( J - L ) Tg ( ins : kaede ) ; Tg ( ins : CFP - NTR ) transgenics , with or without Tg ( bactin : igfbp1a ) , were injected with a control morpholino or a morpholino that knocked down arx , which is necessary for α - cell differentiation and thus glucagon expression ( J - K ) . Scale bars : 20 μm . ( L ) Quantification of the total number of regenerating β cells at 4 dpf ( after β - cell ablation at 2 - 3 dpf ) . * * * * P < 0 . 0001 , * P < 0 . 05 , ns = nonsignificant . n = 42 , 20 , 48 and 20 , respectively .

O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(J-L) Tg(ins:kaede);Tg(ins:CFP-NTR) transgenics, with or without Tg(bactin:igfbp1a), were injected with a control morpholino or a morpholino that knocked down arx, which is necessary for α-cell differentiation and thus glucagon expression (J-K). Scale bars: 20 μm. (L) Quantification of the total number of regenerating β cells at 4 dpf (after β-cell ablation at 2-3 dpf). ****P<0.0001, *P<0.05, ns=nonsignificant. n=42, 20, 48 and 20, respectively.


( A - E ) Control and Tg ( bactin : igfbp1a ) - overexpressing larvae were treated with MTZ from 3 - 4 dpf to ablate β cells , and analyzed at 6 dpf , after 2 days of regeneration . ( A - B ) Representative confocal images of control and Tg ( bactin : igfbp1a ) - overexpressing Tg ( ins : dsRed ) ; Tg ( gcg : GFP ) ; Tg ( ins : Flag - NTR ) larvae . A bihormonal glucagon - and insulin - expressing cell ( gcg + ; ins + ) is indicated by an arrow . Scale bars : 20 μm . ( C - D ) Representative confocal images of control and Tg ( bactin : igfbp1a ) - overexpressing Tg ( gcg : GFP ) ; Tg ( ins : Flag - NTR ) larvae stained for Pdx1 . Pdx1 - and glucagon - expressing cells ( pdx1 + ; gcg + ) are indicated by arrows . Scale bars : 20 μm . ( E ) Quantification of gcg + ; ins + and pdx1 + ; gcg + cells in the control and Tg ( bactin : igfbp1a ) group ; P < 0 . 001 , P = 0 . 0012 ; n = 18 and 18 in the control groups , n = 15 and 10 in the Tg ( bactin : igfbp1a ) groups , for the gcg + ; ins + and pdx1 + ; gcg + quantification , respectively .

O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O

(A-E) Control and Tg(bactin:igfbp1a)-overexpressing larvae were treated with MTZ from 3-4 dpf to ablate β cells, and analyzed at 6 dpf, after 2 days of regeneration. (A-B) Representative confocal images of control and Tg(bactin:igfbp1a)-overexpressing Tg(ins:dsRed);Tg(gcg:GFP);Tg(ins:Flag-NTR) larvae. A bihormonal glucagon- and insulin-expressing cell (gcg+;ins+) is indicated by an arrow. Scale bars: 20 μm. (C-D) Representative confocal images of control and Tg(bactin:igfbp1a)-overexpressing Tg(gcg:GFP);Tg(ins:Flag-NTR) larvae stained for Pdx1. Pdx1- and glucagon-expressing cells (pdx1+;gcg+) are indicated by arrows. Scale bars: 20 μm. (E) Quantification of gcg+;ins+ and pdx1+;gcg+ cells in the control and Tg(bactin:igfbp1a) group; P<0.001, P=0.0012; n=18 and 18 in the control groups, n=15 and 10 in the Tg(bactin:igfbp1a) groups, for the gcg+;ins+ and pdx1+;gcg+ quantification, respectively.


( F - I ) Lineage - tracing evidence supports α - to β - cell transdifferentiation . ( G ) Quantification of insulin - expressing cells that originate from α - cells . Control larvae , n = 13 ; bactin : igfbp1a larvae , n = 16 . P = 0 . 0051 .

O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O B-GENEPROD O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O

(F-I) Lineage-tracing evidence supports α- to β-cell transdifferentiation. (G) Quantification of insulin-expressing cells that originate from α-cells. Control larvae, n=13; bactin:igfbp1a larvae, n=16. P=0.0051.


( F - I ) Lineage - tracing evidence supports α - to β - cell transdifferentiation . ( H - I ) Representative confocal images at 6 dpf ( after 2 days of regeneration ) of control and bactin : igfbp1a - overexpressing larvae stained for insulin , glucagon , to - pro , and H2B - GFP . ins + ; H2B - GFP + cells are indicated by the arrowheads , and H ' and I ' show staining against only insulin and H2B - GFP . Moreover , most insulin + ; H2B - GFP + cells no longer express glucagon . Scale bars : 15 μm .

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

(F-I) Lineage-tracing evidence supports α- to β-cell transdifferentiation. (H-I) Representative confocal images at 6 dpf (after 2 days of regeneration) of control and bactin:igfbp1a-overexpressing larvae stained for insulin, glucagon, to-pro, and H2B-GFP. ins+;H2B-GFP+ cells are indicated by the arrowheads, and H' and I' show staining against only insulin and H2B-GFP. Moreover, most insulin+;H2B-GFP+ cells no longer express glucagon. Scale bars: 15 μm.


( A - H ) PPP , an IGF1R inhibitor , promotes β - cell regeneration . Tg ( ins : H2B - GFP ) ; Tg ( ins : Flag - NTR ) larvae were treated with MTZ from 3 - 4 dpf to ablate the β cells and then treated with EdU and DMSO or with EdU and PPP during regeneration from 4 - 6 dpf . ( A - B ) Representative confocal images at 6 dpf of DMSO - and PPP - treated larvae displaying β cells in green and the β cells that had incorporated EdU as yellow overlap ( arrowheads ) . Scale bars : 20 μm . ( C ) Quantification of the total number of β cells ( green bars ) at 6 dpf , and β cells that had incorporated EdU ( white bars ) from 4 - 6 dpf during β - cell regeneration . P = 0 . 0003 , P = 0 . 8607 respectively . ( D ) Rate of β - cell proliferation , displayed as the percentage of β cells that incorporated EdU . P = 0 . 1194 . n = 18 larvae for the DMSO - treated group , n = 17 larvae for the PPP - treated group . ( E - H ) To examine whether PPP affected β - cell proliferation during regular development , Tg ( ins : H2B - GFP ) larvae were treated with EdU and DMSO or PPP from 4 - 6 dpf . ( E - F ) Representative confocal images of 6 dpf DMSO - and PPP - treated larvae displaying β cells in green and the β cells that had incorporated EdU as yellow overlap . Scale bars : 20 μm . ( G ) Quantification of the total number of β cells ( green bars ) and β cells that had incorporated EdU ( white bars ) per larva from 4 - 6 dpf . P = 0 . 9098 and 0 . 9976 , respectively . ( H ) Rate of β cell proliferation , displayed as the percentage of β cells that incorporated EdU . P = 0 . 7822 . n = 16 larvae for DMSO - treated group , 18 larvae for PPP - treated group .

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

(A-H) PPP, an IGF1R inhibitor, promotes β-cell regeneration. Tg(ins:H2B-GFP);Tg(ins:Flag-NTR) larvae were treated with MTZ from 3-4 dpf to ablate the β cells and then treated with EdU and DMSO or with EdU and PPP during regeneration from 4-6 dpf. (A-B) Representative confocal images at 6 dpf of DMSO- and PPP-treated larvae displaying β cells in green and the β cells that had incorporated EdU as yellow overlap (arrowheads). Scale bars: 20 μm. (C) Quantification of the total number of β cells (green bars) at 6 dpf, and β cells that had incorporated EdU (white bars) from 4-6 dpf during β-cell regeneration. P=0.0003, P=0.8607 respectively. (D) Rate of β-cell proliferation, displayed as the percentage of β cells that incorporated EdU. P=0.1194. n=18 larvae for the DMSO-treated group, n=17 larvae for the PPP-treated group. (E-H) To examine whether PPP affected β-cell proliferation during regular development, Tg(ins:H2B-GFP) larvae were treated with EdU and DMSO or PPP from 4-6 dpf. (E-F) Representative confocal images of 6 dpf DMSO- and PPP-treated larvae displaying β cells in green and the β cells that had incorporated EdU as yellow overlap. Scale bars: 20 μm. (G) Quantification of the total number of β cells (green bars) and β cells that had incorporated EdU (white bars) per larva from 4-6 dpf. P=0.9098 and 0.9976, respectively. (H) Rate of β cell proliferation, displayed as the percentage of β cells that incorporated EdU. P=0.7822. n=16 larvae for DMSO-treated group, 18 larvae for PPP-treated group.


( I - K ) Activation of the Igf pathway reduces β - cell regeneration . Control and bactin : igf2a - overexpressing Tg ( ins : H2B - GFP ) ; Tg ( ins : Flag - NTR ) larvae were treated with MTZ from 3 - 4 dpf to ablate β cells and subsequently let to regenerate from 4 - 6 dpf . ( I - J ) Representative confocal images of 6 dpf control and bactin : igf2a - overexpressing larva displaying β cells in green . Scale bars : 15 μm . ( K ) Quantification of the total number of β cells per larva at 6 dpf following β cell regeneration from 4 - 6 dpf . P < 0 . 0001 . n = 28 larvae for control , n = 15 larvae for bactin : igf2a .

