rabbit polyclonal anti cxcl12  (Proteintech)

Journal: iScience

Article Title: AID-induced CXCL12 upregulation enhances castration-resistant prostate cancer cell metastasis by stabilizing β-catenin expression

doi: 10.1016/j.isci.2023.108523

Figure Lengend Snippet: AID upregulates the invasiveness of PCa cells in vitro (A and B) The expression of AID in prostate cancer cell lines. Overall, the expression of AID in CRPC cells is significantly higher than that in HSPC cells. There is no significant difference of AID expression between C4-2, C4-2B, and PC3 cells (p = 0.4057, 0.5165, and 0.6612, respectively), or between 22RV1 and LNCaP cells (p = 0.5185). (C–E) The expression of CXCL12, E-cadherin, N-cadherin, vimentin, MMP14, and WLS in experimental cell lines. The EMT-related proteins described above express significantly higher in CRPC cells than that in androgen-sensitive prostate cancer cells (p < 0.01); conversely, the expression of E-cadherin in HSPC cells is significantly higher than that in CRPC cells (p < 0.001), and the expression of those proteins has no significant difference between C4-2 and C4-2B cells (p > 0.05). (F and G) Transwell assay shows that the invasiveness of C4-2 and C4-2B cells is significantly stronger than that in LNCaP cells (p < 0.001), and there is no significant difference between C4-2 and C4-2B cells (p = 0.4138). (H and I) The lentivirus-based AICDA-specific shRNA significantly inhibits AID expression in all three experimental cell lines (C4-2 and C4-2B: p < 0.001; LNCaP: p < 0.05, respectively). (J–L) AICDA silencing significantly downregulates CXCL12, N-cadherin, vimentin, MMp14, and WLS and upregulates E-cadherin in C4-2 and C4-2B cell (p < 0.01). (M and N) The expression of MM14 (p < 0.001) and WLS (p < 0.001) in LNCaP cells is significantly inhibited by AICDA silencing. (O–R) Transwell assay shows that shAICDA significantly suppresses the invasiveness of C4-2 (p < 0.05), C4-2B (p < 0.001), and LNCaP cells (p < 0.01). Scale bar, 50 μm. Student’s t test or one-way ANOVA was used to evaluate statistical differences, and data are presented as the means ± standard deviation, all experiments were repeated three times. Two-sided Student’s t test was used for all panels. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n.s, no significance.

Article Snippet: Rabbit polyclonal anti-CXCL12 , Abcam , Cat# ab9797; RRID: AB_296627.

Techniques: In Vitro, Expressing, Transwell Assay, shRNA, Standard Deviation

Journal: iScience

Article Title: AID-induced CXCL12 upregulation enhances castration-resistant prostate cancer cell metastasis by stabilizing β-catenin expression

doi: 10.1016/j.isci.2023.108523

Figure Lengend Snippet: AID enhances the malignant phenotype of C4-2B cells in vivo (A and B) AICDA silencing significantly suppresses the proliferation of C4-2B cells in nude mice (n = 8, p < 0.001). Tumor volume (in mm 3 ) was calculated by the formula, 0.5 × (long diameter) × (short diameter) 2 . (C and D) IHC shows that AID, CXCL12, N-cadherin, vimentin, MMP14, and WLS expression is significantly higher in shCon group than that in shAICDA group, and expression of E-cadherin is conversely (n = 8, p < 0.01). Scale bar, 50 or 20 μm. (E and F) The number of metastatic tumor in shCon group is significantly much more than that in shAICDA group (n = 8, p < 0.01). (G and H) IHC shows that the expression of AID, CXCL12, N-cadherin, vimentin, MMP14, and WLS in shCon group is significantly higher than that in shAICDA group (p < 0.001), and there is no significant difference of E-cadherin expression level between both group (p > 0.05), and almost all metastatic cancer cells express target proteins strongly, except E-cadherin. Scale bar = 100 or 50 μm. (I) HE staining of experimental mice lung shows that all nude mice used in survival time analysis die of C4-2B cell-induced pulmonary alveoli or blood vessel destruction. Scale bar, 100 μm. (J) The Kaplan-Meyer curve was performed to explore the influence caused by AID on survival time of nude mice. AICDA silencing significantly prolongs the survival time of nude mice (n = 17, p < 0.001). Two-sided Student’s t test was used for A and B and E and F, one-way ANOVA was used for C and D and G and H, log rank was used for J, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n.s, no significance.

Article Snippet: Rabbit polyclonal anti-CXCL12 , Abcam , Cat# ab9797; RRID: AB_296627.

