sharpincpdm mouse mammary epithelium in the mammary fat pads (epithelium-free) of virgin wt mice. A. Representative carmine-alum stained images of mouse mammary glands generated from wt or Sharpincpdm epithelial tissue transplants. B. Quantification of transplant growth take-on-rate (n = 17). C. Quantification of fat pad filling rate of grown transplants (n = 7 - 10) in wt hosts.


D . Monitoring of mammary gland differentiation during pregnancy following transplantation of wt or Sharpincpdm mouse mammary epithelium into wt hosts . Host animals were subsequently mated and mammary glands isolated at P15 . Representative Carmine - alum stained mammary gland whole mounts generated from wt and cdpm mammary epithelial transplants ( upper panel ) , and magnifications of the branched ductal epithelium ( lower panel ) are shown ( n = 5 mice ) . Scale bars represent 1 mm .

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D. Monitoring of mammary gland differentiation during pregnancy following transplantation of wt or Sharpincpdm mouse mammary epithelium into wt hosts. Host animals were subsequently mated and mammary glands isolated at P15. Representative Carmine-alum stained mammary gland whole mounts generated from wt and cdpm mammary epithelial transplants (upper panel), and magnifications of the branched ductal epithelium (lower panel) are shown (n = 5 mice). Scale bars represent 1 mm.


E . Representative images of carmine - alum stained mouse mammary gland whole mounts generated from 7 weeks old S100a4 - Cre ; Sharpinfl / fl conditional knockout mice and their littermate controls ( S100a4 - Cre ; Sharpin fl / + ) F . Quantification of the normalized Sharpincpdm and S100a4 - Cre ; Sharpinfl / fl mammary ductal outgrowth area relative to littermate control mice ( wt and Sharpincpdm n = 10 glands ; S100a4 - Cre ; Sharpin fl / + and S100a4 - Cre ; Sharpinfl / fl n = 8 glands ) . G . Quantification of the number of TEBs per gland ( n = 8 - 9 glands ) . Mean ± SEM .

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E. Representative images of carmine-alum stained mouse mammary gland whole mounts generated from 7 weeks old S100a4-Cre; Sharpinfl/fl conditional knockout mice and their littermate controls (S100a4-Cre; Sharpin fl/+) F. Quantification of the normalized Sharpincpdm and S100a4-Cre; Sharpinfl/fl mammary ductal outgrowth area relative to littermate control mice (wt and Sharpincpdm n = 10 glands; S100a4-Cre; Sharpin fl/+ and S100a4-Cre; Sharpinfl/fl n = 8 glands). G. Quantification of the number of TEBs per gland (n = 8 - 9 glands). Mean ± SEM.


A . Second harmonic generation ( SHG ) and unfiltered multiphoton ( MP ) imaging of collagen fibres surrounding TEBs ( dashed line ) in 6 - 7 weeks old wt and Sharpincpdm mouse mammary glands ( n = 6 - 9 mice , 5 - 13 TEBs analysed per mouse ; scale bars represent 100 µm ) . Magnified images of collagen fibres are also shown ( scale bars represent 50 µm ) . B . Frequency of clear collagen bundles around TEBs per mouse ( Mean ± SEM , n = 6 - 9 mice ) .

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A. Second harmonic generation (SHG) and unfiltered multiphoton (MP) imaging of collagen fibres surrounding TEBs (dashed line) in 6-7 weeks old wt and Sharpincpdm mouse mammary glands (n = 6 - 9 mice, 5 - 13 TEBs analysed per mouse; scale bars represent 100 µm). Magnified images of collagen fibres are also shown (scale bars represent 50 µm). B. Frequency of clear collagen bundles around TEBs per mouse (Mean ± SEM, n = 6 - 9 mice).


C . AFM indentation analysis of stromal stiffness in 6 - 7 weeks old wt and Sharpincpdm mouse mammary gland tissue sections ( n = 3 mice ; 3 - 6 sample sections per mouse and 192 indentation curves per sample ) .

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C. AFM indentation analysis of stromal stiffness in 6-7 weeks old wt and Sharpincpdm mouse mammary gland tissue sections (n = 3 mice; 3 - 6 sample sections per mouse and 192 indentation curves per sample).


A . Ingenuity Pathway Analysis of differential gene expression ( fold - change ( FC ) > 1 . 5 , p < 0 . 05 ) in wt and Sharpincpdm MSFs ( passage 0 ) . The differentially expressed genes implicated in the most significantly altered canonical pathways ( leukocyte adhesion and transmigration , and tissue fibrosis ) are shown . Three independent cell isolation replicates were compared pairwise . FC colour scale represents a range from 0 − 4 .