O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O

(I-K) Activation of the Igf pathway reduces β-cell regeneration. Control and bactin:igf2a-overexpressing Tg(ins:H2B-GFP);Tg(ins:Flag-NTR) larvae were treated with MTZ from 3-4 dpf to ablate β cells and subsequently let to regenerate from 4-6 dpf. (I-J) Representative confocal images of 6 dpf control and bactin:igf2a-overexpressing larva displaying β cells in green. Scale bars: 15 μm. (K) Quantification of the total number of β cells per larva at 6 dpf following β cell regeneration from 4-6 dpf. P<0.0001. n=28 larvae for control, n=15 larvae for bactin:igf2a.


( L - P ) No synergistic effect was observed for igfbp1a and PPP . Control and bactin : igfbp1a - overexpressing Tg ( ins : H2B - GFP ) ; Tg ( ins : Flag - NTR ) larvae were treated with MTZ from 3 - 4 dpf to ablate β cells and subsequently treated with DMSO or PPP during regeneration from 4 - 6 dpf . ( L ) Quantification of the total number of β cells per larva . n > 10 ( n = 23 , 20 , 14 , 13 ) . P = 0 . 9546 . ( M - P ) Representative confocal images of 6 dpf control or bactin : igfbp1a overexpressing larvae treated with either DMSO or PPP , displaying β cells after 2 days regeneration . Scale bars : 10 μm . See also Figure EV2 EV3 .

O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O

(L-P) No synergistic effect was observed for igfbp1a and PPP. Control and bactin:igfbp1a-overexpressing Tg(ins:H2B-GFP);Tg(ins:Flag-NTR) larvae were treated with MTZ from 3-4 dpf to ablate β cells and subsequently treated with DMSO or PPP during regeneration from 4-6 dpf. (L) Quantification of the total number of β cells per larva. n>10 (n=23, 20, 14, 13). P=0.9546. (M-P) Representative confocal images of 6 dpf control or bactin:igfbp1a overexpressing larvae treated with either DMSO or PPP, displaying β cells after 2 days regeneration. Scale bars: 10 μm. See also Figure EV2 EV3.


( A - B ) Representative confocal projections of the whole pancreas ( dashed lines ) of 35 - day - old Tg ( ins : H2B - GFP ) ; Tg ( ins : Flag - NTR ) transgenics , with or without Tg ( bactin : igfbp1a ) , that were subjected to β - cell ablation between day 30 - 31 and then allowed to regenerate for 4 days . Scale bars represent 100 μm . ( C ) Body length of control and Tg ( bactin : igfbp1a ) zebrafish ; P < 0 . 0001 . ( D - H ) Quantification of β - cell regeneration was automated with an ImageJ script . ( D ) Insulin - positive area per zebrafish . ( E ) Relative insulin - positive area per body length ; P < 0 . 01 . ( F ) The percentage of the pancreas area that was insulin - positive was significantly larger in Tg ( bactin : igfbp1a ) than in controls ; P < 0 . 0001 . ( G ) The total number of recorded units of adjacent ins : H2B - GFP + pixels ( units ranging from single β cells to β - cell clusters ) did not differ between control and Tg ( bactin : igfbp1a ) zebrafish . ( H ) The size of recorded ins : H2B - GFP + units was on average larger in Tg ( bactin : igfbp1a ) than in controls ; P = 0 . 039 . n = 26 in the control group , n = 27 in the Tg ( bactin : igfbp1a ) group .

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

(A-B) Representative confocal projections of the whole pancreas (dashed lines) of 35-day-old Tg(ins:H2B-GFP);Tg(ins:Flag-NTR) transgenics, with or without Tg(bactin:igfbp1a), that were subjected to β-cell ablation between day 30-31 and then allowed to regenerate for 4 days. Scale bars represent 100 μm. (C) Body length of control and Tg(bactin:igfbp1a) zebrafish; P<0.0001. (D-H) Quantification of β-cell regeneration was automated with an ImageJ script. (D) Insulin-positive area per zebrafish. (E) Relative insulin-positive area per body length; P<0.01. (F) The percentage of the pancreas area that was insulin-positive was significantly larger in Tg(bactin:igfbp1a) than in controls; P<0.0001. (G) The total number of recorded units of adjacent ins:H2B-GFP+ pixels (units ranging from single β cells to β-cell clusters) did not differ between control and Tg(bactin:igfbp1a) zebrafish. (H) The size of recorded ins:H2B-GFP+ units was on average larger in Tg(bactin:igfbp1a) than in controls; P=0.039. n=26 in the control group, n=27 in the Tg(bactin:igfbp1a) group.


Isolated islets from mice were treated for 3 days with recombinant mouseIGFBP1 protein , respectively . ( A - B ) Representative confocal images of control and Igfbp1 - treated mouse islets . gcg + ins + cells are indicated by arrows . Scale bars : 25 μm . ( C ) Quantification of the percentage of gcg + ins + bihormonal cells per ins + cells in mouse islets after treatment with increasing concentrations of IGFBP1 . P = 0 . 7304 , 0 . 0023 , 0 . 0013 , 0 . 0410 , respectively . ( D ) Quantification of β - cell proliferation in mouse islets after treatment with Igfbp1 ; data displayed as relative values so that the proliferation markers EdU and Ki67 can be compared .

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

Isolated islets from mice were treated for 3 days with recombinant mouseIGFBP1 protein, respectively. (A-B) Representative confocal images of control and Igfbp1-treated mouse islets. gcg+ins+ cells are indicated by arrows. Scale bars: 25 μm. (C) Quantification of the percentage of gcg+ins+ bihormonal cells per ins+ cells in mouse islets after treatment with increasing concentrations of IGFBP1. P=0.7304, 0.0023, 0.0013, 0.0410, respectively. (D) Quantification of β-cell proliferation in mouse islets after treatment with Igfbp1; data displayed as relative values so that the proliferation markers EdU and Ki67 can be compared.


Isolated islets from human donors were treated for 3 days with recombinant human IGFBP1 protein , respectively . ( E - F ) Representative confocal images of control or IGFBP1 - treated human islets . A gcg + ins + cell is indicated by an arrow . For clarity , individual colors for denoting insulin ( green ) , glucagon ( red ) , and DAPI ( blue ) are also shown separately . Scale bars : 15 μm . ( G ) Quantification of the percentage of gcg + ins + bihormonal cells per ins + cells in human islets from 5 donors . P = 0 . 0036 . Co - expression of insulin and glucagon is indicative of transdifferentiation , which could reflect either α cells transdifferentiating to β cells or vice versa .

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

Isolated islets from human donors were treated for 3 days with recombinant human IGFBP1 protein, respectively. (E-F) Representative confocal images of control or IGFBP1-treated human islets. A gcg+ins+ cell is indicated by an arrow. For clarity, individual colors for denoting insulin (green), glucagon (red), and DAPI (blue) are also shown separately. Scale bars: 15 μm. (G) Quantification of the percentage of gcg+ins+ bihormonal cells per ins+ cells in human islets from 5 donors. P=0.0036. Co-expression of insulin and glucagon is indicative of transdifferentiation, which could reflect either α cells transdifferentiating to β cells or vice versa.


( A ) Three glycosylated forms of endogenous SMO protein are shown . NIH3T3 cells were harvested and treated with indicated glycosidases . Black bracket indicates mature glycosylated form and grey bracket indicates core glycosylated form . The representative results of three independent experiments are shown .

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(A) Three glycosylated forms of endogenous SMO protein are shown. NIH3T3 cells were harvested and treated with indicated glycosidases. Black bracket indicates mature glycosylated form and grey bracket indicates core glycosylated form. The representative results of three independent experiments are shown.


( C ) PTCH1 inhibited the Endo H - resistant glycosylation of SMO . HEK293T cells were co - transfected with the SMO and PTCH1 expression plasmids . After 48 h , the cells were harvested , lysed , and treated with indicated glycosidases . Results shown are representative of three independent experiments .

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

(C) PTCH1 inhibited the Endo H-resistant glycosylation of SMO. HEK293T cells were co-transfected with the SMO and PTCH1 expression plasmids. After 48 h, the cells were harvested, lysed, and treated with indicated glycosidases. Results shown are representative of three independent experiments.


( D ) Quantification of Endo H - resistant form of SMO shown in ( C ) . Three independent experiments were quantified by Image J and expressed as a percentage of the total SMO protein .

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

(D) Quantification of Endo H-resistant form of SMO shown in (C). Three independent experiments were quantified by Image J and expressed as a percentage of the total SMO protein.


( E ) Shh increased the Endo H - resistant form of SMO . Cells were transfected as indicated for 24 h , and incubated with ShhN conditioned medium for an additional 24 h . Cells were harvested and analyzed as in ( C ) . Results shown are representative of three independent experiments .

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(E) Shh increased the Endo H-resistant form of SMO. Cells were transfected as indicated for 24 h, and incubated with ShhN conditioned medium for an additional 24 h. Cells were harvested and analyzed as in (C). Results shown are representative of three independent experiments.


( F ) Quantification of Endo H - resistant form of SMO shown in ( E ) . Three independent experiments were quantified by Image J and expressed as a percentage of the total SMO protein .

(F) Quantification of Endo H-resistant form of SMO shown in (E). Three independent experiments were quantified by Image J and expressed as a percentage of the total SMO protein.