Techniques: In Vivo, Expressing, Staining

Journal: iScience

Article Title: AID-induced CXCL12 upregulation enhances castration-resistant prostate cancer cell metastasis by stabilizing β-catenin expression

doi: 10.1016/j.isci.2023.108523

Figure Lengend Snippet: AID promotes the EMT process in C4-2 and C4-2B cell lines through CXCL12 demethylation (A and B) AID silencing significantly inhibited the expression of CXCL12 on protein level in C4-2 and C4-2B cells (p < 0.01), and there is no significant difference between shCon and shAICDA group in LNCaP cells (p = 0.508). The expression of CXCL12 in shAICDA+5-aza (50 nM, dissolved in PBS) group is remarkable than that in shAICDA group in C4-2 and C4-2B (p < 0.01) cells, and there is no significant difference between shCon and shAICDA+5-aza group (p = 0.811). Besides, the expression of CXCL12 in LNCaP cells was also upregulated by the demethylation reagent (p < 0.01). AICDA silencing-induced downregulation of CXCL12 was recovered by the treatment of 5-aza (50 nM, dissolved in PBS); furthermore, the expression of CXCL12 in LNCaP cells was also upregulated by the demethylation reagent. (C and D) The shRNA-based specific sequence significantly inhibited the expression of CXCL12 in all experimental cell lines (p < 0.001); however, the expression of AID was not influenced by downregulation of CXCL12 (p > 0.05). (E) RT-PCR demonstrated that the transcription of CXCL12 was remarkably suppressed by AICDA silencing in C4-2 and C4-2B cells (p < 0.001), and there is no significant difference between blank and shCon group (p > 0.05), and transcription of CXCL12 was not influenced by AICDA silencing in LNCaP cells (p > 0.05). (F and G) CXCL12 silencing significantly downregulated the expression of N-cadherin and vimentin and upregulates the expression of E-cadherin in C4-2 (p < 0.01, <0.001 and <0.05) and C4-2B (p < 0.001) cells; however, the same result was not observed in LNCaP cells (p > 0.05). The treatment of 5-aza failed to recover the CXCL12 silencing-induced expression variation of E-cadherin, N-cadherin, and vimentin (p > 0.05). (H–K) The treatment of 5-aza (50 nM, dissolved in PBS) converts the AICDA silencing-induced expression variation of E-cadherin, N-cadherin, and vimentin in C4-2 and C4-2B cells (p < 0.001), and as expected, there is no difference between shCon, shAICDA+5-aza, and shAICDA group (p > 0.05). (L–N) The relative expression of EMT-related proteins including E-cadherin, N-cadherin, and vimentin was detected in shCon, shCXCL12 + OEAICDA, and shCXCL12 group, and OEAICDA failed to upregulate N-cadherin and vimentin, or downregulate E-cadherin in CXCL12-silenced C4-2 and C4-2B cells (p > 0.05). (O and P) Transwell assay shows that both AICDA and CXCL12 silencing depressed the invasiveness of C4-2 (p < 0.001) and C4-2B (p < 0.001) cells, and 5-aza upregulates the invasiveness of AICDA-silenced C4-2 (p < 0.001 and <0.001) and C4-2B (p < 0.001 and <0.001) cells but CXCL12-silenced C4-2 and C4-2B cells (p > 0.05). Nevertheless, the invasiveness of CXCL12-silenced C4-2 and C4-2B cells was enhanced by overexpression of AICDA (p < 0.01), Scale bar, 50 μm. (Q) The gene correlation analysis between CXCL12 and CSNK1A1 (encoding CK1α) based on database GSE46691 . Pearson r values are used to indicate the level of correlation. The Pearson R value between CXCL12 and CSNK1A1 is −0.715 (p < 0.001). (R) The gene correlation analysis between CXCL12 and CTNNB1 (encoding β-catenin) based on TCGA database (497 specimen). The R value is 0.1597 and 0.4043 in biochemical recurrence-free (p = 0.0023) and biochemical recurrence group (p = 0.0016), respectively. Two-sided Student’s t test was used for A–P, Pearson correlation coefficient was used for Q and R. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n.s, no significance.

Article Snippet: Rabbit polyclonal anti-CXCL12 , Abcam , Cat# ab9797; RRID: AB_296627.

Techniques: Expressing, shRNA, Sequencing, Reverse Transcription Polymerase Chain Reaction, Transwell Assay, Over Expression

