Figure 2Immunofluorescence shows that the Vret signals ( green ) co - localize with the Ago3 signals ( red ) . DAPI shows the nuclei ( blue ) . Scale bar : 10 μm . The three panels on the right - hand side show high magnification images of the part indicated by the white dashed line box in the image on the left - hand side . This panel arrangement applies also for panels B − H . The bar chart shows the percentage of Vret - positive nuage in Ago3 - positive nuage . Immunofluorescence shows that the Vret signals ( green ) partially co - localize with the Siwi signals ( red ) . DAPI shows the nuclei ( blue ) . Scale bar : 10 μm . The bar chart shows the percentage of Vret - positive nuage in Siwi - positive nuage . Immunofluorescence showing the Ago3 signals ( green ) in control and Vret - depleted BmN4 cells ( Vret KD ) . Vret is shown in red . DAPI shows the nuclei ( blue ) . Scale bar : 10 μm . Immunofluorescence showing the Siwi signals ( green ) in control and Vret - depleted BmN4 cells ( Vret KD ) . Vret is shown in red . DAPI shows the nuclei ( blue ) . Scale bar : 10 μm . Immunofluorescence showing the Vret signals ( red ) in control and Ago3 - depleted BmN4 cells ( Ago3 KD ) . Ago3 is shown in green . DAPI shows the nuclei ( blue ) . Scale bar : 10 μm . Immunofluorescence showing the Siwi signals ( red ) in control and Ago3 - depleted BmN4 cells ( Ago3 KD ) . Ago3 is shown in green . DAPI shows the nuclei ( blue ) . Scale bar : 10 μm . Immunofluorescence showing Flag - Ago3 WT , KA mutant and DDH mutant signals ( green ) expressed in naïve BmN4 cells ( Control ) . DAPI shows the nuclei ( blue ) . Scale bar : 10 μm . Immunofluorescence showing Flag - Ago3 WT , KA mutant and DDH mutant signals ( green ) in Ago3 - depleted cells ( Ago3 KD ) . DAPI shows the nuclei ( blue ) . Scale bar : 10 μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Immunofluorescence shows that the Vret signals (green) co-localize with the Ago3 signals (red). DAPI shows the nuclei (blue). Scale bar: 10 μm. The three panels on the right-hand side show high magnification images of the part indicated by the white dashed line box in the image on the left-hand side. This panel arrangement applies also for panels B−H. The bar chart shows the percentage of Vret-positive nuage in Ago3-positive nuage. Immunofluorescence shows that the Vret signals (green) partially co-localize with the Siwi signals (red). DAPI shows the nuclei (blue). Scale bar: 10 μm. The bar chart shows the percentage of Vret-positive nuage in Siwi-positive nuage.Immunofluorescence showing the Ago3 signals (green) in control and Vret-depleted BmN4 cells (Vret KD). Vret is shown in red. DAPI shows the nuclei (blue). Scale bar: 10 μm. Immunofluorescence showing the Siwi signals (green) in control and Vret-depleted BmN4 cells (Vret KD). Vret is shown in red. DAPI shows the nuclei (blue). Scale bar: 10 μm.Immunofluorescence showing the Vret signals (red) in control and Ago3-depleted BmN4 cells (Ago3 KD). Ago3 is shown in green. DAPI shows the nuclei (blue). Scale bar: 10 μm. Immunofluorescence showing the Siwi signals (red) in control and Ago3-depleted BmN4 cells (Ago3 KD). Ago3 is shown in green. DAPI shows the nuclei (blue). Scale bar: 10 μm.Immunofluorescence showing Flag-Ago3 WT, KA mutant and DDH mutant signals (green) expressed in naïve BmN4 cells (Control). DAPI shows the nuclei (blue). Scale bar: 10 μm. Immunofluorescence showing Flag-Ago3 WT, KA mutant and DDH mutant signals (green) in Ago3-depleted cells (Ago3 KD). DAPI shows the nuclei (blue). Scale bar: 10 μm.


Figure 3Immunofluorescence showing the Ago3 signals ( green ) in control and Siwi - depleted BmN4 cells ( Siwi KD ) . Siwi is shown in red . DAPI shows the nuclei ( blue ) . Scale bar : 10 μm . The three panels on the right - hand side show high magnification images of the part indicated by the white dashed line box in the images on the left - hand side . This panel arrangement applies also for panels B , H and I . Bar chart indicates dot sizes of > 100 Ago3 particles in each case ( Large : IntDen value > 106 ; Small : IntDen value < 106 ) . Immunofluorescence showing the Vret signals ( green ) in control and Siwi - depleted BmN4 cells ( Siwi KD ) . Siwi is shown in red . DAPI shows the nuclei ( blue ) . Scale bar : 10 μm . Bar chart indicates dot sizes of > 100 Ago3 particles in each case ( Large : IntDen value > 105 ; Small : IntDen value < 105 ) . Western blotting was performed before and after Siwi KD using the anti - Ago3 antibody . Total BmN4 lysate ( Total ) , supernatant ( Sup ) and pellet ( Pellet ) fractions upon centrifugation were used . Western blotting was performed using the anti - Ago3 antibody on Ago3 immunoisolated from Siwi - depleted cells . Ago3 immunoprecipitated was untreated ( − ) or treated with phosphatase ( + ) prior to western botting . Western blot showing the degree of phosphorylation of Flag - Ago3 WT and the 8SA mutant in Siwi - depleted cells ( Siwi KD ) . Western blots showing the degree of phosphorylation of Flag - Ago3 WT and its DDH and KA mutants in Siwi - depleted cells ( Siwi KD ) . The second panel from the top corresponds to the top panel with a longer exposure time . Siwi and β - Tub as a loading control were also detected also by western blotting . Western blotting was performed using anti - Ago3 , anti - Vret and anti - Siwi antibodies on lysates of BmN4 cells before ( Control ) and after Siwi ( Siwi KD ) and Siwi / Vret ( Siwi / Vret KD ) depletion . β - Tub was detected as a loading control . Immunofluorescence showing the Flag - Ago3 signals ( red ) in normal ( Control ) and Siwi - depleted BmN4 cells ( Siwi KD ) . DAPI shows the nuclei ( blue ) . Scale bar : 10 μm . Immunofluorescence showing the Flag - Ago3 8SA and 8SE mutant signals ( red ) in normal ( Control ) and Siwi - depleted BmN4 cells ( Siwi KD ) . DAPI shows the nuclei ( blue ) . Scale bar : 10 μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Immunofluorescence showing the Ago3 signals (green) in control and Siwi-depleted BmN4 cells (Siwi KD). Siwi is shown in red. DAPI shows the nuclei (blue). Scale bar: 10 μm. The three panels on the right-hand side show high magnification images of the part indicated by the white dashed line box in the images on the left-hand side. This panel arrangement applies also for panels B, H and I. Bar chart indicates dot sizes of > 100 Ago3 particles in each case (Large: IntDen value > 106; Small: IntDen value < 106). Immunofluorescence showing the Vret signals (green) in control and Siwi-depleted BmN4 cells (Siwi KD). Siwi is shown in red. DAPI shows the nuclei (blue). Scale bar: 10 μm. Bar chart indicates dot sizes of > 100 Ago3 particles in each case (Large: IntDen value > 105; Small: IntDen value < 105).Western blotting was performed before and after Siwi KD using the anti-Ago3 antibody. Total BmN4 lysate (Total), supernatant (Sup) and pellet (Pellet) fractions upon centrifugation were used.Western blotting was performed using the anti-Ago3 antibody on Ago3 immunoisolated from Siwi-depleted cells. Ago3 immunoprecipitated was untreated (−) or treated with phosphatase (+) prior to western botting.Western blot showing the degree of phosphorylation of Flag-Ago3 WT and the 8SA mutant in Siwi-depleted cells (Siwi KD).Western blots showing the degree of phosphorylation of Flag-Ago3 WT and its DDH and KA mutants in Siwi-depleted cells (Siwi KD). The second panel from the top corresponds to the top panel with a longer exposure time. Siwi and β-Tub as a loading control were also detected also by western blotting.Western blotting was performed using anti-Ago3, anti-Vret and anti-Siwi antibodies on lysates of BmN4 cells before (Control) and after Siwi (Siwi KD) and Siwi/Vret (Siwi/Vret KD) depletion. β-Tub was detected as a loading control.Immunofluorescence showing the Flag-Ago3 signals (red) in normal (Control) and Siwi-depleted BmN4 cells (Siwi KD). DAPI shows the nuclei (blue). Scale bar: 10 μm.Immunofluorescence showing the Flag-Ago3 8SA and 8SE mutant signals (red) in normal (Control) and Siwi-depleted BmN4 cells (Siwi KD). DAPI shows the nuclei (blue). Scale bar: 10 μm.