A. Ingenuity Pathway Analysis of differential gene expression (fold-change (FC) > 1.5, p < 0.05) in wt and Sharpincpdm MSFs (passage 0). The differentially expressed genes implicated in the most significantly altered canonical pathways (leukocyte adhesion and transmigration, and tissue fibrosis) are shown. Three independent cell isolation replicates were compared pairwise. FC colour scale represents a range from 0−4.


B . Analysis of collagen degradation on cross - bow - shaped micropatterns coated with the DQ collagen substrate . Mean intensity projection images of the DQ collagen fluorescence ( top panel ; cell area is outlined by dashed line ; original integer range 0 − 255 ) and single confocal images of the fibrinogen - Alexa647 - marked micropatterns ( bottom panel ) are shown ( n ≥ 53 from four independent experiments ) . Scale bars represent 10 μm .

B. Analysis of collagen degradation on cross-bow-shaped micropatterns coated with the DQ collagen substrate. Mean intensity projection images of the DQ collagen fluorescence (top panel; cell area is outlined by dashed line; original integer range 0 − 255) and single confocal images of the fibrinogen-Alexa647-marked micropatterns (bottom panel) are shown (n ≥ 53 from four independent experiments). Scale bars represent 10 μm.


C - D . Collagen plug contraction assay using wt and Sharpincpdm MSFs or wt MSFs with SHARPIN siRNA silencing C . Representative light microscopy images of the collagen plugs three days post cell culture . D . Quantification of the average collagen plug area 1 − 3 days after seeding ( n = 3 - 6 ) . Scale bars represent 2 mm .

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C-D. Collagen plug contraction assay using wt and Sharpincpdm MSFs or wt MSFs with SHARPIN siRNA silencing C. Representative light microscopy images of the collagen plugs three days post cell culture. D. Quantification of the average collagen plug area 1−3 days after seeding (n = 3-6). Scale bars represent 2 mm.


E . SHARPIN protein level after silencing with negative control siRNA or SHARPIN siRNA in wt MSFs . Tubulin was used as loading control .

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E. SHARPIN protein level after silencing with negative control siRNA or SHARPIN siRNA in wt MSFs. Tubulin was used as loading control.


F - I . Traction force microscopy analysis of wt and Sharpincpdm MSFs on collagen - coated matrices . F . Representative images of traction forces observed in wt and Sharpincpdm cells ( colour scale units : Pa ; cell boundaries are indicated by dashed lines ) . G . Quantification of strain energy per cell ( n = 8 − 11 cells from two independent experiments ) .

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F-I. Traction force microscopy analysis of wt and Sharpincpdm MSFs on collagen-coated matrices. F. Representative images of traction forces observed in wt and Sharpincpdm cells (colour scale units: Pa; cell boundaries are indicated by dashed lines). G. Quantification of strain energy per cell (n = 8 − 11 cells from two independent experiments).


F - I . Traction force microscopy analysis of wt and Sharpincpdm MSFs on collagen - coated matrices . H . Representative images of the traction forces and mCherry fluorescence of mCherry - Ctrl transfected wt cells and mCherry - Ctrl or mCherry - SHARPIN transfected Sharpincpdm cells . Scale bars represent 20 µm . I . Quantification of strain energy per cell ( wt mCherry control n = 26 ; Sharpincpdm mCherry - Ctrl n = 21 ; Sharpincpdm mCherry - SHARPIN n = 24 cells from five independent experiments ) .

F-I. Traction force microscopy analysis of wt and Sharpincpdm MSFs on collagen-coated matrices. H. Representative images of the traction forces and mCherry fluorescence of mCherry-Ctrl transfected wt cells and mCherry-Ctrl or mCherry-SHARPIN transfected Sharpincpdm cells. Scale bars represent 20 µm. I. Quantification of strain energy per cell (wt mCherry control n = 26; Sharpincpdm mCherry-Ctrl n = 21; Sharpincpdm mCherry-SHARPIN n = 24 cells from five independent experiments).


J - K . Focal adhesion dynamics . J . Visualization of GFP - Paxillin dynamics in a wt and a Sharpincpdm cell over time . Colour scale represents a range from early to late occurring focal adhesions . K . Quantification of focal adhesion average size , assembly rate and disassembly rate ( wt n = 17527 , 5250 , 5311 FAs ; and Sharpincpdm n = 9052 , 2790 , 3264 FAs , respectively from two independent experiments ) . Mean ± SEM .

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J-K. Focal adhesion dynamics. J. Visualization of GFP-Paxillin dynamics in a wt and a Sharpincpdm cell over time. Colour scale represents a range from early to late occurring focal adhesions. K. Quantification of focal adhesion average size, assembly rate and disassembly rate (wt n =17527, 5250, 5311 FAs; and Sharpincpdm n= 9052, 2790, 3264 FAs, respectively from two independent experiments). Mean ± SEM.