Journal: iScience

Article Title: AID-induced CXCL12 upregulation enhances castration-resistant prostate cancer cell metastasis by stabilizing β-catenin expression

doi: 10.1016/j.isci.2023.108523

Figure Lengend Snippet: AID-induced upregulation of CXCL12 stabilizes β-catenin to enhance EMT by inhibiting CK1α in CRPC cells (A–C) Expression of CK1α is significantly upregulated by both AICDA and CXCL12 silencing in C4-2 and C4-2B (p < 0.001) cells. (D–H) AICDA and CXCL12 silencing significantly downregulates β-catenin and conversely promotes the expression of pS45-β-catenin and pS33/S37/Thr41-β-catenin in C4-2 (shAICDA: p < 0.001, <0.01 or <0.001; shCXCL12: p < 0.001) and C4-2B (shAICDA: p < 0.0001, <0.001, <0.01; shCXCL12: p < 0.001, <0.01 or <0.01) cell line. (I–O) Pyrvinium significantly inhibits the expression of β-catenin (p < 0.001), N-cadherin (p < 0.001), and vimentin (p < 0.01, <0.001) and promotes the expression of pS45-β-catenin (p < 0.001), pS33/S37/Thr41-β-catenin (p < 0.01, <0.001), and E-cadherin in C4-2 and C4-2B cells (p < 0.001), and its inhibition efficiency was not statistical difference from shCon, shAICDA, shCXCL12, or shAICDA+shCXCL12 group (p > 0.05). (P–R) Transwell assay shows that pyrvinium significantly suppresses the invasiveness of C4-2 and C4-2B cells (p < 0.001), and there is no significantly difference between shCon+ Pyrvinium and shAICDA and shCXCL12 or shAICDA+shCXCL12 group (p > 0.05). Scale bar, 50 μm. (S–U) Overexpression of AID failed to upregulate β-catenin or downregulate pS45-β-catenin and pS33/S37/Thr41-β-Catenin in CXCL12 silenced C4-2 and C4-2B cells (p > 0.05). Student’s t test or one-way ANOVA was used to evaluate statistical differences, and data are presented as the means ± standard deviation, every experiment is repeated three times. Two-sided Student’s t test was used for all panels. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n.s, no significance.

Article Snippet: Rabbit polyclonal anti-CXCL12 , Abcam , Cat# ab9797; RRID: AB_296627.

Techniques: Expressing, Inhibition, Transwell Assay, Over Expression, Standard Deviation

Journal: iScience

Article Title: AID-induced CXCL12 upregulation enhances castration-resistant prostate cancer cell metastasis by stabilizing β-catenin expression

doi: 10.1016/j.isci.2023.108523

Figure Lengend Snippet: AID facilitates the metastasis of PCa cells by upregulating MMP14 and WLS (A and B) AICDA silencing significantly downregulates the expression of MMP14 and WLS in C4-2 (p < 0.001), C4-2B (p < 0.001), and LNCaP (p < 0.05, <0.001) cells. 5-Aza partially rescued AICDA silencing caused by MMP14 depression in C4-2 (p < 0.01) and C4-2B (p < 0.001) cells, and expression of WLS was not influenced by 5-aza significantly in all experimental cell lines (p > 0.05). (C–E) CXCL12 silencing obviously inhibits the expression of MMP14 in C4-2 (p < 0.001) and C4-2B cells (p < 0.001) but not in LNCaP cells (p > 0.05), and the expression of WLS was not influenced by CXCL12 silencing significantly in all experimental cell lines (p > 0.05) and overexpression of AICDA dramatically upregulated the expression of WLS in all three experimental cell lines (p < 0.01). 5-Aza was unable to upregulate the expression of MMP14 and WLS in CXCL12-silenced experimental cell lines (p > 0.05), and overexpression of AICDA partially recover or promote the expression level of MMP14 in CXCL12-silenced C4-2 and C4-2B cells (p < 0.01) or LNCaP (p < 0.01) cells, respectively. (F and G) CK1α-specific activator pyrvinium remarkably downregulated the expression of MMP14 in C4-2 (p < 0.001) and C4-2B (p < 0.001) cells, whereas is unfunctional in upregulated MMP14 in LNCaP cells (p > 0.05). (H and I) Transwell assay showed that AICDA silencing suppressed the invasiveness of LNCaP obviously (p < 0.001), there is no difference of invasiveness between shCon group and pyrvinium, CXCL12 knockdown, 5-aza group (p > 0.05), and overexpression of AICDA dramatically enhanced the invasiveness of LNCaP cells (p < 0.001), Scale bar, 50 μm. Two-sided Student’s t test was used for all panels. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n.s, no significance.

Article Snippet: Rabbit polyclonal anti-CXCL12 , Abcam , Cat# ab9797; RRID: AB_296627.

Techniques: Expressing, Over Expression, Transwell Assay

Journal: iScience

Article Title: AID-induced CXCL12 upregulation enhances castration-resistant prostate cancer cell metastasis by stabilizing β-catenin expression

doi: 10.1016/j.isci.2023.108523

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-CXCL12 , Abcam , Cat# ab9797; RRID: AB_296627.

