Journal: Journal of Inflammation Research

Article Title: Unveiling and Validating the Role of Fatty Acid Metabolism in Ulcerative Colitis

doi: 10.2147/JIR.S479011

Figure Lengend Snippet: ( A and E ) DCA evaluating the prediction efficacy of the nomogram in the testing ( A ) and validation sets ( E ). ( B and F ) Calibration curves evaluating the clinical benefit of the nomogram in the testing ( B ) and validation sets ( F ). ( C and G ) ROC evaluation of the nomogram’s diagnostic performance in the testing ( C ) and validation sets ( G ). ( D and H ) Risk score derived from the model for patients with UC and HC in the testing ( D ) and validation sets ( H ). ( I ) ROC curve analysis for ACAT1, ACOX2, and HADHB in diagnosing UC in the external dataset. ( J ) Expression levels of ACAT1, ACOX2, and HADHB in patients with UC and HC in the external dataset.

Article Snippet: The antibodies used included HADHB (Rabbit, 1:2000, Proteintech, China), ACOX2 (Rabbit, 1:1000, ELK Biotechnology), and ACAT1 (Rabbit, 1:1000, ELK Biotechnology).

Techniques: Diagnostic Assay, Derivative Assay, Expressing

Journal: Journal of Inflammation Research

Article Title: Unveiling and Validating the Role of Fatty Acid Metabolism in Ulcerative Colitis

doi: 10.2147/JIR.S479011

Figure Lengend Snippet: Validation of gene expression in colon tissues. ( A and B ) Relative weight change ( A ) and disease activity index ( B ) in each group ( n = 6). ( C ) Representative morphological images of mouse colons from the control and DSS groups. ( D ) Statistical analysis of colon length. ( E ) Representative H&E staining of colonic tissues from each group. ( F ) Statistical results for histological damage scores in each group ( n = 6). ( G ) Western blot analysis of ACAT1, ACOX2, and HADHB levels in colon tissues. Group identifications are as shown. ( H–J ) Quantitative analysis of Western blot results for ACAT1 ( H ), ACOX2 ( I ), and HADHB ( J ) expression. Data are presented as mean ± SEM.

Article Snippet: The antibodies used included HADHB (Rabbit, 1:2000, Proteintech, China), ACOX2 (Rabbit, 1:1000, ELK Biotechnology), and ACAT1 (Rabbit, 1:1000, ELK Biotechnology).

Techniques: Expressing, Activity Assay, Control, Staining, Western Blot

Journal: Journal of Inflammation Research

Article Title: Unveiling and Validating the Role of Fatty Acid Metabolism in Ulcerative Colitis

doi: 10.2147/JIR.S479011

Figure Lengend Snippet: ( A ) Heatmap illustrating variations in immune cell infiltration in the colon tissues of patients with UC and HC. ( B ) Differences in infiltrated immune cells between patients with UC and HC. ( C ) Heatmap showing correlations among immune cells. ( D–F ) Lollipop charts presenting the correlations between ACAT1 ( D ), ACOX2 ( E ), HADHB ( F ) expression, and immune cells.

Article Snippet: The antibodies used included HADHB (Rabbit, 1:2000, Proteintech, China), ACOX2 (Rabbit, 1:1000, ELK Biotechnology), and ACAT1 (Rabbit, 1:1000, ELK Biotechnology).

Techniques: Expressing

Journal: Journal of Inflammation Research

Article Title: Unveiling and Validating the Role of Fatty Acid Metabolism in Ulcerative Colitis

doi: 10.2147/JIR.S479011

Figure Lengend Snippet: Identification and enrichment analysis of fatty acid metabolism-related subtypes. ( A ) Consensus clustering matrix at k = 2. ( B ) Consensus scores for each subtype at k = 2 to 6. ( C ) PCA plot distinguishing subtype 1 (blue) from subtype 2 (red) samples. ( D ) Split violin plot showing the expression of ACAT1, ACOX2, and HADHB between subtypes. ( E and F ) Volcano plot ( E ) and heatmap ( F ) depicting gene expression patterns between subtypes.

