Figure 4Representative fluorescence microscopy images of CD142 - ASPCs , co - cultured with CD142 + ASPCs ( Aregs ) carrying control ( scr ) or siRNA - mediated knockdowns of selected CD142 + ASPC ( Areg ) - specific candidate genes : F3 , Mgp , and Gdf10 after adipogenic differentiation ( i . e . , after exposure to an adipogenic cocktail , Methods ) Fraction of differentiated CD142 - ASPCs , as quantified by the " adiposcore " , co - cultured with CD142 + ASPCs ( Aregs ) subjected to specific knockdowns ( or scrambled ( scr ) siRNA control ) , as indicated in A ; marker shapes correspond to different biological replicates , n = 10 , 4 biological replicates , 2 - 3 independent wells for each ;

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Figure 4Representative fluorescence microscopy images of CD142- ASPCs, co-cultured with CD142+ ASPCs (Aregs) carrying control (scr) or siRNA-mediated knockdowns of selected CD142+ ASPC (Areg)-specific candidate genes: F3, Mgp, and Gdf10 after adipogenic differentiation (i.e., after exposure to an adipogenic cocktail, Methods)Fraction of differentiated CD142- ASPCs, as quantified by the "adiposcore", co-cultured with CD142+ ASPCs (Aregs) subjected to specific knockdowns (or scrambled (scr) siRNA control), as indicated in A; marker shapes correspond to different biological replicates, n=10, 4 biological replicates, 2-3 independent wells for each;


Figure 5Representative fluorescence microscopy images of CD142 - ASPCs , co - cultured with CD142 + ASPCs ( Aregs ) ( in the top well ) subjected to the indicated compounds at the indicated concentration ; AGN and BMS are antagonists of RAR , DEAB is an antagonist of RALDH enzymes which convert retinaldehyde to RA , and HX is an antagonist of RXR ; Fraction of differentiated CD142 - ASPCs indicated in G , as quantified by the " adiposcore " ; marker shapes correspond to different biological replicates , n = 4 - 15 , 2 - 5 biological replicates , 2 - 3 independent wells for each ; Representative fluorescence microscopy images of CD142 - ASPCs , co - cultured with CD142 + ASPCs ( Aregs ) subjected to control ( scr ) or siRNA - mediated knockdowns of selected RA signalling pathway genes : Rara , Rarb , Rarg , Rbp1 , and Rxrg after adipogenic differentiation ( i . e . , after exposure to an adipogenic cocktail , Methods ) ; Fraction of differentiated CD142 - ASPCs indicated in I , as quantified by the " adiposcore " ; marker shapes correspond to different biological replicates , n = 24 - 29 , 4 biological replicates , 5 - 8 independent wells for each .

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Figure 5Representative fluorescence microscopy images of CD142- ASPCs, co-cultured with CD142+ ASPCs (Aregs) (in the top well) subjected to the indicated compounds at the indicated concentration; AGN and BMS are antagonists of RAR, DEAB is an antagonist of RALDH enzymes which convert retinaldehyde to RA, and HX is an antagonist of RXR;   Fraction of differentiated CD142- ASPCs indicated in G, as quantified by the "adiposcore"; marker shapes correspond to different biological replicates, n=4-15, 2-5 biological replicates, 2-3 independent wells for each;  Representative fluorescence microscopy images of CD142- ASPCs, co-cultured with CD142+ ASPCs (Aregs) subjected to control (scr) or siRNA-mediated knockdowns of selected RA signalling pathway genes: Rara, Rarb, Rarg, Rbp1, and Rxrg after adipogenic differentiation (i.e., after exposure to an adipogenic cocktail, Methods);   Fraction of differentiated CD142- ASPCs indicated in I, as quantified by the "adiposcore"; marker shapes correspond to different biological replicates, n=24-29, 4 biological replicates, 5-8 independent wells for each.


Figure 1Phenotypic changes of HSCs by citral . Phase - contrast images ( left ) , oil red O - stained images ( middle ) , and autofluorescence images ( right ) are shown for HSCs after passage 1 with or without the treatment of citral ( 100 μM ) for 24 h . Scale bar = 10 μm . Western blot analysis of cell lysates from the citral - treated HSCs in ( A ) . α - tubulin was used as a loading control . The quantitative densitometric data are expressed as the percentage of untreated control and represent the means ( SD ) for three independent experiments . * * P - value = 0 . 002 , paired t - test ( compared to control ) . Protein expression of ALDH1A1 and ALDH1A2 during the culture activation of HSCs . Cell lysates were prepared from HSCs at 3 days after plating ( HSCs - d3 ) and from HSCs after passage 1 ( P1 ) , 2 ( P2 ) , and 3 ( P3 ) , and analyzed by Western blotting . Real - time PCR analysis for the expression levels of ALDH1A1 and ALDH1A2 during the culture activation of HSCs . The PCR data are expressed as the percentage of HSCs - d3 . * * P - value , paired t - test ( n = 3 ) ( compared to HSCs - d3 ) , ALDH1A1 / HSCs - P3 : 0 . 003 , ALDH1A2 / HSCs - P1 : 0 . 0018 , ALDH1A2 / HSCs - P3 : 0 . 001 . Effect of ALDH1A suppression on HSC activation . HSCs - P1 were transfected with non - targeting siRNA or siRNA specific to ALDH1A1 and / or ALDH1A2 , and after 48 h , whole - cell lysates were subjected to Western blotting . * * P = 0 . 0023 , paired t - test ( n = 3 ) ( siRNA - ALDH1A1 / 2 compared to control ) . Reverse - phase HPLC analysis of whole - cell lysates from the siRNA - ALDH1A - treated HSCs in ( E ) . Typical chromatograms of all - trans retinoic acid and 13 - cis retinoic acid . The retention time is 14 min for 13 - cis - RA and 23 min for all - trans RA . Phenotypic changes of HSCs by RAR antagonist . HSCs - d3 were incubated with AGN193109 ( 1 μM ) ± all - trans RA ( 10 μM ) for 3 days and subjected to Oil red O staining . Scale bar = 10 μm . Western blot analysis of cell lysates from AGN193109 ± RA - treated HSCs in ( G ) . * * P = 0 . 003 , paired t - test ( n = 3 ) ( AGN193109 compared to control ) .

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Figure 1Phenotypic changes of HSCs by citral. Phase-contrast images (left), oil red O-stained images (middle), and autofluorescence images (right) are shown for HSCs after passage 1 with or without the treatment of citral (100 μM) for 24 h. Scale bar = 10 μm.Western blot analysis of cell lysates from the citral-treated HSCs in (A). α-tubulin was used as a loading control. The quantitative densitometric data are expressed as the percentage of untreated control and represent the means (SD) for three independent experiments. **P-value = 0.002, paired t-test (compared to control).Protein expression of ALDH1A1 and ALDH1A2 during the culture activation of HSCs. Cell lysates were prepared from HSCs at 3 days after plating (HSCs-d3) and from HSCs after passage 1 (P1), 2 (P2), and 3 (P3), and analyzed by Western blotting.Real-time PCR analysis for the expression levels of ALDH1A1 and ALDH1A2 during the culture activation of HSCs. The PCR data are expressed as the percentage of HSCs-d3. **P-value, paired t-test (n = 3) (compared to HSCs-d3), ALDH1A1/HSCs-P3: 0.003, ALDH1A2/HSCs-P1: 0.0018, ALDH1A2/HSCs-P3: 0.001.Effect of ALDH1A suppression on HSC activation. HSCs-P1 were transfected with non-targeting siRNA or siRNA specific to ALDH1A1 and/or ALDH1A2, and after 48 h, whole-cell lysates were subjected to Western blotting. **P = 0.0023, paired t-test (n = 3) (siRNA-ALDH1A1/2 compared to control).Reverse-phase HPLC analysis of whole-cell lysates from the siRNA-ALDH1A-treated HSCs in (E). Typical chromatograms of all-trans retinoic acid and 13-cis retinoic acid. The retention time is 14 min for 13-cis-RA and 23 min for all-trans RA.Phenotypic changes of HSCs by RAR antagonist. HSCs-d3 were incubated with AGN193109 (1 μM) ± all-trans RA (10 μM) for 3 days and subjected to Oil red O staining. Scale bar = 10 μm.Western blot analysis of cell lysates from AGN193109 ± RA-treated HSCs in (G). **P = 0.003, paired t-test (n = 3) (AGN193109 compared to control).


Figure 2Albumin and R - III downregulate RA signaling . HSCs - P1 were either transfected with RARE Cignal reporter and expression plasmid containing vehicle or albumin ( left panel ) or treated with R - III ( 25 - 250 nM ) after transfection with RARE Cignal reporter ( right panel ) . After 36 h , the transfected cells were harvested for luciferase assay . Renilla luciferase activity was normalized to firefly luciferase activity . Ratios were normalized against the cells transfected with vehicle plasmid . * * P - value , two - sample t - test ( n = 9 ) ( compared to control ) , albumin : 0 . 0037 , R - III 125 nM : 0 . 0032 . Phenotypic changes of HSCs by the expression of albumin and / or ALDH1A . HSCs - P1 were transfected with plasmids encoding albumin , ALDH1A1 , and ALDH1A2 , either individually or in combination , and incubated in the presence of all - trans retinoic acid ( 50 nM ) for 18 h . Cells were analyzed by oil red O staining . Scale bar , 20 μm . Western blot analysis of cell lysates from albumin - and / or ALDH1A - transfected HSCs as in ( B ) . * * P = 0 . 002 , paired t - test ( n = 3 ) ( albumin compared to control ) . Reverse - phase HPLC analysis of whole - cell lysates from albumin - and / or ALDH1A - transfected HSCs as in ( B ) . RAR agonist counteracts R - III action . HSCs - P1 were treated with His - tagged R - III ( final concentration , 0 . 15 μM ) in the presence or absence of RAR agonist ( AGN191183 , 1 μM ) for 18 h and analyzed by Western blotting . * * P = 0 . 0024 , paired t - test ( n = 3 ) ( R - III compared to control ) . The separating lines demarcate bands that come from non - consecutive lanes of the same gel .

