Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: A regulatory circuit operated by KDELR1 and KDELR3 fine-tunes the composition of the early secretory pathway

doi: 10.1007/s00018-025-06049-1

Figure Lengend Snippet: Different roles of human KDELRs. A Seventy-two hours after silencing with the indicated KDELR-specific duplexes, the supernatants of cells cultured for six hours were harvested and analysed by WB. Aliquots of the lysates and supernatants (the latter tenfold concentrated) of HeLa Milano cells were resolved under reducing conditions on a 4–12% pre-casted polyacrylamide gel run in MOPS buffer. After transfer to nitrocellulose, the membrane was first stained with anti-KDEL antibodies (clone 10C3, Enzo Life Sciences) and subsequently with anti-ERp44 and anti-AGR2, as indicated. Tubulin was used as a loading control. The simultaneous KD of all KDELRs (lanes 3 and 9) induced the extracellular release of all the ER-resident proteins visualized intracellularly. The identity of the two upper bands recognized by anti-KDEL (denoted X and Y) remains to be established. The intracellular signal corresponding to ERp44 and ERp46 (IN) disappeared almost completely upon downregulation of all KDELRs (lane 3), and partially under KDELR2 KD (lane 5). Noteworthy, instead, AGR2 increased dramatically upon downregulation of ERp44 or KDELR3 (lanes 2 and 6, respectively). See panel C for the relative quantifications. B Visual pulse-chase assays [ , ] (see also Materials and Methods for details) were used to compare the secretion rate of Halo-ERp44ΔRDEL (Halo-ERp44Δ), Halo-ERp46ΔKDEL (Halo-ERp46Δ), Halo-PDIΔKDEL (Halo-PDIΔ) and spHalo . The left panel shows the WB image of one representative experiment out of three, highlighting the signal of the fluorescent Halo-TMR used for the pulse (and visualized with a 532 nm laser). Densitometric quantifications are reported in the right panel (average of three independent experiments). The ratio of secreted/intracellular TMR-labelled signals was calculated for each time point (OUT/IN) and normalized to the ratio obtained for spHalo CTRL at 120 min time point, assumed to represent unassisted secretion . Statistical significance was calculated by Two-way ANOVA. The colours of the asterisks refer to the samples against which the comparison was performed. C Densitometric quantification of four independent WBs like the one shown in panel A. The bands specifically decorated by anti-AGR2 antibodies were quantified and normalized against tubulin. The ratios obtained were normalized to untreated cells. Statistical significance was calculated by an unpaired Student's t-test. D HeLa Milano cells were treated with duplexes specific for silencing ERp44 (44) ERp46 (46), or with control reagents (-) as indicated. Aliquots of their lysates were separated on 4–12% polyacrylamide gels under reducing conditions, blotted onto nitrocellulose and decorated with the antibodies indicated on the right-hand margin. Tubulin was used as loading control. Note the increase of AGR2 upon ERp44 KD but not ERp46 KD (compare lanes 2 and 3). The red asterisk points at a background band recognized by anti-ERp46 antibodies in this experiment. E HeLa Milano (left panel) and mucin-producing Caco-2 cells (right panel) were treated with ERp44- or AGR2- specific duplexes, or with control reagents (-) as indicated. Aliquots of their lysates (IN) and of their culture supernatants (OUT) ten-fold concentrated were separated on 4–12% polyacrylamide gels under reducing conditions, blotted onto nitrocellulose and decorated with the indicated antibodies. Ponceau signals provided loading controls

Article Snippet: AGR2 , Rabbit (Proteintech) , 1:500 in PBS 0.1% Tween.

