Human articular chondrocytes with higher aldehyde dehydrogenase activity have stronger expression of COL2A1 and SOX9 A. Unguryte, E. Bernotiene, E. Bagdonas,

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Human articular chondrocytes with higher aldehyde dehydrogenase activity have stronger expression of COL2A1 and SOX9 A. Unguryte, E. Bernotiene, E. Bagdonas, S. Garberyte, N. Porvaneckas, C. Jorgensen Osteoarthritis and Cartilage Volume 24, Issue 5, Pages 873-882 (May 2016) DOI: 10.1016/j.joca.2015.11.019 Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Typical Aldefluor™ fluorescence plots for flow cytometric analysis of ALDH activity and cell sorting. A – Side scatter (SSC) vs forward scatter (FSC) plot, analyzed population marked as P1. B – Control cells, incubated with Aldefluor™ reagent + DEAB. Gates ALDH− and ALDH+ were created for ALDH activity in presence of DEAB (about 3% in ALDH+ gate). C: Cells, incubated with Aldefluor™ reagent; activity expressed in percent of cells in ALDH+ gate, (control, the 3%, was subtracted afterwards); D: plot and gates for sorting ALDH− and ALDH+ cells. Intermediate fraction named ALDHdim was also collected in some experiments. Osteoarthritis and Cartilage 2016 24, 873-882DOI: (10.1016/j.joca.2015.11.019) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 A – Distribution of ALDHpos cells according patients' age, gender and OA grade (according to DL Holden OA grade classification). Lines with error bars represent mean and 95% CI. B – Amount of ALDH-expressing cells (ALDHpos) in freshly isolated and in monolayer cultured chondrocytes, expressed as percent of P1 population. Dots in the scatter plot represent mean value for every different patient, line with error bars represent mean of all patients and 95% CI. n is number of different patients analyzed. C – Percentage of ALDHpos cells during cultivation for individual cell lines, marked as patient age and gender/OA grade. Osteoarthritis and Cartilage 2016 24, 873-882DOI: (10.1016/j.joca.2015.11.019) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 A – Gate, defining ALDHneg and ALDHpos populations for coexpression analysis (Control cells, incubated with Aldefluor™ reagent + DEAB). B – Ratio of MFI value for every surface marker measured in gate ALDH+ vs value in gate ALDH−. Error bars represent 95% CI; Statistical significance is depicted above the scatterplots (P values exceeding but close to 0.05 are shown in brackets). C-E – Flow cytometry plots for co-expression of ALDH activity with surface markers. Three groups were identified: C – surface markers, stronger expressed on ALDHpos cells, than ALDHneg cells; D – nearly equal distribution between ALDHpos and ALDHneg cells; and E – surface markers, stronger expressed on ALDHneg cells than ALDHpos cells. Osteoarthritis and Cartilage 2016 24, 873-882DOI: (10.1016/j.joca.2015.11.019) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Plots for ALDH activity in ALDH-sorted, then cultured 1 day after sorting (A) and 10 days after sorting (B) cells. Numbers inside the gates indicate percentage of ALDH-positive cells. C – percent of ALDH-expressing cells in ALDH+ and ALDH− sorted fractions 2–3 weeks after sorting. Dots in the scatter plot represent different patients' cell lines (n = 5), error bars represent 95% CI. Osteoarthritis and Cartilage 2016 24, 873-882DOI: (10.1016/j.joca.2015.11.019) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Expression of surface markers CD55 on ALDH-sorted cells 1 day after sorting (A) and 10 days after sorting (B). Numbers inside the gates indicate percentage of CD55-positive cells. Osteoarthritis and Cartilage 2016 24, 873-882DOI: (10.1016/j.joca.2015.11.019) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 Gene expression in ALDH-sorted chondrocytes. A and B – one representative plot for sorted cells, when ALDHdim fraction was also collected. Results are expressed as 2−ΔCt for every fraction separately, normalized to reference gene RPS9 expression; A – genes, related to chondrocyte, osteo- and adipogenic phenotype and stem cells. B –ALDH1A and ALDH2 gene expression, ND – not detected. C-E – gene expression ratio for ALDH+ vs ALDH− sorted chondrocytes (2−ΔΔCt); C – genes, related to chondrocyte phenotype. D – genes, related to osteo-/adipogenic phenotype and stem cells. E – gene expression of four ALDH isoforms. Box plots with whiskers represent interquartile range with median. Statistical significance is depicted above the box plots. n – Number of patients. Osteoarthritis and Cartilage 2016 24, 873-882DOI: (10.1016/j.joca.2015.11.019) Copyright © 2015 Osteoarthritis Research Society International Terms and Conditions