Figure 7 ( A ) Western blottings of GLT - 1a in cortical crude synaptic membranes of P39 WT mice following Cef treatment for 8 days . GLT - 1a levels are significantly increased in mice treated with Cef ( Cef WT , N = 4 ) compared to control saline - injected mice ( Ctr WT , N = 4 ) ( Mann - Withney U test : P = 0 . 028 ) . ( B ) Visualization of GLT - 1a + puncta ( green ) and VGLUT1 + puncta ( red ) in sections from SI of P45 - 46 WT mice that were treated with saline ( Ctr WT ) or Cef ( Cef WT ) . Arrows point to some GLT - 1a / VGLUT1 related puncta . Framed regions ( enlarged below ) are examples of GLT - 1a / VGLUT1 related puncta ( arrowheads ) . Right , Cef treatment increased significantly the size of GLT - 1a + puncta overlaying VGLUT1 ( 142 and 186 GLT - 1a + puncta analyzed from 4 Ctr WT and 4 Cef WT mice , respectively ; 3 sections / animal ) ( Mann - Withney U test : P < 0 . 0001 ) . All microscopic fields are from layers II / III . Scale bars : 3 . 5 µm for left and right panels and 1 µm for enlarged framed areas . ( C ) CSD threshold and CSD velocity in cortical slices from P30 - 33 KI mice that were injected with saline ( Ctr KI , n = 38 ; N = 7 ) or Cef ( Cef KI , n = 31 ; N = 6 ) . CSD threshold is 12 % higher ( Mann - Withney U test : P = 0 . 02 ) in Cef - treated compared to saline - treated KI mice . CSD velocity is not altered by Cef treatment ( P = 0 . 90 ) .

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

Figure 7(A) Western blottings of GLT-1a in cortical crude synaptic membranes of P39 WT mice following Cef treatment for 8 days. GLT-1a levels are significantly increased in mice treated with Cef (Cef WT, N = 4) compared to control saline-injected mice (Ctr WT, N = 4) (Mann-Withney U test: P = 0.028).(B) Visualization of GLT-1a+ puncta (green) and VGLUT1+ puncta (red) in sections from SI of P45-46 WT mice that were treated with saline (Ctr WT) or Cef (Cef WT). Arrows point to some GLT-1a/VGLUT1 related puncta. Framed regions (enlarged below) are examples of GLT-1a/VGLUT1 related puncta (arrowheads). Right, Cef treatment increased significantly the size of GLT-1a+ puncta overlaying VGLUT1 (142 and 186 GLT-1a+ puncta analyzed from 4 Ctr WT and 4 Cef WT mice, respectively; 3 sections/animal) (Mann-Withney U test: P < 0.0001). All microscopic fields are from layers II/III. Scale bars: 3.5 µm for left and right panels and 1 µm for enlarged framed areas.(C) CSD threshold and CSD velocity in cortical slices from P30-33 KI mice that were injected with saline (Ctr KI, n = 38; N=7) or Cef (Cef KI, n = 31; N=6). CSD threshold is 12% higher (Mann-Withney U test: P = 0.02) in Cef-treated compared to saline-treated KI mice. CSD velocity is not altered by Cef treatment (P = 0.90).


Figure 8 ( A ) Distribution of GLT - 1agold particles in PAP of saline - injected ( Ctr KI , N = 4 ) and Cef - treated KI mice ( Cef KI , N = 4 ) ( P45 - 46 ) . Cef treatment does not modify the density of total , cytoplasmic ( arrows ) and membrane - associated ( arrowheads ) gold particles in PAP of KI mice ( cf Table II ) . ( B ) Distribution of GLT - 1agold particles in AxT in SI of Ctr KI and Cef KI KI mice . The density of the membrane - associated gold particles ( arrowheads ) is increased in Cef - treated KI mice ( cf Table II ) . ( C ) Western blottings of GLT - 1a in cortical crude synaptic membranes of P39 KI mice following Cef treatment . GLT - 1a levels are similar in Cef - treated ( Cef KI , N = 4 ) and and saline - injected ( Ctr KI , N = 4 ) ( Mann - Withney U test : P = 0 . 83 ) . ( D ) Visualization of GLT - 1a + puncta ( green ) and VGLUT1 + puncta ( red ) in KI mice that received saline ( Ctr KI ) and in Cef - treated KI mice ( Cef KI ) ( P45 - 46 ) . Arrows point to some GLT - 1a / VGLUT1 related puncta ; framed regions are examples of GLT - 1a / VGLUT1 related puncta ( arrowheads ) . Right , Cef treatment does not increase the size of GLT - 1a + puncta overlaying with VGLUT1 ( 160 and 175 GLT - 1a + puncta analyzed from the same 4 Ctr KI and 4 Cef KI used for post - embedding electron microscopy analysis ; 3 sections / animal ) ( Mann - Withney U test : P = 0 . 31 ) . Scale bar : 3 . 5 µm for left and right panels and 1 µm for enlarged framed areas . All microscopic fields are from layers II / III .

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

Figure 8(A) Distribution of GLT-1agold particles in PAP of saline-injected (Ctr KI, N=4) and Cef-treated KI mice (Cef KI, N=4) (P45-46). Cef treatment does not modify the density of total, cytoplasmic (arrows) and membrane-associated (arrowheads) gold particles in PAP of KI mice (cf Table II).(B) Distribution of GLT-1agold particles in AxT in SI of Ctr KI and Cef KI KI mice. The density of the membrane-associated gold particles (arrowheads) is increased in Cef-treated KI mice (cf Table II).(C) Western blottings of GLT-1a in cortical crude synaptic membranes of P39 KI mice following Cef treatment. GLT-1a levels are similar in Cef-treated (Cef KI, N=4) and and saline-injected (Ctr KI, N=4) (Mann-Withney U test: P = 0.83).(D) Visualization of GLT-1a+ puncta (green) and VGLUT1+ puncta (red) in KI mice that received saline (Ctr KI) and in Cef-treated KI mice (Cef KI) (P45-46). Arrows point to some GLT-1a/VGLUT1 related puncta; framed regions are examples of GLT-1a/VGLUT1 related puncta (arrowheads). Right, Cef treatment does not increase the size of GLT-1a+ puncta overlaying with VGLUT1 (160 and 175 GLT-1a+ puncta analyzed from the same 4 Ctr KI and 4 Cef KI used for post-embedding electron microscopy analysis; 3 sections/animal) (Mann-Withney U test: P = 0.31). Scale bar: 3.5 µm for left and right panels and 1 µm for enlarged framed areas. All microscopic fields are from layers II/III.


Figure 9 ( A ) τdecay of the STC evoked by single pulse stimulation in layer 1 astrocytes in acute cortical slices from P22 - 23 WT mice before ( Ctr WT ) and after application of 2 . 5 µM DL - TBOA ( TBOA 2 . 5 ) ( n = 4 ; N = 2 ) ( right panel ) . The traces on the left are the corresponding average normalized STCs , isolated as in Figure 1C . The STC τdecay in TBOA 2 . 5 is 32 ± 2 % higher than in Ctr WT ( paired t - test : P = 0 . 002 ) . The left top panel shows the time course of τdecay of the transient component ( due to the STC ) of the current recorded in an astrocyte during a representative experiment in which TBOA 2 . 5 was applied at the time indicated by the horizontal bar . The steady - state effect was reached within 10 - 15 minutes from the beginning of the drug perfusion . ( B ) CSD threshold and velocity measured in cortical slices from P22 - 23 WT mice after perfusion for 20 minutes with extracellular solution without ( Ctr WT : n = 23 ; N = 15 ) and with 2 . 5 µM DL - TBOA ( TBOA 2 . 5 : n = 25 ; N = 8 ) . CSD threshold in TBOA 2 . 5 is 36 % lower than in Ctr WT ( Mann - Withney U test test : P < 0 . 0001 ) and CSD velocity is 20 % higher ( unpaired t - test : P < 0 . 0001 ) . ( C ) τdecay of the STC evoked by single pulse stimulation in layer 1 astrocytes in acute cortical slices from P22 - 23 WT mice before ( Ctr WT ) and after application of 1 . 5 µM DL - TBOA ( TBOA 1 . 5 ) ( n = 4 ; N = 4 ) . The STC τdecay in TBOA 1 . 5 is 22 ± 3 % higher than in Ctr WT ( paired t - test : P = 0 . 004 ) . ( D ) CSD threshold and velocity measured in cortical slices from P22 - 23 WT mice after perfusion for 20 minutes with extracellular solution without ( Ctr WT : n = 23 ; N = 15 ) and with 1 . 5 µM DL - TBOA ( TBOA 1 . 5 : n = 18 ; N = 6 ) . CSD threshold in TBOA 1 . 5 is 23 % lower than in Ctr WT ( Mann - Withney U test test : P < 0 . 0001 ) and CSD velocity is 13 % higher ( unpaired t - test : P = 0 . 003 ) .

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

Figure 9(A) τdecay of the STC evoked by single pulse stimulation in layer 1 astrocytes in acute cortical slices from P22-23 WT mice before (Ctr WT) and after application of 2.5 µM DL-TBOA (TBOA 2.5) (n = 4; N=2) (right panel). The traces on the left are the corresponding average normalized STCs, isolated as in Figure 1C. The STC τdecay in TBOA 2.5 is 32 ± 2 % higher than in Ctr WT (paired t-test: P = 0.002). The left top panel shows the time course of τdecay of the transient component (due to the STC) of the current recorded in an astrocyte during a representative experiment in which TBOA 2.5 was applied at the time indicated by the horizontal bar. The steady-state effect was reached within 10-15 minutes from the beginning of the drug perfusion.(B) CSD threshold and velocity measured in cortical slices from P22-23 WT mice after perfusion for 20 minutes with extracellular solution without (Ctr WT: n = 23; N = 15) and with 2.5 µM DL-TBOA (TBOA 2.5: n = 25; N = 8). CSD threshold in TBOA 2.5 is 36 % lower than in Ctr WT (Mann-Withney U test test: P < 0.0001) and CSD velocity is 20% higher (unpaired t-test: P < 0.0001).(C) τdecay of the STC evoked by single pulse stimulation in layer 1 astrocytes in acute cortical slices from P22-23 WT mice before (Ctr WT) and after application of 1.5 µM DL-TBOA (TBOA 1.5) (n = 4; N=4). The STC τdecay in TBOA 1.5 is 22 ± 3 % higher than in Ctr WT (paired t-test: P = 0.004).(D) CSD threshold and velocity measured in cortical slices from P22-23 WT mice after perfusion for 20 minutes with extracellular solution without (Ctr WT: n = 23; N = 15) and with 1.5 µM DL-TBOA (TBOA 1.5: n = 18; N = 6). CSD threshold in TBOA 1.5 is 23 % lower than in Ctr WT (Mann-Withney U test test: P < 0.0001) and CSD velocity is 13 % higher (unpaired t-test: P = 0.003).