( G ) Shh increased the Endo H - resistant form of endogenous SMO in MEF cells . Results shown are representative of three independent experiments .

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(G) Shh increased the Endo H-resistant form of endogenous SMO in MEF cells. Results shown are representative of three independent experiments.


( H ) Densitometric analysis of Endo H - resistant form of SMO shown in ( G ) . Three independent experiments were quantified by Image J and expressed as a percentage of the total SMO protein .

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(H) Densitometric analysis of Endo H-resistant form of SMO shown in (G). Three independent experiments were quantified by Image J and expressed as a percentage of the total SMO protein.


( A ) Biotinylation of SMO by SAR1B - BirA * ( asterisk denotes the R118G mutation in BirA ) in a proximity - dependent biotinylation assay . The cells were transfected with plasmids encoding SMO and SAR1B - BirA * - FLAG . After 48 h , cells were treated with different doses of biotin for 4 h . Cells were harvested and lysates were dialyzed to remove free biotin . Biotinylated proteins were pulled down by streptavidin beads and subjected to immunoblotting analysis . ( B ) The cells were treated with different doses of biotin for 24 h . Cells were harvested , lysed , and subjected to procedure as shown in Fig . 2A .

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(A) Biotinylation of SMO by SAR1B-BirA* (asterisk denotes the R118G mutation in BirA) in a proximity-dependent biotinylation assay. The cells were transfected with plasmids encoding SMO and SAR1B-BirA*-FLAG. After 48 h, cells were treated with different doses of biotin for 4 h. Cells were harvested and lysates were dialyzed to remove free biotin. Biotinylated proteins were pulled down by streptavidin beads and subjected to immunoblotting analysis. (B) The cells were treated with different doses of biotin for 24 h. Cells were harvested, lysed, and subjected to procedure as shown in Fig. 2A.


( B ) The cells were treated with different doses of biotin for 24 h . Cells were harvested , lysed , and subjected to procedure as shown in Fig . 2A .

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(B) The cells were treated with different doses of biotin for 24 h. Cells were harvested, lysed, and subjected to procedure as shown in Fig. 2A.


( C ) The GDP - locked SAR1B ( G37A ) - BirA * failed to label SMO , while the GTP - locked SAR1B ( H79G ) - BirA * could efficiently biotinylate SMO . The cells were transfected with plasmids expressing SMO and indicated SAR1B - BirA * . After 48 h , cells were treated with 15 μM biotin for 4 h . Then experiments were carried out as described in Fig . 2A .

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(C) The GDP-locked SAR1B(G37A)-BirA* failed to label SMO, while the GTP-locked SAR1B(H79G)-BirA* could efficiently biotinylate SMO. The cells were transfected with plasmids expressing SMO and indicated SAR1B-BirA*. After 48 h, cells were treated with 15 μM biotin for 4 h. Then experiments were carried out as described in Fig. 2A.


( D ) PTCH1 decreased SAR1B - BirA * - mediated biotinylation of SMO , but not LMAN1 . The experiments were done similarly in Fig . 2A .

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(D) PTCH1 decreased SAR1B-BirA*-mediated biotinylation of SMO, but not LMAN1. The experiments were done similarly in Fig. 2A.


( C ) The GDP - locked SAR1B ( G37A ) - BirA * failed to label SMO , while the GTP - locked SAR1B ( H79G ) - BirA * could efficiently biotinylate SMO . The cells were transfected with plasmids expressing SMO and indicated SAR1B - BirA * . After 48 h , cells were treated with 15 μM biotin for 4 h . Then experiments were carried out as described in Fig . 2A . ( D ) PTCH1 decreased SAR1B - BirA * - mediated biotinylation of SMO , but not LMAN1 . The experiments were done similarly in Fig . 2A . ( E ) The Shh increased the binding of SAR1B to SMO . After transfection for 24 h , cells were incubated with the ShhN conditioned medium for an additional 24 h . Then 15 μM biotin was added into the culture medium for 4 h . The cells were harvested and analyzed as described in Fig . 2A .

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(C) The GDP-locked SAR1B(G37A)-BirA* failed to label SMO, while the GTP-locked SAR1B(H79G)-BirA* could efficiently biotinylate SMO. The cells were transfected with plasmids expressing SMO and indicated SAR1B-BirA*. After 48 h, cells were treated with 15 μM biotin for 4 h. Then experiments were carried out as described in Fig. 2A. (D) PTCH1 decreased SAR1B-BirA*-mediated biotinylation of SMO, but not LMAN1. The experiments were done similarly in Fig. 2A. (E) The Shh increased the binding of SAR1B to SMO. After transfection for 24 h, cells were incubated with the ShhN conditioned medium for an additional 24 h. Then 15 μM biotin was added into the culture medium for 4 h. The cells were harvested and analyzed as described in Fig. 2A.


( B ) Treatment with glycosidase demonstrated that SMO ( 4A , in which RRTWCR was mutated to AATACA ) was sensitive to Endo H . The experiments were done similarly in Fig . 2A . Black bracket indicates mature glycosylated form .

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(B) Treatment with glycosidase demonstrated that SMO (4A, in which RRTWCR was mutated to AATACA) was sensitive to Endo H. The experiments were done similarly in Fig. 2A. Black bracket indicates mature glycosylated form.


( C ) SAR1B - BirA * - mediated biotinylation of the SMO variants . The experiments were done similarly in Fig . 2A .

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(C) SAR1B-BirA*-mediated biotinylation of the SMO variants. The experiments were done similarly in Fig. 2A.


( D ) In vitro budding of SMO . Membranes were prepared and incubated in vitro in the presence of cytosol , ATP , GTP , and an ATP - regenerating system as described in experimental procedures . Then vesicle and membrane fractions were separated by centrifugation and subjected to immunoblotting analysis .

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(D) In vitro budding of SMO. Membranes were prepared and incubated in vitro in the presence of cytosol, ATP, GTP, and an ATP-regenerating system as described in experimental procedures. Then vesicle and membrane fractions were separated by centrifugation and subjected to immunoblotting analysis.


( E ) In vitro budding of the SMO variants . The experiments were done as same as in ( D ) .

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(E) In vitro budding of the SMO variants. The experiments were done as same as in (D).


( F ) The ciliary localization of SMO variants . Smo - KO NIH3T3 cells were transfected with the EGFP fused SMO variants , incubated with serum starvation medium for 24 h to fully induce cilia formation , and treated with or without ShhN conditioned medium for 4 h . Cells were immunostained with the acetylated tubulin ( red ) . Results shown are representative of three independent experiments . Scale bars : 5μm .

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(F) The ciliary localization of SMO variants. Smo-KO NIH3T3 cells were transfected with the EGFP fused SMO variants, incubated with serum starvation medium for 24 h to fully induce cilia formation, and treated with or without ShhN conditioned medium for 4 h. Cells were immunostained with the acetylated tubulin (red). Results shown are representative of three independent experiments. Scale bars: 5μm.


( G ) Quantitative assessment of the intensity of ciliary SMO in ( F ) by Image J . More than 45 cells were randomly selected and counted for each group .

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(G) Quantitative assessment of the intensity of ciliary SMO in (F) by Image J. More than 45 cells were randomly selected and counted for each group.


( H ) Quantification of the percentage of cells with ciliary SMO . 80 - 90 cilia were analyzed in each group in each experiment .

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(H) Quantification of the percentage of cells with ciliary SMO. 80-90 cilia were analyzed in each group in each experiment.


( I ) Gli - luciferase reporter assay in Smo - KO NIH3T3 cells . The cells were transfected with plasmids encoding SMO or SMO variants , GLI - promoted firefly luciferase and renilla luciferase . After 24 h , cells were incubated with a medium containing different amounts of ShhN for another 24 h .

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(I) Gli-luciferase reporter assay in Smo-KO NIH3T3 cells. The cells were transfected with plasmids encoding SMO or SMO variants, GLI-promoted firefly luciferase and renilla luciferase. After 24 h, cells were incubated with a medium containing different amounts of ShhN for another 24 h.


( A ) Shh increased cholesterylation of SMO . HEK293T cells were transfected , harvested , and

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(A) Shh increased cholesterylation of SMO. HEK293T cells were transfected, harvested, and analyzed


( B ) The SMO cholesterylation of Ptch1 - KO NIH3T3 cells were increased comparing with WT cells .

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(B) The SMO cholesterylation of Ptch1-KO NIH3T3 cells were increased comparing with WT cells.


( C ) The CP - modified SMO was mainly resistant to Endo H . The CP - modified SMO proteins were pulled down by streptavidin beads and digested with Endo H or PNGase F at 37 ℃ . Then proteins were eluted with SDS - PAGE sample buffer and were subjected to immunoblotting analysis . Results shown are representative of two independent experiments .

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(C) The CP-modified SMO was mainly resistant to Endo H. The CP-modified SMO proteins were pulled down by streptavidin beads and digested with Endo H or PNGase F at 37 ℃. Then proteins were eluted with SDS-PAGE sample buffer and were subjected to immunoblotting analysis. Results shown are representative of two independent experiments.


( D ) Cholesterylation of SMO ( 4A ) was inhibited by PTCH1 and increased by ShhN .

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(D) Cholesterylation of SMO(4A) was inhibited by PTCH1 and increased by ShhN.


( E ) The CP - modified SMO ( 4A ) was sensitive to Endo H treatment .