A . Quantification of soluble collagen levels in wt and Sharpincpdm MSF cultures on days 2 , 5 and 7 post serum removal ( n = 6 from two independent experiments ) .

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A. Quantification of soluble collagen levels in wt and Sharpincpdm MSF cultures on days 2, 5 and 7 post serum removal (n = 6 from two independent experiments).


B - C . Analysis of the collagen network in cell - derived matrices ( CDMs ) produced by wt and Sharpincpdm MSFs using COL1A1 immunolabelling . B . Maximum intensity projections of confocal images are shown . Scale bars represent 50 μm . C . Quantification of distinct collagen fibre bundles in wt and Sharpincpdm CDM samples ( n = 5 ) . Mean ± SEM .

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B-C. Analysis of the collagen network in cell-derived matrices (CDMs) produced by wt and Sharpincpdm MSFs using COL1A1 immunolabelling. B. Maximum intensity projections of confocal images are shown. Scale bars represent 50 μm. C. Quantification of distinct collagen fibre bundles in wt and Sharpincpdm CDM samples (n = 5). Mean ± SEM.


Cells were fixed and the viral bait fused with the V5 - tag was visualized by immunofluorescence staining using Alexa Fluor - 488 labeled anti - V5 immunostaining ( yellow ) , actin with Alexa Fluor594 - labeled phalloidin ( magenta ) , and nuclei were stained with DAPI ( cyan ) ( Scale bar : 10 μm ) .

Cells were fixed and the viral bait fused with the V5-tag was visualized by immunofluorescence staining using Alexa Fluor-488 labeled anti-V5 immunostaining (yellow), actin with Alexa Fluor594-labeled phalloidin (magenta), and nuclei were stained with DAPI (cyan) (Scale bar: 10 μm).


A For the image - based drug screening , Calu - 1 cells were infected with SARS - CoV - 2 in the presence of drugs with indicated doses for 48 h , immunostained , imaged and analyzed . The example images of mock - infected cells and virus - infected control cells ( no drugs ) . The viral yield in cells was detected with an antibody for the viral N protein ( virus - infected cells ; upper image ) , and nuclear staining ( mock - infected cells ; lower image ) ( Scale bar : 200 μm ) .

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A For the image-based drug screening, Calu-1 cells were infected with SARS-CoV-2 in the presence of drugs with indicated doses for 48 h, immunostained, imaged and analyzed. The example images of mock-infected cells and virus-infected control cells (no drugs). The viral yield in cells was detected with an antibody for the viral N protein (virus-infected cells; upper image), and nuclear staining (mock-infected cells; lower image) (Scale bar: 200 μm).


B The dose - response curves for six drugs with potential antiviral effects . The drug - induced inhibition of virus infection ( count of N protein positive cells ) was quantified using the area under the curve ( AUC ) values .

B The dose-response curves for six drugs with potential antiviral effects. The drug-induced inhibition of virus infection (count of N protein positive cells) was quantified using the area under the curve (AUC) values.


C The effect of drugs on cell viability in the presence of the virus , e . g . , cytotoxicity was evaluated by the count of nuclei .

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C The effect of drugs on cell viability in the presence of the virus, e.g., cytotoxicity was evaluated by the count of nuclei.


D Inhibition of interactions formed with DDX39B in the presence of MTX using co - IP assay . DDX39B is fused to the C terminus of a V5 tag , and the interaction partner is fused with a Strep - HA tag . The diagram ( upper ) shows the interaction pairs formed with DDX39B used for co - IP assay . Dot - blot results ( lower ) show the interaction pairs ( arrangement as upper panel ) in presence of different concentration of MTX as indicated on the top of the image . The image displayed is representative of 3 replicates ( n = 3 ) .

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D Inhibition of interactions formed with DDX39B in the presence of MTX using co-IP assay. DDX39B is fused to the C terminus of a V5 tag, and the interaction partner is fused with a Strep-HA tag. The diagram (upper) shows the interaction pairs formed with DDX39B used for co-IP assay. Dot-blot results (lower) show the interaction pairs (arrangement as upper panel) in presence of different concentration of MTX as indicated on the top of the image. The image displayed is representative of 3 replicates (n = 3).


E Quantification of DDX39B binding to different interaction partners . Quantification of V5 - tagged DDX39B protein amount was normalized over HA - tagged interaction partner intensity and displayed as a fold change over the ratio of untreated cells . Data that represent the mean of 3 replicates are highlighted in red . A fold change > 0 . 5 is considered significant .

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E Quantification of DDX39B binding to different interaction partners. Quantification of V5-tagged DDX39B protein amount was normalized over HA-tagged interaction partner intensity and displayed as a fold change over the ratio of untreated cells. Data that represent the mean of 3 replicates are highlighted in red. A fold change > 0.5 is considered significant.