Figure 1A . Relative mRNA expression in skeletal muscle from SBMA patients ( white bars , n = 3 ) and controls ( black bars , n = 3 ) ( mean + / − SEM ) . * p < 0 . 05 , * * p < 0 . 01 by Student ' s t test . B . Splicing of XBP1 mRNA was assessed by RT - PCR . Products from unspliced ( XBP1u ) and spliced ( XBP1s ) transcripts were resolved on a nondenaturing polyacrylamide gel and stained with SYBR green . C . Relative mRNA expression in proximal hind limb muscle ( mean + / − SEM ) . Mice evaluated were littermate WT ( n = 6 ) , AR113Q ( n = 6 ) , castrated WT ( C - WT , n = 6 ) and castrated AR113Q males ( C - AR113Q , n = 5 ) on a mixed C57BL / 6J - 129 genetic background . * p < 0 . 05 by ANOVA . D . Western blot of BiP and PDI expression in proximal hind limb muscle . Right panels show quantification of signal relative to loading control ( mean + / − SEM ) . * p < 0 . 05 by Student ' s t test . E . Relative mRNA expression in proximal hind limb muscle of AR21Q ( n = 5 ) and AR113Q ( n = 3 ) males backcrossed to C57BL / 6J . * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t test . F . Relative mRNA expression in spinal cord of AR21Q ( n = 5 ) and AR113Q ( n = 3 ) males ( mean + / − SEM ) . n . s . = not significant by Student ' s t test .

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

Figure 1A. Relative mRNA expression in skeletal muscle from SBMA patients (white bars, n = 3) and controls (black bars, n = 3) (mean +/− SEM). * p<0.05, ** p<0.01 by Student's t test.B. Splicing of XBP1 mRNA was assessed by RT-PCR. Products from unspliced (XBP1u) and spliced (XBP1s) transcripts were resolved on a nondenaturing polyacrylamide gel and stained with SYBR green.C. Relative mRNA expression in proximal hind limb muscle (mean +/− SEM). Mice evaluated were littermate WT (n = 6), AR113Q (n = 6), castrated WT (C-WT, n = 6) and castrated AR113Q males (C-AR113Q, n = 5) on a mixed C57BL/6J-129 genetic background. *p<0.05 by ANOVA.D. Western blot of BiP and PDI expression in proximal hind limb muscle. Right panels show quantification of signal relative to loading control (mean +/− SEM). * p<0.05 by Student's t test.E. Relative mRNA expression in proximal hind limb muscle of AR21Q (n = 5) and AR113Q (n = 3) males backcrossed to C57BL/6J. ** p<0.01, ***p<0.001 by Student's t test. F. Relative mRNA expression in spinal cord of AR21Q (n = 5) and AR113Q (n = 3) males (mean +/− SEM). n. s. = not significant by Student's t test.


Figure 2A . Muscle fiber size ( 100 fibers / mouse ) was quantified from proximal hind limb muscle of AR113Q ( black ) or AR113Q , CHOP − / − mice ( white ) at 12 wks . Left panel shows fiber size distribution , middle panel shows cumulative percent of fibers as a function of fiber area , and right panel shows relative fiber cross sectional area ( mean + / − SEM ) . Left , middle panels , p < 0 . 0001 by Mann - Whitney test . Right panel , p < 0 . 001 by Student ' s t test . B . Representative image of muscle fibers following NADH stain . Bar = 20 µM . C . Distribution of proximal hind limbmuscle fiber size from wt ( black ) and CHOP − / − ( white ) mice at 12 wks . Difference not significant by Mann - Whitney test .

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

Figure 2A. Muscle fiber size (100 fibers/mouse) was quantified from proximal hind limb muscle of AR113Q (black) or AR113Q, CHOP −/− mice (white) at 12 wks. Left panel shows fiber size distribution, middle panel shows cumulative percent of fibers as a function of fiber area, and right panel shows relative fiber cross sectional area (mean +/− SEM). Left, middle panels, p<0.0001 by Mann-Whitney test. Right panel, p<0.001 by Student's t test.B. Representative image of muscle fibers following NADH stain. Bar = 20 µM.C. Distribution of proximal hind limbmuscle fiber size from wt (black) and CHOP −/− (white) mice at 12 wks. Difference not significant by Mann-Whitney test.


Figure 3A . Relative expression of MurRF1 and MAFbx mRNAs in proximal hind limbmuscle of 12 wk mice ( n = 5 - 6 / genotype ) . * p < 0 . 05 by ANOVA , n . s . = not significant . B . LC3 and p62 expression in proximal hind limbmuscle of 12 wk mice was assessed by western blot . Right panels show quantification of signal relative to GAPDH . * * p < 0 . 01 by Student ' s t test . C . Relative expression of mRNAs encoding autophagy regulators in proximal hind limbmuscle . * p < 0 . 05 by Student ' s t test . Androgen receptor protein expression in skeletal muscle of 12 wk mice . Hsp90 serves as a loading control . Right panel shows quantification of relative signal intensity ( n = 3 / genotype ) . D . Proximal hind limb muscles stained for the androgen receptor ( in red ) exhibit intranuclear inclusions . Nuclei are stained by DAPI ( in blue ) . E . P - JNK and P - eIF2 alpha expression ( top , middle ) and XBP1 mRNA splicing ( bottom ) in proximal hind limbmuscle of 12 wk mice . Right panels show quantification of signal relative to loading control . * * p < 0 . 01 by Student ' s t test . n . s . = not significant .

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

Figure 3A. Relative expression of MurRF1 and MAFbx mRNAs in proximal hind limbmuscle of 12 wk mice (n = 5-6/genotype). *p<0.05 by ANOVA, n. s. = not significant.B. LC3 and p62 expression in proximal hind limbmuscle of 12 wk mice was assessed by western blot. Right panels show quantification of signal relative to GAPDH. **p<0.01 by Student's t test.C. Relative expression of mRNAs encoding autophagy regulators in proximal hind limbmuscle. *p<0.05 by Student's t test.Androgen receptor protein expression in skeletal muscle of 12 wk mice. Hsp90 serves as a loading control. Right panel shows quantification of relative signal intensity (n = 3/genotype).D. Proximal hind limb muscles stained for the androgen receptor (in red) exhibit intranuclear inclusions. Nuclei are stained by DAPI (in blue).E. P-JNK and P-eIF2 alpha expression (top, middle) and XBP1 mRNA splicing (bottom) in proximal hind limbmuscle of 12 wk mice. Right panels show quantification of signal relative to loading control. **p<0.01 by Student's t test. n. s. = not significant.


Figure 4Denervated gastrocnemius muscles or contralateral intact controls were harvested at the indicated times following unilateral sciatic nerve transection in 6 wk male mice . A . Western blot shows enhanced eIF2 alpha phosphorylation ( top ) and RT - PCR demonstrates increased XBP1 mRNA splicing ( bottom ) in denervated muscle . Right panels show relative quantification of signal intensity . * p < 0 . 05 by Student ' s t test . . B . Relative expression of BiP , ATF4 and CHOP mRNA ( n = 3 ) . * p < 0 . 05 by Student ' s t test . C . Following surgical denervation of wild type or CHOP − / − mice , LC3 and p62 expression was assessed by western blot . Right panels show quantification of signal relative to GAPDH . * * * p < 0 . 001 by ANOVA , n . s . = not significant . D . Muscle fiber size ( 100 fibers / mouse ) was quantified from wild type ( black , n = 5 ) , CHOP − / − ( white , n = 3 ) and Beclin - 1 + / − ( grey , n = 3 ) mice 7 days post sciatic nerve transection . Shown is relative fiber cross sectional area ( mean + / − SEM ) . * * * p < 0 . 001 by ANOVA . E . Muscle fiber size ( 100 fibers / mouse ) was quantified from CHOP − / − ( black , n = 6 ) or CHOP − / − , Beclin - 1 + / − mice ( white , n = 6 ) 7 days post sciatic nerve transection . Left panel shows fiber size distribution , middle panel shows cumulative percent of fibers as a function of fiber area , and right panel shows relative fiber cross sectional area ( mean + / − SEM ) . Left , middle panels , p < 0 . 0001 by Mann - Whitney test . Right panel , p < 0 . 001 by Student ' s t test .

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

Figure 4Denervated gastrocnemius muscles or contralateral intact controls were harvested at the indicated times following unilateral sciatic nerve transection in 6 wk male mice. A. Western blot shows enhanced eIF2 alpha phosphorylation (top) and RT-PCR demonstrates increased XBP1 mRNA splicing (bottom) in denervated muscle. Right panels show relative quantification of signal intensity. *p<0.05 by Student's t test.. B. Relative expression of BiP, ATF4 and CHOP mRNA (n = 3). *p<0.05 by Student's t test.C. Following surgical denervation of wild type or CHOP−/− mice, LC3 and p62 expression was assessed by western blot. Right panels show quantification of signal relative to GAPDH. ***p<0.001 by ANOVA, n. s. = not significant.D. Muscle fiber size (100 fibers/mouse) was quantified from wild type (black, n = 5), CHOP −/− (white, n = 3) and Beclin-1 +/− (grey, n = 3) mice 7 days post sciatic nerve transection. Shown is relative fiber cross sectional area (mean +/− SEM). ***p<0.001 by ANOVA.E. Muscle fiber size (100 fibers/mouse) was quantified from CHOP −/− (black, n = 6) or CHOP −/−, Beclin-1 +/− mice (white, n = 6) 7 days post sciatic nerve transection. Left panel shows fiber size distribution, middle panel shows cumulative percent of fibers as a function of fiber area, and right panel shows relative fiber cross sectional area (mean +/− SEM). Left, middle panels, p<0.0001 by Mann-Whitney test. Right panel, p<0.001 by Student's t test.


Figure 5A . Muscle fiber size ( 100 fibers / mouse ) was quantified from proximal hind limbmuscle of AR113Q ( red , n = 6 ) or AR113Q , Beclin - 1 + / − ( blue , n = 6 ) mice at 16 wks . Left panel shows fiber size distribution , and right panel shows relative fiber cross sectional area ( mean + / − SEM ) . Left panel , p < 0 . 0001 by Mann - Whitney test . Right panel , p < 0 . 0001 by Student ' s t test . B . AR expression in skeletal muscle of 16 wk mice was assessed by western blot . GAPDH controls for loading . Right panel shows quantification of signal relative to GAPDH ( mean + / − SEM ) . Differences not significant ( n . s . ) .

