U2OS cells were transfected , synchronized at the G1 / S - border by thymidine for 24 hours and then either released into nocodazole for 16 hours ( unperturbed mitotic entry ) or released for 7 hours , treated with 0 . 5 μM Adriamycin for one hour and after 16 hour G2 arrest were induced to recover by addition of caffeine ( checkpoint recovery ) . Mitotic index was scored based on the percentage of Histone H3 - pS10 positive DAPI - nuclei and normalized to the untransfected controls . Black diamonds indicate individual siRNA - targeted genes from the library , light grey diamonds indicate positive ( Wip1 - depleted ) and negative ( untransfected and GAPDH - depleted ) controls and dark grey diamonds indicate the hits based on the two screens . Dotted lines indicate selection criteria for recovery - specific genes .

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U2OS cells were transfected, synchronized at the G1/S-border by thymidine for 24 hours and then either released into nocodazole for 16 hours (unperturbed mitotic entry) or released for 7 hours, treated with 0.5 μM Adriamycin for one hour and after 16 hour G2 arrest were induced to recover by addition of caffeine (checkpoint recovery). Mitotic index was scored based on the percentage of Histone H3-pS10 positive DAPI-nuclei and normalized to the untransfected controls. Black diamonds indicate individual siRNA-targeted genes from the library, light grey diamonds indicate positive (Wip1-depleted) and negative (untransfected and GAPDH-depleted) controls and dark grey diamonds indicate the hits based on the two screens. Dotted lines indicate selection criteria for recovery-specific genes.


A ) U2OS cells were transfected with four independent siRNA ' s from the pools used in the screen , treated as in figure 1 and analyzed for mitotic index . Error bars represent SD , n = 3 . Statistical significance was tested using a paired two - tailed t - test ( NS for P > 0 . 05 , * for P ≤ 0 . 05 , * * for P ≤ 0 . 01 )

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A) U2OS cells were transfected with four independent siRNA's from the pools used in the screen, treated as in figure 1 and analyzed for mitotic index. Error bars represent SD, n=3. Statistical significance was tested using a paired two-tailed t-test (NS for P>0.05, * for P≤0.05, ** for P≤0.01)


B ) U2OS cells were synchronized with a single thymidine block , released into G2 and damaged with 0 . 5 μM Adriamycin for one hour . After a 16 hour G2 arrest cells were harvested for western blot analysis and analyzed for Tlk2 protein levels .

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B) U2OS cells were synchronized with a single thymidine block, released into G2 and damaged with 0.5 μM Adriamycin for one hour. After a 16 hour G2 arrest cells were harvested for western blot analysis and analyzed for Tlk2 protein levels.


C ) Live cell imaging of thymidine synchronized unperturbed or damaged G2 cells . Cumulative percentage of cells entering mitosis were scored and plotted .

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C) Live cell imaging of thymidine synchronized unperturbed or damaged G2 cells. Cumulative percentage of cells entering mitosis were scored and plotted.


D ) U2OS cells were transfected with either a control siRNA or Tlk2 siRNA # 3 , synchronized and damaged in G2 . Cells were either harvested or treated with caffeine for 8 hours before harvest and cell cycle distribution was analyzed by FACS . Percentages of cells in each quadrant are indicated .

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D) U2OS cells were transfected with either a control siRNA or Tlk2 siRNA #3, synchronized and damaged in G2. Cells were either harvested or treated with caffeine for 8 hours before harvest and cell cycle distribution was analyzed by FACS. Percentages of cells in each quadrant are indicated.


E ) Tlk2Δ cells were generated using CRISPR / Cas9 genome editing . Cells were synchronized in G2 by thymidine release , damaged with 0 . 5 μM Adriamycin for one hour . After a 16 - hour G2 arrest cells were induced to recover by addition of caffeine for 8 hours and analyzed by FACS . Error bars represent SD , n = 4 . Statistical significance was tested using a paired two - tailed t - test ( NS for P > 0 . 05 , * for P ≤ 0 . 05 , * * * for P ≤ 0 . 001 ) .

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E) Tlk2Δ cells were generated using CRISPR/Cas9 genome editing. Cells were synchronized in G2 by thymidine release, damaged with 0.5 μM Adriamycin for one hour. After a 16-hour G2 arrest cells were induced to recover by addition of caffeine for 8 hours and analyzed by FACS. Error bars represent SD, n=4. Statistical significance was tested using a paired two-tailed t-test (NS for P>0.05, * for P≤0.05, *** for P≤0.001).


F ) U2TR cells stably expressing Tlk2 siRNA # 3 - insensitive tetracycline - inducible FLAG - Tlk2 - wt or FLAG - Tlk2 - D613A were thymidine synchronized and damaged in G2 . Tetracycline was present form the start of the experiment where indicated .

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F) U2TR cells stably expressing Tlk2 siRNA #3-insensitive tetracycline-inducible FLAG-Tlk2-wt or FLAG-Tlk2-D613A were thymidine synchronized and damaged in G2. Tetracycline was present form the start of the experiment where indicated.


G ) Caffeine induced recovery assay of cell lines shown in 2F . Error bars represent SD , n = 3 . Statistical significance was tested using a paired two - tailed t - test ( NS for P > 0 . 05 , * for P ≤ 0 . 05 , * * for P ≤ 0 . 01 , * * * for P ≤ 0 . 001 ) .

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G) Caffeine induced recovery assay of cell lines shown in 2F. Error bars represent SD, n=3. Statistical significance was tested using a paired two-tailed t-test (NS for P>0.05, * for P≤0.05, ** for P≤0.01, *** for P≤0.001).


A ) U2OS cells were grown on glass coverslips and synchronized in G2 after thymidine release . Cells were fixed at the indicated times after adriamycin - induced DNA damage and stained for DAPI and γH2AX . Scale bar indicates 10 μm . B ) Quantification of cells in 3A . Each dot represents the total signal of γH2AX in the nucleus of a single cell . Bars indicate the mean and error bars indicate the SD of the data points in each condition . Whiskers represent 5 - 95 % of data points . Statistical significance was tested using an unpaired two - tailed t - test ( NS for P > 0 . 05 , * * * * for P ≤ 0 . 0001 ) .

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A) U2OS cells were grown on glass coverslips and synchronized in G2 after thymidine release. Cells were fixed at the indicated times after adriamycin-induced DNA damage and stained for DAPI and γH2AX. Scale bar indicates 10 μm. B) Quantification of cells in 3A. Each dot represents the total signal of γH2AX in the nucleus of a single cell. Bars indicate the mean and error bars indicate the SD of the data points in each condition. Whiskers represent 5-95% of data points. Statistical significance was tested using an unpaired two-tailed t-test (NS for P>0.05, **** for P≤0.0001).


C ) U2OS cells were synchronized in G2 and harvested at the indicated times after induction of adriamycin - induced DNA damage with or without Tlk2 - depletion . Samples were analyzed by western blotting for the indicated proteins .

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C) U2OS cells were synchronized in G2 and harvested at the indicated times after induction of adriamycin-induced DNA damage with or without Tlk2-depletion. Samples were analyzed by western blotting for the indicated proteins.


D ) U2OS cells were synchronized in G2 and damaged with 6 Gy of IR . Samples were taken at the indicated times after induction of damage with or without Tlk2 - depletion . Samples were analyzed by western blot for the indicated proteins . Band signal intensity for Asf1A and Asf1B was measured and corrected for Ponceau S staining .

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D) U2OS cells were synchronized in G2 and damaged with 6 Gy of IR. Samples were taken at the indicated times after induction of damage with or without Tlk2-depletion. Samples were analyzed by western blot for the indicated proteins. Band signal intensity for Asf1A and Asf1B was measured and corrected for Ponceau S staining.


E ) U2OS cells were transfected with the indicated siRNA ' s and treated as in 2A . Mitotic index was analyzed by FACS . Error bars represent SD , n = 3 . Statistical significance was tested using a paired two - tailed t - test ( NS for P > 0 . 05 , * for P ≤ 0 . 05 ) .

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E) U2OS cells were transfected with the indicated siRNA's and treated as in 2A. Mitotic index was analyzed by FACS. Error bars represent SD, n=3. Statistical significance was tested using a paired two-tailed t-test (NS for P>0.05, * for P≤0.05).


F ) U2OS cells were synchronized with a single thymidine block , released into G2 and damaged with 0 . 5 μM Adriamycin for one hour . After a 16 hour G2 arrest cells were harvested for western blot analysis and analyzed for the indicated protein levels . Lanes were all present on the same blot but pasted together for comparison as indicated by the lines .

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F) U2OS cells were synchronized with a single thymidine block, released into G2 and damaged with 0.5 μM Adriamycin for one hour. After a 16 hour G2 arrest cells were harvested for western blot analysis and analyzed for the indicated protein levels. Lanes were all present on the same blot but pasted together for comparison as indicated by the lines.


G ) Cells were synchronized in S - or G2 - phase by a single thymidine block . Damage was induced by 0 . 5 μM Adriamycin for one hour and cells were allowed to incubate for 16 hours afterwards . Samples were fixed at the indicated cell cycle stages and stained for Asf1A - pS166 . Nuclear fluorescence intensity was measured relative to DAPI . Whiskers represent 5 - 95 % of data points .

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G) Cells were synchronized in S- or G2-phase by a single thymidine block. Damage was induced by 0.5 μM Adriamycin for one hour and cells were allowed to incubate for 16 hours afterwards. Samples were fixed at the indicated cell cycle stages and stained for Asf1A-pS166. Nuclear fluorescence intensity was measured relative to DAPI. Whiskers represent 5-95% of data points.


H ) Cells were treated and analyzed as in 2E . Induction of FLAG - Asf1A - 4D was induced using doxycycline . Error bars represent SD , n = 5 . Statistical significance was tested using a paired two - tailed t - test ( NS for P > 0 . 05 , * for P ≤ 0 . 05 ) .