Figure 4Western blot showing Ago3 immunopurified from BmN4 cells before ( Control ) and after Siwi knockdown ( Siwi KD ) . 32P - labeling of RNAs isolated from Ago3 ( a mixture of unphosphorylated and phosphorylated ) immunopurified from Siwi - depleted cells . 15 - 20 - nt RNAs ( indicated by a black line ) were isolated and sequenced . The measurement of distances between the 3 ′ - ends of Target - S ( 15 - 20 - nt RNAs in A ) and the 5 ′ - ends of Ago3 - bound piRNAs . 32P - labeling of RNAs isolated from Ago3 . Target - Ss are isolated from Siwi - depleted BmN4 cells but are not isolated from Siwi / Vret double knockdown cells . Western blot showing Ago3 immunopurified from BmN4 cells before ( Control ) and after Siwi knockdown ( Siwi KD ) . Northern blot showing Bmmar6 - piRNA precursors in the Ago3 complex . 50 - 100 - nt RNAs ( indicated by a black line ) were isolated and sequenced . The measurement of distances between the 5 ′ - ends of Target - L and the 5 ′ - ends of Ago3 - bound piRNAs . The measurement of distances between the 5 ′ - and 3 ′ - ends of Target - L fragments on the same genomic strand . Heat map displays 5 ′ - ends of Target - L in a window around the most abundant 1 , 000 3 ′ - ends of Target - L . The measurement of distances between the 5 ′ - ends and 3 ′ - ends of Target - S fragments on the same genomic strand . The heat map displays 5 ′ - ends of Target - S in a window around the most abundant 1 , 000 3 ′ - ends of Target - S . The measurement of distances between the 5 ′ - ends of Target - L and the 3 ′ - ends of Target - S on the same genomic strand . The heat map displays 5 ′ - ends of Target - L in a window around the most abundant 1 , 000 3 ′ - ends of Target - S . The measurement of distances between the 5 ′ - ends of Target - S and the 3 ′ - ends of Target - L on the same genomic strand . The heat map displays 5 ′ - ends of Target - S in a window around the most abundant 1 , 000 3 ′ - ends of Target - L .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Western blot showing Ago3 immunopurified from BmN4 cells before (Control) and after Siwi knockdown (Siwi KD). 32P-labeling of RNAs isolated from Ago3 (a mixture of unphosphorylated and phosphorylated) immunopurified from Siwi-depleted cells. 15-20-nt RNAs (indicated by a black line) were isolated and sequenced.The measurement of distances between the 3′-ends of Target-S (15-20-nt RNAs in A) and the 5′-ends of Ago3-bound piRNAs.32P-labeling of RNAs isolated from Ago3. Target-Ss are isolated from Siwi-depleted BmN4 cells but are not isolated from Siwi/Vret double knockdown cells.Western blot showing Ago3 immunopurified from BmN4 cells before (Control) and after Siwi knockdown (Siwi KD). Northern blot showing Bmmar6-piRNA precursors in the Ago3 complex. 50-100-nt RNAs (indicated by a black line) were isolated and sequenced.The measurement of distances between the 5′-ends of Target-L and the 5′-ends of Ago3-bound piRNAs.The measurement of distances between the 5′- and 3′-ends of Target-L fragments on the same genomic strand. Heat map displays 5′-ends of Target-L in a window around the most abundant 1,000 3′-ends of Target-L. The measurement of distances between the 5′-ends and 3′-ends of Target-S fragments on the same genomic strand. The heat map displays 5′-ends of Target-S in a window around the most abundant 1,000 3′-ends of Target-S. The measurement of distances between the 5′-ends of Target-L and the 3′-ends of Target-S on the same genomic strand. The heat map displays 5′-ends of Target-L in a window around the most abundant 1,000 3′-ends of Target-S. The measurement of distances between the 5′-ends of Target-S and the 3′-ends of Target-L on the same genomic strand. The heat map displays 5′-ends of Target-S in a window around the most abundant 1,000 3′-ends of Target-L.


Figure 5Immunofluorescence showing the Ago3 signals ( green ) in Siwi - depleted BmN4 cells ( Siwi KD ) and Siwi KD cells where Flag - Siwi ( red ) was expressed . DAPI shows the nuclei ( blue ) . Scale bar : 10 μm . The panels on the right - hand side show high magnification images of the part indicated by the white dashed line box in the images on the left - hand side . This arrangement applies also for panel C . Bar chart indicates dot sizes of > 100 Ago3 particles in each case ( Large : IntDen value > 105 ; Small : IntDen value < 105 ) . A significant difference was found plus / minus Flag - Siwi expression by the Fisher ' s exact test ( P < 0 . 05 ) . Western blots show that the degree of Ago3 phosphorylation was reduced upon Siwi KA mutant as well as Siwi WT and DDH mutant expression . Immunofluorescence showing the Ago3 signals ( green ) in Siwi - depleted BmN4 cells ( Siwi KD ) and Siwi KD cells where Flag - Siwi KA mutant ( red ) was expressed . DAPI shows the nuclei ( blue ) . Scale bar : 10 μm . Bar chart indicates dot sizes of > 100 Ago3 particles in each case ( Large : IntDen value > 105 ; Small : IntDen value < 105 ) . A significant difference was found plus / minus Flag - Siwi expression by the Fisher ' s exact test ( P < 0 . 05 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Immunofluorescence showing the Ago3 signals (green) in Siwi-depleted BmN4 cells (Siwi KD) and Siwi KD cells where Flag-Siwi (red) was expressed. DAPI shows the nuclei (blue). Scale bar: 10 μm. The panels on the right-hand side show high magnification images of the part indicated by the white dashed line box in the images on the left-hand side. This arrangement applies also for panel C. Bar chart indicates dot sizes of > 100 Ago3 particles in each case (Large: IntDen value > 105; Small: IntDen value < 105). A significant difference was found plus/minus Flag-Siwi expression by the Fisher's exact test (P < 0.05).Western blots show that the degree of Ago3 phosphorylation was reduced upon Siwi KA mutant as well as Siwi WT and DDH mutant expression.Immunofluorescence showing the Ago3 signals (green) in Siwi-depleted BmN4 cells (Siwi KD) and Siwi KD cells where Flag-Siwi KA mutant (red) was expressed. DAPI shows the nuclei (blue). Scale bar: 10 μm. Bar chart indicates dot sizes of > 100 Ago3 particles in each case (Large: IntDen value > 105; Small: IntDen value < 105). A significant difference was found plus/minus Flag-Siwi expression by the Fisher's exact test (P < 0.05).