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(E) The CP-modified SMO(4A) was sensitive to Endo H treatment.


( F ) The levels of CP - modified WT SMO and SMO ( 4A ) were compared . 1 . 39 % of SMO ( WT ) pellet samples and 11 . 1 % of SMO ( 4A ) pellet samples were loaded . Results shown are representative of three independent experiments .

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(F) The levels of CP-modified WT SMO and SMO(4A) were compared. 1.39% of SMO(WT) pellet samples and 11.1% of SMO(4A) pellet samples were loaded. Results shown are representative of three independent experiments.


( G ) Quantification of CP - modified SMO shown in ( F ) . Three independent experiments were quantified by Image J and expressed as a percentage of the total SMO .

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(G) Quantification of CP-modified SMO shown in (F). Three independent experiments were quantified by Image J and expressed as a percentage of the total SMO.


( H , I ) The stimulative / inhibitory effect of Shh / PTCH1 on SMO and SAR1B interaction was compromised by D95E mutation . The experiments were done the same as Fig . 2E .

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(H, I) The stimulative /inhibitory effect of Shh/PTCH1 on SMO and SAR1B interaction was compromised by D95E mutation. The experiments were done the same as Fig. 2E.


ALOD4 inhibited the cholesterol modification of SMO Cells were treated with ALOD4 for 1 h , and then incubated with a medium containing ShhN and CP for 2 and 3 h . Cells were harvested and

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ALOD4 inhibited the cholesterol modification of SMO Cells were treated with ALOD4 for 1 h, and then incubated with a medium containing ShhN and CP for 2 and 3 h. Cells were harvested and analyzed


ALOD4 inhibited the cholesterol modification of SMO ( 4A ) . Cells were treated with ALOD4 for 1 h In C , ShhN and CP added for 2 h . Cells were harvested and

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ALOD4 inhibited the cholesterol modification of SMO(4A). Cells were treated with ALOD4 for 1 h In C, ShhN and CP added for 2 h. Cells were harvested and analyzed


( D ) Ciliary localization of endogenous SMO treated with or without ALOD4 . Cells were treated with ALOD4 for 1 h , and then incubated with ShhN conditioned medium for 2 h . Results shown are representative of three independent experiments .

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(D) Ciliary localization of endogenous SMO treated with or without ALOD4. Cells were treated with ALOD4 for 1 h, and then incubated with ShhN conditioned medium for 2 h. Results shown are representative of three independent experiments.


( E ) Quantitative assessment of the intensity of ciliary SMO in ( D ) . More than 80 cells were randomly selected and counted for each group . Data are presented as median with interquartile .

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(E) Quantitative assessment of the intensity of ciliary SMO in (D). More than 80 cells were randomly selected and counted for each group. Data are presented as median with interquartile.


( F ) Quantification of the percentage of cells with ciliary SMO in ( D ) . Data are presented as mean ± SEM from three biological replicates ( * * * * p < 0 . 0001 , one - way ANOVA with Dunnett ' s multiple comparison test ) . 90 - 100 cilia were analyzed in each group in each experiment .

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(F) Quantification of the percentage of cells with ciliary SMO in (D). Data are presented as mean ± SEM from three biological replicates (****p < 0.0001, one-way ANOVA with Dunnett's multiple comparison test). 90-100 cilia were analyzed in each group in each experiment.


( G ) Effect of ALOD4 on the expression of Gli1 was measured by qRT - PCR . The cells were treated with ALOD4 for 1 h , and then incubated with ShhN conditioned medium for 5 h .

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(G) Effect of ALOD4 on the expression of Gli1 was measured by qRT-PCR. The cells were treated with ALOD4 for 1 h, and then incubated with ShhN conditioned medium for 5 h.


( A , B ) GRAMD1B increased cholesterylation of SMO . HEK293T cells were co - transfected with the SMO or SMO ( 4A ) and GRAMD1B protein expression plasmids for 24 h , and then incubated with a cholesterol depletion medium ( DMEM supplemented with 5 % lipoprotein - deficient serum , 1 μM lovastatin and 10 μM mevalonate ) for 12 h , followed by 2 μg / mL of CP treatment . The cells were harvested and analyzed as Results shown are representative of two independent experiments .

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(A, B) GRAMD1B increased cholesterylation of SMO. HEK293T cells were co-transfected with the SMO or SMO (4A) and GRAMD1B protein expression plasmids for 24 h, and then incubated with a cholesterol depletion medium (DMEM supplemented with 5% lipoprotein-deficient serum, 1 μM lovastatin and 10 μM mevalonate) for 12 h, followed by 2 μg/mL of CP treatment. The cells were harvested and analyzed as Results shown are representative of two independent experiments.


( C , D ) GRAMD1B promoted SMO maturation . The expression plasmids of SMO or SMO ( D95E ) and GRAMD1B were transfected into SMO - KO HEK293T cells . After 12 h , cells were switched to a cholesterol depletion medium for 16 h and then added 50 μM MβCD - cholesterol for 8 h . Black bracket indicates mature glycosylated form .

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(C, D) GRAMD1B promoted SMO maturation. The expression plasmids of SMO or SMO(D95E) and GRAMD1B were transfected into SMO-KO HEK293T cells. After 12 h, cells were switched to a cholesterol depletion medium for 16 h and then added 50 μM MβCD-cholesterol for 8 h. Black bracket indicates mature glycosylated form.


( E ) Expression of Gli1 mRNAs measured by qRT - PCR in NIH3T3 cells stably expressing GRAMD1B . After incubating in serum starvation medium for 16 h , cells were depleted of cholesterol by treatment with 1 . 5 % HPCD for 0 . 5 h and then incubated in serum starvation medium containing 1 μM lovastatin , 10 μM mevalonate and 10 μM MβCD - cholesterol , ShhN as indicated for 24 h .

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(E) Expression of Gli1 mRNAs measured by qRT-PCR in NIH3T3 cells stably expressing GRAMD1B. After incubating in serum starvation medium for 16 h, cells were depleted of cholesterol by treatment with 1.5% HPCD for 0.5 h and then incubated in serum starvation medium containing 1 μM lovastatin, 10 μM mevalonate and 10 μM MβCD-cholesterol, ShhN as indicated for 24 h.


( F ) Confocal images of NIH3T3 cells stably expressing mCherry - GRAMD1B . PM was labeled with wheat germ agglutinin ( WGA ) - CF ® 488A conjugate . Cells were incubated in a cholesterol depletion medium for 16 h and switched to the same medium containing 50 μM MβCD - cholesterol for 1 h . Cells were then treated with or without ShhN conditioned medium for another 2 h . Results shown are representative of three independent experiments .

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(F) Confocal images of NIH3T3 cells stably expressing mCherry-GRAMD1B. PM was labeled with wheat germ agglutinin (WGA)-CF®488A conjugate. Cells were incubated in a cholesterol depletion medium for 16 h and switched to the same medium containing 50 μM MβCD-cholesterol for 1 h. Cells were then treated with or without ShhN conditioned medium for another 2 h. Results shown are representative of three independent experiments.


( G ) The percentage of cells with GRAMD1B localization on PM . 60 - 70 cells were analyzed in each group in each experiment .

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(G) The percentage of cells with GRAMD1B localization on PM. 60-70 cells were analyzed in each group in each experiment.


( A ) The cholesterol modification of endogenous SMO was decreased in NIH3T3 cells lacking Gramd1a , 1b , 1c , 2 , and 3 ( Gramd - 5KO ) . The cells were incubated in a cholesterol depletion medium for 12 h , followed by 2 μg / mL of CP and ShhN for an additional 12h .

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(A) The cholesterol modification of endogenous SMO was decreased in NIH3T3 cells lacking Gramd1a, 1b, 1c, 2, and 3 (Gramd-5KO). The cells were incubated in a cholesterol depletion medium for 12 h, followed by 2 μg/mL of CP and ShhN for an additional 12h.


( B ) The glycosylation of endogenous SMO was decreased in Gramd - 5KO NIH3T3 cells . The ShhN conditioned medium was applied for 24 h . Black bracket indicates mature glycosylated form .

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(B) The glycosylation of endogenous SMO was decreased in Gramd-5KO NIH3T3 cells. The ShhN conditioned medium was applied for 24 h. Black bracket indicates mature glycosylated form.


( C ) The ciliary localization of endogenous SMO in WT or Gramd - 5KO NIH3T3 cells . Cells were incubated in serum starvation medium for 24 h to fully induce cilia formation and then incubated with or without ShhN conditioned medium for 2 h . Results shown are representative of three independent experiments .

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(C) The ciliary localization of endogenous SMO in WT or Gramd-5KO NIH3T3 cells. Cells were incubated in serum starvation medium for 24 h to fully induce cilia formation and then incubated with or without ShhN conditioned medium for 2 h. Results shown are representative of three independent experiments.


( D ) Quantitative assessment of the intensity of ciliary SMO in ( C ) . More than 100 cells were randomly selected and counted for each group . Data are presented as median with interquartile .

(D) Quantitative assessment of the intensity of ciliary SMO in (C). More than 100 cells were randomly selected and counted for each group. Data are presented as median with interquartile.


( E ) Quantification of the percentage of cells with ciliary SMO . 86 - 100 cilia were analyzed in each group in each experiment .

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(E) Quantification of the percentage of cells with ciliary SMO. 86-100 cilia were analyzed in each group in each experiment.