Techniques: Cell Culture, Membrane, Staining, Control, Pulse Chase, Comparison

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: A regulatory circuit operated by KDELR1 and KDELR3 fine-tunes the composition of the early secretory pathway

doi: 10.1007/s00018-025-06049-1

Figure Lengend Snippet: Opposite effects of KDELR1 and KDELR3 on AGR2. HeLa Milano cells were silenced as indicated and analysed as described in Figs. and . Proteins were quantified in cell lysates (IN) and in 6h culture supernatants (OUT) as described in legends to Fig. (panels B-C). To quantify mRNAs, RT-qPCR analyses were performed with AGR2-specific oligos (panels A, and D-F). GAPDH was used to normalize the results obtained. A AGR2 mRNAs increases upon ERp44 KD and/or KDELR3 KD . RT-qPCR data are expressed as Log2FC with respect to control (-) conditions (that, by definition, correspond to 0). The data of the single experiments are shown in the plot, together with the average ± SEM. Statistical significance was calculated by an unpaired Student's t-test. B and C . Aliquots from cell lysates (IN) and 10X supernatants (OUT) were analysed as described above. Tubulin was used as a loading control. Note that AGR2 protein accumulates also when both KDELR3 and ERp44 are silenced (panel B), upon combined silencing of all the KDELRs, as well as upon combined silencing of KDELR1 and KDELR2, a condition in which most ERp44 is secreted. D AGR2 mRNAs increase upon double KDELR silencing. The panel shows the AGR2 transcript levels in cells treated as in C. mRNAs were quantified as in panel A. Average of five or more independent experiments ± SEM. Statistical significance was calculated by an unpaired Student's t-test. E ERp44 rescues AGR2 inhibition in HeLa ERp44KO cells via KDELR3. HeLa ERp44KO cells, stably expressing doxycycline-inducible Halo-ERp44, were silenced for 72 h for KDELR3, and then treated with 100 µM doxycycline overnight. Transcripts were quantified as described in panels A and D. Average of three or more independent experiments ± SEM. An unpaired Student's t-test was performed. F Neither Halo-RDEL nor ERp46 activate KDELR3-dependent AGR2 inhibition. HeLa ERp44KO cells were transfected either with an empty vector or with vectors encoding for Halo-ERp44, Halo-ERp46, and Halo-RDEL. After 48 h, RNAs were extracted and quantified by RTq-PCR (see panel A). Average of three independent experiments ± SEM. Statistical significance was calculated by an unpaired Student's t-test

Article Snippet: AGR2 , Rabbit (Proteintech) , 1:500 in PBS 0.1% Tween.

Techniques: Quantitative RT-PCR, Control, Inhibition, Stable Transfection, Expressing, Transfection, Plasmid Preparation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: A regulatory circuit operated by KDELR1 and KDELR3 fine-tunes the composition of the early secretory pathway

doi: 10.1007/s00018-025-06049-1

Figure Lengend Snippet: Transcriptional patterns of HeLa cells upon KDELRs downregulation. A Principal Component Analysis Score plot of PC1 vs PC2. The graph shows clear separation of the five experimental conditions. B Overlap analysis of differentially expressed genes after silencing of KDEL receptors or ERp44. Left panel: genes up-regulated; central panel: genes down-regulated; right panel: genes up-regulated upon silencing of KDELR3 and ERp44 and down-regulated upon KDELR1 (thus sharing the same behaviour as AGR2). C Functional Enrichment using Enrichr of Gene Ontology Cellular Components and the Databases of Jensen Compartments of the 41 genes behaving like AGR2 showing enrichment in “Extracellular Space”, “Extracellular matrix”, and “Endoplasmic Reticulum Lumen”

Article Snippet: AGR2 , Rabbit (Proteintech) , 1:500 in PBS 0.1% Tween.

Techniques: Functional Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: A regulatory circuit operated by KDELR1 and KDELR3 fine-tunes the composition of the early secretory pathway

doi: 10.1007/s00018-025-06049-1

Figure Lengend Snippet: Functional specialization of human KDELRs. As summarized in the right part of the cartoon, ERp44 and KDELR3 act epistatically to inhibit AGR2 transcription, while KDELR1 (on the left) can be stimulated by different chaperones to promote AGR2 transcription. In HeLa cells, the more abundant KDELR2 and KDELR1 sustain the retrieval activity of diverse ER chaperones and enzymes. Considering the essential roles of AGR2 in assisting mucin folding and inhibiting the gut specific ER-stress sensor Ire1ß, this pathway may be important in mucin biogenesis

Article Snippet: AGR2 , Rabbit (Proteintech) , 1:500 in PBS 0.1% Tween.

