Journal: Clinical and Experimental Pharmacology & Physiology

Article Title: Mechanisms of IL‐17A Neutralisation in Alleviating Renal Fibrosis and Inflammation in Spontaneously Hypertensive Rats

doi: 10.1111/1440-1681.70116

Figure Lengend Snippet: IL‐17A NAb inhibited EMT in SHR renal tissues. (A) Representative IHC images showing the expression of E‐cadherin, α‐SMA, and Collagen III, with quantitative analysis of their positive areas. (B) Representative immunoblots and relative expression levels of E‐cadherin, α‐SMA, and Collagen III proteins. (C) mRNA expression levels of E‐cadherin, α‐SMA, and Collagen III. Data are expressed as mean ± SD ( n = 6).

Article Snippet: Sections were then incubated overnight at 4°C with primary antibodies against: E‐cadherin (Boster, China), Collagen III (Boster, China), inducible nitric oxide synthase (iNOS) (Boster, China), CD86 (Boster, China), arginase‐1 (Arg‐1) (Boster, China), CD163 (Boster, China), α‐smooth muscle actin (α‐SMA) (Cell Signalling Technology, USA).

Techniques: Expressing, Western Blot

Journal: Scientific reports

Article Title: Dual attenuation of proteasomal and autophagic BMAL1 degradation in Clock Δ19/+ mice contributes to improved glucose homeostasis.

doi: 10.1038/srep12801

Figure Lengend Snippet: Figure 1. ClockΔ19/+ (Clk/+) mutant mice displayed improved glucose homeostasis and resistance to period lengthening and BMAL1 depletion under high-fat diet (HFD) feeding. Fasting glucose (a) serum insulin (b) glucose tolerance (c) and insulin tolerance (d) in WT and Clk/+ mutant mice after HFD feeding for 12 weeks (n = 9 − 11). For (c) and (d) area under curve (A.U.C) comparison is shown next to the graphs. (e) Representative actograms in each condition were shown. The mice were kept on a 12 h:12 h light:dark cycle (represented in the bar above) for 16 days and then released into constant darkness. (f) Circadian free-running period (tau) was calculated by using the ActiView software (n = 3 − 5). (g) Liver tissues were collected over the circadian time course from mice fed with regular chow (RC) or high-fat diet (HFD). Lysates were prepared and subjected to immunoblotting analysis using antibodies against clock proteins and GAPDH as control. The results were representative of three independent experiments. *p < 0.05, **p < 0.01, one-way ANOVA.

Article Snippet: The primary antibodies used in the study are: Flag-tag (Sigma-Aldrich), Myc-tag, AKT, pAKT (Ser473), S6K, pS6K (Thr389) and FOXO1, pFOXO1 (Thr24) (Cell Signaling), BMAL1 (Cocalico, epitope: aa 381–579), CLOCK (Santa Cruz), LC3B (Novus), p62/SQSTM1 (Boster), REV-ERBα (Cell Signaling), PER256, GADPH (Ambion), α -Tubulin (Santa Cruz) and Lamin B1 (Abcam).

Techniques: Mutagenesis, Comparison, Software, Western Blot, Control

Journal: Scientific reports

Article Title: Dual attenuation of proteasomal and autophagic BMAL1 degradation in Clock Δ19/+ mice contributes to improved glucose homeostasis.