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Figure 2Albumin and R-III downregulate RA signaling. HSCs-P1 were either transfected with RARE Cignal reporter and expression plasmid containing vehicle or albumin (left panel) or treated with R-III (25-250 nM) after transfection with RARE Cignal reporter (right panel). After 36 h, the transfected cells were harvested for luciferase assay. Renilla luciferase activity was normalized to firefly luciferase activity. Ratios were normalized against the cells transfected with vehicle plasmid. **P-value, two-sample t-test (n = 9) (compared to control), albumin: 0.0037, R-III 125 nM: 0.0032.Phenotypic changes of HSCs by the expression of albumin and/or ALDH1A. HSCs-P1 were transfected with plasmids encoding albumin, ALDH1A1, and ALDH1A2, either individually or in combination, and incubated in the presence of all-trans retinoic acid (50 nM) for 18 h. Cells were analyzed by oil red O staining. Scale bar, 20 μm.Western blot analysis of cell lysates from albumin- and/or ALDH1A-transfected HSCs as in (B). **P = 0.002, paired t-test (n = 3) (albumin compared to control).Reverse-phase HPLC analysis of whole-cell lysates from albumin- and/or ALDH1A-transfected HSCs as in (B).RAR agonist counteracts R-III action. HSCs-P1 were treated with His-tagged R-III (final concentration, 0.15 μM) in the presence or absence of RAR agonist (AGN191183, 1 μM) for 18 h and analyzed by Western blotting. **P = 0.0024, paired t-test (n = 3) (R-III compared to control). The separating lines demarcate bands that come from non-consecutive lanes of the same gel.


Figure 3Protein expression of STRA6 during the culture activation of HSCs . Cell lysates were prepared from HSCs at 3 days after plating ( HSCs - 3d ) , and from HSCs after passage 1 ( P1 ) , 2 ( P2 ) , and 3 ( P3 ) , and analyzed by Western blotting . Lysates from NIH3T3 cells were used as a negative control . The separating lines demarcate bands that come from non - consecutive lanes of the same gel . Real - time PCR analysis for the expression level of STRA6 during the culture activation of HSCs . The PCR data are expressed as the percentage of HSCs - 3d . * * P - value , paired t - test ( n = 3 ) ( compared to HSCs - d3 ) , HSCs - P2 : 0 . 002 , HSCs - P3 : 0 . 0022 . R - III uptake parallels STRA6 level . HSCs at different stages of activation were incubated with His - tagged R - III ( final concentration , 0 . 15 μM ) for 30 min , washed , and subjected to Western blotting for R - III uptake . STRA6 - mediated uptake of R - III . HSCs - P1 were transfected with non - targeting siRNA or one of the two different STRA6 - specific siRNAs , and after 48 h , cells were incubated with R - III for 30 min and analyzed by Western blotting .

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Figure 3Protein expression of STRA6 during the culture activation of HSCs. Cell lysates were prepared from HSCs at 3 days after plating (HSCs-3d), and from HSCs after passage 1 (P1), 2 (P2), and 3 (P3), and analyzed by Western blotting. Lysates from NIH3T3 cells were used as a negative control. The separating lines demarcate bands that come from non-consecutive lanes of the same gel.Real-time PCR analysis for the expression level of STRA6 during the culture activation of HSCs. The PCR data are expressed as the percentage of HSCs-3d. **P-value, paired t-test (n = 3) (compared to HSCs-d3), HSCs-P2: 0.002, HSCs-P3: 0.0022.R-III uptake parallels STRA6 level. HSCs at different stages of activation were incubated with His-tagged R-III (final concentration, 0.15 μM) for 30 min, washed, and subjected to Western blotting for R-III uptake.STRA6-mediated uptake of R-III. HSCs-P1 were transfected with non-targeting siRNA or one of the two different STRA6-specific siRNAs, and after 48 h, cells were incubated with R-III for 30 min and analyzed by Western blotting.


Figure 4Representative macroscopic pictures of livers from control and CCl4 - , CCl4 / albumin - , CCl4 / RBP - , and CCl4 / R - III - treated mice . Liver sections were stained with H & amp ; amp ; E and Sirius red , and also subjected to immunohistochemistry for type I collagen . Scale bar , 200 μm .

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Figure 4Representative macroscopic pictures of livers from control and CCl4-, CCl4/albumin-, CCl4/RBP-, and CCl4/R-III-treated mice.Liver sections were stained with H&amp;E and Sirius red, and also subjected to immunohistochemistry for type I collagen. Scale bar, 200 μm.


Figure 5Hydroxyproline content in the livers from control and CCl4 - , CCl4 / albumin - , and CCl4 / R - III - treated mice ( μg / g liver ) . * P = 0 . 037 , two - sample t - test ( n = 10 ) ( CCl4 + R - III compared to CCl4 - treated mice ) . Western blot analysis on liver extracts from the treated mice with antibody against type I collagen . Immunohistochemical analysis of α - SMA on representative liver sections from the treated mice . Scale bar , 200 μm .

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Figure 5Hydroxyproline content in the livers from control and CCl4-, CCl4/albumin-, and CCl4/R-III-treated mice (μg/g liver). *P = 0.037, two-sample t-test (n = 10) (CCl4 + R-III compared to CCl4-treated mice).Western blot analysis on liver extracts from the treated mice with antibody against type I collagen.Immunohistochemical analysis of α-SMA on representative liver sections from the treated mice. Scale bar, 200 μm.


Figure 6Liver sections from control and CCl4 - and CCl4 / R - III - treated mice were immunohistochemically stained for TGF - β and F4 / 80 . Scale bar , 50 μm .

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Figure 6Liver sections from control and CCl4- and CCl4/R-III-treated mice were immunohistochemically stained for TGF-β and F4/80. Scale bar, 50 μm.


Figure 7Liver sections from treated mice were immunohistochemically stained for desmin , His - tag , and α - SMA . His - positive signals largely overlapped with desmin / α - SMA staining . Scale bar , 50 μm .

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Figure 7Liver sections from treated mice were immunohistochemically stained for desmin, His-tag, and α-SMA. His-positive signals largely overlapped with desmin/α-SMA staining. Scale bar, 50 μm.


Figure 8Liver sections from control and CCl4 - and CCl4 / R - III - treated mice were stained with Sirius red . Scale bar , 200 μm . Hydroxyproline content in the livers ( μg / g liver ) . * P = 0 . 034 , two - sample t - test ( n = 10 ) ( CCl4 + R - III compared to CCl4 - treated mice ) .

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Figure 8Liver sections from control and CCl4- and CCl4/R-III-treated mice were stained with Sirius red. Scale bar, 200 μm.Hydroxyproline content in the livers (μg/g liver). *P = 0.034, two-sample t-test (n = 10) (CCl4 + R-III compared to CCl4-treated mice).


Figure 9Liver sections from control , BDL , and BDL / R - III - treated mice were stained with Sirius red . Scale bar , 200 μm . . Hydroxyproline content in the livers ( μg / g liver ) . * P - value , two - sample t - test ( n = 10 ) ( compared to BDL - treated mice ) ; BDL + R - III ( 10 μg ) : 0 . 031 , BDL + R - III ( 5 μg ) : 0 . 026 .

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Figure 9Liver sections from control, BDL, and BDL/R-III-treated mice were stained with Sirius red. Scale bar, 200 μm..Hydroxyproline content in the livers (μg/g liver). *P-value, two-sample t-test (n = 10) (compared to BDL-treated mice); BDL + R-III (10 μg): 0.031, BDL+R-III (5 μg): 0.026.


Figure 1 ( A ) Summary of HMCES recovery upon TAP - MS analyses performed with 293T cells stably expressing SFB - PCNA . Cells were treated with CPT : camptothecin ( 1 μg / ml for 6 h ) ; HU : hydroxyurea ( 2 mM for 16 h ) , UV : ultraviolet light irradiation ( 10 mJ / cm2 harvested after 6 h post - irradiation ) or Thymidine ( 100 mM for 6 h ) . Recovered peptide - spectrum matches ( PSMs ) for PCNA and HMCES are indicated for each condition . ( C ) Immunoprecipitation ( IP ) of SFB - tagged full - length ( FL ) HMCES and indicated HMCES deletion mutants was conducted to decipher the region responsible for PCNA interaction . ( D ) GST - PCNA pulldown assay was used to evaluate the interaction between PCNA and full - length HMCES or indicated HMCES deletion mutants . ( E ) aniPOND - Western blotting analysis revealed the presence of HMCES on replication fork . Cells were pulsed with EdU for 10 mins , and click - reaction was performed . In thymidine chase samples , cells were incubated with thymidine for another 30 mins prior to click - reaction . ( F ) Analysis of HMCES level throughout the cell cycle . Cells were synchronized by double thymidine block and released for the indicated time points . CYCLIN E and CYCLIN B1 were used as markers for cell cycle phases .

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Figure 1(A) Summary of HMCES recovery upon TAP-MS analyses performed with 293T cells stably expressing SFB-PCNA. Cells were treated with CPT: camptothecin (1 μg/ml for 6 h); HU: hydroxyurea (2 mM for 16 h), UV: ultraviolet light irradiation (10 mJ/cm2 harvested after 6 h post-irradiation) or Thymidine (100 mM for 6 h). Recovered peptide-spectrum matches (PSMs) for PCNA and HMCES are indicated for each condition.(C) Immunoprecipitation (IP) of SFB-tagged full-length (FL) HMCES and indicated HMCES deletion mutants was conducted to decipher the region responsible for PCNA interaction.(D) GST-PCNA pulldown assay was used to evaluate the interaction between PCNA and full-length HMCES or indicated HMCES deletion mutants.(E) aniPOND-Western blotting analysis revealed the presence of HMCES on replication fork. Cells were pulsed with EdU for 10 mins, and click-reaction was performed. In thymidine chase samples, cells were incubated with thymidine for another 30 mins prior to click-reaction.(F) Analysis of HMCES level throughout the cell cycle. Cells were synchronized by double thymidine block and released for the indicated time points. CYCLIN E and CYCLIN B1 were used as markers for cell cycle phases.