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Figure 5A. Muscle fiber size (100 fibers/mouse) was quantified from proximal hind limbmuscle of AR113Q (red, n = 6) or AR113Q, Beclin-1 +/− (blue, n = 6) mice at 16 wks. Left panel shows fiber size distribution, and right panel shows relative fiber cross sectional area (mean +/− SEM). Left panel, p<0.0001 by Mann-Whitney test. Right panel, p<0.0001 by Student's t test.B. AR expression in skeletal muscle of 16 wk mice was assessed by western blot. GAPDH controls for loading. Right panel shows quantification of signal relative to GAPDH (mean +/− SEM). Differences not significant (n. s.).


Figure 6A . Left panel , Kaplan - Meyer survival curve of AR113Q males ( red line , n = 12 ) and AR113Q , Beclin - 1 + / − males ( blue line , n = 15 ) . * p < 0 . 05 by log - rank analysis . Right panel , mean survival + / − SEM . * p < 0 . 05 by Student ' s t - test . B , C . Body weight ( panel B ) and grip strength ( panel C ) at different ages for wild type ( wt , green line , n = 7 ) , Beclin - 1 + / − ( yellow line n = 9 ) , AR113Q ( red line , n = 12 ) , and AR113Q , Beclin - 1 + / − ( blue line , n = 15 ) male mice . B , C . Body weight ( panel B ) and grip strength ( panel C ) at different ages for wild type ( wt , green line , n = 7 ) , Beclin - 1 + / − ( yellow line n = 9 ) , AR113Q ( red line , n = 12 ) , and AR113Q , Beclin - 1 + / − ( blue line , n = 15 ) male mice . D . Age of disease onset as measured by a decrease of 5 % or more in forelimb grip strength ( not significant by log rank analysis ) .

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Figure 6A. Left panel, Kaplan-Meyer survival curve of AR113Q males (red line, n = 12) and AR113Q, Beclin-1 +/− males (blue line, n = 15). *p<0.05 by log-rank analysis. Right panel, mean survival +/− SEM. *p<0.05 by Student's t-test.B, C. Body weight (panel B) and grip strength (panel C) at different ages for wild type (wt, green line, n = 7), Beclin-1 +/− (yellow line n = 9), AR113Q (red line, n = 12), and AR113Q, Beclin-1 +/− (blue line, n = 15) male mice.B, C. Body weight (panel B) and grip strength (panel C) at different ages for wild type (wt, green line, n = 7), Beclin-1 +/− (yellow line n = 9), AR113Q (red line, n = 12), and AR113Q, Beclin-1 +/− (blue line, n = 15) male mice.D. Age of disease onset as measured by a decrease of 5% or more in forelimb grip strength (not significant by log rank analysis).


Figure 1 ( A ) The expression of AR was determined using Western blots ( overexpressing AR in J82 cells and knocking down AR in UMUC3 cells ) . ( B ) Relative expression of the top 20 up - regulated circRNAs from the GEO dataset GSE92675 ( BCa tumor tissues compared to para - tumor tissues ) in response to AR modulation in J82 and UMUC3 cells . The # indicates an RNA with a relatively low expression ( qRT - PCR cycle number > 35 ) , thus the circRNA was excluded for further analysis . ( E ) Real - time quantitative PCR ( qRT - PCR ) results show the knock down efficiency for these 4 circRNAs in J82 oe - AR ( J82 - AR ) cells . ( F ) Transwell invasion assays showed that knock down of hsa _ circ _ 0084171 ( circ _ oo84171 ) , but not the other circRNAs , can partly reverse oeAR - increased invasion in J82 cells . Scale bar , 100 μm . ( G ) MTT assays showed that shcirc _ 0084171 can partly reverse oeAR - increased cisplatin chemo - resistance ( 2 μg / ml ) in J82 cells . To calculate the mean , the value of 0 µg / ml cisplatin groups were set as 1 . ( H ) The qRT - PCR results showed that the overexpression ( oe ) efficacy for hsa _ circ _ 0084171 ( oecirc _ 0084171 ) in UMUC3 - shAR cells . ( I ) Transwell invasion assays showed that oecirc _ 0084171 can partly rescue shAR induced invasion deduction in UMUC3 cells . Scale bar , 100 μm . ( J ) MTT assays showed that oecirc _ 0084171 can partly overcome shAR effect for cisplatin chemo - resistance ( 2 μg / ml ) in UMUC3 cells .

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Figure 1(A) The expression of AR was determined using Western blots (overexpressing AR in J82 cells and knocking down AR in UMUC3 cells).(B) Relative expression of the top 20 up-regulated circRNAs from the GEO dataset GSE92675 (BCa tumor tissues compared to para-tumor tissues) in response to AR modulation in J82 and UMUC3 cells. The # indicates an RNA with a relatively low expression (qRT-PCR cycle number > 35), thus the circRNA was excluded for further analysis.(E) Real-time quantitative PCR (qRT-PCR) results show the knock down efficiency for these 4 circRNAs in J82 oe-AR (J82-AR) cells.(F) Transwell invasion assays showed that knock down of hsa_circ_0084171 (circ_oo84171), but not the other circRNAs, can partly reverse oeAR-increased invasion in J82 cells. Scale bar, 100 μm.(G) MTT assays showed that shcirc_0084171 can partly reverse oeAR-increased cisplatin chemo-resistance (2 μg/ml) in J82 cells. To calculate the mean, the value of 0 µg/ml cisplatin groups were set as 1.(H) The qRT-PCR results showed that the overexpression (oe) efficacy for hsa_circ_0084171 (oecirc_0084171) in UMUC3-shAR cells.(I) Transwell invasion assays showed that oecirc_0084171 can partly rescue shAR induced invasion deduction in UMUC3 cells. Scale bar, 100 μm.(J) MTT assays showed that oecirc_0084171 can partly overcome shAR effect for cisplatin chemo-resistance (2 μg/ml) in UMUC3 cells.


Figure 2B ) The relative expression of circFNTA was elevated in multiple BCa cell lines ( T24 , J82 , UMUC3 , and 5637 ) compared to normal bladder epithelial cell line SVHUC as shown by qRT - PCR . C ) The expression of circFNTA and GAPDH mRNA in J82 and UMUC3 cells treated with or without RNase R was detected by qRT - PCR . D ) RNA Fluorescence in situ hybridization ( FISH ) demonstrated that circFNTA was predominantly localized to the cytoplasm of J82 and UMUC3 cells . DAPI was mainly localized to the cytoplasm . Scale bar , 20 μm . E ) Two shcircFNTA plasmids using PLKO . 1 vector were transfected into UMUC3 cells . The knock down efficacy was measured by qRT - PCR . G ) The qRT - PCR results show oecircFNTA efficacy in J82 cells compared with PWPI vector ( left panel ) and oecircFNTA or oelinearFNTA in J82 cells ( right panel ) . Transwell invasion assays showed that shcircFNTA can decrease UMUC3 cell invasion ( H ) more profoundly than oelinearFNTA as compared with the oevector group . Scale bar , 100 μm . Transwell invasion assays showed that shcircFNTA can increase J82 cells invasion ( I ) more profoundly than oelinearFNTA as compared with the oevector group . Scale bar , 100 μm . J ) MTT assays showed that oecircFNTA enhances J82 cells chemo - resistance to cisplatin ( 0 , 1 , 2 , 5 , 10 μg / ml were tested ) . This effect was more obvious in the oecircFNTA group as in the oelinearFNTA group ( upper panel ) . MTT assays showed that shcircFNTA increases UMUC3 cells chemo - sensitivity to cisplatin ( lower panel ) . K ) FISH analyses revealed that circFNTA was significantly upregulated in BCa tissues as compared with the adjacent para - tumor tissues ( N = 41 patients ) and representative images are shown . Scale bar , 250 μm . L ) The qRT - PCR assays also demonstrated that circFNTA is significantly elevated in fresh BCa tissues compared to para - tumor tissues ( N = 23 patients ) .

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Figure 2B) The relative expression of circFNTA was elevated in multiple BCa cell lines (T24, J82, UMUC3, and 5637) compared to normal bladder epithelial cell line SVHUC as shown by qRT-PCR.C) The expression of circFNTA and GAPDH mRNA in J82 and UMUC3 cells treated with or without RNase R was detected by qRT-PCR.D) RNA Fluorescence in situ hybridization (FISH) demonstrated that circFNTA was predominantly localized to the cytoplasm of J82 and UMUC3 cells. DAPI was mainly localized to the cytoplasm. Scale bar, 20 μm.E) Two shcircFNTA plasmids using PLKO.1 vector were transfected into UMUC3 cells. The knock down efficacy was measured by qRT-PCR.G) The qRT-PCR results show oecircFNTA efficacy in J82 cells compared with PWPI vector (left panel) and oecircFNTA or oelinearFNTA in J82 cells (right panel).Transwell invasion assays showed that shcircFNTA can decrease UMUC3 cell invasion (H) more profoundly than oelinearFNTA as compared with the oevector group. Scale bar, 100 μm.Transwell invasion assays showed that shcircFNTA can increase J82 cells invasion (I) more profoundly than oelinearFNTA as compared with the oevector group. Scale bar, 100 μm.J) MTT assays showed that oecircFNTA enhances J82 cells chemo-resistance to cisplatin (0, 1, 2, 5, 10 μg/ml were tested). This effect was more obvious in the oecircFNTA group as in the oelinearFNTA group (upper panel). MTT assays showed that shcircFNTA increases UMUC3 cells chemo-sensitivity to cisplatin (lower panel).K) FISH analyses revealed that circFNTA was significantly upregulated in BCa tissues as compared with the adjacent para-tumor tissues (N=41 patients) and representative images are shown. Scale bar, 250 μm.L) The qRT-PCR assays also demonstrated that circFNTA is significantly elevated in fresh BCa tissues compared to para-tumor tissues (N=23 patients).