Article Snippet: The antibodies used included HADHB (Rabbit, 1:2000, Proteintech, China), ACOX2 (Rabbit, 1:1000, ELK Biotechnology), and ACAT1 (Rabbit, 1:1000, ELK Biotechnology).

Techniques: Expressing

Journal: Nature Communications

Article Title: ATF4 selectively regulates heat nociception and contributes to kinesin-mediated TRPM3 trafficking

doi: 10.1038/s41467-021-21731-1

Figure Lengend Snippet: a – c TRPM3 expression in the DRG membrane fraction ( a ), cytoplasmic fraction ( b ) and total lysate ( c ) from WT and Atf4 +/− mice. n = 3 mice per group. t 4 = 10.3, P = 0.0005 in a . t 4 = 6.043, P = 0.0038 in b . d TRPM3 surface levels were measured in cultured DRG neurons prepared from WT and Atf4 +/− mice using a surface biotinylation assay. n = 3 cultures per group. t 4 = 6.28, P = 0.0033. e Co-IP showing the ATF4/TRPM3 interaction in DRGs. DRG lysates were immunoprecipitated with an ATF4 (top) or TRPM3 (bottom) antibody and immunoblotted with a TRPM3 or ATF4 antibody as indicated. This experiment was repeated three times. f The SIM images show that the colocalization between ATF4 and TRPM3 in DRG neurons. Scale bar, 5 μm. g The GST pull-down assay with two purified proteins, TRPM3-GST-Flag and ATF4, showed a direct interaction between ATF4 and TRPM3. This experiment was repeated three times. h Interaction between TRPM3 and ATF4 mutants. The ATF4 mutants was transiently co-expressed with TRPM3, and the cell lysates were immunoprecipitated with a His antibody and then immunoblotted with a His or Flag antibody as indicated. This experiment was repeated three times. i – k TRPM3 expression in the DRG membrane fraction ( i ), cytoplasmic fraction ( j ) and total lysate ( k ) after ATF4 overexpression. n = 6 mice per group. F (2,15) = 14.31, P = 0.0007 in i . F (2,15) = 30.15, P < 0.0001 in j . l , n , p Time course of a whole-cell patch-clamp recording showing the effect of CIM0216 (3 μM) and isosakuranetin (Iso; 10 μM) on the TRPM3 current in DRG neurons from WT ( l ), Atf4 +/− ( n ) and rescued ( p ) mice. The open circle indicates the outward current recorded at + 75 mV, and the closed circle indicates the inward current recorded at −75 mV. m , o , q I–V relationship of the TRPM3 current at the time points indicated in I , n and p . r Histogram of the TRPM3 current density (outward and inward, current before CIM0216 was subtracted) of DRG neurons from WT, Atf4 +/− and ATF4 rescued mice after CIM0216 treatment. n = 6 neurons per group. F (2,15) = 8.735, P = 0.0049 in WT vs. Atf4 +/− , P = 0.0097 in Atf4 +/− vs. rescue in +75 mV; F (2,15) = 9.158, P = 0.0038 in WT vs. Atf4 +/− , P = 0.0089 in Atf4 +/− vs. rescue in −75 mV. a – d Two-tailed Independent Student’s t test. i , j , k , r , One-way ANOVA followed by Tukey’s multiple comparisons test, * P < 0.05, ** P < 0.01, n.s. means not significant. The error bars indicate the SEMs.

Article Snippet: The membranes were placed in blocking buffer for 1 h at RT and incubated in primary antibodies against ATF4 (goat, 1:1000, GeneTex, catalogue no.: GTX89973), TRPM3 (rabbit, 1:1000, Bioss, catalogue no.: bs-9046R; rabbit, 1:200, Alomone Labs, catalogue no.: ACC-050), TRPV1 (rabbit, 1:200, Alomone Labs, catalogue no.: ACC-030), TRPA1 (rabbit, 1:1000, ABclonal, catalogue no.: A12544), KIF17 (mouse, 1:100, Santa Cruz Biotechnology, catalogue no.: sc-137040), KIF3A (goat, 1:100, Santa Cruz Biotechnology, catalogue no.: sc-18745), KIF3B (rabbit, 1:100, Santa Cruz Biotechnology, catalogue no.: sc-50456), KIF5A (rabbit, 1:1000, Abcam, catalogue no.: ab5628), KIF5B (rabbit, 1:2000, Abcam, catalogue no.: ab167429), KIFC2 (rabbit, 1:100, Abcam, catalogue no.: ab3476), TfR (mouse, 1:1000, Thermo Fisher Scientific, catalogue no.: 13-6800), CXCR4 (rabbit, 1:1000, Abcam, catalogue no.: ab124824), CXCR7 (rabbit, 1:1000, Abcam, catalogue no.: ab72100), β-tubulin (mouse, 1:2000, Arigo, catalogue no.: ARG62347), Flag (rabbit, 1:1000, Cell Signaling Technology, catalogue no.: 14793) or His (rabbit, 1:1000, Cell Signaling Technology, catalogue no.: 12698) overnight at 4 °C.