A αSMA mRNA levels in TGFβ - treated HFL1 fibroblasts and the effect of the IRE1α inhibitor 4μ8C . * P = 0 , 001 .

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A αSMA mRNA levels in TGFβ-treated HFL1 fibroblasts and the effect of the IRE1α inhibitor 4μ8C. *P = 0,001.


B Collagen1α2 mRNA levels in TGFβ - treated HFL1 fibroblasts ± the IRE1α inhibitor 4μ8C . * P = 0 , 01 .

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B Collagen1α2 mRNA levels in TGFβ-treated HFL1 fibroblasts ± the IRE1α inhibitor 4μ8C. *P = 0,01.


C mRNA levels of ER - stress related genes in TGFβ - treated HFL1 fibroblasts . * P CHOP = 0 , 045 , * P Bip = 0 , 023 , * P spliced XBP - 1 P = 0 , 001 , compared to corresponding control .

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C mRNA levels of ER-stress related genes in TGFβ-treated HFL1 fibroblasts. *P CHOP = 0,045, *P Bip = 0,023, *P spliced XBP-1 P = 0,001, compared to corresponding control.


D Western blot of CHOP protein levels in TGFβ - treated HFL1 cells ± the IRE1α inhibitor 4μ8C .

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D Western blot of CHOP protein levels in TGFβ-treated HFL1 cells ± the IRE1α inhibitor 4μ8C.


E Agarose gel showing the presence of spliced and unspliced XBP - 1 mRNA in TGFβ - treated HFL1 fibroblasts ± the IRE1α inhibitor 4μ8C .

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E Agarose gel showing the presence of spliced and unspliced XBP-1 mRNA in TGFβ-treated HFL1 fibroblasts ± the IRE1α inhibitor 4μ8C.


F Immunohistochemistry staining of αSMA in TGFβ - treated HFL1fibroblasts ± the IRE1α inhibitor 4μ8C . Scale bars = 20μm .

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F Immunohistochemistry staining of αSMA in TGFβ-treated HFL1fibroblasts ± the IRE1α inhibitor 4μ8C. Scale bars = 20μm.


G Quantification of secreted soluble collagen proteins produced by TGFβ - treated HFL1 cells ± the IRE1α inhibitor 4μ8C . * P = 0 , 049 .

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G Quantification of secreted soluble collagen proteins produced by TGFβ-treated HFL1 cells ± the IRE1α inhibitor 4μ8C. *P = 0,049.


H αSMA mRNA expression in TGFβ - treated HFL1 cells overexpressing two RNase dead mutants ( K599A and K907A ) of IRE1α . * P K599A = 0 . 013 , * P K907A = 0 . 0008 compared to WT .

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H αSMA mRNA expression in TGFβ-treated HFL1 cells overexpressing two RNase dead mutants (K599A and K907A) of IRE1α. *P K599A = 0.013, *P K907A = 0.0008 compared to WT.


I αSMA mRNA expression in TGFβ - treated IRE1α - / - or WT MEF cells . * P = 0 . 025 .

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I αSMA mRNA expression in TGFβ-treated IRE1α -/- or WT MEF cells. *P = 0.025.


A miR - 150 levels in TGFβ - treated HFL1 fibroblasts and the effect of the IRE1α inhibitor 4μ8C . * P = 0 . 046 compared to control .

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A miR-150 levels in TGFβ-treated HFL1 fibroblasts and the effect of the IRE1α inhibitor 4μ8C. * P = 0.046 compared to control.


B miR - 150 levels in HFL1 fibroblasts overexpressing two RNase dead mutants ( K599A and K907A ) of IRE1α . * P = 0 . 0043 ( K599A ) , P = 0 . 043 ( K907A ) , compared to control .

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B miR-150 levels in HFL1 fibroblasts overexpressing two RNase dead mutants (K599A and K907A) of IRE1α. * P = 0.0043 (K599A), P = 0.043 (K907A), compared to control.


C miR - 150 levels in TGFβ - treated WT ( IRE1α + / + ) and IRE1α - / - MEF cells . * P = 0 . 018 . Results are expressed as fold change compared to the Ctrl .

C miR-150 levels in TGFβ-treated WT (IRE1α +/+) and IRE1α -/- MEF cells. *P = 0.018. Results are expressed as fold change compared to the Ctrl.


D miR - 150 levels in IRE1α - / - MEF cells that were either untransfected ( UT ) or transfected with miRNA inhibitor control ( siCtrl ) or with a miR - 150 - 5p miRNA inhibitor . * P = 0 . 034 compared to UT .

