Journal: Chemical Engineering Journal

Article Title: Self-healing hydrogel reduces inflammation through ANT1/OPTN axis mediated mitophagy for spinal cord injury repair

doi: 10.1016/j.cej.2024.152263

Figure Lengend Snippet: Fig. 6. RNA-seq analysis revealed that ANT1-mediated mitophagy involves in anti-inflammation effects. The top 10 upregulated enriched GO term (a) and the top 10 downregulated enriched GO term (b) of differentially expressed (p < 0.05, |log2FC > 0.58|) mRNA in TPHD@Tc treated zebrafish compared with SCI group. (c) Veen analysis of the differentially expressed mRNA and autophagy-related genes. mRNA expression (d, n = 3), protein expression (e, n = 3) and Immunoflu orescent staining (f) of ANT1 in BV2 cells treated with TPHD, TPHD@TcL, TPHD@Tc and TPHD@TcH for 4 days. mRNA (g, n = 3), protein expression (h, n = 3) and Immunofluorescent staining (i) of NLRP3 Data were expressed by mean with SD. * p < 0.05, ** p < 0.01, **** p < 0.0001, compared with TPHD in b and c.

Article Snippet: The primary antibodies of NLRP3 (1:1000, Rabbit, ab263899, Abcam), ANT1 (1:1000, Rabbit, NBP292642, NOVUS), and the secondary antibodies of HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H + L) (1:5000, SA00001-2, Proteintech) were used.

Techniques: RNA Sequencing, Expressing, Staining

Journal: Chemical Engineering Journal

Article Title: Self-healing hydrogel reduces inflammation through ANT1/OPTN axis mediated mitophagy for spinal cord injury repair

doi: 10.1016/j.cej.2024.152263

Figure Lengend Snippet: Fig. 7. ANT1 enhanced OPTN recruitment promotes mitophagy mediated inflammation inhibition. Immunofluorescence of TOMM20 (red) and OPTN (green) in BV2 cells treated with TPHD (a), TPHD@TcL (b), TPHD@Tc (c), TPHD@TcH (d) for 4 days. LC3B expression in BV2 cells treated with TPHD, TPHD@TcL, TPHD@Tc, TPHD@TcH for 4 days (e) and related quantitative m-cherry labeled area (f, n = 5), GFP labeled area (g, n = 5) and m-cherry and GFP co-labeled area (h, n = 5). NLRP3 mRNA expression (i, n = 3) and protein expression (j, n = 3) in BV2 cells treated with TPHD, TPHD@TcL, TPHD@Tc, TPHD@TcH for 4 days. Immunofluorescent staining of NLRP3 of BV2 cells treated with TPHD, TPHD@TcL, TPHD@Tc, TPHD@TcH for 4 days (k). (l) The schematic diagram of hydrogel containing Tc promoted mitophagy to decrease inflammation. The elevated ANT1 promoted OPTN recruitment, which provided LC3 anchoring for phagophore formation around dysfunctional mitochondria, leading to a regressive inflammation. Data were expressed by mean with SD. * p < 0.05, *** p < 0.001, **** p < 0.0001, compared with TPHD in f-j.

Article Snippet: The primary antibodies of NLRP3 (1:1000, Rabbit, ab263899, Abcam), ANT1 (1:1000, Rabbit, NBP292642, NOVUS), and the secondary antibodies of HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H + L) (1:5000, SA00001-2, Proteintech) were used.

Techniques: Inhibition, Immunofluorescence, Expressing, Labeling, Staining

Journal: PLoS ONE

Article Title: P-Loop Residues Critical for Selectivity in K + Channels Fail to Confer Selectivity to Rabbit HCN4 Channels

doi: 10.1371/journal.pone.0007712

Figure Lengend Snippet: (A) Sequence alignment of mammalian HCN channels, and various K + selective channels shows all mammalian HCN channels have a leucine and cysteine residue in place of the two threonines typically (though not always) present in the underlined K + channel ‘selectivity sequence’. These residues (L478 and C479 in HCN4) were mutated to threonine to create a pore-lining sequence resembling that of a K + selective channel. (B) Sample traces of WT, L478/C479T, and C479T HCN4 currents recorded in a 5 mM K + /135 mM Na + bath solution, elicited by a 3 s prepulse to −130 mV from a holding potential of 0 mV, followed by a 1 s test pulse between +20 to −70 mV (ΔV = 10 mV). (C) Western blots performed from whole-cell lysates of untransfected (UT) CHO-K1 cells, or cells transfected with WT, L478/C479T or C479T constructs, with GAPDH used as a loading control. WT and mutant channels are highly expressed in both N -glycosylated and core bands (←), indicating the absence of L478/C479T currents in expressed cells is not due to mis-trafficking or low protein production.