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H) Cells were treated and analyzed as in 2E. Induction of FLAG-Asf1A-4D was induced using doxycycline. Error bars represent SD, n=5. Statistical significance was tested using a paired two-tailed t-test (NS for P>0.05, * for P≤0.05).


I ) U2OS cells were synchronized in G2 by a single thymidine block and where indicated treated with 0 . 5 μM Adriamycin for one hour and after the indicated time points were pre - extracted and fixed . Cells were stained for total levels of Histone H3 and quantified relative to DAPI . Scale bar indicates 10 μm . Whiskers represent 5 - 95 % of data points .

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I) U2OS cells were synchronized in G2 by a single thymidine block and where indicated treated with 0.5 μM Adriamycin for one hour and after the indicated time points were pre-extracted and fixed. Cells were stained for total levels of Histone H3 and quantified relative to DAPI. Scale bar indicates 10 μm. Whiskers represent 5-95% of data points.


J ) FLAG - Asf1A - 4D inducible cells were treated as in 3I and expression was induced using doxycycline where indicated . Whiskers represent 5 - 95 % of data points . Statistical significance was tested using an unpaired two - tailed t - test ( * * * * for P ≤ 0 . 0001 ) .

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J) FLAG-Asf1A-4D inducible cells were treated as in 3I and expression was induced using doxycycline where indicated. Whiskers represent 5-95% of data points. Statistical significance was tested using an unpaired two-tailed t-test (**** for P≤0.0001).


A ) U2OS cells were treated as in 2B after which mRNA was isolated and analyzed by qPCR . Error bars represent SD , n = 3 . Statistical significance was tested using a paired two - tailed t - test ( NS for P > 0 . 05 , * for P ≤ 0 . 05 ) .

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A) U2OS cells were treated as in 2B after which mRNA was isolated and analyzed by qPCR. Error bars represent SD, n=3. Statistical significance was tested using a paired two-tailed t-test (NS for P>0.05, * for P≤0.05).


B ) Tlk2Δ 9 . 3 . 1 cells were treated and analyzed as in 3A . Error bars represent SD , n = 3 . Statistical significance was tested using a paired two - tailed t - test ( NS for P > 0 . 05 , * for P ≤ 0 . 05 , * * for P ≤ 0 . 01 ) .

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B) Tlk2Δ 9.3.1 cells were treated and analyzed as in 3A. Error bars represent SD, n=3. Statistical significance was tested using a paired two-tailed t-test (NS for P>0.05, * for P≤0.05, ** for P≤0.01).


C ) U2OS cells were synchronized in G2 and harvested or synchronized in G2 and harvested 16 hours after adriamycin - induced DNA damage . Samples were analyzed by western blot for the indicated proteins .

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C) U2OS cells were synchronized in G2 and harvested or synchronized in G2 and harvested 16 hours after adriamycin-induced DNA damage. Samples were analyzed by western blot for the indicated proteins.


D ) U2OS cells were transfected with the indicated siRNA ' s , synchronized in G2 and damaged with 6 Gy of IR . Cells were harvested 24 hours later and the mitotic index was analyzed by FACS . Error bars represent SD , n = 3 . Statistical significance was tested using a paired two - tailed t - test ( NS for P > 0 . 05 , * * for P ≤ 0 . 01 ) .

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D) U2OS cells were transfected with the indicated siRNA's, synchronized in G2 and damaged with 6 Gy of IR. Cells were harvested 24 hours later and the mitotic index was analyzed by FACS. Error bars represent SD, n=3. Statistical significance was tested using a paired two-tailed t-test (NS for P>0.05, ** for P≤0.01).


F ) U2OS cells were transfected with the indicated siRNA ' s , arrested in G2 with Adriamycin and induced to recover by addition of caffeine . Mitotic index was analyzed by FACS . Error bars represent SD , n = 3 . Statistical significance was tested using a paired two - tailed t - test ( NS for P > 0 . 05 , * * for P ≤ 0 . 01 ) .

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F) U2OS cells were transfected with the indicated siRNA's, arrested in G2 with Adriamycin and induced to recover by addition of caffeine. Mitotic index was analyzed by FACS. Error bars represent SD, n=3. Statistical significance was tested using a paired two-tailed t-test (NS for P>0.05, ** for P≤0.01).


G ) U2OS cells were synchronized in G2 and harvested 16 hours after adriamycin - induced DNA damage . Samples were analyzed by western blot for the indicated proteins .

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G) U2OS cells were synchronized in G2 and harvested 16 hours after adriamycin-induced DNA damage. Samples were analyzed by western blot for the indicated proteins.


H ) U2TR cells stably expressing Tlk2 siRNA # 3 - insensitive tetracycline - inducible FLAG - Tlk2 - wt were thymidine synchronized , damaged in G2 and harvested 24 hours afterwards . Tetracycline was either absent ( off ) , added from the start of the experiment ( early ) or for 16 hours after induction of DNA damage ( late ) .

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H) U2TR cells stably expressing Tlk2 siRNA #3-insensitive tetracycline-inducible FLAG-Tlk2-wt were thymidine synchronized, damaged in G2 and harvested 24 hours afterwards. Tetracycline was either absent (off), added from the start of the experiment (early) or for 16 hours after induction of DNA damage (late).


I ) Cells were treated as in 5G and recovery was induced by addition of caffeine for 8 hours . Mitotic index was analyzed by FACS and normalized to the control . Error bars represent SD of three independent experiments . Error bars represent SD , n = 3 . Statistical significance was tested using a paired two - tailed t - test ( NS for P > 0 . 05 , * * for P ≤ 0 . 01 ) .

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I) Cells were treated as in 5G and recovery was induced by addition of caffeine for 8 hours. Mitotic index was analyzed by FACS and normalized to the control. Error bars represent SD of three independent experiments. Error bars represent SD, n=3. Statistical significance was tested using a paired two-tailed t-test (NS for P>0.05, ** for P≤0.01).


B . Left , representative images of Western blot for ARID1A expression in lysates extracted from the cortex of Arid1afl / + ( WT ) , cHet , and cKO mice using the indicated antibodies . Right , quantification of protein levels normalized to β - actin ( n = 3 mice per group ) .

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B. Left, representative images of Western blot for ARID1A expression in lysates extracted from the cortex of Arid1afl/+(WT), cHet, and cKO mice using the indicated antibodies. Right, quantification of protein levels normalized to β-actin (n = 3 mice per group).


C . Left , representative images of Western blot for ARID1A expression in lysates extracted from the hippocampi of Arid1afl / + , cHet , and cKO mice . Right , quantification of protein levels which were normalized to β - actin ( n = 4 mice per group ) .

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C. Left, representative images of Western blot for ARID1A expression in lysates extracted from the hippocampi of Arid1afl/+, cHet, and cKO mice. Right, quantification of protein levels which were normalized to β-actin (n = 4 mice per group).


D . Immunostaining against ARID1A ( red ) in the dentate gyrus ( DG ) , cortex , CA1 , and CA3 regions of Arid1afl / + and cHet mice .

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D. Immunostaining against ARID1A (red) in the dentate gyrus (DG), cortex, CA1, and CA3 regions of Arid1afl/+and cHet mice.


E . cHet mice spent more time reaching the platform during 5 - day training in Morris water maze assay compared to the control group mice . Arid1afl / + ( n = 15 mice ) , cHet ( n = 13 mice ) .

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E. cHet mice spent more time reaching the platform during 5-day training in Morris water maze assay compared to the control group mice. Arid1afl/+ (n = 15 mice), cHet (n = 13 mice).


F . In the probe trial , cHet mice displayed spatial learning disabilities compared with Arid1afl / + mice . Arid1afl / + ( n = 15 mice ) , cHet ( n = 13 mice ) .

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F. In the probe trial, cHet mice displayed spatial learning disabilities compared with Arid1afl/+ mice. Arid1afl/+ (n = 15 mice), cHet (n = 13 mice).


G . cHet mice undertook fewer platform crossings than Arid1afl / + mice in the Morris water maze assay . Arid1afl / + ( n = 15 mice ) , cHet ( n = 13 mice ) .

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G. cHet mice undertook fewer platform crossings than Arid1afl/+ mice in the Morris water maze assay. Arid1afl/+ (n = 15 mice), cHet (n = 13 mice).


H - J . cHet mice were evaluated using the Barnes maze test . During the training phase , the cHet mice ( n = 12 mice ) failed to diminish the latency of the first entrance to the hiding box ( H ) , indicating that Arid1a haploinsufficiency results in impaired spatial memory . Probe trials demonstrated that cHet mice also had spatial memory deficits ( I ) , as they visited the target hole less often than the Arid1afl / + mice ( n = 12 mice ) ( J ) .

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H-J. cHet mice were evaluated using the Barnes maze test. During the training phase, the cHet mice (n = 12 mice) failed to diminish the latency of the first entrance to the hiding box (H), indicating that Arid1a haploinsufficiency results in impaired spatial memory. Probe trials demonstrated that cHet mice also had spatial memory deficits (I), as they visited the target hole less often than the Arid1afl/+ mice (n = 12 mice) (J).


A . Representative traces of spontaneous miniature excitatory postsynaptic currents ( mEPSCs ) in CA1 hippocampal acute slices from Arid1afl / + and cHet mice . B . Quantification of amplitude ( B , left ) and frequency ( B , right ) spontaneous miniature excitatory postsynaptic currents ( mEPSCs ) in Arid1afl / + and cHet hippocampal CA1 neurons . Arid1afl / + ( 17 neurons from n = 3 mice ) and cHet ( 15 neurons from n = 4 mice ) .

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A. Representative traces of spontaneous miniature excitatory postsynaptic currents (mEPSCs) in CA1 hippocampal acute slices from Arid1afl/+ and cHet mice. B. Quantification of amplitude (B, left) and frequency (B, right) spontaneous miniature excitatory postsynaptic currents (mEPSCs) in Arid1afl/+ and cHet hippocampal CA1 neurons. Arid1afl/+ (17 neurons from n =3 mice) and cHet (15 neurons from n = 4 mice).