Figure 1A . Representative images of IHC staining of PCIF1 in three CRC tissues and adjacent noncancerous tissues . Scale bar , 50 μm ( left ) . Analysis of the PCIF1 protein expression in normal and CRC tissues . Each dot represents one sample ( right ) . Data are the mean ± SEM . * * P < 0 . 01 , by Student ' s t - test . B . Kaplan - Meier plots of overall survival of 27 CRC patients with low tumor PCIF1 expression ( 0 - 4 . 5 staining score , n = 18 ) or high tumor PCIF1 expression ( 4 . 6 - 9 . 0 staining score , n = 9 ) . Data are the mean ± SEM . * * * P < 0 . 001 by Student ' s t - test . C . Western blot analysis of PCIF1 in normal colon cells ( CCD841CON ) and various CRC cell lines . GAPDH served as a loading control . D . qRT - PCR analysis of PCIF1 mRNA in the indicated cell lines . Levels were normalized to GAPDH expression . Data are the mean ± SD of n = 3 biological replicates . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by Student ' s t - test . E . Representative immunofluorescence images of HT29 and HCT116 cells stained for endogenous PCIF1 protein ( green ) . Nuclei were stained with DAPI ( blue ) . Scale bar , 30 μm . F . Western blot analysis of PCIF1 in whole extract ( W ) , cytosolic ( C ) , and nuclear ( N ) fractions of HCT116 and HT29 cells . Tubulin and lamin A / C served as cytosolic and nuclear markers , respectively .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A. Representative images of IHC staining of PCIF1 in three CRC tissues and adjacent noncancerous tissues. Scale bar, 50 μm (left). Analysis of the PCIF1 protein expression in normal and CRC tissues. Each dot represents one sample (right). Data are the mean ± SEM. **P < 0.01, by Student's t-test.B. Kaplan-Meier plots of overall survival of 27 CRC patients with low tumor PCIF1 expression (0-4.5 staining score, n = 18) or high tumor PCIF1 expression (4.6-9.0 staining score, n = 9). Data are the mean ± SEM. ***P < 0.001 by Student's t-test.C. Western blot analysis of PCIF1 in normal colon cells (CCD841CON) and various CRC cell lines. GAPDH served as a loading control.D. qRT-PCR analysis of PCIF1 mRNA in the indicated cell lines. Levels were normalized to GAPDH expression. Data are the mean ± SD of n=3 biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001 by Student's t-test.E. Representative immunofluorescence images of HT29 and HCT116 cells stained for endogenous PCIF1 protein (green). Nuclei were stained with DAPI (blue). Scale bar, 30 μm.F. Western blot analysis of PCIF1 in whole extract (W), cytosolic (C), and nuclear (N) fractions of HCT116 and HT29 cells. Tubulin and lamin A/C served as cytosolic and nuclear markers, respectively.


Figure 2A . Western blot analysis of PCIF1 in HT29 and HCT116 cells depleted of PCIF1 using four different sgRNAs . GAPDH served as a loading control . Matrigel invasion assay ( B ) , of the indicated control and PCIF1 - depleted CRC cell lines . Representative images and quantification of cells are shown at the left and right , respectively , of each panel . Data are the mean ± SD of n = 3 replicates / condition . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by Student ' s t - test . fibronectin adhesion assay ( C ) , of the indicated control and PCIF1 - depleted CRC cell lines . Representative images and quantification of cells are shown at the left and right , respectively , of each panel . Data are the mean ± SD of n = 3 replicates / condition . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by Student ' s t - test . colony formation assay ( D ) of the indicated control and PCIF1 - depleted CRC cell lines . Representative images and quantification of cells are shown at the left and right , respectively , of each panel . Data are the mean ± SD of n = 3 replicates / condition . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by Student ' s t - test . wound healing migration assay ( E ) of the indicated control and PCIF1 - depleted CRC cell lines . Representative images and quantification of cells are shown at the left and right , respectively , of each panel . Data are the mean ± SD of n = 3 replicates / condition . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by Student ' s t - test . F . Growth of the indicated HCT116 tumors after subcutaneous injection into athymic nude mice . Tumor volume was recorded on the indicated days . Data are the mean ± SEM of the number of mice / group indicated on each panel . n , the numbers of mice . * P < 0 . 05 , * * P < 0 . 01 by Student ' s t - test . G . Survival analysis of mice injected bearing the indicated HCT116 tumors . Data are the mean ± SEM of the number of mice / group indicated on each panel . n , the numbers of mice . * * P < 0 . 01 , * * * P < 0 . 001 by Student ' s t - test .

O B-PANEL_START O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. Western blot analysis of PCIF1 in HT29 and HCT116 cells depleted of PCIF1 using four different sgRNAs. GAPDH served as a loading control.Matrigel invasion assay (B), of the indicated control and PCIF1-depleted CRC cell lines. Representative images and quantification of cells are shown at the left and right, respectively, of each panel. Data are the mean ± SD of n=3 replicates/condition. *P < 0.05, **P < 0.01, ***P < 0.001 by Student's t-test.fibronectin adhesion assay (C), of the indicated control and PCIF1-depleted CRC cell lines. Representative images and quantification of cells are shown at the left and right, respectively, of each panel. Data are the mean ± SD of n=3 replicates/condition. *P < 0.05, **P < 0.01, ***P < 0.001 by Student's t-test.colony formation assay (D) of the indicated control and PCIF1-depleted CRC cell lines. Representative images and quantification of cells are shown at the left and right, respectively, of each panel. Data are the mean ± SD of n=3 replicates/condition. *P < 0.05, **P < 0.01, ***P < 0.001 by Student's t-test.wound healing migration assay (E) of the indicated control and PCIF1-depleted CRC cell lines. Representative images and quantification of cells are shown at the left and right, respectively, of each panel. Data are the mean ± SD of n=3 replicates/condition. *P < 0.05, **P < 0.01, ***P < 0.001 by Student's t-test.F. Growth of the indicated HCT116 tumors after subcutaneous injection into athymic nude mice. Tumor volume was recorded on the indicated days. Data are the mean ± SEM of the number of mice/group indicated on each panel. n, the numbers of mice. *P < 0.05, **P < 0.01 by Student's t-test.G. Survival analysis of mice injected bearing the indicated HCT116 tumors. Data are the mean ± SEM of the number of mice/group indicated on each panel. n, the numbers of mice. **P < 0.01, ***P < 0.001 by Student's t-test.


Figure 3A . LC - MS / MS identification and quantification of m6Am or m6A levels in mRNA from control and PCIF1 - depleted HCT116 cells . Data are presented as the enrichment of methylated vs unmethylated mRNA . Data are the mean ± SD of n = 3 biological replicates . * * P < 0 . 01 by Student ' s t - test . B . Metagene plots analysis of m6Am enrichment around the transcription start site ( TSS ) of all expressed genes in control and PCIF1 - depleted HCT116 cells . E . Western blot analysis of FOS and PCIF1 in control and PCIF1 - depleted HCT116 and HT29 cells . GAPDH served as a loading control . F . Genome browser views of FOS with m6Am sites . Read coverage of input sample and IP sample are shown in blue and red , respectively , and green rectangle indicates the m6Am peaks located near the TSS . G . m6Am - exo - qPCR analysis of m6Am enrichment in FOS mRNAs from the indicated HCT116 cells . GAPDH served as a negative control . Data are the mean ± SD of n = 3 biological replicates . * * P < 0 . 01 by Student ' s t - test . H . qRT - PCR analysis of FOS mRNA stability in control and PCIF1 - depleted HCT116 and HT29 cells treated with actinomycin D for the indicated times . Data are the mean ± SD of n = 3 biological replicates . * P < 0 . 05 , * * P < 0 . 01 by Student ' s t - test . I . Growth of tumors after subcutaneous injection of the indicated HCT116 cells into athymic nude mice . Tumor volumes were recorded on the indicated days . Data are the mean ± SEM of n = 5 . * P < 0 . 05 by Student ' s t - test .