Figure 3 ( A ) The protein expression of AR and FNTA ( circFNTA host gene ) were determined using Western blot and showed that shAR can decrease FNTA protein expression in UMUC3 cells and oeAR can increase FNTA protein expression in J82 cells . ( B ) qRT - PCR results showed that mRNA levels of FNTA are not changed dramatically in UMUC3 cells with shAR and J82 cells with oeAR . ( C ) The mRNA levels of 4 RNA editing genes ( ADAR , ADAR2 , DHX9 , and QKI ) were tested with manipulated AR ( UMUC3 cells in left panel and J82 cells in right panel ) . Results showed ADAR2 mRNA is negatively correlated with AR expression . ( D ) Western blot analysis also confirmed that ADAR2 can be negatively regulated by AR at the protein level . ( E ) Knock down efficiency of AR and ADAR2 ( shADAR2 # 1 and shADAR # 2 ) in UMUC3 cells , as determined by qRT - PCR . ( F ) shADAR2 # 1 and shADAR # 2 could both reverse shAR - dependent circFNTA reduction in UMUC3 cells . ( G ) Transwell invasion assays confirmed that both shADAR2 constructs can partly reverse shAR - dependent UMUC3 cells invasion reduction . Scale bar , 100 μm . ( H ) MTT assays confirmed that both shADAR2 constructs can partly overcome shAR - dependent effects on cisplatin chemo - resistance ( 2 μg / ml ) in UMUC3 cells . ( J ) ChIP assays confirmed that AR can directly bind to ADAR2 AREI / II ( - 572nt to - 555nt ) . ( K ) ChIP pull - down DNA products were amplified by qRT - PCR reaction . The IgG antibody was used as a negative control . ( M ) Co - transfection of ARE Wild type ( WT ) or Mutant ( Mut ) ADAR2 promoter pGL3 - Luciferase constructs into UMUC3 cells with / without shAR ( left panel ) , and J82 cells with / without oeAR ( right panel ) . The luciferase assay was used to detect promoter activity .

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Figure 3(A) The protein expression of AR and FNTA (circFNTA host gene) were determined using Western blot and showed that shAR can decrease FNTA protein expression in UMUC3 cells and oeAR can increase FNTA protein expression in J82 cells.(B) qRT-PCR results showed that mRNA levels of FNTA are not changed dramatically in UMUC3 cells with shAR and J82 cells with oeAR.(C) The mRNA levels of 4 RNA editing genes (ADAR, ADAR2, DHX9, and QKI) were tested with manipulated AR (UMUC3 cells in left panel and J82 cells in right panel). Results showed ADAR2 mRNA is negatively correlated with AR expression.(D) Western blot analysis also confirmed that ADAR2 can be negatively regulated by AR at the protein level.(E) Knock down efficiency of AR and ADAR2 (shADAR2#1 and shADAR#2) in UMUC3 cells, as determined by qRT-PCR.(F) shADAR2#1 and shADAR#2 could both reverse shAR-dependent circFNTA reduction in UMUC3 cells.(G) Transwell invasion assays confirmed that both shADAR2 constructs can partly reverse shAR-dependent UMUC3 cells invasion reduction. Scale bar, 100 μm.(H) MTT assays confirmed that both shADAR2 constructs can partly overcome shAR-dependent effects on cisplatin chemo-resistance (2 μg/ml) in UMUC3 cells.(J) ChIP assays confirmed that AR can directly bind to ADAR2 AREI/II (-572nt to -555nt).(K) ChIP pull-down DNA products were amplified by qRT-PCR reaction. The IgG antibody was used as a negative control.(M) Co-transfection of ARE Wild type (WT) or Mutant (Mut) ADAR2 promoter pGL3-Luciferase constructs into UMUC3 cells with/without shAR (left panel), and J82 cells with/without oeAR (right panel). The luciferase assay was used to detect promoter activity.


Figure 4 ( A ) Western blot analysis showed that shcircFNTA can decrease FNTA expression in UMUC3 cells ( upper panel ) and oecircFNTA can increase FNTA expression , and that this was more profound than using oelinearFNTA in J82 cells ( lower panel ) . ( B ) A RNA pull - down assay was used to test the binding of circFNTA to 11 miRNAs ( predicted by online database ) in UMUC3 cells . The results showed that miR - 328 - 5p , miR - 370 - 3p and miR - 920 can be pulled - down by circFNTA . ( C ) Relative miRNA expressions were quantified by qRT - PCR to test miRNA overexpression efficacy . Transwell invasion assay ( D ) confirmed that only oemiR - 370 - 3p can reverse oecircFNTA induced invasion in J82 cells , MTT assays ( E ) confirmed that oemiR - 370 - 3p can reverse oecircFNTA induced cisplatin chemo - resistance ( 2 μg / ml ) in J82 cells . Scale bar , 100 μm . Transducing the miR - 370 - 3p inhibitor into UMUC3 cells can reverse FNTA protein reduction by shcircFNTA shown by Western blot ( F ) Transducing the miR - 370 - 3p inhibitor into UMUC3 cells can reverse FNTA protein reduction by shcircFNTA shown by Transwell assay ( G ) . ( H ) MTT assays also confirmed that transducing with a miR - 370 - 3p inhibitor in UMUC3 cells can partly reverse cisplatin chemo - resistance ( 2 μg / ml ) reduction by shcircFNTA . ( J ) The transfection efficacy of oecircFNTA WT and oecircFNTA Mut was tested by qRT - PCR . ( K ) Transfection with oecircFNTA WT could increase J82 cells invasion , while oecircFNTA Mut cannot . Scale bar , 100 μm . ( L ) Tramsfection with oecircFNTA WT could increase J82 cells cisplatin chemo - resistance ( 2 μg / ml ) , while oecircFNTA Mut did not have this effect . ( N ) Luciferase reporter activity after transfection of WT and Mut FNTA 3 ' UTR reporter constructs into UMUC3 cells ( left panel ) , comparing transduced miR - 370 - 3p inhibitor vs Ctrl and J82 cells ( right panel ) , and comparing oemiR - 370 - 3p vs control PLV .

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Figure 4(A) Western blot analysis showed that shcircFNTA can decrease FNTA expression in UMUC3 cells (upper panel) and oecircFNTA can increase FNTA expression, and that this was more profound than using oelinearFNTA in J82 cells (lower panel).(B) A RNA pull-down assay was used to test the binding of circFNTA to 11 miRNAs (predicted by online database) in UMUC3 cells. The results showed that miR-328-5p, miR-370-3p and miR-920 can be pulled-down by circFNTA.(C) Relative miRNA expressions were quantified by qRT-PCR to test miRNA overexpression efficacy.Transwell invasion assay (D) confirmed that only oemiR-370-3p can reverse oecircFNTA induced invasion in J82 cells,MTT assays (E) confirmed that oemiR-370-3p can reverse oecircFNTA induced cisplatin chemo-resistance (2 μg/ml) in J82 cells. Scale bar, 100 μm.Transducing the miR-370-3p inhibitor into UMUC3 cells can reverse FNTA protein reduction by shcircFNTA shown by Western blot (F)Transducing the miR-370-3p inhibitor into UMUC3 cells can reverse FNTA protein reduction by shcircFNTA shown by Transwell assay (G).(H) MTT assays also confirmed that transducing with a miR-370-3p inhibitor in UMUC3 cells can partly reverse cisplatin chemo-resistance (2 μg/ml) reduction by shcircFNTA.(J) The transfection efficacy of oecircFNTA WT and oecircFNTA Mut was tested by qRT-PCR.(K) Transfection with oecircFNTA WT could increase J82 cells invasion, while oecircFNTA Mut cannot. Scale bar, 100 μm.(L) Tramsfection with oecircFNTA WT could increase J82 cells cisplatin chemo-resistance (2 μg/ml), while oecircFNTA Mut did not have this effect.(N) Luciferase reporter activity after transfection of WT and Mut FNTA 3'UTR reporter constructs into UMUC3 cells (left panel), comparing transduced miR-370-3p inhibitor vs Ctrl and J82 cells (right panel), and comparing oemiR-370-3p vs control PLV.


Figure 5 ( A ) Western blots show the knock - down efficiency of shFNTA in J82 and UMUC3 cells . ( B ) Transwell assays indicated that shFNTA can partly reverse oecircFNTA - increased invasion in J82 and UMUC3 cells . Quantifications are shown on the right . Scale bar , 100 μm . ( C ) MTT assays also confirmed that shFNTA can reverse oecircFNTA - increased chemo - resistance to cisplatin ( 2 μg / ml ) in J82 and UMUC3 cells . Transwell assays ( D , with quantitations at the right ) indicated that treatment with Tipifarnib ( farnesyltransferase inhibitor ) can partly reverse oecircFNTA - increased invasion in J82 and UMUC3 cells . Scale bar , 100 μm . MTT assays ( E ) indicated that treatment with Tipifarnib ( farnesyltransferase inhibitor ) can partly reverse oecircFNTA - increased invasion in J82 and UMUC3 cells . Scale bar , 100 μm . ( F ) Western blots showed that shcircFNTA in UMUC3 or oecircFNTA in J82 cells did not change KRAS protein level significantly . ( G ) Raf - 1 RBD GST - pull - down assays confirmed that shcircFNTA can decease KRAS activity in UMUC3 cells ( upper panel ) , and oecircFNTA can increase KRAS activity in J82 cells ( lower panel ) . ( H ) Western blots showed that shcircFNTA can decrease ERK1 / 2 and MEK1 / 2 phosphorylation in UMUC3 cells ( left panel ) , and oecircFNTA can increase their phosphorylation in J82 cells ( right panel ) .

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Figure 5(A) Western blots show the knock-down efficiency of shFNTA in J82 and UMUC3 cells.(B) Transwell assays indicated that shFNTA can partly reverse oecircFNTA-increased invasion in J82 and UMUC3 cells. Quantifications are shown on the right. Scale bar, 100 μm.(C) MTT assays also confirmed that shFNTA can reverse oecircFNTA-increased chemo-resistance to cisplatin (2 μg/ml) in J82 and UMUC3 cells.Transwell assays (D, with quantitations at the right) indicated that treatment with Tipifarnib (farnesyltransferase inhibitor) can partly reverse oecircFNTA-increased invasion in J82 and UMUC3 cells. Scale bar, 100 μm.MTT assays (E) indicated that treatment with Tipifarnib (farnesyltransferase inhibitor) can partly reverse oecircFNTA-increased invasion in J82 and UMUC3 cells. Scale bar, 100 μm.(F) Western blots showed that shcircFNTA in UMUC3 or oecircFNTA in J82 cells did not change KRAS protein level significantly.(G) Raf-1 RBD GST-pull-down assays confirmed that shcircFNTA can decease KRAS activity in UMUC3 cells (upper panel), and oecircFNTA can increase KRAS activity in J82 cells (lower panel).(H) Western blots showed that shcircFNTA can decrease ERK1/2 and MEK1/2 phosphorylation in UMUC3 cells (left panel), and oecircFNTA can increase their phosphorylation in J82 cells (right panel).