C . Representative recording of the paired - pulse ratio at the 50 - ms interpulse interval ms from slices prepared from Arid1afl / + ( n = 4 ) and cHet ( n = 4 ) mice . D . Paired - pulse facilitation ( PPF ) studies across different interpulse intervals ( 20 ms , 40 ms , 50 ms , 100 ms , 200 ms , 300ms , and 500 ms ) revealed a significant difference in paired - pulse ratios at 50 - ms intervals .

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C. Representative recording of the paired-pulse ratio at the 50-ms interpulse interval ms from slices prepared from Arid1afl/+ (n = 4) and cHet (n = 4) mice. D. Paired-pulse facilitation (PPF) studies across different interpulse intervals (20 ms, 40 ms, 50 ms, 100 ms, 200 ms, 300ms, and 500 ms) revealed a significant difference in paired-pulse ratios at 50-ms intervals.


E . Summary plots showing the time course of long - term potentiation ( LTP ) induced by frequency stimulation in the CA1 region from Arid1afl / + or cHet mice . Field excitatory postsynaptic potential ( fEPSP ) traces before ( black ) and after ( black or red ) are shown in the inset above . F . Average amplitude of LTP measured at 55 - 60 min postinduction ( n = 8 slices from 4 mice per group ; 10 fEPSP slope ( % ) values were collected from 1 slice ) .

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E. Summary plots showing the time course of long-term potentiation (LTP) induced by frequency stimulation in the CA1 region from Arid1afl/+or cHet mice. Field excitatory postsynaptic potential (fEPSP) traces before (black) and after (black or red) are shown in the inset above. F. Average amplitude of LTP measured at 55-60 min postinduction (n = 8 slices from 4 mice per group; 10 fEPSP slope (%) values were collected from 1 slice).


G . Representative Western blot images for the expression of several LTP - related proteins in the Arid1afl / + and cHet hippocampi . H . Quantification of the expression of each protein normalized to the β - actin level .

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G. Representative Western blot images for the expression of several LTP-related proteins in the Arid1afl/+ and cHet hippocampi. H. Quantification of the expression of each protein normalized to the β-actin level.


A . Golgi - stained coronal section in the hippocampal CA1 region of 2 - month - old Arid1afl / + and cHet mice . Scale bar , 100µm . B - D . Partial loss of Arid1a significantly decreased dendritic length ( B ) , dendritic nodes ( C ) , and dendritic ends ( D ) in cHet neurons .

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A. Golgi-stained coronal section in the hippocampal CA1 region of 2-month-old Arid1afl/+ and cHet mice. Scale bar, 100µm. B-D. Partial loss of Arid1a significantly decreased dendritic length (B), dendritic nodes (C), and dendritic ends (D) in cHet neurons. E


A . Left , Western blot analysis of H3K27ac in forebrain tissues of Arid1afl / + and cHet mice . Right , relative quantitation of the intensity of H3K27ac normalized to the H3 level ( n = 3 mice per group ) .

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A. Left, Western blot analysis of H3K27ac in forebrain tissues of Arid1afl/+and cHet mice. Right, relative quantitation of the intensity of H3K27ac normalized to the H3 level (n=3 mice per group).


B . Left , Western blot analysis of H3K27ac in forebrain tissues of vehicle - or acetate - treated cHet mice . Right , relative quantitation of the intensity of H3K27ac which was normalized to the H3 level ( n = 4 mice per group ) .

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B. Left, Western blot analysis of H3K27ac in forebrain tissues of vehicle- or acetate-treated cHet mice. Right, relative quantitation of the intensity of H3K27ac which was normalized to the H3 level (n=4 mice per group).


D . Immunofluorescence ( IF ) staining of H3K27ac ( green ) and Vgult1 ( red ) in forebrain tissues of Arid1afl / + and cHet mice , respectively . IF staining was performed on 40 - μm thick floating sections . Relative fluorescence intensities of H3K27ac decreased upon the haploinsufficiency of Arid1a in the cortex . Scale Bar , 20μm . Arid1afl / + ( n = 7 ) and cHet ( n = 4 ) mice .

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D. Immunofluorescence (IF) staining of H3K27ac(green) and Vgult1(red) in forebrain tissues of Arid1afl/+ and cHet mice, respectively. IF staining was performed on 40-μm thick floating sections. Relative fluorescence intensities of H3K27ac decreased upon the haploinsufficiency of Arid1a in the cortex. Scale Bar, 20μm. Arid1afl/+ (n = 7) and cHet (n = 4) mice.


E . Immunofluorescence staining of H3K27ac ( green ) and Vgult1 ( red ) in cortex tissues of vehicle - or acetate - treated cHet mice , respectively . IF staining was performed on 40 - μm thick floating sections . Relative fluorescence intensities of H3K27ac increased after treatment with acetate in the cortex of cHet mice . Scale Bar , 20μm ( n = 3 mice per group ) .

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E. Immunofluorescence staining of H3K27ac (green) and Vgult1 (red) in cortex tissues of vehicle- or acetate-treated cHet mice, respectively. IF staining was performed on 40-μm thick floating sections. Relative fluorescence intensities of H3K27ac increased after treatment with acetate in the cortex of cHet mice. Scale Bar, 20μm (n=3 mice per group).


F - H . Morris water maze test of Arid1afl / + and cHet mice treated with vehicle or acetate . Acquisition task , the latency to find the platform was used to assess learning ability . Latency to the platform was measured during training trials ( F ) . Latency to the platform was measured during probe test ( G ) . ( H ) Retention task , the times in the target crossings were used to assess memory retention . The times in the target crossings were measured in probe tests , n = 10 - 15 mice per group .

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F-H. Morris water maze test of Arid1afl/+ and cHet mice treated with vehicle or acetate. Acquisition task, the latency to find the platform was used to assess learning ability. Latency to the platform was measured during training trials (F). Latency to the platform was measured during probe test (G). (H) Retention task, the times in the target crossings were used to assess memory retention. The times in the target crossings were measured in probe tests, n = 10-15 mice per group.


I - K . Barnes maze test of Arid1afl / + and cHet mice treated with vehicle or acetate . ( I ) During the training phase , the time to find the target in Arid1afl / + and cHet mice that were treated with vehicle or acetate for 28 days . ( J ) In probe test trials , the time of latency to the platform to find the target in Arid1afl / + and cHet mice treated with vehicle or acetate . ( K ) In probe trials , the numbers of target crossings of Arid1afl / + and cHet mice treated with vehicle or acetate ( n = 12 mice )

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I-K. Barnes maze test of Arid1afl/+ and cHet mice treated with vehicle or acetate. (I) During the training phase, the time to find the target in Arid1afl/+ and cHet mice that were treated with vehicle or acetate for 28 days. (J) In probe test trials, the time of latency to the platform to find the target in Arid1afl/+ and cHet mice treated with vehicle or acetate. (K) In probe trials, the numbers of target crossings of Arid1afl/+ and cHet mice treated with vehicle or acetate (n = 12 mice)


E . Representative images of secondary branches of apical dendrites of vehicle - or acetate - treated cHet CA1 pyramidal neurons stained by the Golgi - Cox method ( at least 20 neurons from 4 mice for each group ) . Scale bar , 5 μm . F . Spine density per 10 μm was measured on secondary dendrites in ( E ) .

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E. Representative images of secondary branches of apical dendrites of vehicle- or acetate-treated cHet CA1 pyramidal neurons stained by the Golgi-Cox method (at least 20 neurons from 4 mice for each group). Scale bar, 5 μm. F. Spine density per 10 μm was measured on secondary dendrites in (E).


A . Representative image of vehicle - and acetate - treated neurons derived from WT or ARID1A KO hESCs on day 40 of neural differentiation Scale Bar , 20μm B . Quantification of total neurite length of hESC - derived neurons after treatment with acetate or vehicle . n = at least 34 neurons for each group from 3 independent experiments .

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A. Representative image of vehicle- and acetate-treated neurons derived from WT or ARID1A KO hESCs on day 40 of neural differentiation Scale Bar, 20μm B. Quantification of total neurite length of hESC-derived neurons after treatment with acetate or vehicle. n = at least 34 neurons for each group from 3 independent experiments. C


D . Representative images of dendrites ( MAP2 , green ) showing localization of foci of the pre - and postsynaptic protein complexes , synaptophysin ( red ) , and PSD - 95 ( white ) protein in control , acetate treatment of WT and ARID1A KO hESC - derived neurons on day 55 of neural differentiation . Scale Bar , 5μm .

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D. Representative images of dendrites (MAP2, green) showing localization of foci of the pre- and postsynaptic protein complexes, synaptophysin (red), and PSD-95 (white) protein in control, acetate treatment of WT and ARID1A KO hESC-derived neurons on day 55 of neural differentiation. Scale Bar, 5μm.


E , F . Quantification of the spine numbers from synaptophysin ( E ) and PSD - 95 protein ( F ) stained secondary dendrites of neurons on day 55 of neural differentiation ( Data are representative of three independent experiments . )

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E, F. Quantification of the spine numbers from synaptophysin (E) and PSD-95 protein (F) stained secondary dendrites of neurons on day 55 of neural differentiation (Data are representative of three independent experiments.)


C Genomic PCR analysis indicating the existence of MpFGMYB in both male [ Y ] and female [ X ] genomes of M . polymorpha . Two biological replicates were analyzed . Constitutively expressed MpEF1α was used as a control .

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C Genomic PCR analysis indicating the existence of MpFGMYB in both male [Y] and female [X] genomes of M. polymorpha. Two biological replicates were analyzed. Constitutively expressed MpEF1α was used as a control.


D Real - time RT - PCR analyses indicating preferential accumulation of MpFGMYB transcripts in female sexual organs and the sporophytes . MpEF1α was used for normalization . Measurements of six biological replicates for thalli and sporophyte , and three biological replicates for gametangiophore are plotted . Bars represent mean ± SD . Symbols above the bars indicate grouping by p < 0 . 05 in a Tukey - Kramer test .