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Figure 3A. LC-MS/MS identification and quantification of m6Am or m6A levels in mRNA from control and PCIF1-depleted HCT116 cells. Data are presented as the enrichment of methylated vs unmethylated mRNA. Data are the mean ± SD of n=3 biological replicates. **P < 0.01 by Student's t-test.B. Metagene plots analysis of m6Am enrichment around the transcription start site (TSS) of all expressed genes in control and PCIF1-depleted HCT116 cells.E. Western blot analysis of FOS and PCIF1 in control and PCIF1-depleted HCT116 and HT29 cells. GAPDH served as a loading control.F. Genome browser views of FOS with m6Am sites. Read coverage of input sample and IP sample are shown in blue and red, respectively, and green rectangle indicates the m6Am peaks located near the TSS.G. m6Am-exo-qPCR analysis of m6Am enrichment in FOS mRNAs from the indicated HCT116 cells. GAPDH served as a negative control. Data are the mean ± SD of n=3 biological replicates. **P < 0.01 by Student's t-test.H. qRT-PCR analysis of FOS mRNA stability in control and PCIF1-depleted HCT116 and HT29 cells treated with actinomycin D for the indicated times. Data are the mean ± SD of n=3 biological replicates. *P < 0.05, **P < 0.01 by Student's t-test.I. Growth of tumors after subcutaneous injection of the indicated HCT116 cells into athymic nude mice. Tumor volumes were recorded on the indicated days. Data are the mean ± SEM of n=5. *P < 0.05 by Student's t-test.


Figure 4A . Western blot analysis of FOS in control and FOS - depleted HCT116 and HT29 cells . GAPDH served as a loading control . MTS cell proliferation assay ( B ) , of control and FOS - depleted HCT116 cells . Data are the mean ± SD of n = 3 biological replicates . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by Student ' s t - test . wound healing migration assay ( C ) , of control and FOS - depleted HCT116 cells . Representative images and quantification of colonies are shown to the left and right , respectively . Data are the mean ± SD of n = 3 biological replicates . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by Student ' s t - test . colony formation assay ( D ) of control and FOS - depleted HCT116 cells . Representative images and quantification of colonies are shown to the left and right , respectively . Data are the mean ± SD of n = 3 biological replicates . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by Student ' s t - test . E . Growth of control and FOS - depleted HCT116 tumors after subcutaneous injection into athymic nude mice . Tumor volume was recorded on the indicated days . Data are the mean ± SEM of n = 10 mice / group . * * P < 0 . 01 by Student ' s t - test . F . Survival analysis of mice bearing control and FOS - depleted HCT116 tumors . Data are the mean ± SEM of n = 5 mice / group . * P < 0 . 05 by Student ' s t - test . G . Positive correlation between FOS and PCIF1 protein levels in tumors from 29 patients with CRC ( R2 = 0 . 6902 , P < 0 . 001 ) .

O B-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A. Western blot analysis of FOS in control and FOS-depleted HCT116 and HT29 cells. GAPDH served as a loading control.MTS cell proliferation assay (B), of control and FOS-depleted HCT116 cells. Data are the mean ± SD of n=3 biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001 by Student's t-test.wound healing migration assay (C), of control and FOS-depleted HCT116 cells. Representative images and quantification of colonies are shown to the left and right, respectively. Data are the mean ± SD of n=3 biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001 by Student's t-test.colony formation assay (D) of control and FOS-depleted HCT116 cells. Representative images and quantification of colonies are shown to the left and right, respectively. Data are the mean ± SD of n=3 biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001 by Student's t-test.E. Growth of control and FOS-depleted HCT116 tumors after subcutaneous injection into athymic nude mice. Tumor volume was recorded on the indicated days. Data are the mean ± SEM of n=10 mice/group. **P < 0.01 by Student's t-test.F. Survival analysis of mice bearing control and FOS-depleted HCT116 tumors. Data are the mean ± SEM of n= 5 mice/group. *P < 0.05 by Student's t-test.G. Positive correlation between FOS and PCIF1 protein levels in tumors from 29 patients with CRC (R2 =0.6902, P < 0.001).


Figure 7B . mRNA expression of PCIF1 was quantified by qRT - PCR in the HCT116 cells transfected with gradient amount of control and PCIF1 siRNAs . Data are the mean ± SD of n = 3 biological replicates . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by Student ' s t - test . C . Western blot analysis of PCIF1 in HCT116 cells with control and PCIF1 siRNAs . GAPDH served as a loading control . D . Cell viability assays were performed in in the HCT116 cells transfected with 100pmol control and PCIF1 siRNAs . Data are the mean ± SD of n = 3 biological replicates . * * * P < 0 . 001 by Student ' s t - test . F . Tumor growth of HCT116 cells treated with 1mg / kg control and PCIF1 LNP - siRNAs starting on day 9 and mice were intratumorally treated twice a week . Data are the mean ± SEM of n = 5 mice / group . * P < 0 . 05 , * * P < 0 . 01 by Student ' s t - test . G . Immunoblots of PCIF1 and FOS were carried out from the indicated tumors in four replicates with GAPDH as a loading control .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O

Figure 7B. mRNA expression of PCIF1 was quantified by qRT-PCR in the HCT116 cells transfected with gradient amount of control and PCIF1 siRNAs. Data are the mean ± SD of n=3 biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001 by Student's t-test.C. Western blot analysis of PCIF1 in HCT116 cells with control and PCIF1 siRNAs. GAPDH served as a loading control.D. Cell viability assays were performed in in the HCT116 cells transfected with 100pmol control and PCIF1 siRNAs. Data are the mean ± SD of n=3 biological replicates. ***P < 0.001 by Student's t-test.F. Tumor growth of HCT116 cells treated with 1mg/kg control and PCIF1 LNP-siRNAs starting on day 9 and mice were intratumorally treated twice a week. Data are the mean ± SEM of n=5 mice/group. *P < 0.05, **P < 0.01 by Student's t-test.G. Immunoblots of PCIF1 and FOS were carried out from the indicated tumors in four replicates with GAPDH as a loading control.


Figure 1B - C ' ) Immunofluorescence analysis of active - caspase 3 in the ONL of P347S and P347S / miR - 181a / b + / - . D , E ) TUNEL analysis in the ONL of P347S and P347S / miR - 181a / b + / - . F ) Quantification of immunofluorescence analysis in ( B - C ' ) ( N = 4 eyes P347S and N = 6 eyes P347S / miR - 181a / b + / ) . G ) Quantification of TUNEL analysis in ( D , E ) ( N = 3 eyes / genotype ) .

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Figure 1B-C') Immunofluorescence analysis of active-caspase 3 in the ONL of P347S and P347S/miR-181a/b+/-. D, E) TUNEL analysis in the ONL of P347S and P347S/miR-181a/b+/-. F) Quantification of immunofluorescence analysis in (B-C') (N=4 eyes P347S and N=6 eyes P347S/ miR-181a/b+/). G) Quantification of TUNEL analysis in (D, E) (N=3 eyes/genotype).


Figure 2D - P ) Immunofluorescence analysis showed amelioration of Rhodopsin localization ( D - F ' ; white arrowheads ) ; C - arrestin expression ( G - I ' ) and PR outer segment ( OS ) and inner segment ( IS ) structures , as determined , respectively , by PNA ( J - L ' ) ; and Phalloidin staining ( M - O ' ' ) in P347S / miR - 181a / b + / - vs P347S eyes at p30 . M ' ' - O ' ' show higher magnification of M - O . Scale bars 25 μm in D - O ' ; 5μm in M ' ' - O ' ' . Fluorescence densitometry quantification of each staining is reported in ( P ) , N = 3 eye / genotype for each staining . WT vs P347S p values are reported in red , P347S vs P347S / miR - 181a / b + / - p values are reported in black .

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Figure 2D-P) Immunofluorescence analysis showed amelioration of Rhodopsin localization (D-F'; white arrowheads); C-arrestin expression (G-I') and PR outer segment (OS) and inner segment (IS) structures, as determined, respectively, by PNA (J-L'); and Phalloidin staining (M-O'') in P347S/miR-181a/b+/- vs P347S eyes at p30. M''-O'' show higher magnification of M-O. Scale bars 25 μm in D-O'; 5μm in M''-O''. Fluorescence densitometry quantification of each staining is reported in (P), N=3 eye/genotype for each staining. WT vs P347S p values are reported in red, P347S vs P347S/miR-181a/b+/- p values are reported in black.