Techniques: Virus, Recombinant, Plasmid Preparation, Clone Assay, CCK-8 Assay, shRNA, Software

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Proteolytic inactivation of CXCL12 in the lungs and circulation of COVID-19 patients

doi: 10.1007/s00018-023-04870-0

Figure Lengend Snippet: Optimization of the ISTAMPA method for detection of CXCL12 proteoforms. A Principle of top-down MS/MS fragmentation of CXCL12(1-68) or CXCL12(3-68) through low-energy collision-induced dissociation (CID). Fragmentation occurs selectively at the acid-labile bond between Asp52 (D) and Pro53 (P) after isolation of the (B) precursor ion with the highest intensity in the single MS spectrum [mass-to-charge (m/z) value of 885.4 (9 +) for intact CXCL12(1-68) or 860.3 (9 +) for NH2-terminally truncated CXCL12(3-68)]. C Extracted ion chromatogram (EIC) and MS/MS spectrum for CXCL12(1-68) and CXCL12(3-68). The EIC shows the intensity of the signature fragment ions (with mass accuracy of ± 0.5 mass unit) during chemokine elution from the nano-RP-UPLC column at t = 22–24 min. CID fragmentation generates two specific fragments per CXCL12 proteoform, with each two characteristic fragment ions (indicated by their m/z value and charge on the spectra). The red diamond in the MS/MS spectra indicates the m/z of the precursor ion selected for CID fragmentation

Article Snippet: Afterward, 0.25 μg/mL biotinylated polyclonal rabbit anti-human CXCL12 antibodies (#500-P87ABT; PeproTech) with HRP-conjugated streptavidin (R&D Systems) were employed for detection.

Techniques: Tandem Mass Spectroscopy, Isolation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Proteolytic inactivation of CXCL12 in the lungs and circulation of COVID-19 patients

doi: 10.1007/s00018-023-04870-0

Figure Lengend Snippet: Proteolytic processing of CXCL12 in BAL fluid. Recombinant human CXCL12(1-68) (250 ng) was spiked in 20 µL of BAL fluid from (A–D) COVID-19 patients, grouped according to the proteolytic activity in their BAL fluid (n = 9–12), (E,F) stable lung transplantation patients (n = 6), or (G,H) the non-affected lung of lung cancer patients (n = 4) and incubated for 3 h at 37 °C. A,C,E,G Immediately after spiking (without incubation) and (B,D,F,H) after 3 h of incubation at 37 °C, CXCL12 proteolysis was determined by ISTAMPA. ISTAMPA included a 30-min pre-purification at room temperature (RT) of the CXCL12 proteoforms before nano-LC-MS/MS analysis. Only those proteoforms that were detected at least once were included in the graphs. Total CXCL12 was below the detection limit in 4 out of the 12 samples with high proteolytic activity after 3 h of incubation. CXCL12 proteoforms were presented according to their relative abundance in the sample, expressed as percentage of total CXCL12. Data are shown as dots, representing individual patient samples, in a bar graph, indicating the mean ± SEM

Article Snippet: Afterward, 0.25 μg/mL biotinylated polyclonal rabbit anti-human CXCL12 antibodies (#500-P87ABT; PeproTech) with HRP-conjugated streptavidin (R&D Systems) were employed for detection.

Techniques: Recombinant, Activity Assay, Transplantation Assay, Incubation, Purification, Liquid Chromatography with Mass Spectroscopy

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Proteolytic inactivation of CXCL12 in the lungs and circulation of COVID-19 patients

doi: 10.1007/s00018-023-04870-0

Figure Lengend Snippet: Identification of CXCL12-cleaving proteases in COVID-19 BAL fluid. Recombinant human CXCL12(1-68) (250 ng) was spiked in 20 µL of BAL fluid from COVID-19 patients, in the absence (“not treated”) or presence (“treated”) of different protease inhibitors. After 3 h of incubation at 37 °C, CXCL12 proteolysis was determined by ISTAMPA. Different BAL fluid samples were selected per inhibitor according to the proteolytic processing that was observed with untreated samples. A Neutrophil elastase inhibition with sivelestat in high proteolytic activity (HPA) BAL fluid (n = 4). Total CXCL12 was below the detection limit in two untreated samples. B Serine protease inhibition with AEBSF in HPA BAL fluid (n = 2). Total CXCL12 was below the detection limit in both untreated samples. C Metalloprotease inhibition with EDTA in low proteolytic activity (LPA) BAL fluid samples (n = 3). D CD26 inhibition with sitagliptin in LPA BAL fluid samples (n = 3). E Cysteine protease inhibition with E-64 in LPA BAL fluid samples (n = 2). Only those proteoforms that were detected at least once were included in the graphs. CXCL12 proteoforms were presented according to their relative abundance in the sample, expressed as percentage of total CXCL12. Data are shown as dots, representing individual patient samples, in a bar graph, indicating the mean ± SEM

Article Snippet: Afterward, 0.25 μg/mL biotinylated polyclonal rabbit anti-human CXCL12 antibodies (#500-P87ABT; PeproTech) with HRP-conjugated streptavidin (R&D Systems) were employed for detection.