doi: 10.1038/srep12801

Figure Lengend Snippet: Figure 2. CLOCK and CLOCKΔ19 exerted differential effects on BMAL1 ubiquitination and stability. (a,b) Protein lysates and total RNAs were prepared from wild-type (WT), ClockΔ19/+ (Clk/+ ) and ClockΔ19/Δ19 (Clk/Clk) MEF cells and subjected to Western blot analysis of CLOCK and BMAL1 (a) and real-time qPCR analysis of Bmal1 transcript levels (b). GAPDH was used as a loading control in (a). (c) WT, Clk/+ and Clk/ Clk MEF cells were exposed to cycloheximide (CHX; 25 μ g/ml) for the indicated times, and BMAL1 levels were assessed by immunoblotting analysis. Quantification is presented in the lower panel. Error bars represent mean ± SD (n = 3). Half-life was determined by using the GraphPad Prism software with nonlinear, one-phase exponential decay analysis. The analysis calculated the half-life parameter K at 0.191/h, 0.050/h, and 0.054/h in WT, Clk/+ and Clk/Clk MEF cells, corresponding to half-life at 3.63 h, 14.0 h and 12.9 h respectively. The K values are significantly different among all three conditions: p < 0.0001. Half-life was determined by using nonlinear, one-phase exponential decay analysis. Half-life parameter, K, is significantly different in all three conditions: p < 0.0001. (d) 293T cells transfected with expression vectors for Flag-BMAL1, Flag-CLOCK and/ or Flag-CLOCKΔ 19 were subjected to immunoblotting analysis with antibodies against the Flag-tag and GAPDH (loading control). Quantitation of three independent experiments is presented in the lower panel. (e) Cytoplasmic and nuclear fractions of 293T cells transfected with the indicated expression vectors were subjected to immunoblotting analysis with antibodies against anti-Flag, α -Tubulin and Lamin B1. The data represent the mean ± SD of three independent experiments. (f) 293T cells transfected with the indicated expression vectors for Flag-BMAL1, His-tagged ubiquitin (His-Ub), Flag-CLOCK, and Flag-CLOCKΔ 19 were lysed and immunoprecipitated with anti-Flag antibody prior to immunoblotting analysis with anti-BMAL1 and -CLOCK antibodies. The relative levels of the targeted proteins were measured by densitometry using the Image J program and the ratios were calculated relative to the GAPDH control.

Article Snippet: The primary antibodies used in the study are: Flag-tag (Sigma-Aldrich), Myc-tag, AKT, pAKT (Ser473), S6K, pS6K (Thr389) and FOXO1, pFOXO1 (Thr24) (Cell Signaling), BMAL1 (Cocalico, epitope: aa 381–579), CLOCK (Santa Cruz), LC3B (Novus), p62/SQSTM1 (Boster), REV-ERBα (Cell Signaling), PER256, GADPH (Ambion), α -Tubulin (Santa Cruz) and Lamin B1 (Abcam).

Techniques: Ubiquitin Proteomics, Western Blot, Control, Software, Transfection, Expressing, FLAG-tag, Quantitation Assay, Immunoprecipitation

Journal: Scientific reports

Article Title: Dual attenuation of proteasomal and autophagic BMAL1 degradation in Clock Δ19/+ mice contributes to improved glucose homeostasis.

doi: 10.1038/srep12801

Figure Lengend Snippet: Figure 3. CLOCKΔ19 attenuated BMAL1 degradation via both proteasomal and autophagic pathways. (a) WT, Clk/+ , and Clk/Clk MEF cells were exposed to 10 μ M MG132, 5 mM 3-methyladenine (3-MA) or 25 μ M chloroquine (CQ) for 4, 24 and 4 h, respectively. Cell lysates were subjected to immunoblotting analysis with antibodies against BMAL1, CLOCK and GAPDH (loading control). Representative results from three independent experiments are shown. (b) 293T cells transfected with the indicated expression vectors were treated with MG132 and 3-MA for 4 and 24 h followed by CHX exposure for the indicated times, and subjected to immunoblotting with antibodies against Flag and GAPDH (loading control). (c–e) 293T cells transfected with the indicated expression vectors were incubated in the absence or presence of MG132 (10 μ M) (c), 3-MA (1 mM) (d) or CQ (25 μ M) (e) for 4, 24, and 24 h, respectively. Lysates were subjected to immunoblotting analysis with antibodies against Flag and GAPDH (loading control).