Figure 2 ( A ) Western blotting analysis confirmed HMCES knockout in 293A cells ( 293A HMCES KO ) . Blots also included knockout cells reconstituted with WT SFB - HMCES . ( B , C ) Colony - forming capacity of HMCES KO cells was compared to that of control wild - type 293A cells . ( C ) Box plot showing colony forming efficiencies of 293A wild - type control and HMCES KO cells . Box limits represent 25th and 75th percentile , centerline shows the median and whiskers extend minimum to maximum . Data include more than three independent repeats performed in duplicates . Student ' s t - test were performed for statistical analysis ( * * * * , p < 0 . 0001 ) . ( D ) Colonogenic survival assay of 293A wild - type control and HMCES KO cells treated with various concentrations of hydroxyurea ( HU ) was shown . Seeding density with respect to control was indicated below each image . The images shown are representative of three biological repeats , each performed in duplicate . ( E ) Quantification showing survival fraction of wild - type control ( WT ) and HMCES KO cells upon HU treatment . Data are presented as mean ± s . e . m ( n = 3 , each performed in duplicates ) and student ' s t - test were performed for statistical analysis ( ns , not significant ; * , p < 0 . 05 ) . ( F ) Quantification of colonogenic survival assay upon exposure of WT and HMCES KO cells to ionizing radiation ( IR ) . Data are presented as mean ± s . e . m . Quantifications are from three biological repeats performed in duplicates . Student ' s t - test was performed for statistical analysis ( * , p < 0 . 05 ; * * , p < 0 . 01 ) . ( G , H ) Sensitivity assay and quantification of surviving WT control and HMCES KO cells determined by colony - forming assay upon exposure to H2O2 . Experiments were repeated three independent times , each time performed in duplicates and data are presented as mean ± s . e . m . Statistical significance for the corresponding experiment was analyzed by student ' s t - test ( * , p < 0 . 05 ) . ( I ) Bar graph showing sensitivity of WT , HMCES KO and stably reconstituted WT cells towards H2O2 treatment . Experiment was repeated two independent times with technical repeats .

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Figure 2(A) Western blotting analysis confirmed HMCES knockout in 293A cells (293A HMCES KO). Blots also included knockout cells reconstituted with WT SFB- HMCES.(B, C) Colony-forming capacity of HMCES KO cells was compared to that of control wild-type 293A cells. (C) Box plot showing colony forming efficiencies of 293A wild-type control and HMCES KO cells. Box limits represent 25th and 75th percentile, centerline shows the median and whiskers extend minimum to maximum. Data include more than three independent repeats performed in duplicates. Student's t-test were performed for statistical analysis (****, p<0.0001). (D) Colonogenic survival assay of 293A wild-type control and HMCES KO cells treated with various concentrations of hydroxyurea (HU) was shown. Seeding density with respect to control was indicated below each image. The images shown are representative of three biological repeats, each performed in duplicate.(E) Quantification showing survival fraction of wild-type control (WT) and HMCES KO cells upon HU treatment. Data are presented as mean±s.e.m (n=3, each performed in duplicates) and student's t-test were performed for statistical analysis (ns, not significant; *, p<0.05).(F) Quantification of colonogenic survival assay upon exposure of WT and HMCES KO cells to ionizing radiation (IR). Data are presented as mean±s.e.m. Quantifications are from three biological repeats performed in duplicates. Student's t-test was performed for statistical analysis (*, p<0.05; **, p<0.01).(G, H) Sensitivity assay and quantification of surviving WT control and HMCES KO cells determined by colony-forming assay upon exposure to H2O2. Experiments were repeated three independent times, each time performed in duplicates and data are presented as mean±s.e.m. Statistical significance for the corresponding experiment was analyzed by student's t-test (*, p<0.05).(I) Bar graph showing sensitivity of WT, HMCES KO and stably reconstituted WT cells towards H2O2 treatment. Experiment was repeated two independent times with technical repeats.


Figure 3 ( A , B ) Colonogenic assays were performed by adding increasing concentrations of pemetrexed to control wild - type ( WT ) 293A cells and HMCES knockout ( HMCES KO ) cells ( A ) and quantification of results were shown ( B ) . Increasing numbers of cells were seeded with increasing concentrations of the compound to obtain optimal drug - dose response curve . Seeding density with respect to control is indicated below each image . The images shown are representative of three biological repeats , each performed in duplicate . ( B ) Data are presented as mean ± s . e . m . and student ' s t - test was performed for statistical analysis ( * * * * , p < 0 . 0001 ) . ( C ) Western blot showing APEX2 levels upon 293A cells infected with viruses encoding scrambled or two independent APEX2 shRNAs . ( D ) Colony forming assay was performed to indicate colony forming efficiency in 293A and HMCES KO cells upon infection with viruses encoding scrambled or APEX2 shRNAs . ( E ) Sensitivity assay was conducted with WT and HMCES KO cells upon treatment with AP Endonuclease inhibitor III ( APEi ) . Results are presented from two independent experiments and data are presented as mean ± s . e . m . ( F ) Colony forming assay was performed with WT and HMCES KO cells upon either pemetrexed treatment alone or in combination with APEi . Data are presented as mean ± s . e . m ( n = 2 each with technical repeats ) . ( G , H ) Western blotting revealed levels of POLH ( G ) and REV1 ( H ) in two independent clones upon selection following transfection with indicated shRNA . ( I ) Colony formation assay was conducted to reveal proliferation of WT , POLH KD , REV1 KD , HMCES KO , HMCES KO + POLH KD and HMCES KO + REV1 KD cells . ( J ) Representative images showing proliferation of respective shRNA resistant reconstitution of POLH in 293A and HMCES KO cells stably expressing POLH shRNA . Experiment was repeated two independent times in duplicates . ( K ) Mutagenesis frequency analysis was conducted in 293T scrambled and 293T HMCES shRNA cells using sup F shuttle vector - based assay system . Experiments were repeated three independent times and data are presented as mean ± s . e . m . Student ' s t - test was used to calculate statistical significance ( * * , p < 0 . 01 ) .

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 B-PANEL_START 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 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 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 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 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 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

Figure 3(A, B) Colonogenic assays were performed by adding increasing concentrations of pemetrexed to control wild-type (WT) 293A cells and HMCES knockout (HMCES KO) cells (A) and quantification of results were shown (B). Increasing numbers of cells were seeded with increasing concentrations of the compound to obtain optimal drug-dose response curve. Seeding density with respect to control is indicated below each image. The images shown are representative of three biological repeats, each performed in duplicate. (B) Data are presented as mean±s.e.m. and student's t-test was performed for statistical analysis (****, p<0.0001).(C) Western blot showing APEX2 levels upon 293A cells infected with viruses encoding scrambled or two independent APEX2 shRNAs.(D) Colony forming assay was performed to indicate colony forming efficiency in 293A and HMCES KO cells upon infection with viruses encoding scrambled or APEX2 shRNAs.(E) Sensitivity assay was conducted with WT and HMCES KO cells upon treatment with AP Endonuclease inhibitor III (APEi). Results are presented from two independent experiments and data are presented as mean±s.e.m.(F) Colony forming assay was performed with WT and HMCES KO cells upon either pemetrexed treatment alone or in combination with APEi. Data are presented as mean±s.e.m (n=2 each with technical repeats).(G, H) Western blotting revealed levels of POLH (G) and REV1 (H) in two independent clones upon selection following transfection with indicated shRNA.(I) Colony formation assay was conducted to reveal proliferation of WT, POLH KD, REV1 KD, HMCES KO, HMCES KO + POLH KD and HMCES KO +REV1 KD cells.(J) Representative images showing proliferation of respective shRNA resistant reconstitution of POLH in 293A and HMCES KO cells stably expressing POLH shRNA. Experiment was repeated two independent times in duplicates.(K) Mutagenesis frequency analysis was conducted in 293T scrambled and 293T HMCES shRNA cells using sup F shuttle vector-based assay system. Experiments were repeated three independent times and data are presented as mean±s.e.m. Student's t-test was used to calculate statistical significance (**, p<0.01).


Figure 4 ( B ) Volcano plot showing DDR CRISPR - CAS9 screen for HMCES KO cells . - log10 ( p - value ) is plotted against log2 ( HMCES KO / WT ) fold change . Genes showing significant positive and negative enrichment are colored in red and top enriched candidates are also labeled . ( C ) Plot showing number of sgRNAs that are negatively or positively enriched for each gene in DDR library . Western blots showing levels of CtIP ( D ) upon shRNA mediated knockdown in 293A cells . Western blots showing levels of BRCA2 ( E ) upon shRNA mediated knockdown in 293A cells . Cell proliferation measured using CellTiter - Glo assay in 293A and HMCES KO cells without or with transfection with viruses encoding CtIP ( F ) shRNAs . Each experiment was repeated three independent times done in triplicates each time . Data was represented as mean ± s . e . m . Student ' s t - test was used to calculate statistical significance ( * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 ) . Cell proliferation measured using CellTiter - Glo assay in 293A and HMCES KO cells without or with transfection with viruses encoding BRCA2 ( G ) shRNAs . Each experiment was repeated three independent times done in triplicates each time . Data was represented as mean ± s . e . m . Student ' s t - test was used to calculate statistical significance ( * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 ) .

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Figure 4(B) Volcano plot showing DDR CRISPR-CAS9 screen for HMCES KO cells. -log10(p-value) is plotted against log2(HMCES KO/ WT) fold change. Genes showing significant positive and negative enrichment are colored in red and top enriched candidates are also labeled.(C) Plot showing number of sgRNAs that are negatively or positively enriched for each gene in DDR library.Western blots showing levels of CtIP (D) upon shRNA mediated knockdown in 293A cells.Western blots showing levels of BRCA2 (E) upon shRNA mediated knockdown in 293A cells.Cell proliferation measured using CellTiter-Glo assay in 293A and HMCES KO cells without or with transfection with viruses encoding CtIP (F) shRNAs. Each experiment was repeated three independent times done in triplicates each time. Data was represented as mean±s.e.m. Student's t-test was used to calculate statistical significance (***, p<0.001; ****, p<0.0001).Cell proliferation measured using CellTiter-Glo assay in 293A and HMCES KO cells without or with transfection with viruses encoding BRCA2 (G) shRNAs. Each experiment was repeated three independent times done in triplicates each time. Data was represented as mean±s.e.m. Student's t-test was used to calculate statistical significance (***, p<0.001; ****, p<0.0001).