Techniques: Recombinant, Incubation, Inhibition, Activity Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Proteolytic inactivation of CXCL12 in the lungs and circulation of COVID-19 patients

doi: 10.1007/s00018-023-04870-0

Figure Lengend Snippet: Proteolytic processing of CXCL12 in plasma. Recombinant human CXCL12(1–68) (250 ng) was spiked in 20 µL of platelet-free plasma from (A,B) COVID-19 patients (n = 7) or (C,D) healthy controls (n = 7) and incubated for 3 h at 37 °C. A,C Immediately after spiking (without incubation) and (B,D) after 3 h of incubation at 37 °C, CXCL12 proteolysis was determined by ISTAMPA. ISTAMPA included a 30-min pre-purification at room temperature (RT) of the CXCL12 proteoforms before nano-LC-MS/MS analysis. Only those proteoforms that were detected at least once were included in the graphs. CXCL12 proteoforms were presented according to their relative abundance in the sample, expressed as percentage of total CXCL12. E,F CD26 activity was determined using a colorimetric assay in these plasma samples and in BAL fluids from stable lung transplantation patients (n = 6) and COVID-19 patients (n = 21), separated according to (F) high (n = 12) or low (n = 9) proteolytic activity. Data are shown as dots, representing individual patient samples, in a bar graph, indicating the mean ± SEM. Statistical analysis was performed by Brown–Forsythe and Welch ANOVA tests with a Dunnett’s T3 multiple comparisons test and an unpaired t test. *p ≤ 0.05; **p ≤ 0.01; ****p ≤ 0.0001

Article Snippet: Afterward, 0.25 μg/mL biotinylated polyclonal rabbit anti-human CXCL12 antibodies (#500-P87ABT; PeproTech) with HRP-conjugated streptavidin (R&D Systems) were employed for detection.

Techniques: Clinical Proteomics, Recombinant, Incubation, Purification, Liquid Chromatography with Mass Spectroscopy, Activity Assay, Colorimetric Assay, Transplantation Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Proteolytic inactivation of CXCL12 in the lungs and circulation of COVID-19 patients

doi: 10.1007/s00018-023-04870-0

Figure Lengend Snippet: Proteolytic degradation of CXCL12 in BAL fluid and plasma. Recombinant human CXCL12(1-68) (250 ng) was spiked in 20 µL of (A, B) BAL fluid from COVID-19 (n = 17) or (C) stable lung transplantation patients (n = 6); platelet-free plasma from (D) COVID-19 patients (n = 7) or (E) healthy controls (n = 7); or (F) BAL fluid from COVID-19 patients in the absence (“not treated”) or presence (“treated”) of protease inhibitors (n = 2–4) and incubated for 3 h at 37 °C. A–E Immediately after spiking (at t = 0 min) and after 3 h of incubation at 37 °C, total CXCL12 levels were determined by ELISA. In (B), the fold change in CXCL12 concentration after 3 h of incubation is shown in all COVID-19 BAL fluid samples (n = 17) and separated in high (n = 8) and low proteolytic activity samples (n = 9). In (F), the CXCL12 levels after 3 h of incubation are displayed. The dotted line represents the maximal value of 250 ng CXCL12 that was spiked in the samples. The dashed line represents the lower detection limit of the ELISA assay. Samples were tested in quadruplicate, i.e., at two different dilutions and with two different antibody combinations. Data are shown as dots, representing individual patient samples, in a bar graph indicating median ± interquartile range. Statistical analysis for (A, C–E) paired samples was performed by Wilcoxon matched-pairs signed rank tests and (B) a Mann–Whitney U test for unpaired samples.*p ≤ 0.05; ****p ≤ 0.0001

Article Snippet: Afterward, 0.25 μg/mL biotinylated polyclonal rabbit anti-human CXCL12 antibodies (#500-P87ABT; PeproTech) with HRP-conjugated streptavidin (R&D Systems) were employed for detection.

Techniques: Clinical Proteomics, Recombinant, Transplantation Assay, Incubation, Enzyme-linked Immunosorbent Assay, Concentration Assay, Activity Assay, MANN-WHITNEY

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Proteolytic inactivation of CXCL12 in the lungs and circulation of COVID-19 patients

doi: 10.1007/s00018-023-04870-0

Figure Lengend Snippet: Western blot analysis of CXCL12 degradation in BAL fluid. One low proteolytic activity (LPA) and one high proteolytic activity (HPA) representative COVID-19 BAL fluid sample were incubated, with or without the neutrophil elastase inhibitor sivelestat and recombinant human CXCL12(1-68) before loading on the gel. An FITC-coupled anti-human CD19 rat IgG antibody was added in equal amounts to each sample before SDS-PAGE and used as loading control. Immunofluorescence was detected by polyclonal rabbit anti-human CXCL12 primary antibodies, and IRDye 680RD donkey anti-rabbit + IRDye 800CW goat anti-rat secondary antibodies, measuring fluorescence at λ = 700 nm and λ = 800 nm, respectively. The CXCL12/Light chain of rat IgG fluorescence intensity ratio is used as relative measure of residual CXCL12

Article Snippet: Afterward, 0.25 μg/mL biotinylated polyclonal rabbit anti-human CXCL12 antibodies (#500-P87ABT; PeproTech) with HRP-conjugated streptavidin (R&D Systems) were employed for detection.