Article Snippet: The primary antibodies used in the study are: Flag-tag (Sigma-Aldrich), Myc-tag, AKT, pAKT (Ser473), S6K, pS6K (Thr389) and FOXO1, pFOXO1 (Thr24) (Cell Signaling), BMAL1 (Cocalico, epitope: aa 381–579), CLOCK (Santa Cruz), LC3B (Novus), p62/SQSTM1 (Boster), REV-ERBα (Cell Signaling), PER256, GADPH (Ambion), α -Tubulin (Santa Cruz) and Lamin B1 (Abcam).

Techniques: Western Blot, Control, Transfection, Expressing, Incubation

Journal: Scientific reports

Article Title: Dual attenuation of proteasomal and autophagic BMAL1 degradation in Clock Δ19/+ mice contributes to improved glucose homeostasis.

doi: 10.1038/srep12801

Figure Lengend Snippet: Figure 4. CLOCK and CLOCKΔ19 differentially regulated BMAL1 degradation in a p62-dependent manner. (a,b) 293T cells transfected with expression vectors for Flag-BMAL1, Flag-CLOCK, Flag- CLOCKΔ 19, and Flag-p62 were lysed and subjected to immunoblotting analysis with antibodies against anti-Flag and GAPDH (loading control). (c) Schematic representation of p62 constructs employed were Myc-tagged WT p62; full-length p62 (amino acids 1 to 440), and p62 Δ UBA, a construct missing the UBA domain (amino acids 386 to 440) (upper panel). 293T cells transfected with expression vectors for Flag-BMAL1, Flag-CLOCK, Flag-CLOCKΔ 19, and Myc-p62 (WT or Δ UBA) were lysed and subjected to immunoblotting analysis with antibodies against anti-Flag, anti-Myc and GAPDH (loading control). (d) Protein lysates were prepared from p62+/+ (WT) and p62−/− MEF cells and subjected to immunoblotting analysis of BMAL1 and GAPDH. Ponseau S staining of the membrane following transfer was carried out as an additional loading control. (e) MEFs (p62+/+ or p62−/−) expressing Flag-BMAL1 and Flag-CLOCK were lysed and subjected to immunoblotting analysis with antibodies against anti-Flag, p62, and GAPDH (loading control). (f) 293T cells were transfected with indicated expression vectors in the presence of negative control or p62 siRNA for 48 h and then immunoblotted with the indicated antibodies. The relative levels of the targeted proteins were estimated by densitometry using the ImageJ program and the ratios were calculated relative to the GAPDH control. The data represent the mean ± SD of three independent experiments.

Article Snippet: The primary antibodies used in the study are: Flag-tag (Sigma-Aldrich), Myc-tag, AKT, pAKT (Ser473), S6K, pS6K (Thr389) and FOXO1, pFOXO1 (Thr24) (Cell Signaling), BMAL1 (Cocalico, epitope: aa 381–579), CLOCK (Santa Cruz), LC3B (Novus), p62/SQSTM1 (Boster), REV-ERBα (Cell Signaling), PER256, GADPH (Ambion), α -Tubulin (Santa Cruz) and Lamin B1 (Abcam).

Techniques: Transfection, Expressing, Western Blot, Control, Construct, Staining, Membrane, Negative Control

Journal: Journal of Cancer Research and Clinical Oncology

Article Title: High PER1 expression is associated with STK11 mutation and clinical biomarkers of immunotherapy resistance in lung adenocarcinoma