Figure 6 ( A ) UMUC3 - PLKO and UMUC3 - shcircFNTA cells were used to establish a subcutaneous nude mouse xenograft model , and cisplatin ( 1 mg / kg ) was intraperitoneally administered . Images of tumors are shown after mice were sacrificed ( N = 6 ) . ( B ) Compared with the vector ( PLKO ) group , the tumor growth rate was significantly inhibited in shcircFNTA nude mice . ( C ) Tumor weights also decreased in the shcircFNTA group . ( D - E ) The qRT - PCR results using mouse tumor tissues revealed that circFNTA expression was significantly reduced ( D ) , and the expression of its host gene FNTA was correspondently decreased in the shcircFNTA group as compared to the PLKO group ( E ) . ( F ) Representative immunohistochemistry ( IHC ) images detecting FNTA , p - ERK1 / 2 and p - MEK1 / 2 in PLKO tumurs as compared to shcircFNTA xenografted tumors . Scale bars : 100 μm ( upper panels ) and 25 μm ( lower panels ) . ( G ) Quantification of relative IHC staining intensity revealed that targeting circFNTA led to decreased expression of FNTA , p - ERK1 / 2 and p - MEK1 / 2 . ( H ) The tumor tail vein injection model was established to observe lung metastasis ( N = 6 ) . Representative images of lung metastasis foci in gross and microscopic HE examination . Scale bars : 400 μm ( upper panels ) and 100 μm ( lower panels ) . ( I ) Knocking down circFNTA significantly decreased metastatic foci numbers as compared to the PLKO group . ( J ) Targeting circFNTA significantly reduced lung metastatic foci as compared to the PLKO group .

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

Figure 6(A) UMUC3-PLKO and UMUC3-shcircFNTA cells were used to establish a subcutaneous nude mouse xenograft model, and cisplatin (1 mg/kg) was intraperitoneally administered. Images of tumors are shown after mice were sacrificed (N= 6). (B) Compared with the vector (PLKO) group, the tumor growth rate was significantly inhibited in shcircFNTA nude mice. (C) Tumor weights also decreased in the shcircFNTA group. (D-E) The qRT-PCR results using mouse tumor tissues revealed that circFNTA expression was significantly reduced (D), and the expression of its host gene FNTA was correspondently decreased in the shcircFNTA group as compared to the PLKO group (E).(F) Representative immunohistochemistry (IHC) images detecting FNTA, p-ERK1/2 and p-MEK1/2 in PLKO tumurs as compared to shcircFNTA xenografted tumors. Scale bars: 100 μm (upper panels) and 25 μm (lower panels). (G) Quantification of relative IHC staining intensity revealed that targeting circFNTA led to decreased expression of FNTA, p-ERK1/2 and p-MEK1/2. (H) The tumor tail vein injection model was established to observe lung metastasis (N=6). Representative images of lung metastasis foci in gross and microscopic HE examination. Scale bars: 400 μm (upper panels) and 100 μm (lower panels). (I) Knocking down circFNTA significantly decreased metastatic foci numbers as compared to the PLKO group. (J) Targeting circFNTA significantly reduced lung metastatic foci as compared to the PLKO group.


Figure 1A GS ∆ 44 myoblasts were transfected with an empty vector ( CTRL ) or with a CELF2a overexpressing construct ( OE CELF2A ) ( Martone et al , 2016 ) . Cells were collected after 10 days in differentiation medium ( DM ) and the RNA was analysed by RT - PCR for CELF2a expression and DMD exon 45 skipping . GAPDH was used as loading control . The arrow indicates the DMD exon 45 skipped band . Representative results are shown ( n = 3 ) . B Representative immunofluorescence for Myosin Heavy Chain ( MHC in red ) in combination with DAPI staining ( in blue ) of iPSCs obtained from WT ( WT # 1 ) , UP ∆ 44 ( UP ∆ 44 # 3 ) and ∆ 44 edited clones ( UP ∆ 44 # 3 . 1 and UP ∆ 44 # 3 . 5 ) differentiated for 9 days into myocytes by MYOD / BAF60c overexpression . Scale bar 100 μm . C Histogram represents fusion index quantification . At least 7 randomly chosen microscope fields of two independent biological samples were analysed ( n = 2 ) . Data are presented as mean ± SD of the biological replicates . D RNA extracted from myocytes obtained by differentiation of WT ( WT # 1 ) , UP ∆ 44 ( UP ∆ 44 # 3 ) and edited UP ∆ 44 iPSC ( UP ∆ 44 # 3 . 1 ∆ CELF2a and UP ∆ 44 # 3 . 5 ∆ CELF2a ) clones was analysed by RT - PCR for CELF2a , CELF2b , CELF2c and DMD exon 45 skipping . GAPDH was used as loading control . The arrow indicates the DMD exon 45 skipped band . Representative results are shown ( n = 3 ) . E Western blot on proteins ( 40 μg ) extracted from myocytes obtained by differentiation of WT ( WT # 1 ) , UP ∆ 44 ( UP ∆ 44 # 3 ) and edited UP ∆ 44 iPSC ( UP ∆ 44 # 3 . 1 ∆ CELF2a and UP ∆ 44 # 3 . 5 ∆ CELF2a ) clones probed with antibodies against dystrophin ( DMD ) . Actinin ( ACTN ) was used as a loading control . For the WT 5 % sample 2 μg of proteins were diluted in 38 μg of proteins belonging to UP ∆ 44 # 3 to reach a total amount of 40 μg . Representative results are shown ( n = 3 ) .

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

Figure 1A GS∆44 myoblasts were transfected with an empty vector (CTRL) or with a CELF2a overexpressing construct (OE CELF2A) (Martone et al, 2016). Cells were collected after 10 days in differentiation medium (DM) and the RNA was analysed by RT-PCR for CELF2a expression and DMD exon 45 skipping. GAPDH was used as loading control. The arrow indicates the DMD exon 45 skipped band. Representative results are shown (n=3).B Representative immunofluorescence for Myosin Heavy Chain (MHC in red) in combination with DAPI staining (in blue) of iPSCs obtained from WT (WT#1), UP∆44 (UP∆44#3) and ∆44 edited clones (UP∆44#3.1 and UP∆44#3.5) differentiated for 9 days into myocytes by MYOD/BAF60c overexpression. Scale bar 100 μm.C Histogram represents fusion index quantification. At least 7 randomly chosen microscope fields of two independent biological samples were analysed (n=2). Data are presented as mean ± SD of the biological replicates.D RNA extracted from myocytes obtained by differentiation of WT (WT#1), UP∆44 (UP∆44#3) and edited UP∆44 iPSC (UP∆44#3.1 ∆CELF2a and UP∆44#3.5 ∆CELF2a) clones was analysed by RT-PCR for CELF2a, CELF2b, CELF2c and DMD exon 45 skipping. GAPDH was used as loading control. The arrow indicates the DMD exon 45 skipped band. Representative results are shown (n=3).E Western blot on proteins (40 μg) extracted from myocytes obtained by differentiation of WT (WT#1), UP∆44 (UP∆44#3) and edited UP∆44 iPSC (UP∆44#3.1 ∆CELF2a and UP∆44#3.5 ∆CELF2a) clones probed with antibodies against dystrophin (DMD). Actinin (ACTN) was used as a loading control. For the WT 5% sample 2 μg of proteins were diluted in 38 μg of proteins belonging to UP∆44#3 to reach a total amount of 40 μg. Representative results are shown (n=3).


Figure 2A iPSCs obtained from a control ( WT # 1 ; ( Lenzi et al , 2015 ) , GS ∆ 44 ( GS ∆ 44 # 2 , GS ∆ 44 # 8 ) and GSM ( GSM # 1 ) were differentiated into myocytes by MYOD / BAF60c overexpression . Cells were collected before the induction ( iPSC ) and after 9 days in DM ( Myo ) and RNA was analysed by RT - PCR for CELF2a , CELF2b , CELF2c and DMD expression . GAPDH was used as loading control . Representative results are shown ( n = 3 ) . B Representative immunofluorescence for Myosin Heavy Chain ( MHC in red ) in combination with DAPI staining ( in blue ) of iPSCs obtained from WT # 1 , GS ∆ 44 ( GS ∆ 44 # 8 ) and GS ∆ 44 edited clones ( GS ∆ 44 # 8 . 2 ) differentiated for 9 days into myocytes by MYOD / BAF60c overexpression . Scale bar 100 μm . C Histogram represents fusion index quantification . At least 7 randomly chosen microscope fields of two independent biological samples were analysed ( n = 2 ) . Data are presented as mean ± SD of the biological replicates . D RNA extracted from myocytes obtained by differentiation of WT # 1 , GS ∆ 44 ( GS ∆ 44 # 8 ) and GS ∆ 44 edited clones ( GS ∆ 44 # 8 . 2 ∆ CELF2a ) was analysed by RT - PCR for CELF2a , CELF2b , CELF2c and DMD exon 45 skipping . GAPDH was used as loading control . The arrow indicates the DMD exon 45 skipped band . Representative results are shown ( n = 3 ) . E Western blot on proteins ( 40 μg ) extracted from myocytes obtained by differentiation of WT ( WT # 1 ) , GS ∆ 44 ( GS ∆ 44 # 8 ) and edited GS ∆ 44 ( GS ∆ 44 # 8 . 2 ∆ CELF2a ) iPSC clones probed with antibodies against dystrophin ( DMD ) . Actinin ( ACTN ) was used as a loading control . For the WT 5 % sample , 2 μg of proteins were diluted in 38 μg of proteins belonging from GS ∆ 44 # 8 to reach the total amount of 40 μg . Representative results are shown ( n = 3 ) .

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

Figure 2A iPSCs obtained from a control (WT#1; (Lenzi et al, 2015), GS∆44 (GS∆44#2, GS∆44#8) and GSM (GSM#1) were differentiated into myocytes by MYOD/BAF60c overexpression. Cells were collected before the induction (iPSC) and after 9 days in DM (Myo) and RNA was analysed by RT-PCR for CELF2a, CELF2b, CELF2c and DMD expression. GAPDH was used as loading control. Representative results are shown (n=3).B Representative immunofluorescence for Myosin Heavy Chain (MHC in red) in combination with DAPI staining (in blue) of iPSCs obtained from WT#1, GS∆44 (GS∆44#8) and GS∆44 edited clones (GS∆44#8.2) differentiated for 9 days into myocytes by MYOD/BAF60c overexpression. Scale bar 100 μm. C Histogram represents fusion index quantification. At least 7 randomly chosen microscope fields of two independent biological samples were analysed (n=2). Data are presented as mean ± SD of the biological replicates.D RNA extracted from myocytes obtained by differentiation of WT#1, GS∆44 (GS∆44#8) and GS∆44 edited clones (GS∆44#8.2 ∆CELF2a) was analysed by RT-PCR for CELF2a, CELF2b, CELF2c and DMD exon 45 skipping. GAPDH was used as loading control. The arrow indicates the DMD exon 45 skipped band. Representative results are shown (n=3).E Western blot on proteins (40 μg) extracted from myocytes obtained by differentiation of WT (WT#1), GS∆44 (GS∆44#8) and edited GS∆44 (GS∆44#8.2 ∆CELF2a) iPSC clones probed with antibodies against dystrophin (DMD). Actinin (ACTN) was used as a loading control. For the WT 5% sample, 2 μg of proteins were diluted in 38 μg of proteins belonging from GS∆44#8 to reach the total amount of 40 μg. Representative results are shown (n=3).