Figure 3D - L ) Immunofluorescence analysis of Rhodopsin ( D - E ' ) , C - arrestin ( F - G ' ) , PNA ( H - I ' ) ; and Phalloidin staining ( J - K ' ' ) in P347S and P347S / miR - 181a / b + / - retinas at p90 . J ' ' - K ' ' show higher magnification of J - K . Scale bars 25 μm in D - K ' ; 10μm in J ' ' - K ' ' . Immunofluorescence analysis of Rhodopsin C - arrestin PNA and Phalloidin staining in P347S and P347S / miR - 181a / b + / - retinas at p90 . Fluorescence densitometry quantification of each staining is reported in ( L ) , N = 3 eye / genotype / staining . M ) Measurement of P347S and P347S / miR - 181a / b + / - ONL thickness ( N ≥ 3 eyes / genotype ; N ) Graphical representation of the results of OKR analysis by the optokinetic tracking assays reported as cycles / degree . Visual acuity is preserved in P347S / miR - 181a / b + / - animals with respect to P347S ( N = 4 animals / genotype ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3D-L) Immunofluorescence analysis of Rhodopsin (D-E'), C-arrestin (F-G'), PNA (H-I'); and Phalloidin staining (J-K'') in P347S and P347S/miR-181a/b+/- retinas at p90. J''-K'' show higher magnification of J-K. Scale bars 25 μm in D-K'; 10μm in J''-K''.Immunofluorescence analysis of Rhodopsin C-arrestin PNA and Phalloidin staining in P347S and P347S/miR-181a/b+/- retinas at p90. Fluorescence densitometry quantification of each staining is reported in (L), N=3 eye/genotype/staining.M) Measurement of P347S and P347S/miR-181a/b+/- ONL thickness (N≥3 eyes/genotype;N) Graphical representation of the results of OKR analysis by the optokinetic tracking assays reported as cycles/degree. Visual acuity is preserved in P347S/miR-181a/b+/- animals with respect to P347S (N=4 animals/genotype).


Figure 4B - F ) Electron microscopy analysis shows an increase of mitochondrial fragmentation in P347S PRs with respect to WT and amelioration of the mitochondrial phenotype in P347S / miR - 181a / b + / - vs P347S PRs at p12 and p30 ( N = 2 animals / genotype ) . M , mitochondria . G - I ) The quantitative analysis of mitochondrial phenotype is expressed as measurement of the mitochondria perimeter ( G ) , mitochondria number ( H ) and as the ratio of mitochondria area per the analyzed field area ( I ) . N = 2 animals / genotype .

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Figure 4B-F) Electron microscopy analysis shows an increase of mitochondrial fragmentation in P347S PRs with respect to WT and amelioration of the mitochondrial phenotype in P347S/miR-181a/b+/- vs P347S PRs at p12 and p30 (N=2 animals/genotype). M, mitochondria. G-I) The quantitative analysis of mitochondrial phenotype is expressed as measurement of the mitochondria perimeter (G), mitochondria number (H) and as the ratio of mitochondria area per the analyzed field area (I). N=2 animals/genotype.


Figure 5A ) WB analysis of Drp1 , one of the key proteins involved in the mitochondrial fission pathway , in the optic cup of WT , P347S DMSO - treated , P347S / miR - 181a / b + / - DMSO - treated and of P347S / miR - 181a / b + / - FEDRATINIB - treated ( an inhibitor of the JAK2 / STAT3 pathway ) mice at p30 . B ) Quantification revealed that Drp1 protein levels are increased in P347S with respect to WT , and partially rescued in P347S / miR - 181a / b + / - . Data are normalized to Gapdh . N ≥ 4 eye / genotype . Please note that all compared bands from WT , P347S , P347S / miR - 181a / b + / - and P347S / miR - 181a / b + / - with FEDRATINIB samples are from the same blots , which were cropped and shown organized in the panel for the sake of data presentation clarity ( see source data ) . C - D ) qRT - PCR analysis reveals decreased levels of the Drp1 transcript and increased levels of Stat3 and Jak2 transcripts in the eyes of P347S / miR - 181a / b + / - vs those of P347S animals . N ≥ 4 eyes / genotype . F ) Quantification of WB in A reveals decreased levels of Drp1 in P347S / miR - 181a / b + / - DMSO - treated vs P347S and increased levels in P347S / miR - 181a / b + / - FEDRATINIB - treated ex - vivo retinas . N ≥ 4 eyes / genotype .

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Figure 5A) WB analysis of Drp1, one of the key proteins involved in the mitochondrial fission pathway, in the optic cup of WT, P347S DMSO-treated, P347S/miR-181a/b+/- DMSO-treated and of P347S/miR-181a/b+/- FEDRATINIB-treated (an inhibitor of the JAK2/STAT3 pathway) mice at p30.B) Quantification revealed that Drp1 protein levels are increased in P347S with respect to WT, and partially rescued in P347S/miR-181a/b+/-. Data are normalized to Gapdh. N ≥ 4 eye/genotype. Please note that all compared bands from WT, P347S, P347S/miR-181a/b+/- and P347S/miR-181a/b+/- with FEDRATINIB samples are from the same blots, which were cropped and shown organized in the panel for the sake of data presentation clarity (see source data).C-D) qRT-PCR analysis reveals decreased levels of the Drp1 transcript and increased levels of Stat3 and Jak2 transcripts in the eyes of P347S/miR-181a/b+/- vs those of P347S animals. N≥4 eyes/genotype.F) Quantification of WB in A reveals decreased levels of Drp1 in P347S/miR-181a/b+/- DMSO-treated vs P347S and increased levels in P347S/miR-181a/b+/- FEDRATINIB-treated ex-vivo retinas. N≥4 eyes/genotype.


Figure 6 ( D - U ) Immunofluorescence analysis showed amelioration of Rhodopsin localization ( p30 D - G ' , p70 L - O ' ; white arrowheads ) and C - arrestin ( p30 H - K ' , p70 P - S ' ; white arrowheads ) expression in Sponge - miR - 181a / b - injected eyes vs the corresponding controls also highlighting improvement of the OS structure . At both time points the effect of amelioration was more significant and evident when the AAV2 / 8 . CMV . GFP - Sponge - miR - 181a / b was used . Scale bars are 25 μm . Fluorescence densitometry quantification of each staining is reported in T for p30 and in U for p70 , N = 3 eye / treatment / staining .

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Figure 6(D-U) Immunofluorescence analysis showed amelioration of Rhodopsin localization (p30 D-G', p70 L-O'; white arrowheads) and C-arrestin (p30 H-K', p70 P-S'; white arrowheads) expression in Sponge-miR-181a/b-injected eyes vs the corresponding controls also highlighting improvement of the OS structure. At both time points the effect of amelioration was more significant and evident when the AAV2/8.CMV.GFP-Sponge-miR-181a/b was used. Scale bars are 25 μm. Fluorescence densitometry quantification of each staining is reported in T for p30 and in U for p70, N=3 eye/treatment/staining.