D miR-150 levels in IRE1α -/- MEF cells that were either untransfected (UT) or transfected with miRNA inhibitor control (siCtrl) or with a miR-150-5p miRNA inhibitor. * P = 0.034 compared to UT.


E αSMA mRNA expression in TGFβ - treated IRE1α - / - MEF cells that were either untransfected ( UT ) or transfected with miRNA inhibitor control ( siCtrl ) or with a miR150 - 5p miRNA inhibitor . * P = 0 . 043 compared to UT .

E αSMA mRNA expression in TGFβ-treated IRE1α -/- MEF cells that were either untransfected (UT) or transfected with miRNA inhibitor control (siCtrl) or with a miR150-5p miRNA inhibitor. *P = 0.043 compared to UT.


F miR - 150 levels in HFL1 fibroblasts transfected with a control plasmid ( Ctrl ) or a miR - 150 expression plasmid ( miR150 ) . * P = 0 . 038 .

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F miR-150 levels in HFL1 fibroblasts transfected with a control plasmid (Ctrl) or a miR-150 expression plasmid (miR150). * P = 0.038.


G Western blot of c - Myb in TGFβ - treated HFL1 fibroblasts that were either untransfected ( UT ) , transfected with a control plasmid ( pCtrl ) or a miR150 expression plasmid ( miR150 ) . * P = 0 . 0206 in UT Ctrl vs . UT TGFβ and P = 0 . 0126 in pCtrl Ctrl vs . pCtrl TGFβ .

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G Western blot of c-Myb in TGFβ-treated HFL1 fibroblasts that were either untransfected (UT), transfected with a control plasmid (pCtrl) or a miR150 expression plasmid (miR150). *P = 0.0206 in UT Ctrl vs. UT TGFβ and P = 0.0126 in pCtrl Ctrl vs. pCtrl TGFβ .


H Western blot of αSMA in the same experimental setup as in G . * P = 0 . 0405 in UT Ctrl vs . UT TGFβ , and P = 0 . 0284 in pCtrl Ctrl vs . pCtrl TGFβ .

H Western blot of αSMA in the same experimental setup as in G. * P = 0.0405 in UT Ctrl vs. UT TGFβ, and P = 0.0284 in pCtrl Ctrl vs. pCtrl TGFβ.


I c - Myb mRNA levels in untransfected HFL1 cells ( UT ) and in cells transfected with a control siRNA ( siCtrl ) or an siRNA targeting c - Myb ( sicMyb ) . * P = 0 . 00001 compared to UT .

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I c-Myb mRNA levels in untransfected HFL1 cells (UT) and in cells transfected with a control siRNA (siCtrl) or an siRNA targeting c-Myb (sicMyb). * P = 0.00001 compared to UT.


J αSMA mRNA expression in untransfected HFL1 cells ( UT ) and in cells transfected with a control siRNA ( siCtrl ) or with a siRNA targeting c - Myb ( sicMyb ) . * P = 0 . 0042 in UT Ctrl vs . UT TGFβ .

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J αSMA mRNA expression in untransfected HFL1 cells (UT) and in cells transfected with a control siRNA (siCtrl) or with a siRNA targeting c-Myb (sicMyb). * P = 0.0042 in UT Ctrl vs. UT TGFβ.


L In vitro cleavage assay with recombinant IRE1α and miR - 150 ( left ) or XBP - 1 ( right ) .

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L In vitro cleavage assay with recombinant IRE1α and miR-150 (left) or XBP-1 (right).


M miR - 150 expression in HFL - 1 cells that were pre - treated with TGFβ in the absence of presence of 4μ8C and then treated with Actinomycin D ( ActD ) for 4 and 24 hours . * P = 0 . 027 compared to TGFβ .

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M miR-150 expression in HFL-1 cells that were pre-treated with TGFβ in the absence of presence of 4μ8C and then treated with Actinomycin D (ActD) for 4 and 24 hours. * P = 0.027 compared to TGFβ.


A Analysis of ER size in HFL1 fibroblasts treated with TGFβ in the absence ( Ctrl ) or presence of the IRE1α inhibitor 4μ8C , or in HFL1 cells transfected with a siRNA control siRNA ( siCtrl ) or with siRNA targeting XBP - 1 ( siXBP - 1 ) . The ER was visualized with ER tracker Red and nuclei stained with Hoechst ( left , scale bars = 20 μm ) or by electron microscopy ( right , scale bars = 200 nm ) where red lines mark the ER . B Quantification of ER size relative to the nucleus from the experimental setup in A . * P = 0 . 0001 in Ctrl , and * P = 0 . 0223 in siCtrl .