( F ) The protein levels of Gli1 was significantly reduced in Gramd - 5KO cells . The cells were incubated in a serum starvation medium with or without ShhN for 24 h . Results shown are representative of two independent experiments .

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(F) The protein levels of Gli1 was significantly reduced in Gramd-5KO cells. The cells were incubated in a serum starvation medium with or without ShhN for 24 h. Results shown are representative of two independent experiments.


( G ) GLI - luciferase reporter assay in WT or Gramd - 5KO NIH3T3 cells . Data are presented as mean ± SEM from three technical replicates ( * * * * p < 0 . 0001 ; * * * p < 0 . 001 ; * p < 0 . 05 , two - way ANOVA with Sidak ' s multiple comparison test ) .

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(G) GLI-luciferase reporter assay in WT or Gramd-5KO NIH3T3 cells. Data are presented as mean ± SEM from three technical replicates (****p < 0.0001; ***p < 0.001; *p < 0.05, two-way ANOVA with Sidak's multiple comparison test).


( H ) The Gli1 mRNA levels were measured in WT , Gramd - 5KO , Gramd - 5KO overexpression GRAMD1A , 1B , 1C NIH3T3 cells by qRT - PCR after treatment with or without ShhN conditioned medium for 8 h . Data are expressed as mean ± SEM from three technical replicates ( * * * * p < 0 . 0001 , two - way ANOVA with Tukey ' s multiple comparisons test ) .

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(H) The Gli1 mRNA levels were measured in WT, Gramd-5KO, Gramd-5KO overexpression GRAMD1A, 1B, 1C NIH3T3 cells by qRT-PCR after treatment with or without ShhN conditioned medium for 8 h. Data are expressed as mean ± SEM from three technical replicates (****p < 0.0001, two-way ANOVA with Tukey's multiple comparisons test).


( I ) The GRAMD inhibitor AI3d inhibited cholesterol modification of SMO in NIH3T3 cells . The cells were incubated in cholesterol depletion medium for 16 h and then treated with 3 μM AI3d for 6 h . Next , cells were incubated in the same medium containing ShhN and 2 μg / mL of CP for an additional 2 h .

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(I) The GRAMD inhibitor AI3d inhibited cholesterol modification of SMO in NIH3T3 cells. The cells were incubated in cholesterol depletion medium for 16 h and then treated with 3 μM AI3d for 6 h. Next, cells were incubated in the same medium containing ShhN and 2 μg/mL of CP for an additional 2 h.


( J ) The GRAMD inhibitor AI3d decreased the mRNA levels of Gli1 in NIH3T3 cells . Cells were incubated in serum starvation medium for 16 h , depleted of cholesterol by 1 . 5 % HPCD for 0 . 5 h , and then treated with serum starvation medium containing 1 μM lovastatin , 10 μM mevalonate and 1 μM AI3d for 6 h . Next , cells were incubated in the same medium containing ShhN and 10 μM MβCD - cholesterol for an additional

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(J) The GRAMD inhibitor AI3d decreased the mRNA levels of Gli1 in NIH3T3 cells. Cells were incubated in serum starvation medium for 16 h, depleted of cholesterol by 1.5% HPCD for 0.5 h, and then treated with serum starvation medium containing 1 μM lovastatin, 10 μM mevalonate and 1 μM AI3d for 6 h. Next, cells were incubated in the same medium containing ShhN and 10 μM MβCD-cholesterol for an additional 16


( A ) The degradation of 3HA - Pgc1 was analyzed in cells with the indicated genotype upon inhibition of protein synthesis with cycloheximide ( chx ) . A plasmid - borne 3HA - Pgc1 expressed from the endogenous promoter was used . 3HA - Pgc1 was detected with anti - HA antibodies . Kar2 was used as a loading control and detected with anti - Kar2 antibodies . The graph on the right shows the average of four independent experiments ; error bars represent the standard deviation .

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(A) The degradation of 3HA-Pgc1 was analyzed in cells with the indicated genotype upon inhibition of protein synthesis with cycloheximide (chx). A plasmid-borne 3HA-Pgc1 expressed from the endogenous promoter was used. 3HA-Pgc1 was detected with anti-HA antibodies. Kar2 was used as a loading control and detected with anti-Kar2 antibodies. The graph on the right shows the average of four independent experiments; error bars represent the standard deviation.


( B ) The ubiquitination of 3HA - Pgc1 was analyzed in cells with the indicated genotype and expressing myc - tagged ubiquitin . 3HA - Pgc1 was immunoprecipitated using anti - HA and polyubiquitin - conjugated 3HA - Pgc1 was detected using anti - myc antibody . Input fraction corresponds to 2 % of the total amount used for IP .

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(B) The ubiquitination of 3HA-Pgc1 was analyzed in cells with the indicated genotype and expressing myc-tagged ubiquitin. 3HA-Pgc1 was immunoprecipitated using anti-HA and polyubiquitin-conjugated 3HA-Pgc1 was detected using anti-myc antibody. Input fraction corresponds to 2% of the total amount used for IP.


( C ) The degradation of 3HA - Pgc1 was analyzed as in ( A ) in cells bearing the CDC48 temperature - sensitive allele cdc48 - 6 . Cells were grown 2 , 5 hours at 37C prior addition of cycloheximide . Inactivation of Cdc48 mutant protein was confirmed by stabilization of the ERAD substrate Erg1 in the same cells .

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(C) The degradation of 3HA-Pgc1 was analyzed as in (A) in cells bearing the CDC48 temperature-sensitive allele cdc48-6. Cells were grown 2,5 hours at 37C prior addition of cycloheximide. Inactivation of Cdc48 mutant protein was confirmed by stabilization of the ERAD substrate Erg1 in the same cells.


( D ) Membrane association of ubiquitinated 3HA - Pgc1 was analyzed by density gradient centrifugation followed by immunoprecipitation . Cells of the indicated genotype and expressing myc - tagged ubiquitin were grown as in ( C ) . Lysates were subjected to centrifugation on an optiprep density gradient . Collected fractions were analyzed by western blotting before and after immunoprecipitation with anti - HA antibodies . Dpm1 and Pgk1 were used as markers for membrane bound and soluble fractions respectively . Input fraction corresponds to 10 % of the total amount used for IP . " Top " , " mid " , " bottom " indicate the fractions from the gradient . Asterisk marks a faster migrating unspecific band in the input bottom fraction .

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(D) Membrane association of ubiquitinated 3HA-Pgc1 was analyzed by density gradient centrifugation followed by immunoprecipitation. Cells of the indicated genotype and expressing myc-tagged ubiquitin were grown as in (C). Lysates were subjected to centrifugation on an optiprep density gradient. Collected fractions were analyzed by western blotting before and after immunoprecipitation with anti-HA antibodies. Dpm1 and Pgk1 were used as markers for membrane bound and soluble fractions respectively. Input fraction corresponds to 10% of the total amount used for IP. "Top", "mid", "bottom" indicate the fractions from the gradient. Asterisk marks a faster migrating unspecific band in the input bottom fraction.


( A ) Localization of chromosomally expressed GFP - Pgc1 from the constitutive ADH1 promoter in wt and doa10Δ cells . Arrowheads indicate GFP - Pgc1 labeling at the ER , which is stained by Sec63 - Cherry . LDs were visualized upon staining with the neutral lipid dye MDH . On the bottom , box plot with quantitation of GFP - Pgc1 fluorescence intensity at the nuclear envelope in wt and doa10Δ . GFP - Pgc1 measurements were performed as described in the Materials and Methods section . Scale bar : 5 µm .

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

(A) Localization of chromosomally expressed GFP-Pgc1 from the constitutive ADH1 promoter in wt and doa10Δ cells. Arrowheads indicate GFP-Pgc1 labeling at the ER, which is stained by Sec63-Cherry. LDs were visualized upon staining with the neutral lipid dye MDH. On the bottom, box plot with quantitation of GFP-Pgc1 fluorescence intensity at the nuclear envelope in wt and doa10Δ. GFP-Pgc1 measurements were performed as described in the Materials and Methods section. Scale bar: 5 µm.


( C ) Crude membranes from wt and doa10Δ cells expressing 3HA - Pgc1 were subjected to the indicated treatments and subsequently fractionated into membrane pellet ( P ) and supernatant ( S ) . ( D ) Crude membranes from wt and doa10Δ cells expressing 3HA - GFP - Pgc1275 - 321 were analyzed as in ( C ) .

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(C) Crude membranes from wt and doa10Δ cells expressing 3HA-Pgc1 were subjected to the indicated treatments and subsequently fractionated into membrane pellet (P) and supernatant (S).(D) Crude membranes from wt and doa10Δ cells expressing 3HA-GFP-Pgc1275-321 were analyzed as in (C).


( A ) Localization of GFP - Pgc1 in are1Δ are2Δ lro1Δ dga1Δ cells in presence ( no LDs ) or absence of DOA10 ( no LDs doa10Δ ) . The ER was visualized by expression of Sec63 - Cherry . Scale bar : 5 µm .

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(A) Localization of GFP-Pgc1 in are1Δ are2Δ lro1Δ dga1Δ cells in presence (no LDs) or absence of DOA10 (no LDs doa10Δ). The ER was visualized by expression of Sec63-Cherry. Scale bar: 5 µm.