Techniques: Western Blot, Activity Assay, Incubation, Recombinant, SDS Page, Control, Immunofluorescence, Fluorescence

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Proteolytic inactivation of CXCL12 in the lungs and circulation of COVID-19 patients

doi: 10.1007/s00018-023-04870-0

Figure Lengend Snippet: Overview of the results. In the sequence of CXCL12(1-68), only the proteases likely involved in proteolytic processing of CXCL12 in COVID-19 patient samples are indicated. Abbreviations: BAL broncho-alveolar lavage, CPM/CPN carboxypeptidase M/N, HC healthy control, HPA high proteolytic activity, LPA low proteolytic activity, LTx lung transplant

Article Snippet: Afterward, 0.25 μg/mL biotinylated polyclonal rabbit anti-human CXCL12 antibodies (#500-P87ABT; PeproTech) with HRP-conjugated streptavidin (R&D Systems) were employed for detection.

Techniques: Sequencing, Control, Activity Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Proteolytic inactivation of CXCL12 in the lungs and circulation of COVID-19 patients

doi: 10.1007/s00018-023-04870-0

Figure Lengend Snippet: Optimization of the ISTAMPA method for detection of CXCL12 proteoforms. A Principle of top-down MS/MS fragmentation of CXCL12(1-68) or CXCL12(3-68) through low-energy collision-induced dissociation (CID). Fragmentation occurs selectively at the acid-labile bond between Asp52 (D) and Pro53 (P) after isolation of the (B) precursor ion with the highest intensity in the single MS spectrum [mass-to-charge (m/z) value of 885.4 (9 +) for intact CXCL12(1-68) or 860.3 (9 +) for NH2-terminally truncated CXCL12(3-68)]. C Extracted ion chromatogram (EIC) and MS/MS spectrum for CXCL12(1-68) and CXCL12(3-68). The EIC shows the intensity of the signature fragment ions (with mass accuracy of ± 0.5 mass unit) during chemokine elution from the nano-RP-UPLC column at t = 22–24 min. CID fragmentation generates two specific fragments per CXCL12 proteoform, with each two characteristic fragment ions (indicated by their m/z value and charge on the spectra). The red diamond in the MS/MS spectra indicates the m/z of the precursor ion selected for CID fragmentation

Article Snippet: Immunoblotting was performed through overnight incubation at 4 °C with 0.1 μg/mL polyclonal rabbit anti-human CXCL12 (#500-P87A; PeproTech) followed by 1 h of incubation at room temperature with 1/10,000 diluted IRDye 680RD donkey anti-rabbit IgG (#926–68,073; LI-COR Biosciences) and 1/10,000 diluted IRDye 800CW goat anti-rat IgG (#926–32,219; LI-COR Biosciences).

Techniques: Tandem Mass Spectroscopy, Isolation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Proteolytic inactivation of CXCL12 in the lungs and circulation of COVID-19 patients

doi: 10.1007/s00018-023-04870-0

Figure Lengend Snippet: Proteolytic processing of CXCL12 in BAL fluid. Recombinant human CXCL12(1-68) (250 ng) was spiked in 20 µL of BAL fluid from (A–D) COVID-19 patients, grouped according to the proteolytic activity in their BAL fluid (n = 9–12), (E,F) stable lung transplantation patients (n = 6), or (G,H) the non-affected lung of lung cancer patients (n = 4) and incubated for 3 h at 37 °C. A,C,E,G Immediately after spiking (without incubation) and (B,D,F,H) after 3 h of incubation at 37 °C, CXCL12 proteolysis was determined by ISTAMPA. ISTAMPA included a 30-min pre-purification at room temperature (RT) of the CXCL12 proteoforms before nano-LC-MS/MS analysis. Only those proteoforms that were detected at least once were included in the graphs. Total CXCL12 was below the detection limit in 4 out of the 12 samples with high proteolytic activity after 3 h of incubation. CXCL12 proteoforms were presented according to their relative abundance in the sample, expressed as percentage of total CXCL12. Data are shown as dots, representing individual patient samples, in a bar graph, indicating the mean ± SEM

Article Snippet: Immunoblotting was performed through overnight incubation at 4 °C with 0.1 μg/mL polyclonal rabbit anti-human CXCL12 (#500-P87A; PeproTech) followed by 1 h of incubation at room temperature with 1/10,000 diluted IRDye 680RD donkey anti-rabbit IgG (#926–68,073; LI-COR Biosciences) and 1/10,000 diluted IRDye 800CW goat anti-rat IgG (#926–32,219; LI-COR Biosciences).