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D Real-time RT-PCR analyses indicating preferential accumulation of MpFGMYB transcripts in female sexual organs and the sporophytes. MpEF1α was used for normalization. Measurements of six biological replicates for thalli and sporophyte, and three biological replicates for gametangiophore are plotted. Bars represent mean ± SD. Symbols above the bars indicate grouping by p <0.05 in a Tukey-Kramer test.


E A transcriptional reporter with 5 ′ - and 3 ′ - flanking sequences revealed transcription of MpFGMYB throughout mature archegonia . Scale bar , 10 µm . Magenta , chlorophyll autofluorescence ; green , Citrine fluorescence .

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E A transcriptional reporter with 5′- and 3′-flanking sequences revealed transcription of MpFGMYB throughout mature archegonia. Scale bar, 10 µm. Magenta, chlorophyll autofluorescence; green, Citrine fluorescence.


F MpFGMYB - Citrine fusion proteins expressed using the 5 ′ - and 3 ′ - flanking sequences rescued the Mpfgmybge - 1 mutant and accumulated in the nuclei of the egg and the ventral canal cell ( VCC ) Scale bar , 10 µm . Magenta , chlorophyll autofluorescence ; green , Citrine fluorescence .

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F MpFGMYB-Citrine fusion proteins expressed using the 5′- and 3′-flanking sequences rescued the Mpfgmybge-1 mutant and accumulated in the nuclei of the egg and the ventral canal cell (VCC) Scale bar, 10 µm. Magenta, chlorophyll autofluorescence; green, Citrine fluorescence.


G A transcriptional AtMYB64 reporter ( AtMYB64 - NYG ) is specifically expressed in all four cell types of the A . thaliana embryo sac Scale bar : 25 µm . Green , YFP fluorescence ; white , cell walls .

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G A transcriptional AtMYB64 reporter (AtMYB64-NYG) is specifically expressed in all four cell types of the A. thaliana embryo sac Scale bar: 25 µm. Green, YFP fluorescence; white, cell walls.


H Expression of AtMYB64 - Citrine fusion proteins under the AtMYB64 promoter was detected in the central cells ( CC ) and egg cells ( EC ) of the A . thaliana embryo sac Scale bar : 25 µm . Green , Citrine fluorescence ; white , cell walls .

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H Expression of AtMYB64-Citrine fusion proteins under the AtMYB64 promoter was detected in the central cells (CC) and egg cells (EC) of the A. thaliana embryo sac Scale bar: 25 µm. Green, Citrine fluorescence; white, cell walls.


B Diagnosis of genetic sex using Y - and X - chromosome - linked rbm27 and rhf73 markers , respectively . Two biological replicates were analyzed for each genotype .

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B Diagnosis of genetic sex using Y- and X-chromosome-linked rbm27 and rhf73 markers, respectively. Two biological replicates were analyzed for each genotype.


C - L Gametangiophore morphology ( C - G ) and gamete development ( H - L ) of wild - type and mutant plants . Scale bars , 5 mm ( C - G ) , 10 µm ( H and J ) , 100 µm ( I , K , and L ) .

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C-L Gametangiophore morphology (C-G) and gamete development (H-L) of wild-type and mutant plants. Scale bars, 5 mm (C-G), 10 µm (H and J), 100 µm (I, K, and L).


A - C DAPI - staining visualization of sperm formation in wild - type male ( A ) , Mpfgmyb [ X ] ( B ) , and Mpfgmyb [ Y ] ( C ) plants . Note that background DAPI staining visualizes flagella ( arrows ) in addition to nuclei ( arrowheads ) . ( B ′ ) is an enlarged image of the boxed region in ( B ) , visualizing an incompletely condensed nucleus . Scale bars , 5 µm .

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A-C DAPI-staining visualization of sperm formation in wild-type male (A), Mpfgmyb [X] (B), and Mpfgmyb [Y] (C) plants. Note that background DAPI staining visualizes flagella (arrows) in addition to nuclei (arrowheads). (B′) is an enlarged image of the boxed region in (B), visualizing an incompletely condensed nucleus. Scale bars, 5 µm.


D RT - PCR analysis indicating acquisition of male - like autosomal gene expression patterns in Mpfgmyb [ X ] antheridiophores . Two independent Mpfgmyb [ X ] mutant alleles were analyzed .

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D RT-PCR analysis indicating acquisition of male-like autosomal gene expression patterns in Mpfgmyb [X] antheridiophores. Two independent Mpfgmyb [X] mutant alleles were analyzed.


E , F TEM analyses visualizing the abnormal arrangement of axonemal microtubules in Mpfgmyb [ X ] sperm ( F ) , as compared with those of wild - type males ( E ) . Scale bars , 100 nm .

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E, F TEM analyses visualizing the abnormal arrangement of axonemal microtubules in Mpfgmyb [X] sperm (F), as compared with those of wild-type males (E). Scale bars, 100 nm.


A RNA - seq analysis showing male - specific accumulation of lncRNAs derived from the MpFGMYB 3 ′ region . Diagrams illustrate wild - type and mutant MpFGMYB / SUF loci . Folded lines with a ∆ symbol indicate a deletion .

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A RNA-seq analysis showing male-specific accumulation of lncRNAs derived from the MpFGMYB 3′ region. Diagrams illustrate wild-type and mutant MpFGMYB/SUF loci. Folded lines with a ∆ symbol indicate a deletion.


B RT - PCR analysis of wild - type and genetically male suf mutants revealed loss of SUF expression and gain of MpFGMYB expression after induction of reproductive growth by far - red irradiation . Two independent suf mutant alleles were analyzed . The SUF primer pair used here flanked an intron and the duplicated bands of SUF likely represent spliced and unspliced forms .

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B RT-PCR analysis of wild-type and genetically male suf mutants revealed loss of SUF expression and gain of MpFGMYB expression after induction of reproductive growth by far-red irradiation. Two independent suf mutant alleles were analyzed. The SUF primer pair used here flanked an intron and the duplicated bands of SUF likely represent spliced and unspliced forms.


C - H Gametangiophore morphology ( C , E and G ) and gametangium development ( D , F and H ) of plants with the designated genotypes . Scale bars , 1 mm ( C , E and G ) , 20 µm ( D ) , 50 µm ( F ) , 100 µm ( H ) .

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C-H Gametangiophore morphology (C, E and G) and gametangium development (D, F and H) of plants with the designated genotypes. Scale bars, 1 mm (C, E and G), 20 µm (D), 50 µm (F), 100 µm (H).


A gametangiophore of MpEF1pro : SUF / suf - 30ge [ Y ] , indicating the inability of transgenic SUF overexpression in rescuing the feminization phenotype of sufge [ Y ] . Scale bar , 2mm .

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A gametangiophore of MpEF1pro:SUF/suf-30ge [Y], indicating the inability of transgenic SUF overexpression in rescuing the feminization phenotype of sufge [Y]. Scale bar, 2mm.


B Real - time RT - PCR analyses confirming SUF transcript accumulation in SUF - overexpressing lines . Constitutively expressed MpEF1α was used as control . Measurements of six biological replicates for WT [ Y ] , and three biological replicates for each SUF - overexpressing line are plotted . Data information : bars represent mean ± SD . Asterisks indicate significant differences from WT [ Y ] ( B ) ( p < 0 . 05 , two - tailed Student ' s t - test ) .

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B Real-time RT-PCR analyses confirming SUF transcript accumulation in SUF-overexpressing lines. Constitutively expressed MpEF1α was used as control. Measurements of six biological replicates for WT [Y], and three biological replicates for each SUF-overexpressing line are plotted. Data information: bars represent mean ± SD. Asterisks indicate significant differences from WT [Y] (B) (p <0.05, two-tailed Student's t-test).


D Real - time RT - PCR measurement of MpFGMYB transcript levels . Three biological replicates are analyzed for each line . Data information bars represent mean ± SD . Asterisks indicate significant differences from WT [ X ] ( D ) ( p < 0 . 05 , two - tailed Student ' s t - test ) .

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D Real-time RT-PCR measurement of MpFGMYB transcript levels. Three biological replicates are analyzed for each line. Data information bars represent mean ± SD. Asterisks indicate significant differences from WT [X] (D) (p <0.05, two-tailed Student's t-test).


E - H Gametangiophores ( E , G ) and gametangia ( F , H ) of gMpFGMYB - S [ Y ] ( E , F ) and gMpFGMYB - L [ Y ] ( G , H ) . Scale bars , 1 mm ( E , G ) , 50 μm ( F ) , 100 μm ( H ) .

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E-H Gametangiophores (E, G) and gametangia (F, H) of gMpFGMYB-S [Y] (E, F) and gMpFGMYB-L [Y] (G, H). Scale bars, 1 mm (E, G), 50 μm (F), 100 μm (H).


A ) Transmission electron microscopy ( TEM ) of MoDCs infected with P . gingivalis for 2 , 12 and 24 hours ( left , middle and right panels ) . The sections show the intra - and extra - cellular contents of Cont . ( un - infected ) , Pg381 , Mfa1 + Pg and FimA + Pg infected MoDCs for the different time

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A) Transmission electron microscopy (TEM) of MoDCs infected with P. gingivalis for 2, 12 and 24 hours (left, middle and right panels). The sections show the intra-and extra-cellular contents of Cont. (un-infected), Pg381, Mfa1+Pg and FimA+Pg infected MoDCs for the different time points


B ) The figure shows the intracellular content of P . gingivalis strains after 24 hours of infection . MoDCs infected with Pg381 or isogenic mutants strains were lysed and the survived intracellular bacteria were re - suspended and maintained in anaerobic broth for 5 days . The data represents CFU within MoDCs harvested from three healthy individuals . The means ± standard deviation ( in triplicates ) were analyzed by One - way ANOVA of different groups and Tukey ' s test for multiple group comparisons within 3 different experiments ( * statistical significance at the p < 0 . 001 ) .