Figure 3A DNA accessibility ( ATAC - sequencing ) and chromatin marks ( ChIP - seq ) signals in the Celf2 region ( + / - 50kb ) using the UCSC genome browser . ATAC - seq peaks called in the two conditions are reported below each track . The differential peaks specific for one of the two conditions are reported in the tracks ATACseq _ specific peaks _ WT and ATACseq _ specific peaks _ GS ∆ 44 . ChIP - seq signals for H3K4me3 , H3K27ac and H3K27me3 in WT and GSΔ44 myoblasts ( WT myoblasts in light orange ; GSΔ44 myoblasts in light blue ) . ChIP - seq for MyoD in control cells was obtained from ( MacQuarrie et al , 2013 ) ( GSM1218849 ) . Orange boxes highlight the regions containing the first exon of each isoform ( a , b and c ) . D Chromatins extracted from Control ( WT ) and GS ∆ 44 myoblasts were immunoprecipitated with anti - MyoD and control IgG antibodies . Regions corresponding to the MyoD binding sites present in Peak A ( see Figure EV3D ) and in the MAFA and LSTZ2 genes were analysed by qPCR . Fold enrichments of PeakA over MAFA or LSTZ2 immunoprecipitation ( IP ) are plotted for WT and GS ∆ 44 samples . The mean ± SEM of triplicates from one representative among three experiment is shown . Statistical significance of differences between means was assessed by a two - tailed t - test and P < 0 . 05 was considered significant . * P = 0 . 0339 , * * P = 0 . 0067 .

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

Figure 3A DNA accessibility (ATAC-sequencing) and chromatin marks (ChIP-seq) signals in the Celf2 region (+/- 50kb) using the UCSC genome browser. ATAC-seq peaks called in the two conditions are reported below each track. The differential peaks specific for one of the two conditions are reported in the tracks ATACseq_specific peaks_WT and ATACseq_specific peaks_GS∆44. ChIP-seq signals for H3K4me3, H3K27ac and H3K27me3 in WT and GSΔ44 myoblasts (WT myoblasts in light orange; GSΔ44 myoblasts in light blue). ChIP-seq for MyoD in control cells was obtained from (MacQuarrie et al, 2013) (GSM1218849). Orange boxes highlight the regions containing the first exon of each isoform (a, b and c).D Chromatins extracted from Control (WT) and GS∆44 myoblasts were immunoprecipitated with anti-MyoD and control IgG antibodies. Regions corresponding to the MyoD binding sites present in Peak A (see Figure EV3D) and in the MAFA and LSTZ2 genes were analysed by qPCR. Fold enrichments of PeakA over MAFA or LSTZ2 immunoprecipitation (IP) are plotted for WT and GS∆44 samples. The mean ± SEM of triplicates from one representative among three experiment is shown. Statistical significance of differences between means was assessed by a two-tailed t-test and P<0.05 was considered significant. *P=0.0339, **P=0.0067.


Figure 4B RT - PCR analyses of indicated RNAs in control ( WT ) , ∆ 44 and in GS ∆ 44 myocytes collected after 9 days upon the induction of differentiation . GAPDH was used as control . Representative results are shown ( n = 3 ) . C RT - PCR analyses for CELF2a and DUXAP8 expression in WT and GS ∆ 44 myoblasts ( GM ) and myocytes ( DM ) . GAPDH was used as control . Representative results are shown ( n = 3 ) . D RT - PCR analysis of DUXAP8 expression in RNA extracted from GS ∆ 44 , GS ∆ 44 ' s mother ( GSM ) and GS ∆ 44 ' s father ( F ) fibroblasts . GAPDH was used as control . Lane ( - ) indicates the negative control . Representative results are shown ( n = 3 ) . E RT - PCR analysis for DUXAP8 expression in RNA extracted from WT and GS ∆ 44 myocytes and from WT ( WT # 1 ) , GS ∆ 44 ( GS ∆ 44 # 2 , GS ∆ 44 # 8 ) and GSM ( GSM # 1 ) iPSCs induced to differentiation for 9 days . GAPDH was used as control . Lane ( - ) indicates the negative control . Representative results are shown ( n = 3 ) . F RT - PCR for the indicated RNAs on extracts from GS ∆ 44 myoblasts treated with either control siRNAs ( siSCR ) or siRNAs against DUXAP8 ( siDUXAP8 ) and collected two days after transfection . GAPDH was used as control . Lane ( - ) indicates the negative control . Representative results are shown ( n = 3 ) . G qPCR analysis of DUXAP8 and CELF2a expression in control myoblasts transfected with a plasmid for the overexpression of DUXAP8 ( OE DUXAP8 ) or an empty vector ( Empty ) , and collected after 96h . Relative mRNA levels were calculated with the delta delta Ct method . qPCR reactions were normalized against an internal control ( GAPDH ) and plotted relative to the expression level in the sample treated with the empty vector which was set to a value of 1 . The mean ± SEM of triplicates from one representative experiment is shown ( n = 3 ) .

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

Figure 4B RT-PCR analyses of indicated RNAs in control (WT), ∆44 and in GS∆44 myocytes collected after 9 days upon the induction of differentiation. GAPDH was used as control. Representative results are shown (n=3).C RT-PCR analyses for CELF2a and DUXAP8 expression in WT and GS∆44 myoblasts (GM) and myocytes (DM). GAPDH was used as control. Representative results are shown (n=3).D RT-PCR analysis of DUXAP8 expression in RNA extracted from GS∆44, GS∆44's mother (GSM) and GS∆44's father (F) fibroblasts. GAPDH was used as control. Lane (-) indicates the negative control. Representative results are shown (n=3).E RT-PCR analysis for DUXAP8 expression in RNA extracted from WT and GS∆44 myocytes and from WT (WT#1), GS∆44 (GS∆44#2, GS∆44#8) and GSM (GSM#1) iPSCs induced to differentiation for 9 days. GAPDH was used as control. Lane (-) indicates the negative control. Representative results are shown (n=3).F RT-PCR for the indicated RNAs on extracts from GS∆44 myoblasts treated with either control siRNAs (siSCR) or siRNAs against DUXAP8 (siDUXAP8) and collected two days after transfection. GAPDH was used as control. Lane (-) indicates the negative control. Representative results are shown (n=3).G qPCR analysis of DUXAP8 and CELF2a expression in control myoblasts transfected with a plasmid for the overexpression of DUXAP8 (OE DUXAP8) or an empty vector (Empty), and collected after 96h. Relative mRNA levels were calculated with the delta delta Ct method. qPCR reactions were normalized against an internal control (GAPDH) and plotted relative to the expression level in the sample treated with the empty vector which was set to a value of 1. The mean ± SEM of triplicates from one representative experiment is shown (n=3).


Figure 1 ( A ) Summary and representative analysis of NET formation of pPMNs stimulated by 50 nM PMA for 3 h , detected by fluorescent microplate reader and confocal microscopy , respectively . Scale bar 20 µm . ( B ) Representative confocal microscopy images of pPMNs that were treated without ( control ) or with 50 nM PMA for 3 h , and stained for lamin B and DNA as described in panel B . The light blue arrows indicate the release of decondensed DNA associated with lamin B molecules from ruptured nuclear envelope . Scale bar 10 µm . ( D ) Representative and summary of immunoblots of lamin B of bone marrow neutrophils from WT and Lmnb1TG mice . Vinculin served as loading control . ( E ) Summary analysis of PAF - induced NET formation in mPMNs from WT vs Lmnb1TG mice that were stimulated without or with 10 µM PAF for 3 h , then fixed by 2 % PFA and stained by Sytox Green , following by detection with fluorescent microplate reader . ( F ) The endpoint analysis of NET - DNA release index was detected by coincubation of primary mouse peritoneal mPMNs from WT and Lmnb1TG mice without ( control ) or with 10 µM PAF in medium containing 1 μM Sytox Green dye with recording by a microplate reader at the 3 h time point . The NET - DNA release index was reported in comparison to an assigned value of 100 % for the total DNA released by neutrophils lysed by 0 . 5 % ( v / v ) Triton - X - 100 . ( G , H ) Representative images ( G ) and summary analysis ( H ) of PAF - induced NET formation in mPMNs from WT vs Lmnb1TG mice that were stimulated without or with 10 µM PAF for 3 h , and stained with both cell permeable SYTO Red and cell impermeable Sytox Green , without fixation . Images were taken by Olympus confocal microscopy , followed by automated quantification of NETs on 5 - 6 non - overlapping area per well using ImageJ for calculation of % cells with NET formation . ( G ) Scale bars , 40 μm .

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

Figure 1(A) Summary and representative analysis of NET formation of pPMNs stimulated by 50 nM PMA for 3 h, detected by fluorescent microplate reader and confocal microscopy, respectively. Scale bar 20 µm.(B) Representative confocal microscopy images of pPMNs that were treated without (control) or with 50 nM PMA for 3 h, and stained for lamin B and DNA as described in panel B. The light blue arrows indicate the release of decondensed DNA associated with lamin B molecules from ruptured nuclear envelope. Scale bar 10 µm.(D) Representative and summary of immunoblots of lamin B of bone marrow neutrophils from WT and Lmnb1TG mice. Vinculin served as loading control.(E) Summary analysis of PAF-induced NET formation in mPMNs from WT vs Lmnb1TG mice that were stimulated without or with 10 µM PAF for 3 h, then fixed by 2% PFA and stained by Sytox Green, following by detection with fluorescent microplate reader.(F) The endpoint analysis of NET-DNA release index was detected by coincubation of primary mouse peritoneal mPMNs from WT and Lmnb1TG mice without (control) or with 10 µM PAF in medium containing 1 μM Sytox Green dye with recording by a microplate reader at the 3 h time point. The NET-DNA release index was reported in comparison to an assigned value of 100% for the total DNA released by neutrophils lysed by 0.5% (v/v) Triton-X-100.(G,H) Representative images (G) and summary analysis (H) of PAF-induced NET formation in mPMNs from WT vs Lmnb1TG mice that were stimulated without or with 10 µM PAF for 3 h, and stained with both cell permeable SYTO Red and cell impermeable Sytox Green, without fixation. Images were taken by Olympus confocal microscopy, followed by automated quantification of NETs on 5-6 non-overlapping area per well using ImageJ for calculation of % cells with NET formation. (G) Scale bars, 40 μm.