( B ) GFP - Pgc1 was expressed under the ADH1 promoter ; LD formation was stimulated by galactose - induced expression of DGA1 in are1Δare2Δlro1Δ . Fluorescence microscopy was used to follow GFP - Pgc1 localization over time . LDs were visualized upon staining with the neutral lipid dye MDH . Scale bar : 5 µm .

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(B) GFP-Pgc1 was expressed under the ADH1 promoter; LD formation was stimulated by galactose-induced expression of DGA1 in are1Δare2Δlro1Δ. Fluorescence microscopy was used to follow GFP-Pgc1 localization over time. LDs were visualized upon staining with the neutral lipid dye MDH. Scale bar: 5 µm.


( C ) Photo - conversion of tdEOS - Pgc1 expressed under the ADH1 promoter ; LD formation was stimulated by galactose - induced expression of DGA1 in are1Δare2Δlro1Δdoa10Δ cells . The red square marks the photo - converted region . The time ( in minutes ) after photoconversion is indicated . Arrowheads point LDs containing photoconverted tdEOS - Pgc1 . Scale bar 5µm .

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(C) Photo-conversion of tdEOS-Pgc1 expressed under the ADH1 promoter; LD formation was stimulated by galactose-induced expression of DGA1 in are1Δare2Δlro1Δdoa10Δ cells. The red square marks the photo-converted region. The time (in minutes) after photoconversion is indicated. Arrowheads point LDs containing photoconverted tdEOS-Pgc1. Scale bar 5µm.


( D ) FRAP experiment of GFP - Pgc1 in wt and doa10Δcells . Representative examples are shown . The bleached areas are marked by red squares and include a LD adjacent to ER . The time ( in seconds ) after photobleaching is indicated . Each graph shows average fluorescence intensities for 10 cells normalized to pre - bleached plotted over time . Error bars indicate standard deviation .

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

(D) FRAP experiment of GFP-Pgc1 in wt and doa10Δcells. Representative examples are shown. The bleached areas are marked by red squares and include a LD adjacent to ER. The time (in seconds) after photobleaching is indicated. Each graph shows average fluorescence intensities for 10 cells normalized to pre-bleached plotted over time. Error bars indicate standard deviation.


( A ) The degradation of 3HA - Pgc1 was analyzed in cells with the indicated genotype as in Fig . 1A . The graph shows the average of three independent experiments ; error bars represent the standard deviation .

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

(A) The degradation of 3HA-Pgc1 was analyzed in cells with the indicated genotype as in Fig. 1A. The graph shows the average of three independent experiments; error bars represent the standard deviation.


( B ) The degradation of the Doa10 substrates 3HA - Pgc1 and 3HA - Vma12 - Ndc10C ' was analyzed in cells of the indicated genotypes treated with oleic acid . 3HA - Pgc1 and 3HA - Vma12 - Ndc10C ' were detected with anti - HA antibodies . Dpm1 was used as a loading control and detected with anti - Dpm1 antibodies . The graph shows the average of three independent experiments ; error bars represent the standard deviation .

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(B) The degradation of the Doa10 substrates 3HA-Pgc1 and 3HA-Vma12-Ndc10C' was analyzed in cells of the indicated genotypes treated with oleic acid. 3HA-Pgc1 and 3HA-Vma12-Ndc10C' were detected with anti-HA antibodies. Dpm1 was used as a loading control and detected with anti-Dpm1 antibodies. The graph shows the average of three independent experiments; error bars represent the standard deviation.


( A ) Localization of GFP - tagged Pgc1 derivatives in which the hydrophobic hairpin ( aa275 - 321 ) was replaced by membrane anchor of Scs2 ( Pgc1Scs2MA ) or Bos1 ( Pgc1Bos1MA ) . ER and LDs were visualized by expression of Sec63 - Cherry and MDH staining , respectively . Scale bar : 5 µm .

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(A) Localization of GFP-tagged Pgc1 derivatives in which the hydrophobic hairpin (aa275-321) was replaced by membrane anchor of Scs2 (Pgc1Scs2MA) or Bos1 (Pgc1Bos1MA). ER and LDs were visualized by expression of Sec63-Cherry and MDH staining, respectively. Scale bar: 5 µm.


( B ) The degradation of 3HA - Pgc1 or the indicated chimeras in wt and doa10Δ cells was analyzed as in Fig . 1A . The blot is representative of at least three independent experiments .

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(B) The degradation of 3HA-Pgc1 or the indicated chimeras in wt and doa10Δ cells was analyzed as in Fig. 1A. The blot is representative of at least three independent experiments.


( C ) The degradation of 3HA - GFP - Pgc1275 - 321 in wt and doa10Δ cells was analyzed as in Fig . 1A . The graph shows the average of four independent experiments ; error bars represent the standard deviation .

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(C) The degradation of 3HA-GFP-Pgc1275-321 in wt and doa10Δ cells was analyzed as in Fig. 1A. The graph shows the average of four independent experiments; error bars represent the standard deviation.


( D ) Localization of 3HA - GFP - Pgc1275 - 321 in doa10Δ cells . The ER was visualized by expression of Sec63 - Cherry . Scale bar : 5 µm .

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(D) Localization of 3HA-GFP-Pgc1275-321 in doa10Δ cells. The ER was visualized by expression of Sec63-Cherry. Scale bar: 5 µm.


( A ) The degradation of 3HA - Pgc1 - GPAT160 - 216 , containing GPAT4 hydrophobic hairpin ( aa160 - 216 ) replacing the one of Pgc1 was analyzed in cells with the indicated genotype as in Fig . 1A . The graph shows the average of three independent experiments ; error bars represent the standard deviation .

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(A) The degradation of 3HA-Pgc1-GPAT160-216, containing GPAT4 hydrophobic hairpin (aa160-216) replacing the one of Pgc1 was analyzed in cells with the indicated genotype as in Fig. 1A. The graph shows the average of three independent experiments; error bars represent the standard deviation.


( B ) Localization of GFP - Pgc1 - GPAT4160 - 216 in cells with the indicated genotype . Arrowheads indicate GFP - Pgc1 - GPAT4160 - 216 labeling at the ER , marked with Sec63 - Cherry . LDs were visualized upon staining with MDH . Scale bar : 5 µm .

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(B) Localization of GFP-Pgc1-GPAT4160-216 in cells with the indicated genotype. Arrowheads indicate GFP-Pgc1-GPAT4160-216 labeling at the ER, marked with Sec63-Cherry. LDs were visualized upon staining with MDH. Scale bar: 5 µm.


( C ) The degradation of 3HA - Pgc1 - GPAT160 - 216 in cells with the indicated genotype was analyzed as in Fig . 1A . The graph shows the average of three independent experiments ; error bars represent the standard deviation .

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(C) The degradation of 3HA-Pgc1-GPAT160-216 in cells with the indicated genotype was analyzed as in Fig. 1A. The graph shows the average of three independent experiments; error bars represent the standard deviation.


( D ) Localization of GFP - Pgc1 - GPAT160 - 216 in are1Δ are2Δ lro1Δ dga1Δ doa10Δ mutant ( no LDs doa10Δ ) . The ER was visualized by expression of Sec63 - Cherry . Scale bar : 5 µm .

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(D) Localization of GFP-Pgc1-GPAT160-216 in are1Δ are2Δ lro1Δ dga1Δ doa10Δ mutant (no LDs doa10Δ). The ER was visualized by expression of Sec63-Cherry. Scale bar: 5 µm.


A Time course images of the hippocampal CA1 region of Siglechdtr / dtr mice after DT administration . Sections were stained with an anti - Iba1 antibody ( green ) and DAPI ( cyan ) . Arrow : live microglia with normal nucleus . Double arrow : apoptotic microglia with pyknotic nucleus . Arrowhead : microglial debris with no nucleus . Scale bar , 50 µm . B Number of apoptotic microglia with pyknotic or fragmented nucleus ( n = 5 animals per group , Kruskal - Wallis test with post hoc Dunn ' s test ) . C Number of live microglia with normal nucleus ( n = 5 animals per group , one - way ANOVA with post hoc Tukey ' s test ) . D Number of pieces of microglial debris ( Iba1 + spheres with a diameter > 2 µm and no nucleus ) ( n = 5 animals per group , Kruskal - Wallis test with post hoc Dunn ' s test ) .

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A Time course images of the hippocampal CA1 region of Siglechdtr/dtr mice after DT administration. Sections were stained with an anti-Iba1 antibody (green) and DAPI (cyan). Arrow: live microglia with normal nucleus. Double arrow: apoptotic microglia with pyknotic nucleus. Arrowhead: microglial debris with no nucleus. Scale bar, 50 µm. B Number of apoptotic microglia with pyknotic or fragmented nucleus (n = 5 animals per group, Kruskal-Wallis test with post hoc Dunn's test). C Number of live microglia with normal nucleus (n = 5 animals per group, one-way ANOVA with post hoc Tukey's test). D Number of pieces of microglial debris (Iba1+ spheres with a diameter > 2 µm and no nucleus) (n = 5 animals per group, Kruskal-Wallis test with post hoc Dunn's test).


E A representative image of a survived microglial cell in the hippocampal CA1 region of Siglechdtr / dtr mice 2 days after DT administration . Sections were stained with an anti - Iba1 antibody ( green ) and DAPI ( cyan ) . A 3D image ( rightmost panel ) was reconstructed using Imaris software . Arrow : live microglia . Arrowhead : microglial debris . Scale bar , 10 µm .