Techniques: Functional Assay, Activity Assay

Journal: Frontiers in Immunology

Article Title: To reveal biomarkers related to macrophage and lactic acid metabolism in renal fibrosis and explore their mechanisms

doi: 10.3389/fimmu.2025.1609903

Figure Lengend Snippet: Screening and diagnostic value of AGR3, CD74, and SYT11 in RF. (A) Least absolute shrinkage and selection operator (LASSO) regression analysis screening and cross-validation. (B) Boruta analysis screening results. Blue box: The minimum, average, and maximum Z-values of the shadow property; Red: rejection feature; Yellow: features to be confirmed; Green box: Confirmed feature. (C) Intersection of the two algorithms to obtain key feature genes. Pink represents genes confirmed by Lasso regression, and blue represents genes confirmed by Boruta as relevant. (D) Expression of key feature genes in the training set. (E) Expression of key feature genes in the validation set. (F) Receiver operating characteristic (ROC) analysis of candidate key genes in the training set. (G) ROC analysis of candidate key genes in the validation set.

Article Snippet: After incubation with 10% goat serum at room temperature for blocking, 100 μl of the working solution of CD74 (1:200) (Affinity, China), AGR2 + AGR3 (1:300) (since AGR3 and AGR2 are highly related homologous genes, we chose the AGR2 + AGR3 antibody for subsequent detection, Abcam, USA), and SYT11 (1:200) (Proteintech, China) primary antibodies were added to each tissue section and incubated at 4°C overnight.

Techniques: Diagnostic Assay, Selection, Biomarker Discovery, Expressing

Journal: Frontiers in Immunology

Article Title: To reveal biomarkers related to macrophage and lactic acid metabolism in renal fibrosis and explore their mechanisms

doi: 10.3389/fimmu.2025.1609903

Figure Lengend Snippet: Elucidating the biological mechanisms of biomarkers. (A) Gene set enrichment analysis (GSEA) of AGR3. (B) GSEA of CD74. (C) GSEA of SYT11.

Article Snippet: After incubation with 10% goat serum at room temperature for blocking, 100 μl of the working solution of CD74 (1:200) (Affinity, China), AGR2 + AGR3 (1:300) (since AGR3 and AGR2 are highly related homologous genes, we chose the AGR2 + AGR3 antibody for subsequent detection, Abcam, USA), and SYT11 (1:200) (Proteintech, China) primary antibodies were added to each tissue section and incubated at 4°C overnight.

Techniques:

Journal: Frontiers in Immunology

Article Title: To reveal biomarkers related to macrophage and lactic acid metabolism in renal fibrosis and explore their mechanisms

doi: 10.3389/fimmu.2025.1609903

Figure Lengend Snippet: Validation of key biomarker expression in animal experiments. (A) Representative kidneys of rats in the two groups at the time of kidney tissue collection. (B) Comparison of body weight and renal function between the two groups of rats. (C) HE, Masson and PAS pathological staining of kidneys in the two groups of rats. The black arrow indicates the area of this field of view in the renal tissue. (D) Immunohistochemical staining of AGR3, CD74 and SYT11 in kidney tissues of the two groups of rats. The black arrow indicates the area of this field of view in the renal tissue. (E) Immunofluorescence staining of AGR3, CD74 and SYT11 in kidney tissues of the two groups of rats. The white arrow indicates the area of this field of view in the renal tissue. (F) Western blot detection of differences in protein expression of AGR3, CD74 and SYT11 in kidney tissues of the two groups of rats. (B; mean ± SD, n=5; F, mean ± SD, n=3; Compared with the control group: ns, P ≥ 0.05; * P < 0.05; ** P < 0.01).

Article Snippet: After incubation with 10% goat serum at room temperature for blocking, 100 μl of the working solution of CD74 (1:200) (Affinity, China), AGR2 + AGR3 (1:300) (since AGR3 and AGR2 are highly related homologous genes, we chose the AGR2 + AGR3 antibody for subsequent detection, Abcam, USA), and SYT11 (1:200) (Proteintech, China) primary antibodies were added to each tissue section and incubated at 4°C overnight.

Techniques: Biomarker Discovery, Expressing, Comparison, Staining, Immunohistochemical staining, Immunofluorescence, Western Blot, Control