Figure 1A , Hierarchical clustering analysis of RNA - sequencing data from cells treated with DMSO , 20 Gy IR , 250nM AZD6738 , or AZD6738 + IR ( n = 2 biological replicates ) . C , Heat map of 33 representative inflammatory genes regulated by AZD6738 + IR . D , MCF10A WT cells were pretreated with DMSO or AZD6738 ( 250 nM ) for 2 hours , and then irradiated with 20 Gy , cells were harvested at indicated time points and immunoblotting was performed with indicated antibodies . h , hours ; pSTAT1 , pSTAT1 ( Y701 ) ; pIRF3 , pIRF3 ( S396 ) ; pTBK1 , pTBK1 ( S172 ) . E , MCF10A WT cells were pretreated with DMSO or AZD6738 ( 250 nM ) for 2 hours , and then irradiated with indicated dose . 72 hours later , cells were harvested for immunoblotting with indicated antibodies . F , Same condition as E except the irradiation dose with 20 Gy , mRNA levels of inflammatory genes as indicated were assessed by real - time quantitative PCR , presented as mean + standard error of the mean of three biological replicates .

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Figure 1A, Hierarchical clustering analysis of RNA-sequencing data from cells treated with DMSO, 20 Gy IR, 250nM AZD6738, or AZD6738 + IR (n=2 biological replicates).C, Heat map of 33 representative inflammatory genes regulated by AZD6738 + IR.D, MCF10A WT cells were pretreated with DMSO or AZD6738(250 nM) for 2 hours, and then irradiated with 20 Gy, cells were harvested at indicated time points and immunoblotting was performed with indicated antibodies. h, hours; pSTAT1, pSTAT1(Y701); pIRF3, pIRF3(S396); pTBK1, pTBK1(S172).E, MCF10A WT cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with indicated dose. 72 hours later, cells were harvested for immunoblotting with indicated antibodies.F, Same condition as E except the irradiation dose with 20 Gy, mRNA levels of inflammatory genes as indicated were assessed by real-time quantitative PCR, presented as mean + standard error of the mean of three biological replicates.


Figure 2A , MCF10A WT cells were pretreated with AZD6738 ( 250 nM ) and RO - 3306 ( 9 μM ) for 2 hours , and then irradiated with 20 Gy , 3 days later , cells were harvested for immunoblotting with indicated antibodies . B , Same condition except that RO - 3306 is replaced by PD - 0332991 ( 1µM ) . C , MCF10A WT cells were pretreated with DMSO or AZD6738 ( 250 nM ) for 2 hours , and then irradiated with 20 Gy , 24 hours later , cell cycle distribution was measured by flow cytometry . EdU ( 10 μM ) was added into medium 2 hours before harvesting for flow cytometry . D , MCF10A WT cells were pretreated with DMSO or AZD6738 ( 250 nM ) for 2 hours , and then irradiated with 20 Gy , and cells were harvested at indicated time points for immunoblotting . E , MCF10A control cells and CDC25C - WT / constitutively active mutant CDC25C - S216A over - expressed cells were pretreated with DMSO or AZD6738 ( 250 nM ) for 2 hours , and then irradiated with 20 Gy , 24 hours later , cells were harvested for immunoblotting with indicated antibodies . F , Same condition except that cells were harvested 72 hours later .

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Figure 2A, MCF10A WT cells were pretreated with AZD6738(250 nM) and RO-3306 (9 μM) for 2 hours, and then irradiated with 20 Gy, 3 days later, cells were harvested for immunoblotting with indicated antibodies.B, Same condition except that RO-3306 is replaced by PD-0332991 (1µM).C, MCF10A WT cells were pretreated with DMSO or AZD6738(250 nM) for 2 hours, and then irradiated with 20 Gy, 24 hours later, cell cycle distribution was measured by flow cytometry. EdU (10 μM) was added into medium 2 hours before harvesting for flow cytometry.D, MCF10A WT cells were pretreated with DMSO or AZD6738(250 nM) for 2 hours, and then irradiated with 20 Gy, and cells were harvested at indicated time points for immunoblotting.E, MCF10A control cells and CDC25C-WT/constitutively active mutant CDC25C-S216A over-expressed cells were pretreated with DMSO or AZD6738(250 nM) for 2 hours, and then irradiated with 20 Gy, 24 hours later, cells were harvested for immunoblotting with indicated antibodies.F, Same condition except that cells were harvested 72 hours later.


Figure 3A , MCF10A Wild - type ( WT ) , cGAS knockout ( KO ) , and STING KO cells were treated with cGAMP at indicated concentrations for 4 hours , and cells were harvested for immunoblotting . B , MCF10A WT , cGAS KO , and STING KO cells were transfected with herring testis DNA ( HT - DNA , 2 µg / ml ) for 6 hours with lipofectamine 3000 . Immunoblotting was performed with indicated antibodies . MCF10A WT or two independent clones for cGAS KO cells were pretreated with DMSO or AZD6738 ( 250 nM ) for 2 hours , and then irradiated with 20 Gy , 3 days later , cells were harvested for immunoblotting with indicated antibodies . Data are presented as mean + standard error of the mean of three biological replicates . MCF10A WT or two independent clones for STING KO cells were pretreated with DMSO or AZD6738 ( 250 nM ) for 2 hours , and then irradiated with 20 Gy , 3 days later , cells were harvested for immunoblotting with indicated antibodies . Data are presented as mean + standard error of the mean of three biological replicates . MCF10A WT or two independent clones for cGAS , STING , and IRF3 KO cells were pretreated with DMSO or AZD6738 ( 250 nM ) for 2 hours , and then irradiated with 20 Gy , 3 days later , except that cells were harvested for real - time quantitative PCR . IFNB1 mRNA level was measured and statistical analyzed by two - way ANOVA ( * * * means p value < 0 . 001 ) . Data are presented as mean + standard error of the mean of three biological replicates . MCF10A WT or two independent clones for IRF3 KO cells were pretreated with DMSO or AZD6738 ( 250 nM ) for 2 hours , and then irradiated with 20 Gy , 3 days later , cells were harvested for immunoblotting with indicated antibodies . Data are presented as mean + standard error of the mean of three biological replicates .

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Figure 3A, MCF10A Wild-type (WT), cGAS knockout (KO), and STING KO cells were treated with cGAMP at indicated concentrations for 4 hours, and cells were harvested for immunoblotting.B, MCF10A WT, cGAS KO, and STING KO cells were transfected with herring testis DNA (HT-DNA, 2 µg/ml) for 6 hours with lipofectamine 3000. Immunoblotting was performed with indicated antibodies.MCF10A WT or two independent clones for cGAS KO cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with 20 Gy, 3 days later, cells were harvested for immunoblotting with indicated antibodies. Data are presented as mean + standard error of the mean of three biological replicates.MCF10A WT or two independent clones for STING KO cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with 20 Gy, 3 days later, cells were harvested for immunoblotting with indicated antibodies. Data are presented as mean + standard error of the mean of three biological replicates.MCF10A WT or two independent clones for cGAS, STING, and IRF3 KO cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with 20 Gy, 3 days later, except that cells were harvested for real-time quantitative PCR. IFNB1 mRNA level was measured and statistical analyzed by two-way ANOVA (*** means p value < 0.001). Data are presented as mean + standard error of the mean of three biological replicates.MCF10A WT or two independent clones for IRF3 KO cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with 20 Gy, 3 days later, cells were harvested for immunoblotting with indicated antibodies. Data are presented as mean + standard error of the mean of three biological replicates.


Figure 4 ( A MCF10A WT or two independent clones for MAVS KO cells were pretreated with DMSO or AZD6738 ( 250 nM ) for 2 hours , and then irradiated with 20 Gy , 3 days later , cells were harvested for immunoblotting with indicated antibodies . MCF10A WT or two independent clones for MAVS , RIG - I KO cells were pretreated with DMSO or AZD6738 ( 250 nM ) for 2 hours , and then irradiated with 20 Gy , 3 days later , ( B cells were harvested for real - time quantitative PCR instead of immunoblotting . IFNB1 mRNA level was measured by real - time quantitative PCR . Data are presented as mean + standard error of the mean of three biological replicates . C , MCF10A WT or two independent clones for RIG - I KO cells were pretreated with DMSO or AZD6738 ( 250 nM ) for 2 hours , and then irradiated with 20 Gy , 3 days later , cells were harvested for immunoblotting with indicated antibodies . D , MCF10A WT or two independent clones for MDA5 , KO cells were pretreated with DMSO or AZD6738 ( 250 nM ) for 2 hours , and then irradiated with 20 Gy , 3 days later , cells were harvested for immunoblotting with indicated antibodies . MCF10A WT or two independent clones for MDA5 KO cells were pretreated with DMSO or AZD6738 ( 250 nM ) for 2 hours , and then irradiated with 20 Gy , 3 days later E ) cells were harvested for real - time quantitative PCR instead of immunoblotting . IFNB1 mRNA level was measured by real - time quantitative PCR . Data are presented as mean + standard error of the mean of three biological replicates . F ) MCF10A WT or two independent clones for IRF7 KO cells were pretreated with DMSO or AZD6738 ( 250 nM ) for 2 hours , and then irradiated with 20 Gy , 3 days later , cells were harvested for immunoblotting with indicated antibodies .