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A Analysis of ER size in HFL1 fibroblasts treated with TGFβ in the absence (Ctrl) or presence of the IRE1α inhibitor 4μ8C, or in HFL1 cells transfected with a siRNA control siRNA (siCtrl) or with siRNA targeting XBP-1 (siXBP-1). The ER was visualized with ER tracker Red and nuclei stained with Hoechst (left, scale bars = 20 μm) or by electron microscopy (right, scale bars = 200 nm) where red lines mark the ER.B Quantification of ER size relative to the nucleus from the experimental setup in A. * P = 0.0001 in Ctrl, and * P = 0.0223 in siCtrl.


C XBP - 1 mRNA expression in untransfected HFL1 cells ( UT ) and in cells transfected with control siRNA ( siCtrl ) or siRNA targeting XBP - 1 ( siXBP - 1 ) . * P = 0 , 0001 .

C XBP-1 mRNA expression in untransfected HFL1 cells (UT) and in cells transfected with control siRNA (siCtrl) or siRNA targeting XBP-1 (siXBP-1). * P = 0,0001.


D Collagen protein levels in TGFβ treated HFL1 fibroblasts transfected as described in C . * P = 0 , 005 .

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D Collagen protein levels in TGFβ treated HFL1 fibroblasts transfected as described in C. * P = 0,005.


A The effect of the IRE1α inhibitor 4μ8C on CCl4 induced liver fibrosis in C57BL / 6mice as measured by the histological metavir score . Ctrl ( n = 8 ) ; Ctrl + 4μ8C ( n = 6 ) ; CCl4 ( n = 6 ) ; CCl4 + 4μ8C ( n = 8 ) . * P = 0 , 0077 . B Representative liver tissue sections stained with sirius red for collagen ( top ) and αSMA ( bottom ) . Scale bars = 50 μm . C Percentage of αSMA positive cells in pericentral areas in livers from mice in the experimental setup in A . * P = 0 , 036 .

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A The effect of the IRE1α inhibitor 4μ8C on CCl4 induced liver fibrosis in C57BL/6mice as measured by the histological metavir score. Ctrl (n = 8) ; Ctrl + 4μ8C (n = 6) ; CCl4 (n = 6); CCl4 + 4μ8C (n = 8). * P = 0,0077.B Representative liver tissue sections stained with sirius red for collagen (top) and αSMA (bottom). Scale bars= 50 μm.C Percentage of αSMA positive cells in pericentral areas in livers from mice in the experimental setup in A. * P = 0,036.


D Liver to body weight ratio from the experimental setup in A . * P = 0 , 0031 .

D Liver to body weight ratio from the experimental setup in A. * P = 0,0031.


E BiP mRNA expression in liver tissue from mice presented in A . * P = 0 , 020 .

E BiP mRNA expression in liver tissue from mice presented in A. * P = 0,020.


F Representative gel showing levels of spliced and unspliced XBP - 1 in liver tissue from control and CCl4 - induced cirrhotic mice and the effect of 4μ8C treatment .

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F Representative gel showing levels of spliced and unspliced XBP-1 in liver tissue from control and CCl4-induced cirrhotic mice and the effect of 4μ8C treatment.


G miR150 expression in liver tissue from mice in A . * P = 0 , 032 compared to untreated controls .

G miR150 expression in liver tissue from mice in A. * P = 0,032 compared to untreated controls.


H αSMA mRNA levels in primary stellate cells isolated from mouse liver and then treated or not with TGFβ ± 4μ8C . * P = 0 , 031 , n = 3 .

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H αSMA mRNA levels in primary stellate cells isolated from mouse liver and then treated or not with TGFβ ± 4μ8C. * P = 0,031, n = 3.


I BiP mRNA levels in primary stellate cells isolated from mouse liver and then treated with or without TGFβ ± 4μ8C . * P = 0 , 008 , n = 3 .

I BiP mRNA levels in primary stellate cells isolated from mouse liver and then treated with or without TGFβ ± 4μ8C. * P = 0,008, n = 3.


J Microscopic images of primary mouse hepatic stellate cells stained for BiP ( top ) or αSMA ( bottom ) . Scale bars = 20 μm .

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J Microscopic images of primary mouse hepatic stellate cells stained for BiP (top) or αSMA (bottom). Scale bars = 20 μm.


K Western blot showing BiP protein expression in primary mouse hepatic stellate cells .

K Western blot showing BiP protein expression in primary mouse hepatic stellate cells.


L Quantification of mRNA expression levels of CTGF and collagen in primary human hepatic stellate cells stimulated with TGFβ ± 4μ8C .

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L Quantification of mRNA expression levels of CTGF and collagen in primary human hepatic stellate cells stimulated with TGFβ ±4μ8C.