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B) The figure shows the intracellular content of P. gingivalis strains after 24 hours of infection. MoDCs infected with Pg381 or isogenic mutants strains were lysed and the survived intracellular bacteria were re-suspended and maintained in anaerobic broth for 5 days. The data represents CFU within MoDCs harvested from three healthy individuals. The means ±standard deviation (in triplicates) were analyzed by One-way ANOVA of different groups and Tukey's test for multiple group comparisons within 3 different experiments (* statistical significance at the p<0.001).


A ) DC - SIGN mRNA expression in P . gingivalis - infected MoDCs at 0 . 1 , 1 and 10 MOIs . The figure shows the gene expression after 12 hours of Pg381 and mutant strains infections . The target gene ( DC - SIGN ) was normalized using the endogenous control GAPDH ( ΔCt ) and fold regulations were calculated using 2 - ( ΔΔCt ) method . The statistical analysis was performed using the t - test , which accounts for the clustering of infected and un - infected controls within 3 different experiments ( * p < 0 . 001 ) .

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A) DC-SIGN mRNA expression in P. gingivalis-infected MoDCs at 0.1, 1 and 10 MOIs. The figure shows the gene expression after 12 hours of Pg381 and mutant strains infections. The target gene (DC-SIGN) was normalized using the endogenous control GAPDH (ΔCt) and fold regulations were calculated using 2-(ΔΔCt) method. The statistical analysis was performed using the t-test, which accounts for the clustering of infected and un-infected controls within 3 different experiments (* p<0.001).


B ) Immuno - electron microscopy of un - infected MoDCs ( Cont . ) ( upper panel ) , MoDCs infected with Pg381 ( middle panel ) and Mfa1 + Pg mutants ( lower panel ) . Gold particles ( marked with red rings ) for positive DC - SIGN were detected in the cell membrane and cytoplasm of cells infected with Mfa1 + Pg strains . Minimal positive staining for DC - SIGN was detected in the membranes of MoDCs infected with Pg381 , while no cytoplasmic gold labeling was detected in these cells .

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B) Immuno-electron microscopy of un-infected MoDCs (Cont.) (upper panel), MoDCs infected with Pg381 (middle panel) and Mfa1+Pg mutants (lower panel). Gold particles (marked with red rings) for positive DC-SIGN were detected in the cell membrane and cytoplasm of cells infected with Mfa1+Pg strains. Minimal positive staining for DC-SIGN was detected in the membranes of MoDCs infected with Pg381, while no cytoplasmic gold labeling was detected in these cells.


C ) Flow cytometry analysis of surface DC - SIGN in human MoDCs after infection with Pg381 , Mfa1 + Pg and FimA + Pg . The analysis of the intensity used Kruskal - Wallis test analysis of different groups and Dunn ' s test for multiple comparisons 3 different experiments ( * p < 0 . 01 ) .

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C) Flow cytometry analysis of surface DC-SIGN in human MoDCs after infection with Pg381, Mfa1+Pg and FimA+Pg. The analysis of the intensity used Kruskal-Wallis test analysis of different groups and Dunn's test for multiple comparisons 3 different experiments (* p<0.01).


A ) Pg381 and mutant strains count after 24 hr incubation with human MoDCs with / without Rapamycin treatment . The survived bacteria were measured after maintaining the lysed MoDCs suspension in anaerobic broth for 5 days . The plot represents the means ± standard deviation of CFU within MoDCs harvested from three healthy individuals ( * P < 0 . 001 ) . The analysis of readings used One - way AVOVA analysis of different groups and Tukey ' s test for multiple comparisons .

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A) Pg381 and mutant strains count after 24 hr incubation with human MoDCs with/without Rapamycin treatment. The survived bacteria were measured after maintaining the lysed MoDCs suspension in anaerobic broth for 5 days. The plot represents the means ±standard deviation of CFU within MoDCs harvested from three healthy individuals (* P<0.001). The analysis of readings used One-way AVOVA analysis of different groups and Tukey's test for multiple comparisons.


B ) Epifluorescence microscopy images of MoDCs treated with Rapamycin 1 hour after P . gingivalis infections . LC3 - II ( red - fluorescent dye ) and the bacterial strains ( green CFSE ) were studied in MoDCs 11 hours after Rapamycin treatment ( 12 hours after infections ) .

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B) Epifluorescence microscopy images of MoDCs treated with Rapamycin 1 hour after P. gingivalis infections. LC3-II (red-fluorescent dye) and the bacterial strains (green CFSE) were studied in MoDCs 11 hours after Rapamycin treatment (12 hours after infections).


C ) Quantifications of the fluorescent intensity of CFSE - labeled P . gingivalis and LC3 - II signals within infected MoDCs using NIS - Elements BR software . One - way ANOVA analysis was used to compare the means of intensity of different groups and Tukey ' s test for multiple comparisons ( * P < 0 . 001 ) .

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C) Quantifications of the fluorescent intensity of CFSE-labeled P. gingivalis and LC3-II signals within infected MoDCs using NIS-Elements BR software. One-way ANOVA analysis was used to compare the means of intensity of different groups and Tukey's test for multiple comparisons (* P<0.001).


A ) Survival of P . gingivalis strains within MoDCs after 6 , 12 , 24 and 48 hours . Blue lines show P . gingivalis strains survival within MoDCs and their survival in anaerobic condition in the absence of DCs are showed in black lines .

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A) Survival of P. gingivalis strains within MoDCs after 6, 12, 24 and 48 hours. Blue lines show P. gingivalis strains survival within MoDCs and their survival in anaerobic condition in the absence of DCs are showed in black lines.


The effect of rapamycin on Mfa1 + Pg , Pg381 and FimA + Pg survival within MoDCs are shown in figures B , C and D respectively . A three - factor repeated measures ANOVA using mixed models was used to test the effect of strain and rapamycin treatment over time on OD reading . The survival curves for the strains are showed in blue , while the effect of rapamycin treatments are in red . Bacterial survivals in the absence of MoDCs with and without rapamycin are plotted in grey and black , respectively . Statistical analysis showed that the strain by rapamycin treatment overtime interaction indicates the pattern of means in each strain ( Mfa1 + Pg , Pg381 and FimA + Pg ) between treated ( rapamycin ) and untreated were significantly different overtime ( p - value < 0 . = 001 ) .

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The effect of rapamycin on Mfa1+Pg, Pg381 and FimA+Pg survival within MoDCs are shown in figures B, C and D respectively. A three-factor repeated measures ANOVA using mixed models was used to test the effect of strain and rapamycin treatment over time on OD reading. The survival curves for the strains are showed in blue, while the effect of rapamycin treatments are in red. Bacterial survivals in the absence of MoDCs with and without rapamycin are plotted in grey and black, respectively. Statistical analysis showed that the strain by rapamycin treatment overtime interaction indicates the pattern of means in each strain (Mfa1+Pg, Pg381 and FimA+Pg) between treated (rapamycin) and untreated were significantly different overtime (p-value <0.=001).


A ) Epifluorescence microscopy images of MoDCs infected with Pg381 , Mfa1 + Pg , FimA + Pg and MFB strains after 12 hours . LC3 - II was detected in red - fluorescent ( Texas red ) dye and the bacterial strains were pre - labeled with CFSE ( green ) . Co - localization of P . gingivalis and LC3 - II showed in the right panels .

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A) Epifluorescence microscopy images of MoDCs infected with Pg381, Mfa1+Pg, FimA+Pg and MFB strains after 12 hours. LC3-II was detected in red-fluorescent (Texas red) dye and the bacterial strains were pre-labeled with CFSE (green). Co-localization of P. gingivalis and LC3-II showed in the right panels.


B ) Quantifications of the fluorescent intensity of LC3 - II within infected MoDCs using NIS - Elements BR software . One - way ANOVA analysis was used to compare the means of intensity of different groups and Tukey ' s test for multiple comparisons of three different experiments ( * p < 0 . 001 , # p < 0 . 01 ) .

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B) Quantifications of the fluorescent intensity of LC3-II within infected MoDCs using NIS-Elements BR software. One-way ANOVA analysis was used to compare the means of intensity of different groups and Tukey's test for multiple comparisons of three different experiments (* p<0.001, # p<0.01).


C ) Bacterial uptake by MoDCs was determined by CFSE fluorescent intensity ( * p < 0 . 001 , # p < 0 . 01 ) .

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C) Bacterial uptake by MoDCs was determined by CFSE fluorescent intensity (* p<0.001, # p<0.01).


A ) Immuno - fluorescence images of LC3 - II and P . gingivalis within MoDCs pre - treated with GP120 ( DC - SIGN blocker ) after 12 hours of infection . LC3 - II was detected in red - fluorescent dye and the bacterial strains were pre - labeled with green CFSE .

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A) Immuno-fluorescence images of LC3-II and P. gingivalis within MoDCs pre-treated with GP120 (DC-SIGN blocker) after 12 hours of infection. LC3-II was detected in red-fluorescent dye and the bacterial strains were pre-labeled with green CFSE.


B ) Fluorescent intensity of LC3 - II and P . gingivalis strains signal ( in triplicates ) were statistically analyzed ( * and # p < 0 . 001 ) .

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B) Fluorescent intensity of LC3-II and P. gingivalis strains signal (in triplicates) were statistically analyzed (* and # p<0.001).


C ) Flow cytometry of MoDCs treated with siRNA for DC - SIGN ( si - DC - SIGN ) after 12 hours . Left panels show the decrease of DC - SIGN in Cont . ( un - infected ) , Pg381 and Mfa1 + Pg - infected MoDCs .

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C) Flow cytometry of MoDCs treated with siRNA for DC-SIGN (si-DC-SIGN) after 12 hours. Left panels show the decrease of DC-SIGN in Cont. (un-infected), Pg381 and Mfa1+Pg-infected MoDCs.


D ) Epifluorescence microscopy images of MoDCs ( si - DC - SIGN ) infected with Pg381 and Mfa1 + Pg . LC3 - II was detected in red - fluorescent dye and the bacterial strains were pre - labeled with green CFSE .

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D) Epifluorescence microscopy images of MoDCs (si-DC-SIGN) infected with Pg381 and Mfa1+Pg. LC3-II was detected in red-fluorescent dye and the bacterial strains were pre-labeled with green CFSE.