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 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 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 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

Figure 4(A MCF10A WT or two independent clones for MAVS KO cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with 20 Gy, 3 days later, cells were harvested for immunoblotting with indicated antibodies.MCF10A WT or two independent clones for MAVS, RIG-I KO cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with 20 Gy, 3 days later, (B cells were harvested for real-time quantitative PCR instead of immunoblotting. IFNB1 mRNA level was measured by real-time quantitative PCR. Data are presented as mean + standard error of the mean of three biological replicates.C, MCF10A WT or two independent clones for RIG-I KO cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with 20 Gy, 3 days later, cells were harvested for immunoblotting with indicated antibodies.D, MCF10A WT or two independent clones for MDA5, KO cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with 20 Gy, 3 days later, cells were harvested for immunoblotting with indicated antibodies.MCF10A WT or two independent clones for MDA5 KO cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with 20 Gy, 3 days later E) cells were harvested for real-time quantitative PCR instead of immunoblotting. IFNB1 mRNA level was measured by real-time quantitative PCR. Data are presented as mean + standard error of the mean of three biological replicates.F) MCF10A WT or two independent clones for IRF7 KO cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with 20 Gy, 3 days later, cells were harvested for immunoblotting with indicated antibodies.


Figure 5A , 4T1 cells were pretreated with AZD6738 at indicated concentrations for 2 hours , and then irradiated with 10 Gy . 4 days later , Ifnb1 mRNA level was measured by real - time quantitative PCR . Data are presented as mean + standard error of the mean of three biological replicates . B , Ifnb1 mRNA levels were measured by real - time quantitative PCR in WT , Sting KO , Mavs KO , and Irf3 KO 4T1 cells . Data are presented as mean + standard error of the mean of three biological replicates . C , MC38 cells were pretreated with AZD6738 at indicated concentrations for 2 hours , and then irradiated with 20 Gy . 3 days later , Ifnb1 mRNA level was measured by real - time quantitative PCR . Data are presented as mean + standard error of the mean of three biological replicates . D , Ifnb1 mRNA levels were measured by real - time quantitative PCR in WT , Sting KO , Mavs KO , and Irf3 KO MC38 cells . Data are presented as mean + standard error of the mean of three biological replicates .

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 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 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 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 5A, 4T1 cells were pretreated with AZD6738 at indicated concentrations for 2 hours, and then irradiated with 10 Gy. 4 days later, Ifnb1 mRNA level was measured by real-time quantitative PCR. Data are presented as mean + standard error of the mean of three biological replicates.B, Ifnb1 mRNA levels were measured by real-time quantitative PCR in WT, Sting KO, Mavs KO, and Irf3 KO 4T1 cells. Data are presented as mean + standard error of the mean of three biological replicates.C, MC38 cells were pretreated with AZD6738 at indicated concentrations for 2 hours, and then irradiated with 20 Gy. 3 days later, Ifnb1 mRNA level was measured by real-time quantitative PCR. Data are presented as mean + standard error of the mean of three biological replicates.D, Ifnb1 mRNA levels were measured by real-time quantitative PCR in WT, Sting KO, Mavs KO, and Irf3 KO MC38 cells. Data are presented as mean + standard error of the mean of three biological replicates.


Figure 6A , MCF10A WT , cGAS KO , STING KO , and MAVS KO cells were treated with poly ( dA - dT ) / LyoVec at indicated concentrations for 24 hours . Immunoblotting was performed with indicated antibodies . B , 4T1 WT , Sting KO , Mavs KO , and Irf3 KO cells were treated with poly ( dA - dT ) / LyoVec at indicated concentrations for 24 hours . Immunoblotting was performed with indicated antibodies . C , END - seq was used to map double - strand breaks ( DSBs ) associated with TA dinucleotide repeats in MCF10A cells with the indicated treatments . ATR inhibitor ( ATRi ) AZD6738 ( 250nM ) was used with ionizing radiation ( IR , 10 Gy ) for 6 hours . NT , not treated . D , MCF10A WT , MDA5 KO , RIG - I KO , and MAVS KO cells were transfected with AT - rich oligos ( 2 μg / ml ) with LyoVec for 24 hours . Immunoblotting was performed with indicated antibodies . E , MCF10A WT and STING KO cells were transfected with AT - rich and non - AT - rich oligos ( 2 μg / ml ) with lipofectamine 3000 for 4 hours . Immunoblotting was performed with indicated antibodies . F , 4T1 WT , Sting KO , and Irf3 KO cells were transfected with AT - rich and non - AT - rich oligos ( 2 μg / ml ) with LyoVec for 24 hours . Immunoblotting was performed with indicated antibodies .

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 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 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 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 6A, MCF10A WT, cGAS KO, STING KO, and MAVS KO cells were treated with poly(dA-dT)/LyoVec at indicated concentrations for 24 hours. Immunoblotting was performed with indicated antibodies.B, 4T1 WT, Sting KO, Mavs KO, and Irf3 KO cells were treated with poly(dA-dT)/LyoVec at indicated concentrations for 24 hours. Immunoblotting was performed with indicated antibodies.C, END-seq was used to map double-strand breaks (DSBs) associated with TA dinucleotide repeats in MCF10A cells with the indicated treatments. ATR inhibitor (ATRi) AZD6738 ( 250nM) was used with ionizing radiation (IR, 10 Gy) for 6 hours. NT, not treated.D, MCF10A WT, MDA5 KO, RIG-I KO, and MAVS KO cells were transfected with AT-rich oligos (2 μg/ml) with LyoVec for 24 hours. Immunoblotting was performed with indicated antibodies.E, MCF10A WT and STING KO cells were transfected with AT-rich and non-AT-rich oligos (2 μg/ml) with lipofectamine 3000 for 4 hours. Immunoblotting was performed with indicated antibodies.F, 4T1 WT, Sting KO, and Irf3 KO cells were transfected with AT-rich and non-AT-rich oligos (2 μg/ml) with LyoVec for 24 hours. Immunoblotting was performed with indicated antibodies.


Figure 1A cdc14 ‐ 3 temperature ‐ sensitive cells reconstituted with CDC14 or CDC14 ‐ NLS were released from a G1 arrest at the restrictive temperature and stained for Cdc14 and the spindle ( Tub1 ) once cells had reached late anaphase . B Wild ‐ type , cdc14 ‐ 3 , + CDC14 and + CDC14 ‐ NLS cells were released from an α ‐ factor ‐ induced G1 arrest and re ‐ arrested in the next G1 . Samples were harvested at the indicated time points and stained with DAPI and for the nucleolar protein Nop1 ( n > 100 ) . Photographs of examples of cells without ( top ) and with ( bottom ) rDNA segregation defects at 80 min after release are shown together with quantification over time . C Cells from the experiment in ( B ) were stained for the spindle ( n > 100 ) . Cells without ( top ) and with ( bottom ) anaphase spindles at 100 min after release are shown together with their quantification over time . D FACS analysis of DNA content and Western blot analysis of cell cycle markers of the samples from ( B ) . Tub1 served 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 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 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 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

Figure 1A cdc14‐3 temperature‐sensitive cells reconstituted with CDC14 or CDC14‐NLS were released from a G1 arrest at the restrictive temperature and stained for Cdc14 and the spindle (Tub1) once cells had reached late anaphase.B Wild‐type, cdc14‐3, +CDC14 and +CDC14‐NLS cells were released from an α‐factor‐induced G1 arrest and re‐arrested in the next G1. Samples were harvested at the indicated time points and stained with DAPI and for the nucleolar protein Nop1 (n > 100). Photographs of examples of cells without (top) and with (bottom) rDNA segregation defects at 80 min after release are shown together with quantification over time.C Cells from the experiment in (B) were stained for the spindle (n > 100). Cells without (top) and with (bottom) anaphase spindles at 100 min after release are shown together with their quantification over time.D FACS analysis of DNA content and Western blot analysis of cell cycle markers of the samples from (B). Tub1 served as a loading control.


Figure 2A + CDC14 and + CDC14 ‐ NLS cells were grown at 35 . 5 ° C for 5 h and sonicated , and differential interference contrast ( DIC ) images were acquired . B Cells from a synchronous culture ( as in Fig B ) were treated with zymolyase to remove the cell wall . The fraction of remaining budded cells is shown over time ( n = 100 ) . C Cells expressing the plasma membrane marker GFP ‐ Spo2051 - 91 , exemplifying the indicated bud neck phenotypes and stages of cytokinesis . D Bud neck phenotypes of synchronized GFP ‐ Spo2051 - 91 ‐ expressing + CDC14 or + CDC14 ‐ NLS cells during the progression of a cell cycle ( n > 75 ) . E + CDC14 or + CDC14 ‐ NLS cells expressing Spc42 ‐ YFP and GFP fusions with the indicated cytokinetic protein were synchronized in G1 using α ‐ factor . Upon release at 35 . 5 ° C , cells were imaged every 2 min and the time from anaphase onset to the indicated events was recorded . Error bars represent SD ; Student ' s t ‐ test was applied to analyze differences between the indicated conditions ; * * P ≤ 0 . 01 ; * * * * P ≤ 0 . 0001 . For representative image sequences , see Supplementary Fig S2C .

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Figure 2A +CDC14 and +CDC14‐NLS cells were grown at 35.5°C for 5 h and sonicated, and differential interference contrast (DIC) images were acquired.B Cells from a synchronous culture (as in Fig B) were treated with zymolyase to remove the cell wall. The fraction of remaining budded cells is shown over time (n = 100).C Cells expressing the plasma membrane marker GFP‐Spo2051-91, exemplifying the indicated bud neck phenotypes and stages of cytokinesis.D Bud neck phenotypes of synchronized GFP‐Spo2051-91‐expressing +CDC14 or +CDC14‐NLS cells during the progression of a cell cycle (n > 75).E +CDC14 or +CDC14‐NLS cells expressing Spc42‐YFP and GFP fusions with the indicated cytokinetic protein were synchronized in G1 using α‐factor. Upon release at 35.5°C, cells were imaged every 2 min and the time from anaphase onset to the indicated events was recorded. Error bars represent SD; Student's t‐test was applied to analyze differences between the indicated conditions; **P ≤ 0.01; ****P ≤ 0.0001. For representative image sequences, see Supplementary Fig S2C.