Techniques: Expressing, Cell Culture, Surface Biotinylation Assay, Co-Immunoprecipitation Assay, Immunoprecipitation, Pull Down Assay, Purification, Over Expression, Patch Clamp, Two Tailed Test

Journal: Nature Communications

Article Title: ATF4 selectively regulates heat nociception and contributes to kinesin-mediated TRPM3 trafficking

doi: 10.1038/s41467-021-21731-1

Figure Lengend Snippet: a The diagram shows the experimental procedure. The hindpaws of mice were soaked in a 43 °C water bath for 30 s, and the mice were placed at RT for 2, 5, 10, 20 or 40 min. Then, behavioural, co-IP and membrane protein immunoblotting experiments were performed. b , c ATF4/TRPM3 interactions ( b ) and TRPM3 membrane expression ( c ) in mice were evaluated at different time points after heat stimulation. The experiment was repeated four times in b . n = 6 mice per group in c . F (5,18) = 20.17, P = 0.0007 in 2 min, P < 0.0001 in 5 min, P < 0.0001 in 10 min, P = 0.0044 in 20 min in b . F (5,30) = 10.59, P = 0.0277 in 2 min, P = 0.0022 in 5 min, P < 0.0001 in 10 min, P = 0.0027 in 20 min in c . * P < 0.05, ** P < 0.01 versus the RT group. d The diagram shows the experimental procedure. Isolate d DRG neurons were placed in a 43 °C water bath for 30 s and placed at RT for 2, 5, 10, 20 or 40 min. The neurons were then lysed to detect the membrane abundance of TRPM3. e TRPM3 membrane expression in isolated DRG neurons was evaluated at different time points after direct heat stimulation. n = 3. F (5,12) = 12.3, P = 0.0358 in 2 min, P = 0.0100 in 5 min, P < 0.0001 in 10 min, P = 0.0085 in 20 min. * P < 0.05, ** P < 0.01 versus the RT group. f – h Naïve ( f ), ATF4 siRNA-injected ( g ) and Atf4 +/− ( h ) mice were subjected to the Hargreaves test at different time points after heat stimulation. n = 12 mice per group in f , g . n = 6 mice per group in h . F (5,66) = 14.36, P = 0.0004 in 2 min, P < 0.0001 in 5 min, P < 0.0001 in 10 min, P = 0.0057 in 20 min. ** P < 0.01 versus RT group. One-way ANOVA followed by Tukey’s multiple comparisons test. The error bars indicate the SEMs.

Article Snippet: The membranes were placed in blocking buffer for 1 h at RT and incubated in primary antibodies against ATF4 (goat, 1:1000, GeneTex, catalogue no.: GTX89973), TRPM3 (rabbit, 1:1000, Bioss, catalogue no.: bs-9046R; rabbit, 1:200, Alomone Labs, catalogue no.: ACC-050), TRPV1 (rabbit, 1:200, Alomone Labs, catalogue no.: ACC-030), TRPA1 (rabbit, 1:1000, ABclonal, catalogue no.: A12544), KIF17 (mouse, 1:100, Santa Cruz Biotechnology, catalogue no.: sc-137040), KIF3A (goat, 1:100, Santa Cruz Biotechnology, catalogue no.: sc-18745), KIF3B (rabbit, 1:100, Santa Cruz Biotechnology, catalogue no.: sc-50456), KIF5A (rabbit, 1:1000, Abcam, catalogue no.: ab5628), KIF5B (rabbit, 1:2000, Abcam, catalogue no.: ab167429), KIFC2 (rabbit, 1:100, Abcam, catalogue no.: ab3476), TfR (mouse, 1:1000, Thermo Fisher Scientific, catalogue no.: 13-6800), CXCR4 (rabbit, 1:1000, Abcam, catalogue no.: ab124824), CXCR7 (rabbit, 1:1000, Abcam, catalogue no.: ab72100), β-tubulin (mouse, 1:2000, Arigo, catalogue no.: ARG62347), Flag (rabbit, 1:1000, Cell Signaling Technology, catalogue no.: 14793) or His (rabbit, 1:1000, Cell Signaling Technology, catalogue no.: 12698) overnight at 4 °C.