Techniques: Recombinant, Activity Assay, Transplantation Assay, Incubation, Purification, Liquid Chromatography with Mass Spectroscopy

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Proteolytic inactivation of CXCL12 in the lungs and circulation of COVID-19 patients

doi: 10.1007/s00018-023-04870-0

Figure Lengend Snippet: Identification of CXCL12-cleaving proteases in COVID-19 BAL fluid. Recombinant human CXCL12(1-68) (250 ng) was spiked in 20 µL of BAL fluid from COVID-19 patients, in the absence (“not treated”) or presence (“treated”) of different protease inhibitors. After 3 h of incubation at 37 °C, CXCL12 proteolysis was determined by ISTAMPA. Different BAL fluid samples were selected per inhibitor according to the proteolytic processing that was observed with untreated samples. A Neutrophil elastase inhibition with sivelestat in high proteolytic activity (HPA) BAL fluid (n = 4). Total CXCL12 was below the detection limit in two untreated samples. B Serine protease inhibition with AEBSF in HPA BAL fluid (n = 2). Total CXCL12 was below the detection limit in both untreated samples. C Metalloprotease inhibition with EDTA in low proteolytic activity (LPA) BAL fluid samples (n = 3). D CD26 inhibition with sitagliptin in LPA BAL fluid samples (n = 3). E Cysteine protease inhibition with E-64 in LPA BAL fluid samples (n = 2). Only those proteoforms that were detected at least once were included in the graphs. CXCL12 proteoforms were presented according to their relative abundance in the sample, expressed as percentage of total CXCL12. Data are shown as dots, representing individual patient samples, in a bar graph, indicating the mean ± SEM

Article Snippet: Immunoblotting was performed through overnight incubation at 4 °C with 0.1 μg/mL polyclonal rabbit anti-human CXCL12 (#500-P87A; PeproTech) followed by 1 h of incubation at room temperature with 1/10,000 diluted IRDye 680RD donkey anti-rabbit IgG (#926–68,073; LI-COR Biosciences) and 1/10,000 diluted IRDye 800CW goat anti-rat IgG (#926–32,219; LI-COR Biosciences).

Techniques: Recombinant, Incubation, Inhibition, Activity Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Proteolytic inactivation of CXCL12 in the lungs and circulation of COVID-19 patients

doi: 10.1007/s00018-023-04870-0

Figure Lengend Snippet: Proteolytic processing of CXCL12 in plasma. Recombinant human CXCL12(1–68) (250 ng) was spiked in 20 µL of platelet-free plasma from (A,B) COVID-19 patients (n = 7) or (C,D) healthy controls (n = 7) and incubated for 3 h at 37 °C. A,C Immediately after spiking (without incubation) and (B,D) after 3 h of incubation at 37 °C, CXCL12 proteolysis was determined by ISTAMPA. ISTAMPA included a 30-min pre-purification at room temperature (RT) of the CXCL12 proteoforms before nano-LC-MS/MS analysis. Only those proteoforms that were detected at least once were included in the graphs. CXCL12 proteoforms were presented according to their relative abundance in the sample, expressed as percentage of total CXCL12. E,F CD26 activity was determined using a colorimetric assay in these plasma samples and in BAL fluids from stable lung transplantation patients (n = 6) and COVID-19 patients (n = 21), separated according to (F) high (n = 12) or low (n = 9) proteolytic activity. Data are shown as dots, representing individual patient samples, in a bar graph, indicating the mean ± SEM. Statistical analysis was performed by Brown–Forsythe and Welch ANOVA tests with a Dunnett’s T3 multiple comparisons test and an unpaired t test. *p ≤ 0.05; **p ≤ 0.01; ****p ≤ 0.0001

Article Snippet: Immunoblotting was performed through overnight incubation at 4 °C with 0.1 μg/mL polyclonal rabbit anti-human CXCL12 (#500-P87A; PeproTech) followed by 1 h of incubation at room temperature with 1/10,000 diluted IRDye 680RD donkey anti-rabbit IgG (#926–68,073; LI-COR Biosciences) and 1/10,000 diluted IRDye 800CW goat anti-rat IgG (#926–32,219; LI-COR Biosciences).