Figure 3A MET3pr ‐ CDC20cdc15 ‐ as1 cells were arrested in metaphase by Cdc20 depletion and subsequently treated with 1NM ‐ PP1 to inhibit Cdc15 ‐ as1 . Cells were released from the metaphase arrest by Cdc20 re ‐ induction ( left graph ) , or Cdc14 and / or Sic1 expression were induced from the GAL1 promoter while cells remained arrested ( right graph ) . The fraction of large ‐ budded cells after spheroplastation ( n = 100 ) was quantified . Representative photographs 120 min after galactose addition are shown . Scale bar , 10 μm . B As ( A ) , the plasma membrane marker GFP ‐ Spo2051 - 91 was visualized following Cdc14 induction ( n = 100 ) . C Samples from the experiment in ( B ) were processed for Western blot analysis of the indicated cell cycle proteins . Arrowheads indicate endogenous and exogenous forms of Cdc14 . Tub1 served as a loading control . A schematic of downstream processing for the phosphoproteome analysis is shown on the right .

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 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

Figure 3A MET3pr‐CDC20cdc15‐as1 cells were arrested in metaphase by Cdc20 depletion and subsequently treated with 1NM‐PP1 to inhibit Cdc15‐as1. Cells were released from the metaphase arrest by Cdc20 re‐induction (left graph), or Cdc14 and/or Sic1 expression were induced from the GAL1 promoter while cells remained arrested (right graph). The fraction of large‐budded cells after spheroplastation (n = 100) was quantified. Representative photographs 120 min after galactose addition are shown. Scale bar, 10 μm.B As (A), the plasma membrane marker GFP‐Spo2051-91 was visualized following Cdc14 induction (n = 100).C Samples from the experiment in (B) were processed for Western blot analysis of the indicated cell cycle proteins. Arrowheads indicate endogenous and exogenous forms of Cdc14. Tub1 served as a loading control. A schematic of downstream processing for the phosphoproteome analysis is shown on the right.


Figure 4A Phospho ‐ peptide abundance during Cdc14 ‐ induced mitotic exit . Examples of phospho ‐ peptides that disappear with early ( red ) , intermediate ( orange ) and late ( green ) timing or that remain stable ( black ) are shown . This color coding is used throughout all panels of this figure . B Numbers of unique peptides and corresponding proteins that do or do not contain a minimal Cdk consensus motif are shown , categorized according to timing of disappearance as described in ( A ) . C Protein stability data for proteins corresponding to disappearing phospho ‐ peptides ( blue ) and for all other proteins ( black ) . D The fractions of peptides that contain a minimal Cdk consensus motif ( [ S / T ] ‐ P ) within the timing categories shown in ( B ) were calculated . The ratio and P ‐ value of enrichment of each category relative to the combined stable / rest category ( gray ) are shown . E Similar to ( D ) , the enrichment for reported physical Cdc14 interactors is assessed within [ S / T ] ‐ P ‐ containing phospho ‐ peptides . F Peptides that contain the indicated Cdk consensus sequence motifs and that have disappeared over time are represented as a fraction of all phospho ‐ peptides containing that motif . G Unique phospho ‐ peptides of Spa2 , as an example , are categorized based on their dephosphorylation timing ( graph ) . Enrichment of clustered timing for all proteins covered by phospho ‐ peptides was compared to random for Cdk or non ‐ Cdk consensus site ‐ containing peptides ( table ) . Fisher ' s exact tests were used to determine significance .

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Figure 4A Phospho‐peptide abundance during Cdc14‐induced mitotic exit. Examples of phospho‐peptides that disappear with early (red), intermediate (orange) and late (green) timing or that remain stable (black) are shown. This color coding is used throughout all panels of this figure.B Numbers of unique peptides and corresponding proteins that do or do not contain a minimal Cdk consensus motif are shown, categorized according to timing of disappearance as described in (A).C Protein stability data for proteins corresponding to disappearing phospho‐peptides (blue) and for all other proteins (black).D The fractions of peptides that contain a minimal Cdk consensus motif ([S/T]‐P) within the timing categories shown in (B) were calculated. The ratio and P‐value of enrichment of each category relative to the combined stable/rest category (gray) are shown.E Similar to (D), the enrichment for reported physical Cdc14 interactors is assessed within [S/T]‐P‐containing phospho‐peptides.F Peptides that contain the indicated Cdk consensus sequence motifs and that have disappeared over time are represented as a fraction of all phospho‐peptides containing that motif.G Unique phospho‐peptides of Spa2, as an example, are categorized based on their dephosphorylation timing (graph). Enrichment of clustered timing for all proteins covered by phospho‐peptides was compared to random for Cdk or non‐Cdk consensus site‐containing peptides (table). Fisher's exact tests were used to determine significance.


Figure 5B Western blot analysis of cells expressing conditional Clb2m ( + ) or Clb2mΔCdk ( Δ ) fusions to the indicated proteins , before ( − ) and 5 h after ( + ) β ‐ estradiol treatment . Tub1 served as a loading control . c , control cells . C FACS analysis of DNA content after conditional Clb2m or Clb2mΔCdk fusion to the indicated proteins . Quantification of the fraction of cells with > 2C DNA content is shown in the bar graph for two independent biological replicates , with red dots representing individual and bars representing average values . Bnr1 fusions were included as controls for cytokinetic defects . Student ' s t ‐ test was applied to compare matched Clb2m versus Clb2mΔCdk fusion strains . ‐ P > 0 . 05 ; * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; * * * P ≤ 0 . 001 . D Serial dilutions of cells from ( C ) were plated 6 h following β ‐ estradiol treatment and grown for 1 day at 36 ° C . E The length of cell chains containing a common cytoplasm was scored 6 h after β ‐ estradiol treatment using the GFP ‐ Spo2051 - 91 plasma membrane marker ( n > 150 ) . c , control cells ; scale bars , 10 μm .

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Figure 5B Western blot analysis of cells expressing conditional Clb2m (+) or Clb2mΔCdk (Δ) fusions to the indicated proteins, before (−) and 5 h after (+) β‐estradiol treatment. Tub1 served as a loading control. c, control cells.C FACS analysis of DNA content after conditional Clb2m or Clb2mΔCdk fusion to the indicated proteins. Quantification of the fraction of cells with > 2C DNA content is shown in the bar graph for two independent biological replicates, with red dots representing individual and bars representing average values. Bnr1 fusions were included as controls for cytokinetic defects. Student's t‐test was applied to compare matched Clb2m versus Clb2mΔCdk fusion strains. ‐P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.D Serial dilutions of cells from (C) were plated 6 h following β‐estradiol treatment and grown for 1 day at 36°C.E The length of cell chains containing a common cytoplasm was scored 6 h after β‐estradiol treatment using the GFP‐Spo2051-91 plasma membrane marker (n > 150). c, control cells; scale bars, 10 μm.


Figure 6A Genes corresponding to the indicated proteins were replaced by wild ‐ type ( wt ) or Cdk phospho ‐ site mutant ( xA ) sequences and then conditionally fused to Clb2m or Clb2mΔCdk . Cytokinetic defects were scored by FACS analysis of DNA content as in Fig C . The individual values from two independent clones ( red dots ) as well as their average ( bars ) are shown . Student ' s t ‐ test was applied to compare matched wt and xA strains . c , control cells . B Cell chain formation in the samples from ( A ) . The fraction of cells with > 2 cell bodies for each clone are represented with red dots and their averages by the black bar ( n > 150 ) . Significance is determined by Student ' s t ‐ tests for the indicated conditions . Scale bars , 5 μm . C The indicated strains were spotted in serial dilutions onto auxin ‐ containing plates and grown for 2 days . D Auxin was added to cultures of the indicated strains , and after 6 h , cells were sonicated and the number of attached cell bodies was counted ( n > 200 ) . Significance was calculated using a Wilcoxon rank ‐ sum test . E The fraction of cells in asynchronously proliferating cultures of the indicated strains with a DNA content of > 2C was determined by FACS analysis ( n = 3 ) . Error bars represent SD .

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Figure 6A Genes corresponding to the indicated proteins were replaced by wild‐type (wt) or Cdk phospho‐site mutant (xA) sequences and then conditionally fused to Clb2m or Clb2mΔCdk. Cytokinetic defects were scored by FACS analysis of DNA content as in Fig C. The individual values from two independent clones (red dots) as well as their average (bars) are shown. Student's t‐test was applied to compare matched wt and xA strains. c, control cells.B Cell chain formation in the samples from (A). The fraction of cells with > 2 cell bodies for each clone are represented with red dots and their averages by the black bar (n > 150). Significance is determined by Student's t‐tests for the indicated conditions. Scale bars, 5 μm.C The indicated strains were spotted in serial dilutions onto auxin‐containing plates and grown for 2 days.D Auxin was added to cultures of the indicated strains, and after 6 h, cells were sonicated and the number of attached cell bodies was counted (n > 200). Significance was calculated using a Wilcoxon rank‐sum test.E The fraction of cells in asynchronously proliferating cultures of the indicated strains with a DNA content of > 2C was determined by FACS analysis (n = 3). Error bars represent SD.