E ) The figure shows the CFU counts of the P . gingivalis strains with MoDCs that lack DC - SIGN ( si - DC - SIGN ) . The analysis of readings used One - way ANOVA analysis of different groups and Tukey ' s test for multiple comparisons ( * p < 0 . 001 ) .

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E) The figure shows the CFU counts of the P. gingivalis strains with MoDCs that lack DC-SIGN (si-DC-SIGN). The analysis of readings used One-way ANOVA analysis of different groups and Tukey's test for multiple comparisons (* p<0.001).


A ) Western blot detection of LC3 in MoDCs infected with Pg381 and mutant strains for 12 hours . B ) The mean ± standard deviation of LC3 - II intensity represents three different experiments and the values were analyzed using Kruskal - Wallis test analysis of different groups and Dunn ' s test for multiple comparisons .

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A) Western blot detection of LC3 in MoDCs infected with Pg381 and mutant strains for 12 hours. B) The mean ±standard deviation of LC3-II intensity represents three different experiments and the values were analyzed using Kruskal-Wallis test analysis of different groups and Dunn's test for multiple comparisons.


C ) Blot shows the autophagy flux test after blocking the lysosomal fusion with Bafilomycin in MoDCs . Cells were treated with Bafilomycin for 4 hours at 4nM . D ) LC3 - II intensity in MoDCs infected with Pg381 strains with and without Bafilomycin . The analysis of the intensity of three different experiments used Kruskal - Wallis test analysis of different groups and Dunn ' s test for multiple comparisons .

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C) Blot shows the autophagy flux test after blocking the lysosomal fusion with Bafilomycin in MoDCs. Cells were treated with Bafilomycin for 4 hours at 4nM. D) LC3-II intensity in MoDCs infected with Pg381 strains with and without Bafilomycin. The analysis of the intensity of three different experiments used Kruskal-Wallis test analysis of different groups and Dunn's test for multiple comparisons.


A ) Flow cytometry of CD83 on MoDCs after incubation of TLR4 ligand ( E . coli LPS ) and TLR1 and 2 ligand ( Pam3csk4 ) for 4 hour .

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A) Flow cytometry of CD83 on MoDCs after incubation of TLR4 ligand (E. coli LPS) and TLR1 and 2 ligand (Pam3csk4) for 4 hour.


B ) Immuno - fluorescence images of LC3 - II ( red ) within MoDCs after incubation with TLR4 and TLR1 & amp ; amp ; 2 ligands ( E . coli LPS and Pam3csk4 )

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B) Immuno-fluorescence images of LC3-II (red) within MoDCs after incubation with TLR4 and TLR1&amp;2 ligands (E. coli LPS and Pam3csk4)


C ) The plot represents the means ± standard deviation of CFU within MoDCs harvested from three healthy individuals after 24 hours ( * * p < 0 . 001 ) .

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C) The plot represents the means ±standard deviation of CFU within MoDCs harvested from three healthy individuals after 24 hours (** p<0.001).


A ) Scanning electron microscopy ( SEM ) of MoDCs of the early interaction of MoDCs with Pg381 and Mfa1 + Pg ( upper panel 10000x and lower panel 25000x ) . B ) SEM for MoDCs interacting with Pg381 ( green stains for bacteria are computer generated ) .

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A) Scanning electron microscopy (SEM) of MoDCs of the early interaction of MoDCs with Pg381 and Mfa1+Pg (upper panel 10000x and lower panel 25000x). B) SEM for MoDCs interacting with Pg381 (green stains for bacteria are computer generated).


C ) Transmission electron microscopy ( TEM ) of autophagosome like structures within MoDCs infected with Pg381 , Mfa1 + Pg and FimA + Pg for 12 hours ( upper , middle and lower panels , respectively ) . The right and left sections show the different magnifications of randomly selected sections . Pg381 and FimA + Pg strains are mostly enclosed in the characteristics double - membrane intracellular vesicles ( Orange arrows ) . Contrary , Mfa1 + Pg escaped these autophagic ( double - membrane ) vesicles and enclosed within single membrane structures or freely occupy the cytoplasm ( Green arrows ) .

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C) Transmission electron microscopy (TEM) of autophagosome like structures within MoDCs infected with Pg381, Mfa1+Pg and FimA+Pg for 12 hours (upper, middle and lower panels, respectively). The right and left sections show the different magnifications of randomly selected sections. Pg381 and FimA+Pg strains are mostly enclosed in the characteristics double-membrane intracellular vesicles (Orange arrows). Contrary, Mfa1+Pg escaped these autophagic (double-membrane) vesicles and enclosed within single membrane structures or freely occupy the cytoplasm (Green arrows).


D ) The ratio of bacteria included in the double membrane were compared to total number of bacteria within MoDCs and plotted as percentage . Counting of the bacteria included in single or doubled membrane vesicles after 12 hours of infection . Each strain was counted in three randomly selected grids for each sample . The analysis of the bacterial counts used Kruskal - Wallis test of different groups and Dunn ' s test for multiple comparisons ( * p < 0 . 01 ) .

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D) The ratio of bacteria included in the double membrane were compared to total number of bacteria within MoDCs and plotted as percentage. Counting of the bacteria included in single or doubled membrane vesicles after 12 hours of infection. Each strain was counted in three randomly selected grids for each sample. The analysis of the bacterial counts used Kruskal-Wallis test of different groups and Dunn's test for multiple comparisons (*p<0.01).


A ) Epifluorescence microscopy images of MoDCs infected with pre - labeled bacteria ( green - CFSE ) . LAMP1 detected by transduction of red fluorescent protein ( RFP ) chimera using baculovirus transgenes to MoDCs .

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A) Epifluorescence microscopy images of MoDCs infected with pre-labeled bacteria (green-CFSE). LAMP1 detected by transduction of red fluorescent protein (RFP) chimera using baculovirus transgenes to MoDCs.


B ) The mean of fluorescent intensities ± standard deviation of LAMP1 were quantified in three different experiments ( * p < 0 . 001 ) .

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B) The mean of fluorescent intensities ±standard deviation of LAMP1 were quantified in three different experiments (* p<0.001).


C ) The upper scattered plot shows the co - localization of red and green signals in sections of MoDCs infected with Pg381 and mutants strains . The lower plot shows the average of Pearson ' s correlation measures of three randomly selected region of interest ( ROI ) within each field within 3 different experiments ( * p < 0 . 0001 ) ( Table 4 and 5 ) . All analysis of fluorescence intensity used One - way ANOVA analysis of different groups and Tukey ' s test for multiple comparisons .

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C) The upper scattered plot shows the co-localization of red and green signals in sections of MoDCs infected with Pg381 and mutants strains. The lower plot shows the average of Pearson's correlation measures of three randomly selected region of interest (ROI) within each field within 3 different experiments (* p<0.0001) (Table 4 and 5). All analysis of fluorescence intensity used One-way ANOVA analysis of different groups and Tukey's test for multiple comparisons.


A Representative Gli1ZF / DNA structure extrapolated from MD trajectories . Gli1ZF is shown as blue cartoon , residues involved in binding to DNA ( based on single point mutation study ) are shown as magenta sticks , and Zn ions as gray spheres .

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A Representative Gli1ZF/DNA structure extrapolated from MD trajectories. Gli1ZF is shown as blue cartoon, residues involved in binding to DNA (based on single point mutation study) are shown as magenta sticks, and Zn ions as gray spheres.


B Effect of Gli1ZF mutants on the binding affinity to DNA as predicted by in silico alanine scanning . The ∆ ∆ G was calculated along MD trajectories as the difference between the ∆ G of each Gli1ZF alanine mutant and Gli1ZF ‐ WT . Results are shown as ∆ ∆ G values in kcal / mol calculated by means of the MM ‐ PBSA methods ± SEM .

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B Effect of Gli1ZF mutants on the binding affinity to DNA as predicted by in silico alanine scanning. The ∆∆G was calculated along MD trajectories as the difference between the ∆G of each Gli1ZF alanine mutant and Gli1ZF‐WT. Results are shown as ∆∆G values in kcal/mol calculated by means of the MM‐PBSA methods ± SEM.


C Effect of Gli1ZF mutants on Gli1 ‐ dependent transcriptional activation . Luciferase assay was performed in HEK293T cells transfected with 12XGliBS ‐ Luc ( GliBS , Gli binding site ) , pRL ‐ TK Renilla ( normalization control ) , Flag ‐ Gli1 WT or the indicated Flag ‐ Gli1 mutants . Data show the mean ± SD of three independent experiments . * P 0 . 01 ; * * P 0 . 05 versus Gli1 WT . Western blot analysis of Flag ‐ Gli1 WT or the indicated Flag ‐ Gli1 mutant expression levels ( bottom panel ) .

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C Effect of Gli1ZF mutants on Gli1‐dependent transcriptional activation. Luciferase assay was performed in HEK293T cells transfected with 12XGliBS‐Luc (GliBS, Gli binding site), pRL‐TK Renilla (normalization control), Flag‐Gli1 WT or the indicated Flag‐Gli1 mutants. Data show the mean ± SD of three independent experiments. *P 0.01; **P 0.05 versus Gli1 WT. Western blot analysis of Flag‐Gli1 WT or the indicated Flag‐Gli1 mutant expression levels (bottom panel).


E Gli1 / DNA binding . Double ‐ stranded oligonucleotide containing the canonical GliBS sequence ( 5 ′ - TTGCCTACCTGGGTGGTCTCTCCACTT - 3 ′ ) or mutated GliBS sequence used as control ( 5 ′ - TTGCCTACCTCCCACTTCTCTCCACTT - 3 ′ ) was used as probe ( P ) in EMSA experiments . The assay was performed using recombinant GST ‐ Gli1ZF ‐ WT ( Gli1 zinc finger fragment : aa 242 - 424 ) , GST ‐ Gli1ZF ‐ K350A and GST ‐ Gli1ZF ‐ K340A . The graph on the right indicates ratio ( mean arbitrary units ± SD from three independent experiments ) of GST ‐ Gli1ZF ‐ WT or GST ‐ Gli1ZF mutants bound to the labeled GliBS probe / GliBS ‐ free probe normalized to the amount of GST ‐ Gli1ZF ‐ WT / DNA binding ( as described in ) . * P 0 . 05 versus Gli1 WT .