Article Snippet: Membranes were blotted with the polyclonal anti-HCN4 antibodies (Alomone Labs, Israel) (1∶500 dilution) in PBST supplemented with 5% BSA.

Techniques: Sequencing, Residue, Western Blot, Transfection, Construct, Control, Mutagenesis

Journal: PLoS ONE

Article Title: P-Loop Residues Critical for Selectivity in K + Channels Fail to Confer Selectivity to Rabbit HCN4 Channels

doi: 10.1371/journal.pone.0007712

Figure Lengend Snippet: E rev and P X /P K estimates in WT and C479T HCN4 channels.

Article Snippet: Membranes were blotted with the polyclonal anti-HCN4 antibodies (Alomone Labs, Israel) (1∶500 dilution) in PBST supplemented with 5% BSA.

Techniques:

Journal: PLoS ONE

Article Title: P-Loop Residues Critical for Selectivity in K + Channels Fail to Confer Selectivity to Rabbit HCN4 Channels

doi: 10.1371/journal.pone.0007712

Figure Lengend Snippet: (A) Sample traces of C479T, S475D/C479T, S475E/C479T and F471W/K472W/C479T HCN4 currents (labeled S475D, S475E and WW for simplicity) recorded in a 5 mM K + /135 mM Na + bath solution, elicited by a 3 s prepulse to −130 mV from a holding potential of 0 mV, followed by a 1 s test pulse between +20 to −70 mV (ΔV = 10 mV). S475E and WW channels did not express currents. (B) Representative instantaneous I–V relationships from tail currents of C479T (•) and S475D (◊) channels from recordings performed in a 5 mM K + /135 mM Na + bath solution. P X /P K 's were determined to be −17.0±1.9 (n = 4) and −16.2±2.0 (n = 5) for C479T and S475D channels respectively which were not statistically significant. (C) Western blots performed from whole-cell lysates of untransfected (UT) CHO-K1 cells, or cells transfected with C479T, S475D, S475E or WW constructs, with GAPDH used as a loading control (bottom of panel). All mutant channels are highly expressed in both N -glycosylated and core bands (←), thus, the lack of current observed from S475E and WW channels is not due to mistrafficking, or low protein production, but rather non-functional protein at the plasma membrane.

Article Snippet: Membranes were blotted with the polyclonal anti-HCN4 antibodies (Alomone Labs, Israel) (1∶500 dilution) in PBST supplemented with 5% BSA.

Techniques: Labeling, Western Blot, Transfection, Construct, Control, Mutagenesis, Functional Assay, Clinical Proteomics, Membrane

Journal: PLoS ONE

Article Title: P-Loop Residues Critical for Selectivity in K + Channels Fail to Confer Selectivity to Rabbit HCN4 Channels

doi: 10.1371/journal.pone.0007712

Figure Lengend Snippet: (A) Single-channel WT and C479T currents recorded at −120, −130, −140 and −150 mV in 140 mM extracellular K + . The solid line denotes zero current in the closed channel. (B) Sample histograms used to estimate unitary currents ( i ) of WT and C479T channels at −150 mV. Histograms were fit as a dual Gaussian distribution to obtain values for i . (C) Unitary-current-Voltage ( i -V) relationship of WT (▪) and C479T ( ) channels between −120 and −150 mV. The unitary conductance (γ), estimated from the slope of a line through the origin, was larger (P<0.05) in C479T channels (1.7±0.1 pS) compared to WT HCN4 (1.0±0.1 pS) (n≥3 for each voltage; *P<0.05).

Article Snippet: Membranes were blotted with the polyclonal anti-HCN4 antibodies (Alomone Labs, Israel) (1∶500 dilution) in PBST supplemented with 5% BSA.

Techniques:

Journal: PLoS ONE

Article Title: P-Loop Residues Critical for Selectivity in K + Channels Fail to Confer Selectivity to Rabbit HCN4 Channels

doi: 10.1371/journal.pone.0007712

Figure Lengend Snippet: A space filled model of the pore sequence in WT and C479T HCN4 channels based on the model of mHCN2 (Giorgetti et al. , 2005). The top two panels depict a side view of this sequence in these channels with the intracellular side of the channel on the left. The bottom panels depict a bottom up view of the pore in these channels. The bulk of the −SH group (yellow) in WT HCN4 would limit the pore diameter, and provide a steric barrier thereby limiting ionic permeation and selectivity based on hydrated ionic radius. The C479T mutation increases the pore diameter from 5.4 Å in WT (at the edge of the electron clouds) to 6.2 Å and reduces the steric barrier for ion permeation and ionic selectivity based on effective radius.

Article Snippet: Membranes were blotted with the polyclonal anti-HCN4 antibodies (Alomone Labs, Israel) (1∶500 dilution) in PBST supplemented with 5% BSA.

Techniques: Sequencing, Mutagenesis