Techniques: Co-Immunoprecipitation Assay, Western Blot, Expressing, Isolation, Injection

Journal: Nature Communications

Article Title: ATF4 selectively regulates heat nociception and contributes to kinesin-mediated TRPM3 trafficking

doi: 10.1038/s41467-021-21731-1

Figure Lengend Snippet: a – f Co-IP showing the ATF4/KIF interaction in the DRG. DRG lysates were immunoprecipitated with an ATF4 antibody and immunoblotted with a KIF17, KIF3A, KIF3B, KIF5A, KIF5B, KIFC2 or ATF4 antibody as indicated. This experiment was repeated three times. g SIM images show that the colocalization between ATF4 and KIF17 in DRG neurons. Scale bar, 5 μm. h The GST pull-down assay with two purified proteins, KIF17-GST-Flag and ATF4, showed a direct interaction between ATF4 and KIF17. This experiment was repeated three times. i Interaction between KIF17 and ATF4 mutants. The ATF4 mutants were transiently co-expressed with KIF17, and the cell lysates were immunoprecipitated with a His antibody and then immunoblotted with a His or Flag antibody as indicated. This experiment was repeated three times. j The interaction level between TRPM3 and KIF17 was examined by co-IP in scrambled and ATF4 siRNA-treated mice DRG lysates. DRG lysates were immunoprecipitated with a TRPM3 antibody and immunoblotted with a KIF17 or TRPM3 antibody as indicated. This experiment was repeated three times. t 4 = 12.88, P = 0.0002. k SIM images showed the colocalization between TRPM3 and KIF17 in DRG neurons from naïve, ATF4 siRNA and ATF4-overexpresing mice. Quantification data showed the colocalization rates of KIF17 with TRPM3 (colocalized yellow spots/total KIF17 positive spots) and those of TRPM3 with KIF17 (colocalized yellow spots/total TRPM3 positive spots) in DRG neurons. n = 3 mice per group. F (2,6) = 27.17, P = 0.0224 in naïve vs. ATF4-siRNA, P = 0.0255 in naïve vs. ATF4-over in KIF17 with TRPM3. F (2,6) = 33.5, P = 0.0069 in naïve vs. ATF4-siRNA, P = 0.0375 in naïve vs. ATF4-over in TRPM3 with KIF17. Scale bar, 10 μm. j Two-tailed Independent Student’s t test. k One-way ANOVA followed by Tukey’s multiple comparisons test, * P < 0.05, ** P < 0.01. The error bars indicate the SEMs.

Article Snippet: The membranes were placed in blocking buffer for 1 h at RT and incubated in primary antibodies against ATF4 (goat, 1:1000, GeneTex, catalogue no.: GTX89973), TRPM3 (rabbit, 1:1000, Bioss, catalogue no.: bs-9046R; rabbit, 1:200, Alomone Labs, catalogue no.: ACC-050), TRPV1 (rabbit, 1:200, Alomone Labs, catalogue no.: ACC-030), TRPA1 (rabbit, 1:1000, ABclonal, catalogue no.: A12544), KIF17 (mouse, 1:100, Santa Cruz Biotechnology, catalogue no.: sc-137040), KIF3A (goat, 1:100, Santa Cruz Biotechnology, catalogue no.: sc-18745), KIF3B (rabbit, 1:100, Santa Cruz Biotechnology, catalogue no.: sc-50456), KIF5A (rabbit, 1:1000, Abcam, catalogue no.: ab5628), KIF5B (rabbit, 1:2000, Abcam, catalogue no.: ab167429), KIFC2 (rabbit, 1:100, Abcam, catalogue no.: ab3476), TfR (mouse, 1:1000, Thermo Fisher Scientific, catalogue no.: 13-6800), CXCR4 (rabbit, 1:1000, Abcam, catalogue no.: ab124824), CXCR7 (rabbit, 1:1000, Abcam, catalogue no.: ab72100), β-tubulin (mouse, 1:2000, Arigo, catalogue no.: ARG62347), Flag (rabbit, 1:1000, Cell Signaling Technology, catalogue no.: 14793) or His (rabbit, 1:1000, Cell Signaling Technology, catalogue no.: 12698) overnight at 4 °C.

Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation, Pull Down Assay, Purification, Two Tailed Test

Journal: Nature Communications

Article Title: ATF4 selectively regulates heat nociception and contributes to kinesin-mediated TRPM3 trafficking

doi: 10.1038/s41467-021-21731-1

Figure Lengend Snippet: a Colocalization of the KIF17, ATF4 and TRPM3 proteins in mouse DRG sections. Scale bar, 200 μm. b Colocalization of KIF17 mRNA , ATF4 mRNA and TRPM3 mRNA in mouse DRG sections. Scale bar, 20 μm. c Colocalization of the KIF17, ATF4 and TRPM3 proteins in DRG neurons was detected by SIM. Scale bar, 5 μm. d , e Changes in the membrane expression of TRPM3 in the DRG after KIF17 knockdown ( d ) or KIF17 overexpression ( e ). n = 6 mice per group. F (2,15) = 34.36, P = 0.0002 in d . F (2,15) = 20.48, P = 0.0005 in e . f The behaviours of KIF17 siRNA- and scrambled siRNA-injected mice were evaluated by the Hargreaves test. n = 6 mice per group. t 10 = 3.485, P = 0.0059. g The behaviours of KIF17-overexpressing and control mice were evaluated by the Hargreaves test. n = 6 mice per group. t 10 = 6.776, P < 0.0001. h ATF4 knockdown suppressed the increased expression of TRPM3 in the membrane induced by KIF17 overexpression. n = 6 mice per group. F (2,15) = 16.19, P = 0.0002 in naïve vs. KIF17-over, P = 0.0014 in KIF17-over vs. KIF17-over + ATF4-siRNA. i KIF17 knockdown inhibited the increase in TRPM3 expression on the cell surface induced by ATF4 overexpression. n = 6 mice per group. F (2,15) = 12.76, P = 0.0025 in naïve vs. ATF4-over, P = 0.0010 in ATF4-over vs. ATF4-over + KIF17-siRNA. ** P < 0.01. d , e , h , i One-way ANOVA followed by Tukey’s multiple comparisons test. f , g Two-tailed Independent Student’s t test. The error bars indicate the SEMs.

Article Snippet: The membranes were placed in blocking buffer for 1 h at RT and incubated in primary antibodies against ATF4 (goat, 1:1000, GeneTex, catalogue no.: GTX89973), TRPM3 (rabbit, 1:1000, Bioss, catalogue no.: bs-9046R; rabbit, 1:200, Alomone Labs, catalogue no.: ACC-050), TRPV1 (rabbit, 1:200, Alomone Labs, catalogue no.: ACC-030), TRPA1 (rabbit, 1:1000, ABclonal, catalogue no.: A12544), KIF17 (mouse, 1:100, Santa Cruz Biotechnology, catalogue no.: sc-137040), KIF3A (goat, 1:100, Santa Cruz Biotechnology, catalogue no.: sc-18745), KIF3B (rabbit, 1:100, Santa Cruz Biotechnology, catalogue no.: sc-50456), KIF5A (rabbit, 1:1000, Abcam, catalogue no.: ab5628), KIF5B (rabbit, 1:2000, Abcam, catalogue no.: ab167429), KIFC2 (rabbit, 1:100, Abcam, catalogue no.: ab3476), TfR (mouse, 1:1000, Thermo Fisher Scientific, catalogue no.: 13-6800), CXCR4 (rabbit, 1:1000, Abcam, catalogue no.: ab124824), CXCR7 (rabbit, 1:1000, Abcam, catalogue no.: ab72100), β-tubulin (mouse, 1:2000, Arigo, catalogue no.: ARG62347), Flag (rabbit, 1:1000, Cell Signaling Technology, catalogue no.: 14793) or His (rabbit, 1:1000, Cell Signaling Technology, catalogue no.: 12698) overnight at 4 °C.