A The effect of the IRE1α inhibitor 4μ8C on TGFβ - induced skin fibrosis in C57BL / 6 mice as measured by the percentage of sirius red areas in the hypodermis . Ctrl ( n = 6 ) ; Ctrl + 4μ8C ( n = 5 ) ; TGFβ ( n = 8 ) ; TGFβ + 4μ8C ( n = 8 ) . * P = 0 , 046 . B Representative skin tissue sections stained with sirius red for collagen ( top ) and αSMA ( bottom ) . Scale bars = 50 μm . C Percentage αSMA - positive cells in the hypodermis of mice from the experimental setup in A . * P = 0 , 024 .

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A The effect of the IRE1α inhibitor 4μ8C on TGFβ-induced skin fibrosis in C57BL/6 mice as measured by the percentage of sirius red areas in the hypodermis. Ctrl (n = 6); Ctrl + 4μ8C (n = 5); TGFβ (n = 8); TGFβ + 4μ8C (n = 8). * P = 0,046.B Representative skin tissue sections stained with sirius red for collagen (top) and αSMA (bottom). Scale bars = 50 μm.C Percentage αSMA-positive cells in the hypodermis of mice from the experimental setup in A. * P = 0,024.


D Representative image of the area selected for laser capture microdissection ( scale bars = 100 μm ) . E Expression levels of αSMA mRNA in samples of hypodermis obtained by laser capture microdissection . Ctrl ( n = 5 ) , Ctrl + 4μ8C ( n = 5 ) , TGFβ ( n = 5 ) , TGFβ + 4μ8C ( n = 5 ) . * P = 0 , 044 . F miR - 150 expression levels in the same samples as shown in E .

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D Representative image of the area selected for laser capture microdissection (scale bars = 100 μm).E Expression levels of αSMA mRNA in samples of hypodermis obtained by laser capture microdissection. Ctrl (n = 5), Ctrl + 4μ8C (n = 5), TGFβ (n = 5), TGFβ + 4μ8C (n = 5). * P = 0,044.F miR-150 expression levels in the same samples as shown in E.


G Immunohistochemistry staining of αSMA in primary skinfibroblasts isolated from healthy mice after treatment with TGFβ ± 4μ8C . Scale bars = 20 μm .

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G Immunohistochemistry staining of αSMA in primary skinfibroblasts isolated from healthy mice after treatment with TGFβ ± 4μ8C. Scale bars = 20 μm.


H αSMA mRNA expression in primary mouse skin fibroblasts after treatment with TGFβ ± 4μ8C . * P = 0 , 049 , n = 4 .

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H αSMA mRNA expression in primary mouse skin fibroblasts after treatment with TGFβ ± 4μ8C. * P = 0,049, n = 4.


I Agarose gel showing levels of spliced and unspliced XBP - 1 in primary mouse skin fibroblasts treated as indicated with TGFβ and 4μ8C .

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I Agarose gel showing levels of spliced and unspliced XBP-1 in primary mouse skin fibroblasts treated as indicated with TGFβ and 4μ8C.


J BiP mRNA expression in primary mouse skin fibroblasts after treatment with TGFβ in the absence and presence of 4μ8C . * P = 0 , 00007 for TGFβ vs . Ctrl and P = 0 , 00002 for TGFβ vs . TGFβ + 4μ8C . n = 4 .

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J BiP mRNA expression in primary mouse skin fibroblasts after treatment with TGFβ in the absence and presence of 4μ8C. * P = 0,00007 for TGFβ vs. Ctrl and P = 0,00002 for TGFβ vs. TGFβ + 4μ8C. n=4.


K Western blot showing CHOP protein expression in primary mouse skin fibroblasts treated as indicated with TGFβ and 4μ8C .

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K Western blot showing CHOP protein expression in primary mouse skin fibroblasts treated as indicated with TGFβ and 4μ8C.


L miR - 150 mRNA levels in primary mouse skin fibroblasts treated as indicated with TGFβ and 4μ8C . * P = 0 , 00031 compared to untreated control , n = 3 .

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L miR-150 mRNA levels in primary mouse skin fibroblasts treated as indicated with TGFβ and 4μ8C. * P = 0,00031 compared to untreated control, n = 3.


A CTGF mRNA expression levels in skin ( dSSc ) and lung ( LSSc ) fibroblasts isolated from scleroderma patients and treated with the IRE1α inhibitor 4μ8C . * P = 0 , 018 for dSSc ( n = 6 ) and P = 0 , 003 for LSSc ( n = 3 ) .

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A CTGF mRNA expression levels in skin (dSSc) and lung (LSSc) fibroblasts isolated from scleroderma patients and treated with the IRE1α inhibitor 4μ8C. * P = 0,018 for dSSc (n = 6) and P = 0,003 for LSSc (n = 3).


B Collagen1α2 mRNA expression levels in the same samples as in A . * P = 0 , 0030 for dSSC and P = 0 , 0036 for LSSc .

