Journal: bioRxiv

Article Title: Role of Klhl14 in senescence and epithelial-to-mesenchymal transition via TGF-β modulation

doi: 10.1101/2025.09.12.675818

Figure Lengend Snippet: ( a ) General experimental setup for mTGO culture: cells are isolated from mice thyroid glands by enzymatic and mechanical digestion, then seeded in a solid BMM and cultured for 5, 7, or 11 days. ( b ) qPCR expression of Klhl14 in 5-, 7-, and 11-days old organoids. ( c ) Immunofluorescent staining of KLHL14 protein levels in 5-, 7-, and 11-days old organoids. Scalebar 25 μm. ( d ) Bulk RNA sequencing of 5-, 7-, and 11-days old organoids. The heatmap represents the count-per-million Z-scores of the expression of genes related to thyroid maturation and differentiation at the three considered time points, highlighting how a higher definition of a thyroid profile accompanies the growth of the three-dimensional structure. Data are shown as mean ± SEM. Group differences were evaluated with two-tailed Student’s t -tests, n=3. * p < 0.05

Article Snippet: KLHL14 and GAPDH primary antibodies (Proteintech, 16693-1-AP; Fitzgeral, 10R-G109a) were used respectively with a 1:1000 and 1:10000 dilution in BSA 3% PBS-Tween20; secondary antibodies were used with a 1:3000 dilution in BSA 3% PBS-Tween20.

Techniques: Isolation, Cell Culture, Expressing, Staining, RNA Sequencing, Two Tailed Test

Journal: bioRxiv

Article Title: Role of Klhl14 in senescence and epithelial-to-mesenchymal transition via TGF-β modulation

doi: 10.1101/2025.09.12.675818

Figure Lengend Snippet: ( a ) Experimental setup: single cells obtained from 7-day-old organoids were transduced with lentiviruses after 24 hours, seeded in BMM domes, and analyzed one week later. ( b ) Western blot analysis to validate KLHL14 silencing, using GAPDH as housekeeping. On the right is the relative normalized quantification of the bands. ( c ) Microscope images of organoids grown from transduced cells for one week, using the GFP channel, 4x objective. Scalebar 100 μm. ( d ) Data obtained from the Incucyte Live-Cell Analysis system exploiting the GFP expression of the organoids: on the left, the green integrated intensity. On the right, the ratio between the green integrated intensity and the total object count indicates the organoids’ dimension. ( e ) 7-day-old organoids obtained from transduced cells were collected after the disruption of the BMM domes, and relative OFE was calculated by counting organoids with a size > 50 mm (left graph). After trypsinization of the collected organoids, single cells were counted to calculate the population doublings (right graph) according to the formula described in the Materials and Methods. Data are shown as mean ± SEM. Group differences were evaluated with two-tailed Student’s t -tests. N=4. ** p < 0.01

Article Snippet: KLHL14 and GAPDH primary antibodies (Proteintech, 16693-1-AP; Fitzgeral, 10R-G109a) were used respectively with a 1:1000 and 1:10000 dilution in BSA 3% PBS-Tween20; secondary antibodies were used with a 1:3000 dilution in BSA 3% PBS-Tween20.

Techniques: Transduction, Western Blot, Microscopy, Cell Analysis, Expressing, Disruption, Two Tailed Test

Journal: bioRxiv

Article Title: Role of Klhl14 in senescence and epithelial-to-mesenchymal transition via TGF-β modulation

doi: 10.1101/2025.09.12.675818

Figure Lengend Snippet: ( a ) PCA plot of 7 days-old shSCRA and shKlhl14 treated organoids obtained from proteomic data. ( b ) Normalized results from the whole proteomic analysis were used to evaluate significantly dysregulated proteins in 7-day-old shKlhl14 organoids: 83 molecules are overrepresented, whereas 194 are downregulated. ( c ) GSEA indicated key processes associated with the two groups of differentially expressed proteins, depicted with their significance scores (green shades) and the number of proteins belonging to each term. ( d ) Top 15 overexpressed genes in Klhl14 KD organoids with their respective -AVG Log2 ratio. N=3

Article Snippet: KLHL14 and GAPDH primary antibodies (Proteintech, 16693-1-AP; Fitzgeral, 10R-G109a) were used respectively with a 1:1000 and 1:10000 dilution in BSA 3% PBS-Tween20; secondary antibodies were used with a 1:3000 dilution in BSA 3% PBS-Tween20.