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E A representative image of a survived microglial cell in the hippocampal CA1 region of Siglechdtr/dtr mice 2 days after DT administration. Sections were stained with an anti-Iba1 antibody (green) and DAPI (cyan). A 3D image (rightmost panel) was reconstructed using Imaris software. Arrow: live microglia. Arrowhead: microglial debris. Scale bar, 10 µm.


A Histological analysis of RFP + cells in Siglechdtr / dtr : Ccr2RFP / + mice . Lumber spinal cord after EAE induction , and hippocampal CA1 2 or 4 days after PBS or DT injection were analyzed . Iba1 immunoreactivity ( green ) and RFP fluorescence ( red ) are shown . Images were acquired using the same laser power and sensitivity , and image processing was the same for all RFP signals ( red ) . Scale bar , 50 µm .

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A Histological analysis of RFP+ cells in Siglechdtr/dtr:Ccr2RFP/+ mice. Lumber spinal cord after EAE induction, and hippocampal CA1 2 or 4 days after PBS or DT injection were analyzed. Iba1 immunoreactivity (green) and RFP fluorescence (red) are shown. Images were acquired using the same laser power and sensitivity, and image processing was the same for all RFP signals (red). Scale bar, 50 µm.


B Flow cytometric analysis of RFP + Ly6Chigh inflammatory monocytes in peripheral blood of Siglechdtr / dtr : Ccr2RFP / + mice 2 days after PBS or DT administration . Representative data and a quantification graph ( n = 5 animals per group , two - tailed unpaired Student ' s t - test ) are shown .

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B Flow cytometric analysis of RFP+Ly6Chigh inflammatory monocytes in peripheral blood of Siglechdtr/dtr:Ccr2RFP/+ mice 2 days after PBS or DT administration. Representative data and a quantification graph (n = 5 animals per group, two-tailed unpaired Student's t-test) are shown.


C Immunohistochemical localization of CD206 + perivascular macrophages ( arrows ) in hippocampal CA1 of Siglechdtr / dtr mice . Sections prepared 2 days after administration of PBS or DT were stained with anti - Iba1 ( green ) , anti - CD206 ( red ) and anti - CD31 ( blue ) antibodies . Scale bar , 50 µm . D Number of perivascular macrophages in hippocampal CA1 of Siglechdtr / dtr mice 2 days after PBS or DT administration ( n = 5 animals per group , two - tailed unpaired Student ' s t - test ) .

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C Immunohistochemical localization of CD206+ perivascular macrophages (arrows) in hippocampal CA1 of Siglechdtr/dtr mice. Sections prepared 2 days after administration of PBS or DT were stained with anti-Iba1 (green), anti-CD206 (red) and anti-CD31 (blue) antibodies. Scale bar, 50 µm. D Number of perivascular macrophages in hippocampal CA1 of Siglechdtr/dtr mice 2 days after PBS or DT administration (n = 5 animals per group, two-tailed unpaired Student's t-test).


A qPCR analysis of marker molecules for CNS cell types . The hippocampus of WT and Siglechdtr / dtr mice 2 days after PBS or DT administration was analyzed ( n = 3 animals per group , one - way ANOVA with post hoc Tukey ' s test ) . Results are normalized to Gapdh , and are shown as ratios to the value of WT mice injected with PBS .

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A qPCR analysis of marker molecules for CNS cell types. The hippocampus of WT and Siglechdtr/dtr mice 2 days after PBS or DT administration was analyzed (n = 3 animals per group, one-way ANOVA with post hoc Tukey's test). Results are normalized to Gapdh, and are shown as ratios to the value of WT mice injected with PBS.


B Immunohistochemical detection of astrocytes in Siglechdtr / dtr mice . Hippocampal CA1 sections were prepared 2 days after PBS or DT administration , and stained with an anti - GFAP antibody . Images were acquired using the same laser power and sensitivity , and image processing was the same . Scale bar , 30 µm . C Astrocyte number in hippocampal CA1 of Siglechdtr / dtr mice 2 days after PBS or DT administration ( n = 5 animals , two - tailed unpaired Student ' s t - test ) .

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B Immunohistochemical detection of astrocytes in Siglechdtr/dtr mice. Hippocampal CA1 sections were prepared 2 days after PBS or DT administration, and stained with an anti-GFAP antibody. Images were acquired using the same laser power and sensitivity, and image processing was the same. Scale bar, 30 µm. C Astrocyte number in hippocampal CA1 of Siglechdtr/dtr mice 2 days after PBS or DT administration (n = 5 animals, two-tailed unpaired Student's t-test).


A Representative images of microglial debris surrounded by astrocyte processes in hippocampal CA1 . Sections were prepared from Siglechdtr / dtr mice 2 days after PBS or DT administration , and stained with anti - GFAP ( green ) and anti - CD11b ( red ) antibodies . 3D images ( lower row ) were reconstructed from confocal images ( upper row ) using Imaris software . Scale bar , 10 µm .

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A Representative images of microglial debris surrounded by astrocyte processes in hippocampal CA1. Sections were prepared from Siglechdtr/dtr mice 2 days after PBS or DT administration, and stained with anti-GFAP (green) and anti-CD11b (red) antibodies. 3D images (lower row) were reconstructed from confocal images (upper row) using Imaris software. Scale bar, 10 µm.


B CLEM analysis of a phagocytic astrocyte in hippocampal CA1 of Siglechdtr / dtr mice 2 days after DT administration . A phagocytic astrocyte , which engulfed degenerated microglial components , was identified by immunohistochemistry using anti - S100β ( green ) and anti - CD11b ( red ) antibodies , and DAPI ( cyan ) ( upper and middle panels ) . Then , a phagocytic cup of the same cell was analyzed by electron microscopy ( lower panel ) . Degenerated microglial cytoplasm / plasma membrane ( arrow ) and nucleus ( double arrow ) in a phagocytic cup of the astrocyte , and a piece of microglial debris internalized in astrocytic cytoplasm ( arrowhead ) are indicated . Scale bar , 50 µm ( upper panel ) , 10 µm ( upper panel [ inset ] and middle panel ) , and 2 µm ( lower panel ) .

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B CLEM analysis of a phagocytic astrocyte in hippocampal CA1 of Siglechdtr/dtr mice 2 days after DT administration. A phagocytic astrocyte, which engulfed degenerated microglial components, was identified by immunohistochemistry using anti-S100β (green) and anti-CD11b (red) antibodies, and DAPI (cyan) (upper and middle panels). Then, a phagocytic cup of the same cell was analyzed by electron microscopy (lower panel). Degenerated microglial cytoplasm/plasma membrane (arrow) and nucleus (double arrow) in a phagocytic cup of the astrocyte, and a piece of microglial debris internalized in astrocytic cytoplasm (arrowhead) are indicated. Scale bar, 50 µm (upper panel), 10 µm (upper panel [inset] and middle panel), and 2 µm (lower panel).


C A representative image of a phagocytic astrocyte in hippocampal CA1 of Siglechdtr / dtr mice 2 days after DT administration . Sections were stained with anti - S100β ( green ) and anti - CD11b ( red ) antibodies , and confocal images were reconstructed using Imaris software . The lower two panels are higher magnification images of contacted microglial debris . Arrow : microglial debris contacted by astrocyte processes ( coverage : approximately 30 or 50 % ) . Arrowhead : microglial debris internalized in astrocyte cytoplasm . Scale bar , 10 µm ( upper panel ) and 2 µm ( lower panels ) . D , E Quantification of the pieces of debris ( CD11b + spheres with > 0 . 5 µm diameter ) contacted with ( coverage > 30 % ) ( D ) and internalized by ( E ) a single astrocyte within 30 µm - thick sections ( n = 75 cells from five animals per group ) . The number of debris pieces was counted in 3D images reconstructed using Imaris software . All confocal images were acquired using the same laser power and sensitivity , and image processing was the same .

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C A representative image of a phagocytic astrocyte in hippocampal CA1 of Siglechdtr/dtr mice 2 days after DT administration. Sections were stained with anti-S100β (green) and anti-CD11b (red) antibodies, and confocal images were reconstructed using Imaris software. The lower two panels are higher magnification images of contacted microglial debris. Arrow: microglial debris contacted by astrocyte processes (coverage: approximately 30 or 50%). Arrowhead: microglial debris internalized in astrocyte cytoplasm. Scale bar, 10 µm (upper panel) and 2 µm (lower panels). D, E Quantification of the pieces of debris (CD11b+ spheres with > 0.5 µm diameter) contacted with (coverage > 30%) (D) and internalized by (E) a single astrocyte within 30 µm-thick sections (n = 75 cells from five animals per group). The number of debris pieces was counted in 3D images reconstructed using Imaris software. All confocal images were acquired using the same laser power and sensitivity, and image processing was the same.


B Expression level of phagocytic receptors and their extracellular adaptor molecules ( n = 3 independent experiments ) . Values show the mean ± SEM . N . S . : no significance ; * * * q < 0 . 001 . The probability values for differential expression analysis were computed with edgeR followed by a correction of multiple testing with q - value .

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B Expression level of phagocytic receptors and their extracellular adaptor molecules (n = 3 independent experiments). Values show the mean ± SEM. N.S.: no significance; ***q < 0.001. The probability values for differential expression analysis were computed with edgeR followed by a correction of multiple testing with q-value.