Techniques: Clinical Proteomics, Recombinant, Incubation, Purification, Liquid Chromatography with Mass Spectroscopy, Activity Assay, Colorimetric Assay, Transplantation Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Proteolytic inactivation of CXCL12 in the lungs and circulation of COVID-19 patients

doi: 10.1007/s00018-023-04870-0

Figure Lengend Snippet: Proteolytic degradation of CXCL12 in BAL fluid and plasma. Recombinant human CXCL12(1-68) (250 ng) was spiked in 20 µL of (A, B) BAL fluid from COVID-19 (n = 17) or (C) stable lung transplantation patients (n = 6); platelet-free plasma from (D) COVID-19 patients (n = 7) or (E) healthy controls (n = 7); or (F) BAL fluid from COVID-19 patients in the absence (“not treated”) or presence (“treated”) of protease inhibitors (n = 2–4) and incubated for 3 h at 37 °C. A–E Immediately after spiking (at t = 0 min) and after 3 h of incubation at 37 °C, total CXCL12 levels were determined by ELISA. In (B), the fold change in CXCL12 concentration after 3 h of incubation is shown in all COVID-19 BAL fluid samples (n = 17) and separated in high (n = 8) and low proteolytic activity samples (n = 9). In (F), the CXCL12 levels after 3 h of incubation are displayed. The dotted line represents the maximal value of 250 ng CXCL12 that was spiked in the samples. The dashed line represents the lower detection limit of the ELISA assay. Samples were tested in quadruplicate, i.e., at two different dilutions and with two different antibody combinations. Data are shown as dots, representing individual patient samples, in a bar graph indicating median ± interquartile range. Statistical analysis for (A, C–E) paired samples was performed by Wilcoxon matched-pairs signed rank tests and (B) a Mann–Whitney U test for unpaired samples.*p ≤ 0.05; ****p ≤ 0.0001

Article Snippet: Immunoblotting was performed through overnight incubation at 4 °C with 0.1 μg/mL polyclonal rabbit anti-human CXCL12 (#500-P87A; PeproTech) followed by 1 h of incubation at room temperature with 1/10,000 diluted IRDye 680RD donkey anti-rabbit IgG (#926–68,073; LI-COR Biosciences) and 1/10,000 diluted IRDye 800CW goat anti-rat IgG (#926–32,219; LI-COR Biosciences).

Techniques: Clinical Proteomics, Recombinant, Transplantation Assay, Incubation, Enzyme-linked Immunosorbent Assay, Concentration Assay, Activity Assay, MANN-WHITNEY

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Proteolytic inactivation of CXCL12 in the lungs and circulation of COVID-19 patients

doi: 10.1007/s00018-023-04870-0

Figure Lengend Snippet: Western blot analysis of CXCL12 degradation in BAL fluid. One low proteolytic activity (LPA) and one high proteolytic activity (HPA) representative COVID-19 BAL fluid sample were incubated, with or without the neutrophil elastase inhibitor sivelestat and recombinant human CXCL12(1-68) before loading on the gel. An FITC-coupled anti-human CD19 rat IgG antibody was added in equal amounts to each sample before SDS-PAGE and used as loading control. Immunofluorescence was detected by polyclonal rabbit anti-human CXCL12 primary antibodies, and IRDye 680RD donkey anti-rabbit + IRDye 800CW goat anti-rat secondary antibodies, measuring fluorescence at λ = 700 nm and λ = 800 nm, respectively. The CXCL12/Light chain of rat IgG fluorescence intensity ratio is used as relative measure of residual CXCL12

Article Snippet: Immunoblotting was performed through overnight incubation at 4 °C with 0.1 μg/mL polyclonal rabbit anti-human CXCL12 (#500-P87A; PeproTech) followed by 1 h of incubation at room temperature with 1/10,000 diluted IRDye 680RD donkey anti-rabbit IgG (#926–68,073; LI-COR Biosciences) and 1/10,000 diluted IRDye 800CW goat anti-rat IgG (#926–32,219; LI-COR Biosciences).

Techniques: Western Blot, Activity Assay, Incubation, Recombinant, SDS Page, Control, Immunofluorescence, Fluorescence

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Proteolytic inactivation of CXCL12 in the lungs and circulation of COVID-19 patients

doi: 10.1007/s00018-023-04870-0

Figure Lengend Snippet: Overview of the results. In the sequence of CXCL12(1-68), only the proteases likely involved in proteolytic processing of CXCL12 in COVID-19 patient samples are indicated. Abbreviations: BAL broncho-alveolar lavage, CPM/CPN carboxypeptidase M/N, HC healthy control, HPA high proteolytic activity, LPA low proteolytic activity, LTx lung transplant

Article Snippet: Immunoblotting was performed through overnight incubation at 4 °C with 0.1 μg/mL polyclonal rabbit anti-human CXCL12 (#500-P87A; PeproTech) followed by 1 h of incubation at room temperature with 1/10,000 diluted IRDye 680RD donkey anti-rabbit IgG (#926–68,073; LI-COR Biosciences) and 1/10,000 diluted IRDye 800CW goat anti-rat IgG (#926–32,219; LI-COR Biosciences).

Techniques: Sequencing, Control, Activity Assay