Figure 7A - I ' ) Immunofluorescence analysis on central retinal sections at p30 showed ONL thickness increase , amelioration of Rhodopsin localization ( A - D ' ) and C - arrestin expression ( E - H ' ) in AAV2 / 8 . CMV . GFP - Sponge - miR - 181a / b - injected vs AAV2 / 8 . CMV . GFP - injected rd10 eyes . Scale bars are 25 μm . Fluorescence densitometry quantification of each staining is reported in I , N = 3 eye / treatment / staining . Analysis of retinal function based on pupillary light responses ( PLR ) Water Maze tests . ( J ) PLR showed a significantly higher pupil constriction in rd10 mice injected with AAV2 / 8 . CMV . GFP - Sponge - miR - 181a / b than in AAV2 / 8 . CMV . GFP - injected rd10 eyes , at both 10 lux and 100 lux intensity stimuli ( N ≥ 6 eyes / treatment and N = 9 eyes / treatment , respectively ) . Data are presented as mean ± SEM . Student ' s t - test , unpaired . Analysis of retinal function based on pupillary light responses ( PLR ) Water Maze tests . ( K ) Water Maze test highlighted a shorter latency in reaching the platform in AAV2 / 8 . CMV . GFP - Sponge - miR - 181a / b - injected animals respect to control - injected ones ( N ≥ 6 eyes / treatment ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A-I') Immunofluorescence analysis on central retinal sections at p30 showed ONL thickness increase, amelioration of Rhodopsin localization (A-D') and C-arrestin expression (E-H') in AAV2/8.CMV.GFP-Sponge-miR-181a/b-injected vs AAV2/8.CMV.GFP-injected rd10 eyes. Scale bars are 25 μm. Fluorescence densitometry quantification of each staining is reported in I, N=3 eye/treatment/staining.Analysis of retinal function based on pupillary light responses (PLR) Water Maze tests. (J) PLR showed a significantly higher pupil constriction in rd10 mice injected with AAV2/8.CMV.GFP-Sponge-miR-181a/b than in AAV2/8.CMV.GFP-injected rd10 eyes, at both 10 lux and 100 lux intensity stimuli (N≥6 eyes/treatment and N=9 eyes/treatment, respectively). Data are presented as mean ± SEM. Student's t-test, unpaired.Analysis of retinal function based on pupillary light responses (PLR) Water Maze tests. (K) Water Maze test highlighted a shorter latency in reaching the platform in AAV2/8.CMV.GFP-Sponge-miR-181a/b-injected animals respect to control-injected ones (N≥6 eyes/treatment).


Figure 1 ( A ) HEK cells transfected with STIM1 - CFP , Orai1 - mCherry , and with ( red ) or without ( black ) untagged ANO8 were used to measure CRAC current with pipette solution ( cytoplasmic buffer ) containing 3 mM EGTA . The columns show the averaged effect of ANO8 on current density and on the slop of slow Ca2 + - dependent inactivation ( SCDI ) . The results are mean ± s . e . m and difference were analyzed by unpaired t test . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed . ( B ) Fura2 - loaded HEK cells transfected with YFP ( control , black ) or ANO8 - YFP ( red ) were used to measure store - mediated Ca2 + influx . Stores were depleted by treatment with the SERCA inhibitor CPA and Ca2 + influx was measured by Ca2 + add - back . The results are mean ± s . e . m and difference were analyzed by unpaired t test . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed . ( C ) FRET efficiency was measured with HEK cells transfected with STIM1 - CFP , ANO8 - YFP , and with ( red ) and without ( black ) Orai1 - HA before and after store depletion . Here and all other FRET measurements , representative images of the FRET signal under each condition are shown next to the traces . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed . ( D ) Summary of FRET efficiency measurements at the indicated conditions . The results are mean ± s . e . m and difference were analyzed by unpaired t test . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed . ( E ) FRET efficiency was measured with HEK cells transfected with STIM1 - CFP , STIM1 - YFP ( blue ) , and co - transfected with untagged ANO8 ( green ) or treated with siANO8 ( red ) before and after store depletion . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed . ( F ) Resting ( R ) or store - depleted cells ( S ) HEK cells transfected with STIM1 - YFP and myc - STIM1 and with or without ANO8 or treated with siANO8 , were used to immunoprecipitate ( IP ) STIM1 - YFP and blot ( B ) for mys - STIM1 . The inputs ( In ) are with anti - myc , anti - YFP or anti - ANO8 . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed . ( G ) Confocal and bright field images of basal ( upper ) and store depleted cells ( lower ) of HEK cells transfected with ANO8 - YFP alone . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed . The experiments represent at least 4 separate experiments with similar results . ( H ) TIRF images ( upper ) and TIRF - Z scan ( lower ) of the same resting and store depleted cells HEK cells transfected with ANO8 - YFP alone . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed . The experiments represent at least 4 separate experiments with similar results . ( I ) : Confocal images of resting cells transfected with ANO8 - YFP , STIM1 - CFP and Orai1 - mCherry . ( J ) Confocal images of store depleted cells transfected with ANO8 - YFP , STIM1 - CFP and Orai1 - mCherry . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed . The experiments represent at least 4 separate experiments with similar results . ( K ) TIRF - Z scan of resting ( upper ) and store depleted ( lower ) cells transfected with ANO8 - YFP and STIM1 - mCherry demonstrating the relationship between STIM1 and ANO8 puncta at the TIRF field . Cell surface is on top . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed . The experiments represent at least 4 separate experiments with similar results .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) HEK cells transfected with STIM1-CFP, Orai1-mCherry, and with (red) or without (black) untagged ANO8 were used to measure CRAC current with pipette solution (cytoplasmic buffer) containing 3 mM EGTA. The columns show the averaged effect of ANO8 on current density and on the slop of slow Ca2+-dependent inactivation (SCDI). The results are mean±s.e.m and difference were analyzed by unpaired t test. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.(B) Fura2-loaded HEK cells transfected with YFP (control, black) or ANO8-YFP (red) were used to measure store-mediated Ca2+ influx. Stores were depleted by treatment with the SERCA inhibitor CPA and Ca2+ influx was measured by Ca2+ add-back. The results are mean±s.e.m and difference were analyzed by unpaired t test. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.(C) FRET efficiency was measured with HEK cells transfected with STIM1-CFP, ANO8-YFP, and with (red) and without (black) Orai1-HA before and after store depletion. Here and all other FRET measurements, representative images of the FRET signal under each condition are shown next to the traces. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.(D) Summary of FRET efficiency measurements at the indicated conditions. The results are mean±s.e.m and difference were analyzed by unpaired t test. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.(E) FRET efficiency was measured with HEK cells transfected with STIM1-CFP, STIM1-YFP (blue), and co-transfected with untagged ANO8 (green) or treated with siANO8 (red) before and after store depletion. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.(F) Resting (R) or store-depleted cells (S) HEK cells transfected with STIM1-YFP and myc-STIM1 and with or without ANO8 or treated with siANO8, were used to immunoprecipitate (IP) STIM1-YFP and blot (B) for mys-STIM1. The inputs (In) are with anti-myc, anti-YFP or anti-ANO8. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.(G) Confocal and bright field images of basal (upper) and store depleted cells (lower) of HEK cells transfected with ANO8-YFP alone. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed. The experiments represent at least 4 separate experiments with similar results.(H) TIRF images (upper) and TIRF-Z scan (lower) of the same resting and store depleted cells HEK cells transfected with ANO8-YFP alone. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed. The experiments represent at least 4 separate experiments with similar results.(I): Confocal images of resting cells transfected with ANO8-YFP, STIM1-CFP and Orai1-mCherry. (J) Confocal images of store depleted cells transfected with ANO8-YFP, STIM1-CFP and Orai1-mCherry. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed. The experiments represent at least 4 separate experiments with similar results.(K) TIRF-Z scan of resting (upper) and store depleted (lower) cells transfected with ANO8-YFP and STIM1-mCherry demonstrating the relationship between STIM1 and ANO8 puncta at the TIRF field. Cell surface is on top. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed. The experiments represent at least 4 separate experiments with similar results.