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E Gli1/DNA binding. Double‐stranded oligonucleotide containing the canonical GliBS sequence (5′-TTGCCTACCTGGGTGGTCTCTCCACTT-3′) or mutated GliBS sequence used as control (5′-TTGCCTACCTCCCACTTCTCTCCACTT-3′) was used as probe (P) in EMSA experiments. The assay was performed using recombinant GST‐Gli1ZF‐WT (Gli1 zinc finger fragment: aa 242-424), GST‐Gli1ZF‐K350A and GST‐Gli1ZF‐K340A. The graph on the right indicates ratio (mean arbitrary units ± SD from three independent experiments) of GST‐Gli1ZF‐WT or GST‐Gli1ZF mutants bound to the labeled GliBS probe/GliBS‐free probe normalized to the amount of GST‐Gli1ZF‐WT/DNA binding (as described in ). *P 0.05 versus Gli1 WT.


A Inhibition of Gli1 ‐ induced transcription in transfected HEK293T cells . HEK293T cells were transfected with 12XGliBS ‐ Luc and pRL ‐ TK Renilla ( normalization control ) plus control ( empty ) or Gli1 vector and treated with increasing concentrations of GlaB or GANT61 . Treatment time was 24 h , and control cells were treated with DMSO only .

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A Inhibition of Gli1‐induced transcription in transfected HEK293T cells. HEK293T cells were transfected with 12XGliBS‐Luc and pRL‐TK Renilla (normalization control) plus control (empty) or Gli1 vector and treated with increasing concentrations of GlaB or GANT61. Treatment time was 24 h, and control cells were treated with DMSO only.


B GlaB chemical structure with numbering scheme for NMR analysis .

B GlaB chemical structure with numbering scheme for NMR analysis.


C Inhibition of Gli1 ‐ induced transcription in transfected Smo − / − MEF cells . Smo − / − MEF cells were transfected with 12XGliBS ‐ Luc and pRL ‐ TK Renilla ( normalization control ) plus control ( empty ) or Gli1 vector and treated for 24 h with increasing concentrations of GlaB or DMSO only as control .

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C Inhibition of Gli1‐induced transcription in transfected Smo−/−MEF cells. Smo−/−MEF cells were transfected with 12XGliBS‐Luc and pRL‐TK Renilla (normalization control) plus control (empty) or Gli1 vector and treated for 24 h with increasing concentrations of GlaB or DMSO only as control.


D The graphs show the Hh target gene expression levels in Ptch1 − / − MEFs treated for 48 h with GlaB and DMSO as a control . mRNA levels were determined by quantitative real ‐ time PCR ( qRT - PCR ) normalized to endogenous control ( β2 ‐ microglobulin and HPRT ) . Pfkfb3 gene was used as a negative control .

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D The graphs show the Hh target gene expression levels in Ptch1−/−MEFs treated for 48 h with GlaB and DMSO as a control. mRNA levels were determined by quantitative real‐time PCR (qRT-PCR) normalized to endogenous control (β2‐microglobulin and HPRT). Pfkfb3 gene was used as a negative control.


F SuFu − / − MEFs were treated for 48 h with GlaB and DMSO as a control . Gli1 and Ptch1mRNA levels were determined by qRT - PCR normalized to β2 ‐ microglobulin and HPRT expression . Pfkfb3 gene was used as a negative control .

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F SuFu−/−MEFs were treated for 48 h with GlaB and DMSO as a control. Gli1 and Ptch1mRNA levels were determined by qRT-PCR normalized to β2‐microglobulin and HPRT expression. Pfkfb3 gene was used as a negative control.


Promoter occupancy of Gli1 is prevented by GlaB treatment . MEF WT cells were transfected with Flag ‐ tagged Gli1 or empty vectors , and chromatin immunoprecipitation ( ChIP ) was carried out . qRT - PCR was performed using primers encompassing the Gli ‐ BS of mouse Ptch1 promoter ( right , schematic representation ) . Results are indicated as fold difference , relative to empty ( pcDNA3 ) control .

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Promoter occupancy of Gli1 is prevented by GlaB treatment. MEF WT cells were transfected with Flag‐tagged Gli1 or empty vectors, and chromatin immunoprecipitation (ChIP) was carried out. qRT-PCR was performed using primers encompassing the Gli‐BS of mouse Ptch1 promoter (right, schematic representation). Results are indicated as fold difference, relative to empty (pcDNA3) control.


H Ptch1 mRNA expression levels ( left panel ) were determined by qRT - PCR in Daoy cells transfected with siRNA specific for Gli1 and Gli2 ( siGli1 / 2 ) or a non ‐ specific control siRNA ( siCtr ) and treated for 24 h with GlaB or DMSO as a control . ( right panel ) The graph shows Gli1 and Gli2mRNA expression levels determined by qRT - PCR in Daoy cells transfected with siGli1 / 2 or siCtr . Results are expressed as fold repression relative to control , and data were normalized to GAPDH and HPRT expression .

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H Ptch1 mRNA expression levels (left panel) were determined by qRT-PCR in Daoy cells transfected with siRNA specific for Gli1 and Gli2 (siGli1/2) or a non‐specific control siRNA (siCtr) and treated for 24 h with GlaB or DMSO as a control. (right panel) The graph shows Gli1 and Gli2mRNA expression levels determined by qRT-PCR in Daoy cells transfected with siGli1/2 or siCtr. Results are expressed as fold repression relative to control, and data were normalized to GAPDH and HPRT expression.


A The predicted binding mode of GlaB ( green sticks ) to Gli1ZF ( blue cartoons ) . Residues highlighted by the single ‐ point mutation study to impact on Gli1 binding to DNA are shown as blue sticks ; K340 and K350 of the GlaB binding site are colored magenta . GlaB protons highlighted by the NMR study are shown as orange spheres .

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A The predicted binding mode of GlaB (green sticks) to Gli1ZF (blue cartoons). Residues highlighted by the single‐point mutation study to impact on Gli1 binding to DNA are shown as blue sticks; K340 and K350 of the GlaB binding site are colored magenta. GlaB protons highlighted by the NMR study are shown as orange spheres.


B Detail of GlaB binding to Gli1ZF ‐ WT , Gli1ZF ‐ K340A , GLI1ZF ‐ K350A , GLI1ZF ‐ K340A / K350A mutants and respective theoretical affinity values . Gli1 is shown as blue transparent surface ; K340 and K350 are colored magenta . GlaB is shown as green sticks .

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B Detail of GlaB binding to Gli1ZF‐WT, Gli1ZF‐K340A, GLI1ZF‐K350A, GLI1ZF‐K340A/K350A mutants and respective theoretical affinity values. Gli1 is shown as blue transparent surface; K340 and K350 are colored magenta. GlaB is shown as green sticks.


C Inhibition of Gli1 / DNA binding by GlaB . EMSA using recombinant GST ‐ Gli1ZF ‐ WT or GST ‐ Gli1ZF ‐ K340A in the presence of different concentrations of GlaB or with DMSO only . The shifted complex is competed with a 50 × excess of cold probe . The graph on the right indicates ratio ( mean arbitrary units ± SD from three independent experiments ) of GST ‐ Gli1ZF ‐ WT or GST ‐ Gli1ZF ‐ K340A bound to the labeled GliBS probe / GliBS ‐ free probe normalized to the amount of GST ‐ Gli1ZF ‐ WT / DNA binding in absence of GlaB . * P 0 . 05 versus DMSO ; * * P 0 . 05 K340A + GlaB versus Gli1 WT + GlaB .

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C Inhibition of Gli1/DNA binding by GlaB. EMSA using recombinant GST‐Gli1ZF‐WT or GST‐Gli1ZF‐K340A in the presence of different concentrations of GlaB or with DMSO only. The shifted complex is competed with a 50× excess of cold probe. The graph on the right indicates ratio (mean arbitrary units ± SD from three independent experiments) of GST‐Gli1ZF‐WT or GST‐Gli1ZF‐K340A bound to the labeled GliBS probe/GliBS‐free probe normalized to the amount of GST‐Gli1ZF‐WT/DNA binding in absence of GlaB. *P 0.05 versus DMSO; **P 0.05 K340A + GlaB versus Gli1 WT + GlaB.


D HEK293T cells were transfected with 12XGliBS ‐ Luc and pRL ‐ TK Renilla ( normalization control ) plus control ( empty vector ) or Gli1 or Gli1K340A mutant and treated with increasing concentrations of GlaB . Treatment time was 24 h , and control cells were treated with DMSO only . Data show the mean ± SD of three independent experiments . * P 0 . 05 versus DMSO ; * * P 0 . 05 K340A versus Gli1 WT .

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D HEK293T cells were transfected with 12XGliBS‐Luc and pRL‐TK Renilla (normalization control) plus control (empty vector) or Gli1 or Gli1K340A mutant and treated with increasing concentrations of GlaB. Treatment time was 24 h, and control cells were treated with DMSO only. Data show the mean ± SD of three independent experiments. *P 0.05 versus DMSO; **P 0.05 K340A versus Gli1 WT.


A qRT - PCR shows Hh target mRNA expression levels determined in 6 ‐ day ‐ old mouse cerebellar progenitors after s . c . injections of GlaB .

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A qRT-PCR shows Hh target mRNA expression levels determined in 6‐day‐old mouse cerebellar progenitors after s.c. injections of GlaB.


B Representative EGLHE staining of 6 ‐ day ‐ old mousecerebellum after s . c . injections of GlaB ( upper panel ) ; immunohistochemistry shows Gli1 ( middle panel ) and Ki67 stainings ( lower panel ) . In all images , the scale bar represents 50 μm .