doi: 10.1007/s00432-025-06269-9

Figure Lengend Snippet: PER1 expression is increased with loss of LKB1. A PER1 mRNA expression in control (C), sh STK11 (L), KRAS G12V (K), and KRAS G12V /sh STK11 (KL) invading HBEC3-KT organoids; notated with adjusted p-values from ordinary one-way ANOVA followed by Šídák's multiple comparisons tests, C vs L p < 0.0001, K vs KL p < 0.0001, L vs KL p = 0.0052. B PER1 mRNA expression in LKB1-wildtype (WT) and LKB1-knockout (KO) HBEC3-KT cells; two-tailed Student’s t-test, p < 0.0001. C Western blot of PER1, LKB1, pAMPK T172, AMPK, and Actin in LKB1-WT and LKB1-KO HBEC3-KT cells, with and without glucose. D Western blot of PER1, LKB1, and Lamin A/C in LKB1-WT and LKB1-KO HBEC3-KT cells. E Western blot of PER1, LKB1, and Actin in K and KL HBEC3-KT cells. F Western blot of PER1, LKB1, and Actin in empty-vector control A549 cells and with addback of wildtype LKB1. G PER1 mRNA expression in KRAS -mutant (K) and KRAS / STK11 -mutant (KL) tumors from the TCGA LUAD study; two-tailed Student’s t-test, p = 0.0224. H) PER1 protein abundance ratio in KRAS -mutant (K) and KRAS / STK11 -mutant (KL) tumors from the CPTAC 2020 study; two-tailed Student’s t-test, p = 0.0191. I) PER1 mRNA expression in LKB1-wildtype (WT) and LKB1-mutant (MUT) tumors from the TCGA LUAD study; two-tailed Student’s t-test, p < 0.0001. J) PER1 protein abundance ratio in LKB1-wildtype (WT) and LKB1-mutant (MUT) tumors from the CPTAC 2020 study; two-tailed Student’s t-test, p = 0.0422

Article Snippet: The following primary antibodies were diluted in 5% BSA in tris-buffered saline (Cell Signaling, 12498S) supplemented with 0.01% Tween-20 (TBS-T; ChemCruz, sc-29113B) and used at the indicated dilutions: PER1 (1:1000, Boster Bio, A00876, RRID:AB_3086701), LKB1 (1:1000, Cell Signaling, #3050, RRID:AB_823559), JAG1 (1:1000, Cell Signaling, #70,109, RRID:AB_2799774), pAMPK-T172 (1:1000, Cell Signaling, #2535, RRID:AB_331250), AMPK (1:1000, Cell Signaling, #2532, RRID:AB_330331), Actin (1:500, DSHB, JLA20, RRID:AB_528068; 1:1000, Sigma-Aldrich, A5441, RRID:AB_476744), Lamin A/C (1:1000, DSHB, MANLAC3(4C10), RRID:AB_2618205).

Techniques: Expressing, Control, Knock-Out, Two Tailed Test, Western Blot, Plasmid Preparation, Mutagenesis, Quantitative Proteomics

Journal: Journal of Cancer Research and Clinical Oncology

Article Title: High PER1 expression is associated with STK11 mutation and clinical biomarkers of immunotherapy resistance in lung adenocarcinoma

doi: 10.1007/s00432-025-06269-9

Figure Lengend Snippet: PER1 knockdown decreases proliferation of cells lacking LKB1. A Left, CCK-8 cell proliferation assay in KRAS G12V (K) and KRAS G12V /sh STK11 (KL) HBEC3-KT cells treated with siRNA for non-targeting control (NTC) and PER1 (si PER1 ). Right, Alternate presentation of the same growth curves, showing K and KL separately. B Statistical analysis of growth curves from A, showing the effect of si PER1 versus siNTC on cell proliferation of K (two-tailed Student’s t-test, p = 0.7547) and KL (two-tailed Student’s t-test, p = 0.0002) HBEC3-KT cells at 96 h. C Cell counting cell proliferation assay of siNTC- and si PER1 -treated KL HBEC3-KT cells; two-tailed Student’s t-test with correction for multiple comparisons, p = 0.035 at 48 h and p = 0.008 at 72 h. D Cell counting cell proliferation assay of siNTC- and si Per1 -treated JK-43-M cells; two-tailed Student’s t-test with correction for multiple comparisons, p = 0.0001 at 48 h and p = 0.0051 at 72 h. E Effect of siPER1 versus siNTC on relative cell number of A549 cells at 96 h; two-tailed Student’s t-test, p = 0.0263