Techniques:

Journal: bioRxiv

Article Title: Role of Klhl14 in senescence and epithelial-to-mesenchymal transition via TGF-β modulation

doi: 10.1101/2025.09.12.675818

Figure Lengend Snippet: ( a ) Experimental setup: single cells obtained from 7-day-old organoids were transduced with lentiviruses after 24 hours, seeded in BMM domes, and analyzed one week later. ( b ) Western blot analysis to validate KLHL14 silencing, using GAPDH as housekeeping. On the right is the relative normalized quantification of the bands. ( c ) Microscope images of organoids grown from transduced cells for one week, using the GFP channel, 4x objective. Scalebar 100 μm. ( d ) Data obtained from the Incucyte Live-Cell Analysis system exploiting the GFP expression of the organoids: on the left, the green integrated intensity. On the right, the ratio between the green integrated intensity and the total object count indicates the organoids’ dimension. ( e ) 7-day-old organoids obtained from transduced cells were collected after the disruption of the BMM domes, and relative OFE was calculated by counting organoids with a size > 50 mm (left graph). After trypsinization of the collected organoids, single cells were counted to calculate the population doublings (right graph) according to the formula described in the Materials and Methods. Data are shown as mean ± SEM. Group differences were evaluated with two-tailed Student’s t -tests. N=4. ** p < 0.01

Article Snippet: KLHL14 and GAPDH primary antibodies (Proteintech, 16693-1-AP; Fitzgeral, 10R-G109a) were used respectively with a 1:1000 and 1:10000 dilution in BSA 3% PBS-Tween20; secondary antibodies were used with a 1:3000 dilution in BSA 3% PBS-Tween20.

Techniques: Transduction, Western Blot, Microscopy, Cell Analysis, Expressing, Disruption, Two Tailed Test

Journal: Gastroenterology Report

Article Title: Targeting transglutaminase 2: pathways to celiac disease therapies

doi: 10.1093/gastro/goaf086

Figure Lengend Snippet: Effects of different TG2 inhibitors on the cross-linking activity of recombinant TG2, cell viability, and extracellular TG2 activity. (A) Fluorometric quantitation of the cross-linking of biotin-conjugated gliadin peptide P56-88 by recombinant TG2 ( n = 3, three technical replicates per experiment). (B) Fluorometric quantitation of cell viability after 24 h of incubation with the corresponding substances using the resazurin-based assay ( n = 3, two technical replicates per experiment). (C) Fluorometric quantitation of extracellular cross-linking of the TG2 substrate 5BP by Caco-2 cells ( n = 3, with three technical replicates per experiment). All data are shown as mean ± SD. Statistical significance was tested by using univariate ANOVA (A) or Student’s t -test with Welch correction. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. 5BP = 5-(biotinamido)-pentylamine, ZnCl 2 = zinc chloride, AA = ascorbic acid.

Article Snippet: LDN27219 , , GTP antagonism, stabilization of closed (inactive) conformation of TG2 , HY-16693, MedChemExpress, Monmouth Junction, NJ, USA.