Figure 2 ( A ) FRET ratio was measured with HEK cells transfected with STIM1 - CFP , Orai1 - YFP ( black ) and co - transfected with untagged ANO8 ( red ) before and after store depletion with 25 µM CPA in Ca2 + - free solution . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed . TIRF microscopy was used to quantify puncta formation at the TIRF field in cells transfected with STIM1 - CFP , ANO8 - YFP and Orai1 - mCherry before and after store depletion . ( A ) Example images . ( B ) Time course of STIM1 puncta formation in cells transfected with STIM1 and Orai1 alone ( black ) or together with ANO8 ( red ) . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed . ( C - E ) Effect of ANO8 on the rate of STIM1 - STIM1 clustering ( C ) , number of STIM1 puncta ( D ) and number of Orai1 puncta ( E ) . The results are mean ± s . e . m and difference were analyzed by unpaired t test . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed . ( F ) HEK cells were used to immunoprecipitate ( IP ) the native ANO8 ( A8 ) or Orai1 ( O1 ) and blotted ( B ) for STIM1 . R = resting , S = stimulated . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed . ( G ) HEK cells treated with scrambled ( siScr ) or siANO8 ( siA8 ) were used to IP the native Orai1 ( O1 ) or STIM1 ( S1 ) and blotted ( B ) for the native STIM1 , Orai1 or ANO8 , as indicated . The columns show the mean ± s . e . m of 4 independent experiments . The results are mean ± s . e . m and difference were analyzed by unpaired t test . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed . ( H ) Co - IP of myc - STIM1 and Orai1 - HA , myc - STIM1 and ANO8 - YFP , and Orai1 - HA and ANO8 - YFP was measured in transfected HEK cells before ( R , resting ) and in response to store depletion ( S , stimulated ) . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed . ( I ) Effect of ANO8 - YFP on myc - STIM1 and Orai1 - HA at the ER / PM junction . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed . ( J - L ) Junctional Orai1 ( J ) , STIM1 ( K ) and ANO8 ( L ) in resting ( R ) and store - depleted cells ( S ) . The results are mean ± s . e . m of 4 experiments and difference were analyzed by unpaired t test . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed . ( M ) Example EM images recorded from HEK cells transfected with empty vector ( left ) or ANO8 ( right ) . Red arrows mark ER / PM junctions . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed . ( N , O ) The size ( N ) and density ( O ) of ER / PM junctions in vector - transfected ( control ) and ANO8 - transfected cells . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) FRET ratio was measured with HEK cells transfected with STIM1-CFP, Orai1-YFP (black) and co-transfected with untagged ANO8 (red) before and after store depletion with 25 µM CPA in Ca2+-free solution. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.TIRF microscopy was used to quantify puncta formation at the TIRF field in cells transfected with STIM1-CFP, ANO8-YFP and Orai1-mCherry before and after store depletion. (A) Example images. (B) Time course of STIM1 puncta formation in cells transfected with STIM1 and Orai1 alone (black) or together with ANO8 (red). Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.(C-E) Effect of ANO8 on the rate of STIM1-STIM1 clustering (C), number of STIM1 puncta (D) and number of Orai1 puncta (E). The results are mean±s.e.m and difference were analyzed by unpaired t test. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.(F) HEK cells were used to immunoprecipitate (IP) the native ANO8 (A8) or Orai1 (O1) and blotted (B) for STIM1. R=resting, S=stimulated. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.(G) HEK cells treated with scrambled (siScr) or siANO8 (siA8) were used to IP the native Orai1 (O1) or STIM1 (S1) and blotted (B) for the native STIM1, Orai1 or ANO8, as indicated. The columns show the mean±s.e.m of 4 independent experiments. The results are mean±s.e.m and difference were analyzed by unpaired t test. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.(H) Co-IP of myc-STIM1 and Orai1-HA, myc-STIM1 and ANO8-YFP, and Orai1-HA and ANO8-YFP was measured in transfected HEK cells before (R, resting) and in response to store depletion (S, stimulated). Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.(I) Effect of ANO8-YFP on myc-STIM1 and Orai1-HA at the ER/PM junction. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.(J-L) Junctional Orai1 (J), STIM1 (K) and ANO8 (L) in resting (R) and store-depleted cells (S). The results are mean±s.e.m of 4 experiments and difference were analyzed by unpaired t test. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.(M) Example EM images recorded from HEK cells transfected with empty vector (left) or ANO8 (right). Red arrows mark ER/PM junctions. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.(N, O) The size (N) and density (O) of ER/PM junctions in vector-transfected (control) and ANO8-transfected cells. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


Figure 3 ( A , B ) Knockdown of ANO8 ( siA8 ) reduced CRAC current in cells transfected with Orai1 ( O1 ) and STIM1 ( S1 ) and buffered with 3 mM EGTA . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed and difference were analyzed by unpaired t test . ( C ) Knockdown of ANO8 reduces the native store - dependent Ca2 + influx measured in store depleted cells by Ca2 + add - back . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed and difference were analyzed by unpaired t test . ( D , E ) Knockdown of ANO8 reduced the number of store - dependent STIM1 puncta at the TIRF plane in cells expressing STIM1 and Orai1 . Panel ( D ) shows representative images and ( E ) is the summary of 7 experiments . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed and difference were analyzed by unpaired t test . ( F , G ) Current was measured with pipette solution contacting the fast and strong Ca2 + buffer 10 mM BAPTA in HEK cells transfected with STIM1 , Orai1 , and with ( red ) or without ANO8 ( black ) . Panel ( G ) shows the increase in current density at peak current . Note the prominent current inactivation in the presence of ANO8 . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed and difference were analyzed by unpaired t test . ( H ) Knockdown of SARAF ( red ) in wild - type cells had no effect on current inactivation in the presence of 10 mM BAPTA . ( I ) Knockdown of SARAF did not prevent the ANO8 - dependent current inactivation in the presence of 10 mM BAPTA . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed and difference were analyzed by unpaired t test .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A, B) Knockdown of ANO8 (siA8) reduced CRAC current in cells transfected with Orai1 (O1) and STIM1 (S1) and buffered with 3 mM EGTA. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed and difference were analyzed by unpaired t test.(C) Knockdown of ANO8 reduces the native store-dependent Ca2+ influx measured in store depleted cells by Ca2+ add-back. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed and difference were analyzed by unpaired t test.(D,E) Knockdown of ANO8 reduced the number of store-dependent STIM1 puncta at the TIRF plane in cells expressing STIM1 and Orai1. Panel (D) shows representative images and (E) is the summary of 7 experiments. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed and difference were analyzed by unpaired t test.(F, G) Current was measured with pipette solution contacting the fast and strong Ca2+ buffer 10 mM BAPTA in HEK cells transfected with STIM1, Orai1, and with (red) or without ANO8 (black). Panel (G) shows the increase in current density at peak current. Note the prominent current inactivation in the presence of ANO8. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed and difference were analyzed by unpaired t test.(H) Knockdown of SARAF (red) in wild-type cells had no effect on current inactivation in the presence of 10 mM BAPTA. (I) Knockdown of SARAF did not prevent the ANO8-dependent current inactivation in the presence of 10 mM BAPTA. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed and difference were analyzed by unpaired t test.