Figure 7A Cyk3 ‐ GFP signal intensity at the GFP relative to the onset of actomyosin ring contraction ( t = 0 ) . The mean and SEM from at least 20 cells are shown . B Inn1 ‐ Clb2m , Inn1 ‐ Clb2mΔCdk and control cells before Clb2m fusion were arrested in G1 using α ‐ factor , released and harvested 90 - 100 min later at the time of cytokinesis . Indirect immunofluorescence microscopy was used to determine Inn1bud neck staining and the spindle status . Representative images of full spindles and partially and fully disassembled spindles are shown . nr , non ‐ recombined cells . Scale bars , 5 μm . C The time from anaphase onset until the GFP appearance of Inn1 was determined by filming synchronized Inn1 ‐ Clb2m ‐ GFP and Inn1 ‐ Clb2mΔCdk ‐ GFP cells containing Spc42 ‐ YFP ‐ labeled spindle pole bodies . Error bars represent SD . D As in ( C ) , now the persistence of a full actomyosin ring was measured in cells expressing Inn1 ‐ Clb2m , Inn1 ‐ Clb2mΔCdk or their respective non ‐ phosphorylatable mutants together with Myo1 ‐ GFP and Spc42 ‐ YFP . Error bars represent SD . E Montage of medial view projections of actomyosin rings in Inn1 ‐ Clb2m and Inn1 ‐ Clb2mΔCdk cells . Frames are shown in 2 ‐ min intervals . F Actomyosin ring contraction phenotypes from at least 21 cells filmed as in ( E ) were blindly scored as having a normal ( " constriction " ) or aberrant ( " disintegration " ) cytokinetic phenotype . G Cytokinetic defect and cell viability of Inn1 ‐ Clb2m or Inn1 ‐ Clb2mΔCdk cells in the absence or presence of Chs2V377I . In the bar graph , red dots represent values of duplicate biological replicates with bars representing the average . Significance is calculated using Student ' s t ‐ test . 10 ‐ fold spot dilution assays are represented , together with the Western blotting analysis for Inn1 before and after its fusion to Clb2m . Tub1 served as a loading control .

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Figure 7A Cyk3‐GFP signal intensity at the GFP relative to the onset of actomyosin ring contraction (t = 0). The mean and SEM from at least 20 cells are shown.B Inn1‐Clb2m, Inn1‐Clb2mΔCdk and control cells before Clb2m fusion were arrested in G1 using α‐factor, released and harvested 90-100 min later at the time of cytokinesis. Indirect immunofluorescence microscopy was used to determine Inn1bud neck staining and the spindle status. Representative images of full spindles and partially and fully disassembled spindles are shown. nr, non‐recombined cells. Scale bars, 5 μm.C The time from anaphase onset until the GFP appearance of Inn1 was determined by filming synchronized Inn1‐Clb2m‐GFP and Inn1‐Clb2mΔCdk‐GFP cells containing Spc42‐YFP‐labeled spindle pole bodies. Error bars represent SD.D As in (C), now the persistence of a full actomyosin ring was measured in cells expressing Inn1‐Clb2m, Inn1‐Clb2mΔCdk or their respective non‐phosphorylatable mutants together with Myo1‐GFP and Spc42‐YFP. Error bars represent SD.E Montage of medial view projections of actomyosin rings in Inn1‐Clb2m and Inn1‐Clb2mΔCdk cells. Frames are shown in 2‐min intervals.F Actomyosin ring contraction phenotypes from at least 21 cells filmed as in (E) were blindly scored as having a normal ("constriction") or aberrant ("disintegration") cytokinetic phenotype.G Cytokinetic defect and cell viability of Inn1‐Clb2m or Inn1‐Clb2mΔCdk cells in the absence or presence of Chs2V377I. In the bar graph, red dots represent values of duplicate biological replicates with bars representing the average. Significance is calculated using Student's t‐test. 10‐fold spot dilution assays are represented, together with the Western blotting analysis for Inn1 before and after its fusion to Clb2m. Tub1 served as a loading control.


Figure 1 ( A ) Overlay of 15N - 1H HSQC spectra of 15N - labeled Ca2 + / CaM by itself ( red peaks ) and after addition of saturating , unlabeled PSD - 95 ( 1 - 21 ) ( blue peaks ) or saturating , unlabeled PSD - 95 _ pT19 ( green peaks ) . All NMR experiments were recorded at 600 MHz 1H frequency . ( B ) Chemical shift perturbation of CaM amide resonances in the absence vs . presence of PSD - 95 _ pT19 . Chemical shift difference ( CSD ) plotted on the vertical axis was defined as CSD = { ( HNA - HNB ) 2 + ( 15NA - 15NB ) 2 } 1 / 2 , where " A " and " B " designate free and bound states of CaM . HN and 15N represent amide 1H and 15N chemical shifts , respectively . Perturbations greater than the average were used as active ambiguous restraints in the Haddock docking calculation ( see Methods ) . ( C ) Representative data from IPAP [ 15N - 1H ] - HSQC spectra of 15N - labeled Ca2 + / CaM bound to PSD - 95 _ pT19 in the absence ( blue ) and presence of 17 mg / ml filamentous bacteriophage Pf1 ( red ) . Residual dipolar couplings ( RDCs ) for backbone HN resonances are calculated ( in Hertz ) as the difference in splitting for the 15N - { 1H } doublet components ( red peaks ) relative to the isotropic 1JNH coupling ( blue peaks ) . RDC values are calculated to be + 40 , + 30 , + 20 , + 20 , and - 20 Hz for I100 , I27 , N137 , D64 , and I130 , respectively . ( D ) Correlation between observed RDCs ( panel C ) versus RDCs calculated from the final NMR derived structure of Ca2 + / CaM bound to PSD - 95 _ pT19 . The correlation coefficient ( R ) equals 0 . 99 and Q is 0 . 11 .

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 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 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

Figure 1(A) Overlay of 15N-1H HSQC spectra of 15N-labeled Ca2+/CaM by itself (red peaks) and after addition of saturating, unlabeled PSD-95 (1-21) (blue peaks) or saturating, unlabeled PSD-95_pT19 (green peaks). All NMR experiments were recorded at 600 MHz 1H frequency.(B) Chemical shift perturbation of CaM amide resonances in the absence vs. presence of PSD-95_pT19. Chemical shift difference (CSD) plotted on the vertical axis was defined as CSD = {(HNA - HNB)2 + (15NA - 15NB)2}1/2 , where "A" and "B" designate free and bound states of CaM. HN and 15N represent amide 1H and 15N chemical shifts, respectively. Perturbations greater than the average were used as active ambiguous restraints in the Haddock docking calculation (see Methods).(C) Representative data from IPAP [15N-1H]-HSQC spectra of 15N-labeled Ca2+/CaM bound to PSD-95_pT19 in the absence (blue) and presence of 17 mg/ml filamentous bacteriophage Pf1 (red). Residual dipolar couplings (RDCs) for backbone HN resonances are calculated (in Hertz) as the difference in splitting for the 15N-{1H} doublet components (red peaks) relative to the isotropic 1JNH coupling (blue peaks). RDC values are calculated to be +40, +30, +20, +20, and -20 Hz for I100, I27, N137, D64, and I130, respectively.(D) Correlation between observed RDCs (panel C) versus RDCs calculated from the final NMR derived structure of Ca2+/CaM bound to PSD-95_pT19. The correlation coefficient (R) equals 0.99 and Q is 0.11.


Figure 2 ( A ) NMR structure of Ca2 + / CaM ( grey ) bound to unphosphorylated PSD - 95 . PSD - 95 residues D2 ( depicted as starting point ) to R13 are resolved and form an - helix ( cyan ) ( Zhang et al . , 2014 ) . Residues 14 - 19 of unphosphorylated PSD - 95 are unstructured and not shown . Ca2 + ions bound to CaM are depicted by orange spheres . ( B ) NMR structure of Ca2 + / CaM bound to PSD - 95 phosphorylated at T19 ( PDB accession no . 5J7J ) . Residues 14 - 19 of phosphorylated PSD - 95 adopt a hook - like structure ( highlighted in red ) not seen in unphosphorylated PSD - 95 . This hook - like structure is stabilized by PSD - 95 residues E17 and pT19 forming salt bridges with R126 and K115 , respectively . Side - chain atoms are depicted by a stick representation for PSD - 95 residues Q15 , E17 and T19 ( red ) and CaM residues K115 , T117 , and R126 ( blue ) .

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 O O O O O O O O O O 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) NMR structure of Ca2+/CaM (grey) bound to unphosphorylated PSD-95. PSD-95 residues D2 (depicted as starting point) to R13 are resolved and form an -helix (cyan) (Zhang et al., 2014). Residues 14-19 of unphosphorylated PSD-95 are unstructured and not shown. Ca2+ ions bound to CaM are depicted by orange spheres.(B) NMR structure of Ca2+/CaM bound to PSD-95 phosphorylated at T19 (PDB accession no. 5J7J). Residues 14-19 of phosphorylated PSD-95 adopt a hook-like structure (highlighted in red) not seen in unphosphorylated PSD-95. This hook-like structure is stabilized by PSD-95 residues E17 and pT19 forming salt bridges with R126 and K115, respectively. Side-chain atoms are depicted by a stick representation for PSD-95 residues Q15, E17 and T19 (red) and CaM residues K115, T117, and R126 (blue).


Figure 3 ( A ) FP titrations showing the binding of wild - type ( WT ) CaM to PSD - 95 ( 1 - 21 ) peptides , either WT ( blue ) , Q15A ( salmon ) , E17R ( black ) , T19E ( green ) , or T19K ( yellow ) . The solid lines represent optimal theoretical curves calculated using a one - site binding model described in Methods . The calculated dissociation constants in each case are given in Table 1 . ( B ) FP titrations showing the binding of R126E CaM to PSD - 95 peptides , either WT ( blue ) or E17R ( black ) . ( C ) FP titrations showing the binding of K115E CaM to PSD - 95 peptides T19K ( yellow ) or T19E ( green ) .

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 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 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

Figure 3(A) FP titrations showing the binding of wild-type (WT) CaM to PSD-95 (1-21) peptides, either WT (blue), Q15A (salmon), E17R (black), T19E (green), or T19K (yellow). The solid lines represent optimal theoretical curves calculated using a one-site binding model described in Methods. The calculated dissociation constants in each case are given in Table 1.(B) FP titrations showing the binding of R126E CaM to PSD-95 peptides, either WT (blue) or E17R (black).(C) FP titrations showing the binding of K115E CaM to PSD-95 peptides T19K (yellow) or T19E (green).


Figure 4 Cultured cortical neurons were treated with BIC ( 50 μM ) at 15 DIV for 48h or at 17 DIV for 2 h or left untreated before harvesting , extraction , and analysis of palmitoylation by the biotin switch method and pull down with NeutrAvidin - agarose beads . ( A ) Representative immunoblots of pull down samples ( top two panels ) and total lysate ( bottom two panels ) for PSD - 95 and , as control , the trimeric G protein subunit G - i3 . Omission of NH2OH before biotinylation resulted in no NeutrAvidin pull down as negative control for non - specific pull down . ( B ) Quantification of PSD - 95 palmitoylation . Seven samples from three independent sets of cultures were analyzed per condition ( * * p < 0 . 01 ; t - test ) .