Techniques: Activity Assay, Recombinant, Quantitation Assay, Incubation, Resazurin Assay

Journal: Gastroenterology Report

Article Title: Targeting transglutaminase 2: pathways to celiac disease therapies

doi: 10.1093/gastro/goaf086

Figure Lengend Snippet: Effect of IFN-γ on the expression of ERp57, TRX and TG2 in Caco-2 cells. Analysis of relative expression of (A) ERp57, (B) TRX, and (C) TG2 by Western blotting after stimulation with different concentrations of IFN-γ over 48 h. No significant change in the expression of ERp57 and TRX. Dose-dependent increase in TG2 expression. All data are shown as mean ± SD. Statistical significance was tested by using Student’s t -test. n = 4. * P < 0.05.

Article Snippet: LDN27219 , , GTP antagonism, stabilization of closed (inactive) conformation of TG2 , HY-16693, MedChemExpress, Monmouth Junction, NJ, USA.

Techniques: Expressing, Western Blot

Journal: Gastroenterology Report

Article Title: Targeting transglutaminase 2: pathways to celiac disease therapies

doi: 10.1093/gastro/goaf086

Figure Lengend Snippet: SiRNA knockdown of ERp57, TRX, and TG2, and the effect on cellular/extracellular TG2 activity. Caco-2 cells were incubated with specific siRNAs or mock siRNA for 48 h. The efficacy of the siRNA knockdown was determined by using Western blotting. Expression of ERp57, TRX, and TG2 was normalized against the housekeeping protein GAPDH. (A–C) SiRNA treatment significantly reduced expression of (A) ERp57, (B) TRX, and (C) TG2. Incubation with mock siRNA did not affect protein expression ( n = 4). (D) Treatment with ERp57-siRNA und TRX-siRNA did not show any off-target effects on TG2 expression ( n = 3). (E) Cellular expression of ERp57, TRX, and TG2 determined by fluorometry after siRNA knockdown. Protein levels of all three target proteins were reduced by siRNA-mediated knockdown. (F) Cellular TG2 activity was determined by fluorometry after siRNA treatment. SiRNA knockdown of TRX and TG2 reduced cellular TG2 activity ( n = 5). (G) Extracellular expression of ERp57, TRX, and TG2 was investigated in unpermeabilized Caco-2 cells by fluorometry. Protein levels of all three target proteins were significantly reduced by siRNA-mediated knockdown. (H) Extracellular TG2 activity was investigated in unpermeabilized Caco-2 cells by fluorometry. SiRNA knockdown of TRX and TG2 significantly reduced extracellular TG2 activity ( n = 5). All data are shown as mean ± SD. Statistical significance was tested by using Student’s t -test with Welch correction where appropriate. * P < 0.05, ** P < 0.01, *** P < 0.001. GAPDH = glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: LDN27219 , , GTP antagonism, stabilization of closed (inactive) conformation of TG2 , HY-16693, MedChemExpress, Monmouth Junction, NJ, USA.

Techniques: Knockdown, Activity Assay, Incubation, Western Blot, Expressing

Journal: Gastroenterology Report

Article Title: Targeting transglutaminase 2: pathways to celiac disease therapies

doi: 10.1093/gastro/goaf086

Figure Lengend Snippet: TG2 is expressed in the lamina propria and the epithelium of CD biopsies. (A) Fluorescence staining revealed TG2 to be nearly absent in the epithelium of control biopsies and low levels were expressed in the lamina propria (arrows). In CD biopsies, TG2 expression was increased in the epithelium (arrowheads) and the lamina propria (arrows). (B) Detailed images of the duodenal mucosa reveal high levels of TG2 in the lamina propria (arrows) and the epithelium (arrowheads) of CD patients. (C) TG2 expression was significantly increased in the epithelium and the lamina propria of CD patients. All data are shown as median. Statistical significance was tested by using Student’s t -test. n = 5. * P < 0.05; scale: 20 µm.

Article Snippet: LDN27219 , , GTP antagonism, stabilization of closed (inactive) conformation of TG2 , HY-16693, MedChemExpress, Monmouth Junction, NJ, USA.

Techniques: Fluorescence, Staining, Control, Expressing