Figure 4 ( A , B ) Depletion of plasma membrane PI ( 4 , 5 ) P2 with the FRB / FKBP system prevented the ANO8 - mediated increase in STIM1 - Orai1 current measured in pipette solutions containing 3 mM EGTA ( A ) or 10 mM BAPTA ( B ) . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed . ( C ) HEK cells transfected with ANO8 - YFP , STIM1 - CFP Orai1 - HA and FRB and mCherry - FKBP PI ( 4 , 5 ) P2 depleting constructs were treated with 0 . 2 µM rapamycin ( rapa ) for 5 min and then with 25 µM CPA for 10 min before fixation and imaging by confocal microscopy . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed . ( D ) Mutating ANO8 R948 , R950 , R951 in RPRRP , a region that is predicted to include an ANO8 PI ( 4 , 5 ) P2 binding site , prevented increased STIM1 - Orai1 current and inactivation . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed . ( E ) Mutating ANO8 R949 , 950 , 951 to Q reduced translocation of ANO8 . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A, B) Depletion of plasma membrane PI(4,5)P2 with the FRB/FKBP system prevented the ANO8-mediated increase in STIM1-Orai1 current measured in pipette solutions containing 3 mM EGTA (A) or 10 mM BAPTA (B). Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.(C) HEK cells transfected with ANO8-YFP, STIM1-CFP Orai1-HA and FRB and mCherry-FKBP PI(4,5)P2 depleting constructs were treated with 0.2 µM rapamycin (rapa) for 5 min and then with 25 µM CPA for 10 min before fixation and imaging by confocal microscopy. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.(D) Mutating ANO8 R948, R950, R951 in RPRRP, a region that is predicted to include an ANO8 PI(4,5)P2 binding site, prevented increased STIM1-Orai1 current and inactivation. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.(E) Mutating ANO8 R949,950,951 to Q reduced translocation of ANO8. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


Figure 5 ( A ) Plot of the slope of inactivation as a function of current density in the presence ( red ) and absence of ANO8 ( black ) . Increasing current density was obtained by varying external Ca2 + between 2 - 50 mM . The results are mean ± s . e . m and difference were analyzed by unpaired t test . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed . ( B , C ) CRAC current was measured in HEK cells expressing STIM1 and Orai1 and in pipette solution containing 3 mM EGTA ( b ) or 10 mM BAPTA ( c ) . SERCA pump was inhibited with 25 µM CPA in the red traces where indicated and 100 µM IP3 was included in the pipette solution ( green in c ) . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed . ( D , E ) Experimental protocol and conditions except that cells were also transfected with ANO8 . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed . ( F , G ) HEK cells expressing STIM1 , Orai1 , and ANO8 were used to measure current in pipette solution containing 10 mM BAPTA and with ( red ) or without ( black ) 100 µM IP3 or 10 mM of the ER Ca2 + chelator TPEN ( green ) . The current density ( F ) and the normalized current ( G ) are shown , illustrating the delay in current inactivation by IP3 and TPEN . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Plot of the slope of inactivation as a function of current density in the presence (red) and absence of ANO8 (black). Increasing current density was obtained by varying external Ca2+ between 2-50 mM. The results are mean±s.e.m and difference were analyzed by unpaired t test. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.(B, C) CRAC current was measured in HEK cells expressing STIM1 and Orai1 and in pipette solution containing 3 mM EGTA (b) or 10 mM BAPTA (c). SERCA pump was inhibited with 25 µM CPA in the red traces where indicated and 100 µM IP3 was included in the pipette solution (green in c). Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.(D, E) Experimental protocol and conditions except that cells were also transfected with ANO8. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.(F, G) HEK cells expressing STIM1, Orai1, and ANO8 were used to measure current in pipette solution containing 10 mM BAPTA and with (red) or without (black) 100 µM IP3 or 10 mM of the ER Ca2+ chelator TPEN (green). The current density (F) and the normalized current (G) are shown, illustrating the delay in current inactivation by IP3 and TPEN. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.


Figure 6 ( A , B ) : ER Ca2 + content was measured with ER - GECO1 in the periphery ( A ) and the center ( B ) or cells transfected with M3 receptors and with ( red , green ) or without ( black , blue ) ANO8 . Receptor mediated store depletion was initiated by stimulating the cells with 0 . 5 mM carbachol in Ca2 + - free solution . Cell stimulation was terminated with 10 µM atropine and ER Ca2 + uptake was initiated by perfusing the cells with a solution containing 5 mM Ca2 + . The fluorescence was measured as F / F0 and normalized to the initial fluorescence in the presence of ANO8 . Panel ( C ) shows traces of ER Ca2 + uptake at expanded time scale in the periods marked by rectangular in ( A and B ) and the averaged slopes of ER Ca2 + influx and ( D ) shows the averaged slope of Ca2 + release at the cell periphery ( Peri ) and cell center ( Center ) . In ( C ) the traces were aligned along the Y axis to better show the difference in uptake rate . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed , and difference were analyzed by unpaired t test . ( E ) Enhancement by ANO8 of the Co - IP of expressed STIM1 with PMCA4a and SERCA2 and of Orai1 with SERCA2 in resting and store depleted cells . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed , and difference were analyzed by unpaired t test . ( F ) Effect of ANO8 expression on the Co - IP of the native SERCA2 and STIM1 in resting and store depleted cells . The columns are the mean ± s . e . m of 3 experiments . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed , and difference were analyzed by unpaired t test . ( G ) FRET ratio between STIM1 and SERCA2 in response to store depletion and its enhancement by ANO8 . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed , and difference were analyzed by unpaired t test . ( H , I ) Simultaneous measurements of ER Ca2 + with ER - GECO1 and PI ( 4 , 5 ) P2 hydrolysis with PHPLCδ - GFP in response to cell stimulation of M3 receptors . Traces are shown in ( h ) and the average slopes are in ( i ) . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed , and difference were analyzed by unpaired t test . ( J ) Effect of ANO8 on the Co - IP of the native STIM1 and IP3Rs . The columns are the mean ± s . e . m of 3 experiments . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed , and difference were analyzed by unpaired t test . ( K ) FRET ratio between expressed STIM1 and IP3R3 in response to store depletion and its enhancement by ANO8 . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed , and difference were analyzed by unpaired t test . ( L ) FRET ratio between expressed STIM1 and PMCA4 in response to store depletion and its enhancement by ANO8 . Data information : The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed . All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed , and difference were analyzed by unpaired t test .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A, B): ER Ca2+ content was measured with ER-GECO1 in the periphery (A) and the center (B) or cells transfected with M3 receptors and with (red, green) or without (black, blue) ANO8. Receptor mediated store depletion was initiated by stimulating the cells with 0.5 mM carbachol in Ca2+-free solution. Cell stimulation was terminated with 10 µM atropine and ER Ca2+ uptake was initiated by perfusing the cells with a solution containing 5 mM Ca2+. The fluorescence was measured as F/F0 and normalized to the initial fluorescence in the presence of ANO8. Panel (C) shows traces of ER Ca2+ uptake at expanded time scale in the periods marked by rectangular in (A and B) and the averaged slopes of ER Ca2+ influx and (D) shows the averaged slope of Ca2+ release at the cell periphery (Peri) and cell center (Center). In (C) the traces were aligned along the Y axis to better show the difference in uptake rate. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed, and difference were analyzed by unpaired t test.(E) Enhancement by ANO8 of the Co-IP of expressed STIM1 with PMCA4a and SERCA2 and of Orai1 with SERCA2 in resting and store depleted cells. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed, and difference were analyzed by unpaired t test.(F) Effect of ANO8 expression on the Co-IP of the native SERCA2 and STIM1 in resting and store depleted cells. The columns are the mean±s.e.m of 3 experiments. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed, and difference were analyzed by unpaired t test.(G) FRET ratio between STIM1 and SERCA2 in response to store depletion and its enhancement by ANO8. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed, and difference were analyzed by unpaired t test.(H, I) Simultaneous measurements of ER Ca2+ with ER-GECO1 and PI(4,5)P2 hydrolysis with PHPLCδ-GFP in response to cell stimulation of M3 receptors. Traces are shown in (h) and the average slopes are in (i). Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed, and difference were analyzed by unpaired t test.(J) Effect of ANO8 on the Co-IP of the native STIM1 and IP3Rs. The columns are the mean±s.e.m of 3 experiments. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed, and difference were analyzed by unpaired t test.(K) FRET ratio between expressed STIM1 and IP3R3 in response to store depletion and its enhancement by ANO8. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed, and difference were analyzed by unpaired t test.(L) FRET ratio between expressed STIM1 and PMCA4 in response to store depletion and its enhancement by ANO8. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed, and difference were analyzed by unpaired t test.