Techniques: Expressing, Over Expression, Injection, Two Tailed Test

Journal: Nature Communications

Article Title: ATF4 selectively regulates heat nociception and contributes to kinesin-mediated TRPM3 trafficking

doi: 10.1038/s41467-021-21731-1

Figure Lengend Snippet: a Cultured DRG neurons were treated with CXCL12 (1 μg/mL) for 120, 240 or 360 min, and then ATF4 expression was measured. n = 6. F (3,20) = 4.642, P = 0.0462 in 120 min, P = 0.0154 in 240 min, P = 0.0128 in 360 min. * P < 0.05 versus 0 min. b CXCL12 (1 μg in 10 μL PBS + 0.5% BSA) was intrathecally injected three times every 3 h, and ATF4 expression was measured in the DRG 2 h after the last injection. n = 6 mice per group. t 10 = 5.151 , P = 0.0004. ** P < 0.01. c ATF4 siRNA signifi c antly relieved CXCL12-induced thermal hyperalgesia. n = 12 mice per group. F (2,33) = 15.68, P < 0.0001 in vehicle vs. CXCL12, P = 0.0101 in CXCL12 vs. CXCL12 + ATF4-siRNA. * P < 0.05, ** P < 0.01. d CXCR4 siRNA but not CXCR7 siRNA abolished the increase in ATF4 expression in the DRG induced by CXCL12 in vivo. n = 6 mice per group. F (3,20) = 54.47, P < 0.0001 in CXCL12, CXCL12 + CXCR4-siRNA and CXCL12 + CXCR7-siRNA. ** P < 0.01 versus the vehicle group, ## P < 0.01 versus the CXCL12 group. e Hypothetical model illustrating that ATF4 interacts with TRPM3 and KIF17 to form a complex to regulate the membrane trafficking of TRPM3 in sensory neurons and thus contributes to thermal sensitivity. a , c , d One-way ANOVA followed by Tukey’s multiple comparisons test. b Two-tailed Independent Student’s t test. The error bars indicate the SEMs.

Article Snippet: The membranes were placed in blocking buffer for 1 h at RT and incubated in primary antibodies against ATF4 (goat, 1:1000, GeneTex, catalogue no.: GTX89973), TRPM3 (rabbit, 1:1000, Bioss, catalogue no.: bs-9046R; rabbit, 1:200, Alomone Labs, catalogue no.: ACC-050), TRPV1 (rabbit, 1:200, Alomone Labs, catalogue no.: ACC-030), TRPA1 (rabbit, 1:1000, ABclonal, catalogue no.: A12544), KIF17 (mouse, 1:100, Santa Cruz Biotechnology, catalogue no.: sc-137040), KIF3A (goat, 1:100, Santa Cruz Biotechnology, catalogue no.: sc-18745), KIF3B (rabbit, 1:100, Santa Cruz Biotechnology, catalogue no.: sc-50456), KIF5A (rabbit, 1:1000, Abcam, catalogue no.: ab5628), KIF5B (rabbit, 1:2000, Abcam, catalogue no.: ab167429), KIFC2 (rabbit, 1:100, Abcam, catalogue no.: ab3476), TfR (mouse, 1:1000, Thermo Fisher Scientific, catalogue no.: 13-6800), CXCR4 (rabbit, 1:1000, Abcam, catalogue no.: ab124824), CXCR7 (rabbit, 1:1000, Abcam, catalogue no.: ab72100), β-tubulin (mouse, 1:2000, Arigo, catalogue no.: ARG62347), Flag (rabbit, 1:1000, Cell Signaling Technology, catalogue no.: 14793) or His (rabbit, 1:1000, Cell Signaling Technology, catalogue no.: 12698) overnight at 4 °C.

Techniques: Cell Culture, Expressing, Injection, In Vivo, Two Tailed Test