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

Figure 4 Cultured cortical neurons were treated with BIC (50 μM) at 15 DIV for 48h or at 17 DIV for 2 h or left untreated before harvesting, extraction, and analysis of palmitoylation by the biotin switch method and pull down with NeutrAvidin-agarose beads. (A) Representative immunoblots of pull down samples (top two panels) and total lysate (bottom two panels) for PSD-95 and, as control, the trimeric G protein subunit G-i3. Omission of NH2OH before biotinylation resulted in no NeutrAvidin pull down as negative control for non-specific pull down.(B) Quantification of PSD-95 palmitoylation. Seven samples from three independent sets of cultures were analyzed per condition (**p<0.01; t-test).


Figure 5 Cultured hippocampal neurons were infected at 14 DIV with lentivirus for expression of both shRNA against PSD - 95 ( sh95 ) and sh - resistant PSD - 95 - EGFP . Thus endogenous PSD - 95 was replaced with either wild - type ( WT ) , E17R , or T19K PSD - 95 . Cultures were treated with or without BIC ( 50 μM ) at 17 DIV for 24 - 48 h or left untreated before fixation and staining for surface GluA1 . ( A ) Representative confocal microscopic images of PSD - 95 - EGFP fluorescence ( green ) and surface GluA1 immunostaining ( red ) at low ( top panels ; scale bar : 20 µm ) and high enlargement ( bottom panels ; scale bar : 5 µm ) . ( B ) Quantification of surface GluA1 intensity from ( A ) showed reduction in AMPAR surface levels during scaling down in neurons expressing WT PSD - 95 but not in those expressing either E17R or T19K mutant . Three dendritic segments from each of 31 - 42 neurons from three independent sets of cultures were analyzed per condition ( * * p < 0 . 01 ; n . s . , not significant ; one - way ANOVA followed by Bonferroni ' s post hoc test ) .

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

Figure 5 Cultured hippocampal neurons were infected at 14 DIV with lentivirus for expression of both shRNA against PSD-95 (sh95) and sh-resistant PSD-95-EGFP. Thus endogenous PSD-95 was replaced with either wild-type (WT), E17R, or T19K PSD-95. Cultures were treated with or without BIC (50 μM) at 17 DIV for 24-48 h or left untreated before fixation and staining for surface GluA1. (A) Representative confocal microscopic images of PSD-95-EGFP fluorescence (green) and surface GluA1 immunostaining (red) at low (top panels; scale bar: 20 µm) and high enlargement (bottom panels; scale bar: 5 µm). (B) Quantification of surface GluA1 intensity from (A) showed reduction in AMPAR surface levels during scaling down in neurons expressing WT PSD-95 but not in those expressing either E17R or T19K mutant. Three dendritic segments from each of 31-42 neurons from three independent sets of cultures were analyzed per condition (**p<0.01; n.s., not significant; one-way ANOVA followed by Bonferroni's post hoc test).


Figure 6 Cultured hippocampal neurons were co - transfected with mCherry and EGFP - tagged PSD - 95 wild - type ( WT ) , E17R , or T19K at 10 - 11 DIV and treated with BIC ( 50 μM ) or left untreated at 17 DIV for 24 - 48 h before fixation . ( A ) Representative confocal microscopic images of PSD - 95 - EGFP ( green ) and mCherry ( red ) used as " fill " to visualize the dendrite with spine heads ( scale bar : 2 µm ) after control ( top ) and BIC treatment ( bottom ) . Individual channels are shown in grayscale . ( B ) BIC treatment reduced spine enrichment of WT but not E17R or T19K PSD - 95 as quantified as the ratio of EGFP intensity in spine heads relative to that in the adjacent dendritic shaft . Values were normalized to control treatments , which were set to equal 100 % for each PSD - 95 construct . ( C ) mCherry showed no enrichment in spines and BIC had no effect on its localization . ( D - F ) Spine enrichment and puncta size and intensity of PSD - 95 were comparable under basal conditions for WT , E17R , and T19K PSD - 95 ( A . U . , arbitrary unit ) . ( G - I ) Spine density , head diameter , and length were comparable under basal conditions for neurons expressing WT , E17R , and T19K PSD - 95 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START 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 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 6 Cultured hippocampal neurons were co-transfected with mCherry and EGFP-tagged PSD-95 wild-type (WT), E17R, or T19K at 10-11 DIV and treated with BIC (50 μM) or left untreated at 17 DIV for 24-48 h before fixation. (A) Representative confocal microscopic images of PSD-95-EGFP (green) and mCherry (red) used as "fill" to visualize the dendrite with spine heads (scale bar: 2 µm) after control (top) and BIC treatment (bottom). Individual channels are shown in grayscale. (B) BIC treatment reduced spine enrichment of WT but not E17R or T19K PSD-95 as quantified as the ratio of EGFP intensity in spine heads relative to that in the adjacent dendritic shaft. Values were normalized to control treatments, which were set to equal 100% for each PSD-95 construct.(C) mCherry showed no enrichment in spines and BIC had no effect on its localization.(D-F) Spine enrichment and puncta size and intensity of PSD-95 were comparable under basal conditions for WT, E17R, and T19K PSD-95 (A.U., arbitrary unit).(G-I) Spine density, head diameter, and length were comparable under basal conditions for neurons expressing WT, E17R, and T19K PSD-95.


Figure 7 Cultured hippocampal neurons were transfected at 10 - 11 DIV with the indicated CaM - IRES - ECFP plasmids and infected at 14 DIV with lentivirus expressing shRNA against PSD - 95 ( sh95 ) and sh - resistant wild - type ( WT ) or E17R PSD - 95 - EGFP . Cultures were treated with BIC ( 50 μM ) or left untreated at 17 DIV for 24 - 48 h before fixation . ( A ) Representative confocal microscopic images of intrinsic ECFP and EGFP fluorescence , and surface GluA1 immunostaining at low ( top panels ; scale bar : 20 µm ) and high enlargement ( bottom panels ; scale bar : 5 µm ) . ( B ) Quantification of surface GluA1 intensity from ( A ) showed impaired AMPAR downscaling in neurons expressing PSD - 95 - E17R in presence of wild - type CaM ( CaM - WT ) that was rescued by coexpression of CaM - R126E . Three dendritic segments from each of 18 - 26 neurons from three independent sets of cultures were analyzed per condition ( * * p < 0 . 01 , * p < 0 . 05 vs . untreated basal levels ; one - way ANOVA followed by Bonferroni ' s post hoc test ) .

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

Figure 7 Cultured hippocampal neurons were transfected at 10-11 DIV with the indicated CaM-IRES-ECFP plasmids and infected at 14 DIV with lentivirus expressing shRNA against PSD-95 (sh95) and sh-resistant wild-type (WT) or E17R PSD-95-EGFP. Cultures were treated with BIC (50 μM) or left untreated at 17 DIV for 24-48 h before fixation. (A) Representative confocal microscopic images of intrinsic ECFP and EGFP fluorescence, and surface GluA1 immunostaining at low (top panels; scale bar: 20 µm) and high enlargement (bottom panels; scale bar: 5 µm). (B) Quantification of surface GluA1 intensity from (A) showed impaired AMPAR downscaling in neurons expressing PSD-95-E17R in presence of wild-type CaM (CaM-WT) that was rescued by coexpression of CaM-R126E. Three dendritic segments from each of 18-26 neurons from three independent sets of cultures were analyzed per condition (**p<0.01,*p<0.05 vs. untreated basal levels; one-way ANOVA followed by Bonferroni's post hoc test).


Figure 8Cultured hippocampal neurons were co - transfected at 10 - 11 DIV with RFP and the indicated CaM - IRES - ECFP plasmids and infected at 12 - 13 DIV with lentivirus expressing shRNA against PSD - 95 ( sh95 ) and sh - resistant wild - type ( WT ) or E17R PSD - 95 - EGFP . mEPSC recordings were performed at DIV 17 - 19 , following 48 h of control or BIC ( 50 μM ) treatment . ( A ) Representative traces of AMPAR mEPSC recordings ( left ) and averages of all mEPSC events from single representative neurons ( right ) drawn to scale ( top ) and normalized to peak ( bottom ) . ( B ) Quantification of mEPSC amplitude revealed downscaling by BIC in neurons expressing PSD - 95WT / CaMWT or PSD - 95E17R / CaMR126E but not PSD - 95WT / CaMR126E ( left ) over the full range of amplitude distributions ( right ) . ( C , D ) Mini - EPSC frequency ( C ) and decay time ( D ) were comparable among all conditions ( n = 11 - 25 neurons per condition from 8 - 14 independent sets of cultures prepared from the embryos of 8 - 14 dams ; * p < 0 . 05 ; two - way ANOVA followed by Bonferroni ' s 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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

Figure 8Cultured hippocampal neurons were co-transfected at 10-11 DIV with RFP and the indicated CaM-IRES-ECFP plasmids and infected at 12-13 DIV with lentivirus expressing shRNA against PSD-95 (sh95) and sh-resistant wild-type (WT) or E17R PSD-95-EGFP. mEPSC recordings were performed at DIV 17-19, following 48 h of control or BIC (50 μM) treatment. (A) Representative traces of AMPAR mEPSC recordings (left) and averages of all mEPSC events from single representative neurons (right) drawn to scale (top) and normalized to peak (bottom).(B) Quantification of mEPSC amplitude revealed downscaling by BIC in neurons expressing PSD-95WT / CaMWT or PSD-95E17R / CaMR126E but not PSD-95WT / CaMR126E (left) over the full range of amplitude distributions (right).(C,D) Mini-EPSC frequency (C) and decay time (D) were comparable among all conditions (n=11-25 neurons per condition from 8-14 independent sets of cultures prepared from the embryos of 8-14 dams;* p<0.05; two-way ANOVA followed by Bonferroni's test).