Figure 2 ( A , B ) Representative and summary of immunoblots of full - length lanes of lamin B in human dPMNs that were treated with PMA for 0 , 0 . 5 , 1 , 2 , 3 h during NET formation , or apoptotic dPMNs that were induced by PMA for longer term ( 12 h ) treatment ( A ) . β - actin served as loading control ( C , D ) Representative and summary immunoblots of lamin B in human dPMNs that were treated with PAF for 0 , 0 . 5 , 1 , 2 , 3 h during NET formation . β - actin served as loading control ( E ) Representative confocal microscopy image of a group of pPMNs with NET formation ( enclosed by a light blue trapezoid ) and their extracellular NETs ( indicated by a purple rectangle square ) released by these cells , as well as the immunoblot analysis of un - cleaved lamin B and cleaved histone H3 with the whole cell lysates , as well as immunoblot analysis of un - cleaved lamin B with lysate of the NETs isolated from conditioned medium of pPMNs with NET formation that were induced by 3 h PMA treatment . The anti - human lamin B was used in all immunoblots confocal image ( E ) , and the latter was further detected by FITC - labeled 2nd Ab , scale bar , 20 μm . ( F ) Representative immunoblots with full - length lanes for lamin B analysis , and pro - caspase - 3 and its activated form caspase - 3 in human dPMNs with NET formation that were treated with PMA for 0 , 0 . 5 , 1 , 2 , 3 h . Anti - human caspase - 3 was used ( F , G ) , and β - actin served as loading control ( G ) Representative immunoblots display full - length lamin B ( 69 kDa ) and their cleaved fragments ( 45 and 25 kDa ) , and pro - caspase - 3 and its activated form caspase - 3 in the apoptotic human dPMNs that were treated with PMA for 12 , 24h . Anti - human caspase - 3 was used ( F , G ) , and β - actin served as loading

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

Figure 2(A,B) Representative and summary of immunoblots of full-length lanes of lamin B in human dPMNs that were treated with PMA for 0, 0.5, 1, 2, 3 h during NET formation, or apoptotic dPMNs that were induced by PMA for longer term (12 h) treatment (A). β-actin served as loading control(C,D) Representative and summary immunoblots of lamin B in human dPMNs that were treated with PAF for 0, 0.5, 1, 2, 3 h during NET formation. β-actin served as loading control(E) Representative confocal microscopy image of a group of pPMNs with NET formation (enclosed by a light blue trapezoid) and their extracellular NETs (indicated by a purple rectangle square) released by these cells, as well as the immunoblot analysis of un-cleaved lamin B and cleaved histone H3 with the whole cell lysates, as well as immunoblot analysis of un-cleaved lamin B with lysate of the NETs isolated from conditioned medium of pPMNs with NET formation that were induced by 3 h PMA treatment. The anti-human lamin B was used in all immunoblots confocal image (E), and the latter was further detected by FITC-labeled 2nd Ab, scale bar, 20 μm.(F) Representative immunoblots with full-length lanes for lamin B analysis, and pro-caspase-3 and its activated form caspase-3 in human dPMNs with NET formation that were treated with PMA for 0, 0.5, 1, 2, 3 h. Anti-human caspase-3 was used (F,G), and β-actin served as loading control(G) Representative immunoblots display full-length lamin B (69 kDa) and their cleaved fragments (45 and 25 kDa), and pro-caspase-3 and its activated form caspase-3 in the apoptotic human dPMNs that were treated with PMA for 12, 24h. Anti-human caspase-3 was used (F,G), and β-actin served as loading control


Figure 3 ( A ) Representative images for the time course of PKCα nuclear translocation , subsequent nuclear envelope rupture , and DNA release in human dPMNs exposed to 50 nM PMA for 5 , 15 , 30 , 60 , and 180 min , then stained concomitantly for DNA ( DAPI ) , nuclear lamin B ( primary anti - lamin B , and FITC - labeled secondary antibody ) , and PKCα ( primary anti - human PKCα , and PE - labeled secondary antibody ) , following by confocal fluorescent microscopy analysis . White - empty arrows indicate cytoplasmic distribution of PKCα at 5 , 15 , and 30 min , yellow arrows indicate site of discontinuity / rupture of nuclear envelope at 60 min , light blue arrows display the sites of nuclear envelope rupture and chromatin release at 180 min ( A ) . Scale bars , 20 μm . The time course started 5 min after PMA stimulation in order to allow the adherence of neutrophils on the bottom of dish for immunocytostaining . ( B ) Summary analysis of the nuclear envelope continuity was analyzed based on staining of the nuclear envelope with primary anti - lamin B , and FITC - labeled secondary antibody . ( C Representative immunoblots of total PKCα and p - PKCα in primary human pPMNs that were treated by PMA ( C ) for 0 , 0 . 5 , 1 , 2 , 3 h . D ) Representative immunoblots of total PKCα and p - PKCα in primary human pPMNs that were treated by PAF ( D ) for 0 , 0 . 5 , 1 , 2 , 3 h .

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

Figure 3(A) Representative images for the time course of PKCα nuclear translocation, subsequent nuclear envelope rupture, and DNA release in human dPMNs exposed to 50 nM PMA for 5, 15, 30, 60, and 180 min, then stained concomitantly for DNA (DAPI), nuclear lamin B (primary anti-lamin B, and FITC-labeled secondary antibody), and PKCα (primary anti-human PKCα, and PE-labeled secondary antibody), following by confocal fluorescent microscopy analysis. White-empty arrows indicate cytoplasmic distribution of PKCα at 5, 15, and 30 min, yellow arrows indicate site of discontinuity/rupture of nuclear envelope at 60 min, light blue arrows display the sites of nuclear envelope rupture and chromatin release at 180 min (A). Scale bars, 20 μm. The time course started 5 min after PMA stimulation in order to allow the adherence of neutrophils on the bottom of dish for immunocytostaining.(B) Summary analysis of the nuclear envelope continuity was analyzed based on staining of the nuclear envelope with primary anti-lamin B, and FITC-labeled secondary antibody.(C Representative immunoblots of total PKCα and p-PKCα in primary human pPMNs that were treated by PMA (C) for 0, 0.5, 1, 2, 3 h.D) Representative immunoblots of total PKCα and p-PKCα in primary human pPMNs that were treated by PAF (D) for 0, 0.5, 1, 2, 3 h.


Figure 4 ( A ) Representative immunoblots of p - PKCα and total PKCα of isolated nuclei and the corresponding status of lamin B in human dPMNs that were treated with PMA for 0 or 2 h . ( B ) Representative sectional images of nuclei of two neutrophils treated by PMA , stained for lamin B ( primary anti - lamin B , and FITC - labeled secondary antibody ) , p - PKCα ( primary anti - human p - PKCαS657 , and PE - labeled secondary antibody ) , and nuclear DNA ( DAPI ) . The accumulation of p - PKCα ( red arrow pointed ) at the site of ruptured / interrupted ( green arrow ) nuclear lamin B , and swollen / de - condensed DNA ( blue arrow ) were indicated ( b1 ) . A neutrophil ( white empty arrow ) with NET formation that contained the mixture of DNA with p - PKCα and lamin B ( white arrow ) was also demonstrated ( b2 ) . Scale bars , 10 µm . ( C ) Immunoblot images of lamin B , then stripped and probed for p - PKCα , of the homogenates of NETs isolated from conditioned medium of primary human pPMNs with NET formation that were induced by PMA . ( D , E ) Representative and summary immunoblot ( IB ) detection of phospho - lamin B and total lamin B with the lamin B protein purified by immunoprecipitation ( IP ) with anti - lamin B from human dPMNs that were treated either by PMA ( D , E ) for 0 or 3 h . Data represent mean ± SD ( n = 3 - 5 biological replicates ) for E . P * < 0 . 05 between groups as indicated . Comparison between two groups was analyzed by student t test .

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

Figure 4(A) Representative immunoblots of p-PKCα and total PKCα of isolated nuclei and the corresponding status of lamin B in human dPMNs that were treated with PMA for 0 or 2 h.(B) Representative sectional images of nuclei of two neutrophils treated by PMA, stained for lamin B (primary anti-lamin B, and FITC-labeled secondary antibody), p-PKCα (primary anti-human p-PKCαS657, and PE-labeled secondary antibody), and nuclear DNA (DAPI). The accumulation of p-PKCα (red arrow pointed) at the site of ruptured/interrupted (green arrow) nuclear lamin B, and swollen/de-condensed DNA (blue arrow) were indicated (b1). A neutrophil (white empty arrow) with NET formation that contained the mixture of DNA with p-PKCα and lamin B (white arrow) was also demonstrated (b2). Scale bars, 10 µm.(C) Immunoblot images of lamin B, then stripped and probed for p-PKCα, of the homogenates of NETs isolated from conditioned medium of primary human pPMNs with NET formation that were induced by PMA.(D,E) Representative and summary immunoblot (IB) detection of phospho-lamin B and total lamin B with the lamin B protein purified by immunoprecipitation (IP) with anti-lamin B from human dPMNs that were treated either by PMA (D,E) for 0 or 3 h. Data represent mean ± SD (n=3-5 biological replicates) for E. P*<0.05 between groups as indicated. Comparison between two groups was analyzed by student t test.