Cultured astrocytes engulf microglial debris . CAG - EGFP mouse - derived primary astrocytes were co - cultured with or without fluorescence - labelled apoptotic microglia . ( A ) Fluorescence images of GFP ( green ) , microglial debris ( red ) and GFAP immunoreactivity ( blue ) . Images were acquired using the same laser power and sensitivity , and image processing was the same in the two images ( red ) . Scale bar , 50 µm .

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Cultured astrocytes engulf microglial debris. CAG-EGFP mouse-derived primary astrocytes were co-cultured with or without fluorescence-labelled apoptotic microglia. (A) Fluorescence images of GFP (green), microglial debris (red) and GFAP immunoreactivity (blue). Images were acquired using the same laser power and sensitivity, and image processing was the same in the two images (red). Scale bar, 50 µm.


( B ) Fluorescence images of GFP ( green ) , microglial debris ( red ) and LAMP - 1 immunoreactivity ( cyan ) . Scale bar , 30 µm and 5 µm ( inset ) .

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(B) Fluorescence images of GFP (green), microglial debris (red) and LAMP-1 immunoreactivity (cyan). Scale bar, 30 µm and 5 µm (inset).


C Flow cytometric analysis of uptake of microglial debris by cultured astrocytes . Fluorescence of internalized microglial debris was measured in GLAST + astrocytes co - cultured with or without fluorescence - labelled apoptotic microglia .

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C Flow cytometric analysis of uptake of microglial debris by cultured astrocytes. Fluorescence of internalized microglial debris was measured in GLAST+ astrocytes co-cultured with or without fluorescence-labelled apoptotic microglia.


D Expression of TAM family members and their knockdown by siRNA in cultured astrocytes . Tyro3 , Axl and Metk in cultured astrocytes or hippocampal tissue were examined by western blotting . Arrows indicate the corresponding protein band ( s ) . The secondary antibody against goat IgG generates a ~ 115 kDa non - specific band . The ~ 115 kDa band in the Axl image is the Axl - specific band overlapping the non - specific band . The ~ 115 kDa band in the Mertk image is the non - specific band . Axl and Mertk expression was knocked down by two siRNAs . GAPDH expression is shown as an internal control . E Quantification of siRNA knockdown in cultured astrocytes ( n = 3 independent experiments , one - way ANOVA with repeated measures ) . Knockdown of Tyro3 mRNA was examined by qPCR because of the very low expression level even in control astrocytes . Knockdown efficiency of Axl and Mertk was quantified from western blots . Results are normalized to Gapdh or GAPDH , and are shown as ratios to the value of non - transfected astrocytes .

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D Expression of TAM family members and their knockdown by siRNA in cultured astrocytes. Tyro3, Axl and Metk in cultured astrocytes or hippocampal tissue were examined by western blotting. Arrows indicate the corresponding protein band(s). The secondary antibody against goat IgG generates a ~115 kDa non-specific band. The ~115 kDa band in the Axl image is the Axl-specific band overlapping the non-specific band. The ~115 kDa band in the Mertk image is the non-specific band. Axl and Mertk expression was knocked down by two siRNAs. GAPDH expression is shown as an internal control. E Quantification of siRNA knockdown in cultured astrocytes (n = 3 independent experiments, one-way ANOVA with repeated measures). Knockdown of Tyro3 mRNA was examined by qPCR because of the very low expression level even in control astrocytes. Knockdown efficiency of Axl and Mertk was quantified from western blots. Results are normalized to Gapdh or GAPDH, and are shown as ratios to the value of non-transfected astrocytes.


F , G Flow cytometric analysis of microglial debris uptake by cultured astrocytes . After knockdown of TAM family members , astrocytes were co - cultured with fluorescence - labelled apoptotic microglia . Fluorescence of internalized microglial debris in GLAST + astrocytes was quantified by flow cytometry ( n = 4 independent experiments , one - way ANOVA with repeated measures ) . Results are shown as ratios to the value of non - transfected astrocytes .

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F, G Flow cytometric analysis of microglial debris uptake by cultured astrocytes. After knockdown of TAM family members, astrocytes were co-cultured with fluorescence-labelled apoptotic microglia. Fluorescence of internalized microglial debris in GLAST+ astrocytes was quantified by flow cytometry (n = 4 independent experiments, one-way ANOVA with repeated measures). Results are shown as ratios to the value of non-transfected astrocytes.


B Representative images of microglial distribution in the cerebral cortex of WT and mutant mice at 3 w . Sections were stained with anti - Iba1 or anti - CD11b antibodies ( red ) . Scale bar , 100 µm . C Quantification of microglial number ( n = 5 animals per group , one - way ANOVA ) . Note that Irf8 - / - mice were analyzed by CD11b immunostaining , while other strains were analyzed by Iba1 staining .

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B Representative images of microglial distribution in the cerebral cortex of WT and mutant mice at 3 w. Sections were stained with anti-Iba1 or anti-CD11b antibodies (red). Scale bar, 100 µm. C Quantification of microglial number (n = 5 animals per group, one-way ANOVA). Note that Irf8-/- mice were analyzed by CD11b immunostaining, while other strains were analyzed by Iba1 staining.


D Representative images of a spontaneous apoptotic cell and surrounding glial cells in the cerebral cortex of WT and Irf8 - / - mice at 3 w . Sections were stained with anti - GFAP ( green ) , anti - CD11b ( red ) and anti - cleaved caspase - 3 ( blue ) antibodies . 3D images ( lower row ) were reconstructed from confocal images ( upper row ) using Imaris software . Arrows indicate cleaved caspase - 3 + apoptotic cells . Insets are higher magnification images around apoptotic cells . Scale bar , 20 µm and 5 µm ( insets ) . E Percentage of spontaneous apoptotic cells surrounded by microglia in WT and mutant mice . 3D images reconstructed from confocal images were analyzed ( n = 36 - 50 cells from five animals per group , Kruskal - Wallis test with post hoc Dunn ' s test ) . F Percentage of spontaneous apoptotic cells surrounded by astrocyte processes in WT and mutant mice ( n = 36 - 50 cells from five animals per group , one - way ANOVA with post hoc Tukey ' s test ) .

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D Representative images of a spontaneous apoptotic cell and surrounding glial cells in the cerebral cortex of WT and Irf8-/- mice at 3 w. Sections were stained with anti-GFAP (green), anti-CD11b (red) and anti-cleaved caspase-3 (blue) antibodies. 3D images (lower row) were reconstructed from confocal images (upper row) using Imaris software. Arrows indicate cleaved caspase-3+ apoptotic cells. Insets are higher magnification images around apoptotic cells. Scale bar, 20 µm and 5 µm (insets). E Percentage of spontaneous apoptotic cells surrounded by microglia in WT and mutant mice. 3D images reconstructed from confocal images were analyzed (n = 36-50 cells from five animals per group, Kruskal-Wallis test with post hoc Dunn's test). F Percentage of spontaneous apoptotic cells surrounded by astrocyte processes in WT and mutant mice (n = 36-50 cells from five animals per group, one-way ANOVA with post hoc Tukey's test).


D Representative images of a spontaneous apoptotic cell and surrounding glial cells in the cerebral cortex of WT and Irf8 - / - mice at 3 w . Sections were stained with anti - GFAP ( green ) , anti - CD11b ( red ) and anti - cleaved caspase - 3 ( blue ) antibodies . 3D images ( lower row ) were reconstructed from confocal images ( upper row ) using Imaris software . Arrows indicate cleaved caspase - 3 + apoptotic cells . Insets are higher magnification images around apoptotic cells . Scale bar , 20 µm and 5 µm ( insets ) .

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D Representative images of a spontaneous apoptotic cell and surrounding glial cells in the cerebral cortex of WT and Irf8-/- mice at 3 w. Sections were stained with anti-GFAP (green), anti-CD11b (red) and anti-cleaved caspase-3 (blue) antibodies. 3D images (lower row) were reconstructed from confocal images (upper row) using Imaris software. Arrows indicate cleaved caspase-3+ apoptotic cells. Insets are higher magnification images around apoptotic cells. Scale bar, 20 µm and 5 µm (insets).


( C ) pS65 - Ub ( left ) and total Ub ( right ) immunoblots of mitochondria - enriched fractions from wild - type flies treated 3 days with either paraquat or vehicle ( sucrose ) .

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(C) pS65-Ub (left) and total Ub (right) immunoblots of mitochondria-enriched fractions from wild-type flies treated 3 days with either paraquat or vehicle (sucrose).


( D ) Immunoblots for the indicated antibodies following subcellular fractionation of flies treated with paraquat for 3 days . T , total lysate ; N , nuclear - enriched fraction ; M , mitochondria - enriched fraction ; S , post - mitochondrial supernatant .

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(D) Immunoblots for the indicated antibodies following subcellular fractionation of flies treated with paraquat for 3 days. T, total lysate; N, nuclear-enriched fraction; M, mitochondria-enriched fraction; S, post-mitochondrial supernatant.


( E ) Immunoblots for the indicated antibodies of ( T ) total lysates or ( M ) mitochondria - enriched fractions from fly heads ( with proboscises removed ) and bodies ( thoraces and abdomens ) .

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(E) Immunoblots for the indicated antibodies of (T) total lysates or (M) mitochondria-enriched fractions from fly heads (with proboscises removed) and bodies (thoraces and abdomens).