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B Collagen1α2 mRNA expression levels in the same samples as in A. * P = 0,0030 for dSSC and P = 0,0036 for LSSc.


C Quantification of collagen protein levels in the same samples as in A . * P = 0 , 010 .

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C Quantification of collagen protein levels in the same samples as in A. * P = 0,010.


D Immunohistochemistry staining to detect BiP protein in fibroblasts isolated from scleroderma patients treated or not with 4μ8C .

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D Immunohistochemistry staining to detect BiP protein in fibroblasts isolated from scleroderma patients treated or not with 4μ8C.


E mRNA levels of αSMA in primary lung fibroblasts isolated from patients with either cystic fibrosis ( CF ) , chronic obstructive pulmonary disease ( COPD ) or asthma . * P = 0 , 00001 for asthma vs . healthy controls , and P = 0 , 00003 for ctrl vs . 4μ8C in asthma patients .

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E mRNA levels of αSMA in primary lung fibroblasts isolated from patients with either cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD) or asthma. * P = 0,00001 for asthma vs. healthy controls, and P = 0,00003 for ctrl vs. 4μ8C in asthma patients.


F Quantification of mRNA levels for BiP in same samples as in E . * P = 0 , 0023 between asthma and healthy controls and P = 0 , 0031 between 4μ8C - treated and control treatment in fibroblasts from asthma patients .

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F Quantification of mRNA levels for BiP in same samples as in E. * P = 0,0023 between asthma and healthy controls and P = 0,0031 between 4μ8C-treated and control treatment in fibroblasts from asthma patients.


A , B Virus titers in the blood from mice ( n = 5 ) fed an NSD ( 0 . 45 % NaCl ) or HSD ( 4 % NaCl ) for 30 days ( A ) or 7 days ( B ) and then intraperitoneally infected with VSV ( 1x108 PFU per gram body , 48 hrs ) were analyzed by the TCID50 assay . Data information : Data represent mean and SEM of five biological replicates For all statistical testing , p values were calculated using two - tailed unpaired Student ' s t - test . NS , not significant ( p > 0 . 05 ) . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 .

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A, B Virus titers in the blood from mice (n=5) fed an NSD (0.45% NaCl) or HSD (4% NaCl) for 30 days (A) or 7 days (B) and then intraperitoneally infected with VSV (1x108 PFU per gram body, 48 hrs) were analyzed by the TCID50 assay. Data information: Data represent mean and SEM of five biological replicates For all statistical testing, p values were calculated using two-tailed unpaired Student's t-test. NS, not significant (p > 0.05). *p < 0.05, **p < 0.01 and ***p < 0.001.


H RT - qPCR analysis of viral RNA levels in U937 , THP1 , HT1080 , 2fTGH and A549 cells infected with VSV ( MOI = 1 . 0 , 24 hrs ) immediately after addition of NaCl ( + 34 mM ) . I RAW264 . 7 cells were infected with VSV / H1N1 / SeV ( MOI = 1 . 0 , 24 hrs ) immediately after addition of NaCl ( + 34 mM ) , or were transfected with HBV - replicon or HCV - replicon and then treated with additional NaCl ( + 34 mM ) for 12 hrs . Viral RNA levels were analyzed by RT - qPCR . Data information : Data show the mean and SD of four or three biological replicates . For all statistical testing , p values were calculated using two - tailed unpaired Student ' s t - test . NS , not significant ( p > 0 . 05 ) . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 .

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H RT-qPCR analysis of viral RNA levels in U937, THP1, HT1080, 2fTGH and A549 cells infected with VSV (MOI=1.0, 24 hrs) immediately after addition of NaCl (+34 mM). I RAW264.7 cells were infected with VSV/H1N1/SeV (MOI=1.0, 24 hrs) immediately after addition of NaCl (+34 mM), or were transfected with HBV-replicon or HCV-replicon and then treated with additional NaCl (+34 mM) for 12 hrs. Viral RNA levels were analyzed by RT-qPCR. Data information: Data show the mean and SD of four or three biological replicates. For all statistical testing, p values were calculated using two-tailed unpaired Student's t-test. NS, not significant (p > 0.05). *p < 0.05, **p < 0.01 and ***p < 0.001.


A Western blot analysis of VSV - G protein levels in Ifnar1 + / + or Ifnar1 - / - MEF cells infected with VSV ( MOI = 1 . 0 , 24 hrs ) immediately after addition of NaCl ( + 34 mM ) . Data information : are representative of at least two biological

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A Western blot analysis of VSV-G protein levels in Ifnar1+/+ or Ifnar1-/- MEF cells infected with VSV (MOI=1.0, 24 hrs) immediately after addition of NaCl (+34 mM). Data information: are representative of at least two biological replicates