Figure 5 ( A ) Summary and representative immunoblots of total PKCα and p - PKCα in dPMNs that were pretreated without or with PKC inhibitor Go6976 for 1 h , and then treated without or with PMA ( A ) for 3 h . Panels A is summary analyses of immunoblots that were calculated based on the arbitrary unit ( A ) ( B ) Immunoblot ( IB ) detection of the phospho - lamin B and total lamin B with lamin B protein purified by immunoprecipitation ( IP ) with anti - lamin B from human dPMNs that were pretreated without or with PKC inhibitor Go6976 for 1 h , and then treated by PMA ( B ) for 3 h . ( C ) Confocal microscopy images of human dPMNs that were pretreated without or with PKCα Go6976 for 1 h , and then treated without or with PMA for 3 h , followed by staining of lamin B ( primary anti - lamin B , and FITC - labeled secondary antibody ) , and phosphorylated PKCα ( primary anti - human p - PKCαS657 , and PE - labeled secondary antibody ) . The green / white arrows indicate the site of nuclear envelope rupture . Scale bars , 10 μm ( C ) . ( E ) The endpoint analysis of % cells with NET formation was detected with dPMNs that were transfected with plasmids of either wildtype lamin B ( WT control ) , or mutants with single or multiple defined point mutations at PKCα - consensus - phosphorylation sites ( S395A , S405A , S408A ) of lamin B , and treated for 3 h by 10 μM PAF in medium containing cell - permeable dye SYTO Red ( 500 nM ) for the total cell count , while the cells with NET formation were stained by cell impermeable dye SYTOX Blue ( 5 μM ) . Then the images were taken with Olympus confocal microscopy , followed by automated quantification of NETs using ImageJ for quantification of % cells with NET formation . percentage of cells with NET formation by immunofluorescent imaging quantification using ImageJ ( E )

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

Figure 5(A) Summary and representative immunoblots of total PKCα and p-PKCα in dPMNs that were pretreated without or with PKC inhibitor Go6976 for 1 h, and then treated without or with PMA (A) for 3 h. Panels A is summary analyses of immunoblots that were calculated based on the arbitrary unit (A)(B) Immunoblot (IB) detection of the phospho-lamin B and total lamin B with lamin B protein purified by immunoprecipitation (IP) with anti-lamin B from human dPMNs that were pretreated without or with PKC inhibitor Go6976 for 1 h, and then treated by PMA (B) for 3 h.(C) Confocal microscopy images of human dPMNs that were pretreated without or with PKCα Go6976 for 1 h, and then treated without or with PMA for 3 h, followed by staining of lamin B (primary anti-lamin B, and FITC-labeled secondary antibody), and phosphorylated PKCα (primary anti-human p-PKCαS657, and PE-labeled secondary antibody). The green/white arrows indicate the site of nuclear envelope rupture. Scale bars, 10 μm (C).(E) The endpoint analysis of % cells with NET formation was detected with dPMNs that were transfected with plasmids of either wildtype lamin B (WT control), or mutants with single or multiple defined point mutations at PKCα-consensus-phosphorylation sites (S395A, S405A, S408A) of lamin B, and treated for 3 h by 10 μM PAF in medium containing cell-permeable dye SYTO Red (500 nM) for the total cell count, while the cells with NET formation were stained by cell impermeable dye SYTOX Blue (5 μM). Then the images were taken with Olympus confocal microscopy, followed by automated quantification of NETs using ImageJ for quantification of % cells with NET formation. percentage of cells with NET formation by immunofluorescent imaging quantification using ImageJ (E)


Figure 6 ( A ) Summary and representative immunoblots of PKCα expressions in mouse bone marrow neutrophils from PKCα - / - , PKCα + / - , and their littermate WT ( PKCα + / + ) mice . Vinculin served as loading control . ( B ) Representative immunoblot ( IB ) detection of the phospho - lamin B ( Ser ) and total lamin B with lamin B protein purified by immunoprecipitation ( IP ) with anti - lamin B from mouse peritoneal mPMNs from WT vs PKCα - / - mice , stimulated without or with PAF for 3 h . ( C ) Summary analyses of NET formation in peritoneal mPMNs , from WT , PKCα + / - , or PKCα - / - mice , which were treated without or with either 5 µM or 10 µM PAF for 3 h following by 2 % PFA fixation and fluorometric microplate analysis . ( D ) The endpoint analysis of NET - DNA release index was detected by coincubation of primary mouse peritoneal mPMNs from WT and PKCα - / - mice without ( control ) or with 10 µM PAF in medium containing 1 μM Sytox Green dye with recording by a microplate reader at the end of the 3 h time point . The NET - DNA release index was reported in comparison to an assigned value of 100 % for the total DNA released by neutrophils lysed by 0 . 5 % ( v / v ) Triton - X - 100 . ( E , F ) ( E ) Representative and ( F ) summary analysis of NET formation of mouse peritoneal mPMNs from WT vs PKCα - / - mice stimulated without or with 10 µM PAF for 3 h , and stained with both cell permeable SYTO Red dye and cell impermeable Sytox Green dye , without fixation . Images were taken by Olympus confocal microscopy , followed by automated quantification of NETs on 5 - 6 non - overlapping area per well using ImageJ for calculation of % cells with NET formation . ( E ) Scale bars , 50 μm . % cells with NET formation by image analysis using ImageJ ( F )

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

Figure 6(A) Summary and representative immunoblots of PKCα expressions in mouse bone marrow neutrophils from PKCα-/-, PKCα+/-, and their littermate WT (PKCα+/+) mice. Vinculin served as loading control.(B) Representative immunoblot (IB) detection of the phospho-lamin B (Ser) and total lamin B with lamin B protein purified by immunoprecipitation (IP) with anti-lamin B from mouse peritoneal mPMNs from WT vs PKCα-/- mice, stimulated without or with PAF for 3 h.(C) Summary analyses of NET formation in peritoneal mPMNs, from WT, PKCα+/-, or PKCα-/- mice, which were treated without or with either 5 µM or 10 µM PAF for 3 h following by 2% PFA fixation and fluorometric microplate analysis.(D) The endpoint analysis of NET-DNA release index was detected by coincubation of primary mouse peritoneal mPMNs from WT and PKCα-/- mice without (control) or with 10 µM PAF in medium containing 1 μM Sytox Green dye with recording by a microplate reader at the end of the 3 h time point. The NET-DNA release index was reported in comparison to an assigned value of 100% for the total DNA released by neutrophils lysed by 0.5% (v/v) Triton-X-100.(E, F) (E) Representative and (F) summary analysis of NET formation of mouse peritoneal mPMNs from WT vs PKCα-/- mice stimulated without or with 10 µM PAF for 3 h, and stained with both cell permeable SYTO Red dye and cell impermeable Sytox Green dye, without fixation. Images were taken by Olympus confocal microscopy, followed by automated quantification of NETs on 5-6 non-overlapping area per well using ImageJ for calculation of % cells with NET formation. (E) Scale bars, 50 μm. % cells with NET formation by image analysis using ImageJ (F)


Figure 7 ( A , B ) representative analysis of confocal fluorescent microscopy images of neutrophils with NET formation in the skin tissue of WT ( A ) vs Lmnb1TG ( B ) mice with UVB exposure , in which DNA was stained by DAPI , citrullinated histone H3 was probed by rabbit anti - mouse citrullinated Histone H3 , following stained by Alexa Fluor - 488 labeled donkey anti - rabbit secondary antibody , while neutrophil surface marker Ly6B was probed by rat anti - mouse Ly6B Ab following stained by Alexa Fluor - 647 conjugated goat anti - rat secondary antibody . The images of different staining and their overlays are shown ( A , B ) . The parental neutrophils ( stained by neutrophil membrane surface marker Ly6B ) and their released extracellular NET structures ( co - stained by anti - cit H3 and DAPI ) are indicated by red arrows and light blue arrows respectively . The white arrows indicated neutrophils without NET release . Scale bar , 50 µm . ( C ) summary analysis of confocal fluorescent microscopy images of neutrophils with NET formation in the skin tissue of WT ( A ) vs Lmnb1TG ( B ) mice with UVB exposure , in which DNA was stained by DAPI , citrullinated histone H3 was probed by rabbit anti - mouse citrullinated Histone H3 , following stained by Alexa Fluor - 488 labeled donkey anti - rabbit secondary antibody , while neutrophil surface marker Ly6B was probed by rat anti - mouse Ly6B Ab following stained by Alexa Fluor - 647 conjugated goat anti - rat secondary antibody . The images of different staining and their overlays are shown ( A , B ) . The parental neutrophils ( stained by neutrophil membrane surface marker Ly6B ) and their released extracellular NET structures ( co - stained by anti - cit H3 and DAPI ) are indicated by red arrows and light blue arrows respectively . The white arrows indicated neutrophils without NET release . Scale bar , 50 µm . ( D , E ) Summary analysis for staining area of neutrophils with NET formation and their exhibition of IL - 17A ( D ) or TNFα ( E ) , in the skin tissue of WT vs Lmnb1TG mice without ( sham ) or with UVB exposure , by co - staining of Ly6B ( stained as described in panels A - C ) with IL - 17A ( D ) or TNFα ( E ) using FITC - conjugated rat anti - mouse IL - 17A , or FITC conjugated rat anti - mouse TNFα Abs .

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

Figure 7(A,B) representative analysis of confocal fluorescent microscopy images of neutrophils with NET formation in the skin tissue of WT (A) vs Lmnb1TG (B) mice with UVB exposure, in which DNA was stained by DAPI, citrullinated histone H3 was probed by rabbit anti-mouse citrullinated Histone H3, following stained by Alexa Fluor-488 labeled donkey anti-rabbit secondary antibody, while neutrophil surface marker Ly6B was probed by rat anti-mouse Ly6B Ab following stained by Alexa Fluor-647 conjugated goat anti-rat secondary antibody. The images of different staining and their overlays are shown (A,B). The parental neutrophils (stained by neutrophil membrane surface marker Ly6B) and their released extracellular NET structures (co-stained by anti-cit H3 and DAPI) are indicated by red arrows and light blue arrows respectively. The white arrows indicated neutrophils without NET release. Scale bar, 50 µm.(C) summary analysis of confocal fluorescent microscopy images of neutrophils with NET formation in the skin tissue of WT (A) vs Lmnb1TG (B) mice with UVB exposure, in which DNA was stained by DAPI, citrullinated histone H3 was probed by rabbit anti-mouse citrullinated Histone H3, following stained by Alexa Fluor-488 labeled donkey anti-rabbit secondary antibody, while neutrophil surface marker Ly6B was probed by rat anti-mouse Ly6B Ab following stained by Alexa Fluor-647 conjugated goat anti-rat secondary antibody. The images of different staining and their overlays are shown (A,B). The parental neutrophils (stained by neutrophil membrane surface marker Ly6B) and their released extracellular NET structures (co-stained by anti-cit H3 and DAPI) are indicated by red arrows and light blue arrows respectively. The white arrows indicated neutrophils without NET release. Scale bar, 50 µm.(D,E) Summary analysis for staining area of neutrophils with NET formation and their exhibition of IL-17A (D) or TNFα (E), in the skin tissue of WT vs Lmnb1TG mice without (sham) or with UVB exposure, by co-staining of Ly6B (stained as described in panels A-C) with IL-17A (D) or TNFα (E) using FITC-conjugated rat anti-mouse IL-17A, or FITC conjugated rat anti-mouse TNFα Abs.