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B Representative EGLHE staining of 6‐day‐old mousecerebellum after s.c. injections of GlaB (upper panel); immunohistochemistry shows Gli1 (middle panel) and Ki67 stainings (lower panel). In all images, the scale bar represents 50 μm.


C The graph shows the percentage of EGL thickness reduction in GlaB ‐ treated mouse cerebella in comparison with vehicle mouse cerebella . Data show the mean ± SD of cerebella ( n = 10 ) for each treatment . * P 0 . 05 versus Ctr .

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C The graph shows the percentage of EGL thickness reduction in GlaB‐treated mouse cerebella in comparison with vehicle mouse cerebella. Data show the mean ± SD of cerebella (n = 10) for each treatment. *P 0.05 versus Ctr.


D BrdU assay in GCPs . Cerebellargranule cell progenitors ( GCPs ) isolated from 4 ‐ day ‐ old mice were treated with Shh ( recombinant mouseSonic Hedgehog , amino ‐ terminal peptide , ShhN , 3 μg / ml ) and with different concentrations of GlaB , as indicated , for 48 h . Inhibition of cell proliferation was measured as percentage of BrdU incorporation in comparison with DMSO ‐ treated sample . Shown is the mean of three independent experiments . Error bars indicate SD . * P 0 . 05 Shh versus DMSO ; * * P 0 . 05 Shh + GlaB versus Shh .

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D BrdU assay in GCPs. Cerebellargranule cell progenitors (GCPs) isolated from 4‐day‐old mice were treated with Shh (recombinant mouseSonic Hedgehog, amino‐terminal peptide, ShhN, 3 μg/ml) and with different concentrations of GlaB, as indicated, for 48 h. Inhibition of cell proliferation was measured as percentage of BrdU incorporation in comparison with DMSO‐treated sample. Shown is the mean of three independent experiments. Error bars indicate SD. *P 0.05 Shh versus DMSO; **P 0.05 Shh + GlaB versus Shh.


E qRT - PCR and show Hh and proliferation target mRNA and protein expression levels determined in ex vivo GCP culture derived from 6 ‐ day ‐ old mousecerebella after s . c . injections of GlaB . In all qRT - PCR experiments , the results were normalized to endogenous control ( β2 ‐ microglobulin and HPRT ) . * P 0 . 05 versus Ctr .

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E qRT-PCR and show Hh and proliferation target mRNA and protein expression levels determined in ex vivo GCP culture derived from 6‐day‐old mousecerebella after s.c. injections of GlaB. In all qRT-PCR experiments, the results were normalized to endogenous control (β2‐microglobulin and HPRT). *P 0.05 versus Ctr.


F Western blot analysis show Hh and proliferation target mRNA and protein expression levels determined in ex vivo GCP culture derived from 6 ‐ day ‐ old mousecerebella after s . c . injections of GlaB .

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F Western blot analysis show Hh and proliferation target mRNA and protein expression levels determined in ex vivo GCP culture derived from 6‐day‐old mousecerebella after s.c. injections of GlaB.


A , B Ex vivo cell cultures from Ptch1 + / − mice MBs were treated with GlaB ( 5 μM ) , GANT61 ( 10 μM ) or DMSO only . After the indicated times , a trypan blue count was performed ( A ) to determine the growth rate of viable cells .

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A, B Ex vivo cell cultures from Ptch1+/−mice MBs were treated with GlaB (5 μM), GANT61 (10 μM) or DMSO only. After the indicated times, a trypan blue count was performed (A) to determine the growth rate of viable cells.


A , B Ex vivo cell cultures from Ptch1 + / − miceMBs were treated with GlaB ( 5 μM ) , GANT61 ( 10 μM ) or DMSO only . After the indicated times , Gli1 mRNA expression levels were determined by qRT - PCR ( B ) normalized to endogenous control ( β2 ‐ microglobulin and HPRT ) .

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A, B Ex vivo cell cultures from Ptch1+/−miceMBs were treated with GlaB (5 μM), GANT61 (10 μM) or DMSO only. After the indicated times, Gli1 mRNA expression levels were determined by qRT-PCR (B) normalized to endogenous control (β2‐microglobulin and HPRT).


C - F GlaB inhibits MB ‐ SCs ' self ‐ renewal and proliferation . ( C ) Suspension of single MB ‐ SCs isolated from Ptch1 + / − mice . MBs were cultured in stem cell medium to allow the formation of primary neurospheres . Primary neurospheres were dissociated and treated with increasing concentrations of GlaB or DMSO only . After 7 days of treatment , the number of secondary neurospheres derived from a known number of single cells was counted . The self ‐ renewal MB ‐ SCs ' capability is expressed as percentage of neurosphere ‐ forming cells ( left ) . Representative bright field images of tumor neurospheres after GlaB treatment are also shown ( right ) .

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C-F GlaB inhibits MB‐SCs' self‐renewal and proliferation. (C) Suspension of single MB‐SCs isolated from Ptch1+/−mice. MBs were cultured in stem cell medium to allow the formation of primary neurospheres. Primary neurospheres were dissociated and treated with increasing concentrations of GlaB or DMSO only. After 7 days of treatment, the number of secondary neurospheres derived from a known number of single cells was counted. The self‐renewal MB‐SCs' capability is expressed as percentage of neurosphere‐forming cells (left). Representative bright field images of tumor neurospheres after GlaB treatment are also shown (right).


C - F GlaB inhibits MB ‐ SCs ' self ‐ renewal and proliferation . ( D , E ) MB ‐ SCs isolated from Ptch1 + / − miceMBs were treated for 48 h with GlaB ( 5 μM ) or DMSO only . qRT - PCR analysis show Hh , proliferation and stemness target mRNA expression levels . For qRT - PCR , results were normalized to endogenous control ( β2 ‐ microglobulin and HPRT ) .

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C-F GlaB inhibits MB‐SCs' self‐renewal and proliferation. (D, E) MB‐SCs isolated from Ptch1+/−miceMBs were treated for 48 h with GlaB (5 μM) or DMSO only. qRT-PCR analysis show Hh, proliferation and stemness target mRNA expression levels. For qRT-PCR, results were normalized to endogenous control (β2‐microglobulin and HPRT).


C - F GlaB inhibits MB ‐ SCs ' self ‐ renewal and proliferation . ( D , E ) MB ‐ SCs isolated from Ptch1 + / − miceMBs were treated for 48 h with GlaB ( 5 μM ) or DMSO only . Western blot analysis show Hh , proliferation and stemness target protein expression levels .

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C-F GlaB inhibits MB‐SCs' self‐renewal and proliferation. (D, E) MB‐SCs isolated from Ptch1+/−miceMBs were treated for 48 h with GlaB (5 μM) or DMSO only. Western blot analysis show Hh, proliferation and stemness target protein expression levels.


C - F GlaB inhibits MB ‐ SCs ' self ‐ renewal and proliferation . ( F ) BrdU assay in MB ‐ SCs treated with GlaB ( 5 μM ) for 24 or 48 h and plated on polylysinated chamber slides . Inhibition of cell proliferation was measured as percentage of BrdU incorporation in comparison with DMSO ‐ treated sample .

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C-F GlaB inhibits MB‐SCs' self‐renewal and proliferation. (F) BrdU assay in MB‐SCs treated with GlaB (5 μM) for 24 or 48 h and plated on polylysinated chamber slides. Inhibition of cell proliferation was measured as percentage of BrdU incorporation in comparison with DMSO‐treated sample.


G TUNEL assay in MB ‐ SCs . MB ‐ SCs isolated from Ptch1 + / − miceMBs were treated with GlaB ( 5 μM ) and compared to DMSO ‐ treated sample . Bottom panel shows a Western blot of caspase ‐ 3 in GlaB ‐ treated versus control cells .

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G TUNEL assay in MB‐SCs. MB‐SCs isolated from Ptch1+/−miceMBs were treated with GlaB (5 μM) and compared to DMSO‐treated sample. Bottom panel shows a Western blot of caspase‐3 in GlaB‐treated versus control cells.


A Change of tumor volume during GlaB or vehicle treatment period ( 18 days ) .

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A Change of tumor volume during GlaB or vehicle treatment period (18 days).


B Representative flank allografts average volumes . Error bars indicate SD . * P 0 . 05 .

B Representative flank allografts average volumes. Error bars indicate SD. *P 0.05.


C Representative Masson ' s trichromestaining of tumors ; immunohistochemistry of Gli1 , Ki67 and TUNEL stainings . Scale bars represent 35 μm for Masson ' s trichromestaining , 25 μm for Gli1 and TUNEL , and 10 μm for Ki67 stainings .

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C Representative Masson's trichromestaining of tumors; immunohistochemistry of Gli1, Ki67 and TUNEL stainings. Scale bars represent 35 μm for Masson's trichromestaining, 25 μm for Gli1 and TUNEL, and 10 μm for Ki67 stainings.


D Quantification of Ki67 and TUNEL stainings from immunohistochemistry shown in ( C ) . Error bars indicate SD . * P 0 . 05 .

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D Quantification of Ki67 and TUNEL stainings from immunohistochemistry shown in (C). Error bars indicate SD. *P 0.05.


E qRT - PCR analysis show Hh and proliferation target mRNA expression levels . For qRT - PCR , results were normalized to endogenous control ( β2 ‐ microglobulin and HPRT ) . Data show the mean ± SD of tumor ( n = 6 ) for each treatment . * P 0 . 05 versus Ctr .

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E qRT-PCR analysis show Hh and proliferation target mRNA expression levels. For qRT-PCR, results were normalized to endogenous control (β2‐microglobulin and HPRT). Data show the mean ± SD of tumor (n = 6) for each treatment. *P 0.05 versus Ctr.


F Western blot analysis show Hh and proliferation target protein expression levels . Data show the mean ± SD of tumor ( n = 6 ) for each treatment . * P 0 . 05 versus Ctr .

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F Western blot analysis show Hh and proliferation target protein expression levels. Data show the mean ± SD of tumor (n = 6) for each treatment. *P 0.05 versus Ctr.