Article Snippet: The following primary antibodies were diluted in 5% BSA in tris-buffered saline (Cell Signaling, 12498S) supplemented with 0.01% Tween-20 (TBS-T; ChemCruz, sc-29113B) and used at the indicated dilutions: PER1 (1:1000, Boster Bio, A00876, RRID:AB_3086701), LKB1 (1:1000, Cell Signaling, #3050, RRID:AB_823559), JAG1 (1:1000, Cell Signaling, #70,109, RRID:AB_2799774), pAMPK-T172 (1:1000, Cell Signaling, #2535, RRID:AB_331250), AMPK (1:1000, Cell Signaling, #2532, RRID:AB_330331), Actin (1:500, DSHB, JLA20, RRID:AB_528068; 1:1000, Sigma-Aldrich, A5441, RRID:AB_476744), Lamin A/C (1:1000, DSHB, MANLAC3(4C10), RRID:AB_2618205).

Techniques: Knockdown, CCK-8 Assay, Proliferation Assay, Control, Two Tailed Test, Cell Counting

Journal: Journal of Cancer Research and Clinical Oncology

Article Title: High PER1 expression is associated with STK11 mutation and clinical biomarkers of immunotherapy resistance in lung adenocarcinoma

doi: 10.1007/s00432-025-06269-9

Figure Lengend Snippet: PER1 knockdown decreases invasion in low-LKB1 H1299 leader cells. A Western blot of PER1, LKB1, JAG1, and Actin in Parental, Leader, and Follower H1299 cells. B Western blot PER1, LKB1, JAG1, Lamin A/C, and Tubulin in cytosolic (cyto) and nuclear (nuc) subcellular fractionation lysates from Parental (P), Leader (L), and Follower (F) H1299 cells. C Left, STK11 mRNA expression in Parental, Leader, and Follower H1299 cells; one-way ANOVA with multiple comparisons tests, Parental vs Leader p = 0.0133, Parental vs Follower p = 0.0512, Leader vs Follower p = 0.0008. Right, PER1 mRNA expression in Parental, Leader, and Follower H1299 cells; one-way ANOVA with multiple comparisons tests, Parental vs Leader p = 0.0852, Parental vs Follower p = 0.0080, Leader vs Follower p = 0.0007. D Effect of si PER1 versus siNTC on 48 h 3D invasion of Leader and Follower H1299 spheroids (n = 11–12); 2-way ANOVA with multiple comparisons, Leaders p < 0.0001, Followers p = 0.4328. E Representative images from H1299 spheroid invasion assays. Green lines indicate the spheroid cores and pink lines indicate the invasive edges

Article Snippet: The following primary antibodies were diluted in 5% BSA in tris-buffered saline (Cell Signaling, 12498S) supplemented with 0.01% Tween-20 (TBS-T; ChemCruz, sc-29113B) and used at the indicated dilutions: PER1 (1:1000, Boster Bio, A00876, RRID:AB_3086701), LKB1 (1:1000, Cell Signaling, #3050, RRID:AB_823559), JAG1 (1:1000, Cell Signaling, #70,109, RRID:AB_2799774), pAMPK-T172 (1:1000, Cell Signaling, #2535, RRID:AB_331250), AMPK (1:1000, Cell Signaling, #2532, RRID:AB_330331), Actin (1:500, DSHB, JLA20, RRID:AB_528068; 1:1000, Sigma-Aldrich, A5441, RRID:AB_476744), Lamin A/C (1:1000, DSHB, MANLAC3(4C10), RRID:AB_2618205).

Techniques: Knockdown, Western Blot, Fractionation, Expressing

Journal: Journal of Cancer Research and Clinical Oncology

Article Title: High PER1 expression is associated with STK11 mutation and clinical biomarkers of immunotherapy resistance in lung adenocarcinoma

doi: 10.1007/s00432-025-06269-9

Figure Lengend Snippet: Clinical significance of high PER1 expression in lung adenocarcinoma. A–B Top ten most differentially altered genes between the highest and lowest PER1 mRNA expression quartiles of lung adenocarcinoma samples in the TCGA LUAD study. C–D Top ten most differentially altered genes between the highest and lowest PER1 protein expression quartiles of lung adenocarcinoma samples in the CPTAC 2020 study. E Ragnum (p < 10 −10 , q < 10 −10 ), Buffa (p < 10 −10 , q < 10 −10 ), and Winter (p < 10 −10 , q = 1.06 −10 ) hypoxia scores as calculated in cBioPortal in Low PER1 versus High PER1 mRNA expression quartiles in the TCGA LUAD study. F Expression of SLC2A1 (GLUT1) in: Low PER1 and High PER1 expression quartiles in the TCGA LUAD study (two-tailed Student’s t-test, p < 0.0001), Low PER1 and High PER1 expression quartiles in the CPTAC-GDC study (two-tailed Student’s t-test, p = 0.0006), and Low PER1 and High PER1 expression quartiles in the CPTAC 2020 study (two-tailed Student’s t-test, p = 0.2034). G Expression of ANLN in: Low PER1 and High PER1 expression quartiles in the TCGA LUAD study (two-tailed Student’s t-test, p < 0.0001), Low PER1 and High PER1 expression quartiles in the CPTAC-GDC study (two-tailed Student’s t-test, p = 0.0006), and Low PER1 and High PER1 expression quartiles in the CPTAC 2020 study (two-tailed Student’s t-test, p = 0.0114). H Expression of HIF3A in: Low PER1 and High PER1 expression quartiles in the TCGA LUAD study (two-tailed Student’s t-test, p < 0.0001), Low PER1 and High PER1 expression quartiles in the CPTAC-GDC study (two-tailed Student’s t-test, p = 0.0049), and Low PER1 and High PER1 expression quartiles in the CPTAC 2020 study (two-tailed Student’s t-test, p = 0.0590). I Overall (p = 2.76 −4 , q = 0.0146), stromal (p = 2.001 −3 , q = 0.0323), and immune (p = 2.73 −3 , q = 0.0323) ESTIMATE scores as calculated in cBioPortal in Low PER1 versus High PER1 protein expression quartiles in the CPTAC 2020 study. J CD274 (PD-L1) mRNA expression in Low PER1 versus High PER1 expression quartiles in the TCGA LUAD study; two-tailed Student’s t-test, p = 0.0126. K CD274 (PD-L1) mRNA expression in Low PER1 versus High PER1 mRNA expression quartiles in the CPTAC GDC study; two-tailed Student’s t-test, p = 0.0005. L CD274 mRNA expression in Low PER1 versus High PER1 protein expression quartiles in the CPTAC 2020 study; two-tailed Student’s t-test, p = 0.0139. M) PD-L1 protein expression (as measured by protein abundance ratio) in the Low PER1 and High PER1 protein expression quartiles of the CPTAC 2020 study; two-tailed Student’s t-test, p = 0.0386

Article Snippet: The following primary antibodies were diluted in 5% BSA in tris-buffered saline (Cell Signaling, 12498S) supplemented with 0.01% Tween-20 (TBS-T; ChemCruz, sc-29113B) and used at the indicated dilutions: PER1 (1:1000, Boster Bio, A00876, RRID:AB_3086701), LKB1 (1:1000, Cell Signaling, #3050, RRID:AB_823559), JAG1 (1:1000, Cell Signaling, #70,109, RRID:AB_2799774), pAMPK-T172 (1:1000, Cell Signaling, #2535, RRID:AB_331250), AMPK (1:1000, Cell Signaling, #2532, RRID:AB_330331), Actin (1:500, DSHB, JLA20, RRID:AB_528068; 1:1000, Sigma-Aldrich, A5441, RRID:AB_476744), Lamin A/C (1:1000, DSHB, MANLAC3(4C10), RRID:AB_2618205).

Techniques: Expressing, Two Tailed Test, Quantitative Proteomics