Journal: bioRxiv

Article Title: Schwann cells modified to secrete MANF is a potential cellular therapy for peripheral nerve regeneration

doi: 10.1101/2025.03.14.642904

Figure Lengend Snippet: (A) Western blot shows the expression of MANF in the normal and injured (3-day transection injury) adult rat DRGs. GAPDH is used as the loading control. (B) Quantification of ‘A’ shows the induction of MANF in injured DRGs (data presented as mean ± SE; standard ‘t’ test; n=3). (C) Co-immunostaining of IB4 (yellow arrows), NF200 (white arrows) and MANF (red arrows) in normal and injured DRGs shows the preferential expression of MANF in IB4 positive non-peptidergic sensory neurons (yellow arrows in the merged view) (scale bar, 50µm).

Article Snippet: ELISA was performed using the commercially available MANF ELISA Kit (Cat No. EKL58888- 96T, Biomatik) according to the manufacturer’s instructions.

Techniques: Western Blot, Expressing, Control, Immunostaining

Journal: bioRxiv

Article Title: Schwann cells modified to secrete MANF is a potential cellular therapy for peripheral nerve regeneration

doi: 10.1101/2025.03.14.642904

Figure Lengend Snippet: (A) Western blot shows the expression of MANF in normal and injured (3-day transection injury) proximal and distal sciatic nerve segments of adult rats. GAPDH is used as the loading control. (B) Quantification of ‘A’ shows no significant change in the expression of MANF in injured nerves compared to the control (data presented as mean ± SE; One-Way ANOVA; n=3). (C) Co- immunostaining of βIII tubulin and MANF in normal and injured distal sciatic nerve of adult rats shows MANF expression in a subpopulation of βIII tubulin stained axons (yellow arrows in the merged view) (scale bar, 100µm). (D) Co-immunostaining of NF200 and MANF in normal and injured distal sciatic nerve of adult rats shows no remarkable co-localization of MANF with NF200 positive axons (scale bar, 100µm). (E) Co-immunostaining of GFAP and MANF in normal and injured distal sciatic nerve of adult rats shows expression of MANF in a few populations of SCs (yellow arrows in the merged view) (scale bar, 100µm).

Article Snippet: ELISA was performed using the commercially available MANF ELISA Kit (Cat No. EKL58888- 96T, Biomatik) according to the manufacturer’s instructions.

Techniques: Western Blot, Expressing, Control, Immunostaining, Staining

Journal: bioRxiv

Article Title: Schwann cells modified to secrete MANF is a potential cellular therapy for peripheral nerve regeneration

doi: 10.1101/2025.03.14.642904

Figure Lengend Snippet: (A) βIII tubulin staining shows that the supplementation of 50 and 100 ng/ml MANF promotes neurite outgrowth of injured and normal adult primary sensory neurons, respectively (scale bar, 100µm). (B) Quantification of neurite outgrowth using WIS-NeuroMath Software shows significant induction of neurite outgrowth in both normal and injured βIII tubulin positive neurons after MANF supplementation (data presented as mean ± SE; Standard ‘t’ test; n=3; **p<0.01, ***p<0.001). (C) NF200 staining shows that the supplementation of 50 and 100 ng/ml MANF promotes neurite outgrowth of injured and normal adult primary sensory neurons, respectively (scale bar, 100µm). (D) Quantification of neurite outgrowth using WIS-NeuroMath Software shows significant induction of neurite outgrowth in both normal and injured NF200 positive neurons after MANF supplementation (data presented as mean ± SE; Standard ‘t’ test; n=4; *p<0.05, **p<0.01).

Article Snippet: ELISA was performed using the commercially available MANF ELISA Kit (Cat No. EKL58888- 96T, Biomatik) according to the manufacturer’s instructions.

Techniques: Staining, Software

Journal: bioRxiv

Article Title: Schwann cells modified to secrete MANF is a potential cellular therapy for peripheral nerve regeneration

doi: 10.1101/2025.03.14.642904

Figure Lengend Snippet: (A) Immunostaining of GFAP in adult rat primary SCs (scale bar, 100µm). (B) Immunostaining of MANF in adult rat primary SCs. Yellow arrows show intense expression, and white arrows show weak to null expression (scale bar, 100µm). (C) MTT assay using adult rat primary SCs shows increased proliferation of cells in response to exogenous MANF supplementation (data presented as mean ± SE; One-Way ANOVA (Tukey’s multiple comparisons test); n=6; *p<0.05). (D) Brightfield images of the scratch assay using adult rat primary SCs show a time-dependent gap closure in control and MANF (50 ng/ml) supplemented groups (scale bar, 100µm). (E) Quantification of percentage gap closure in scratch assay shows increased gap closure in MANF (50 ng/ml) supplemented group compared to control (data presented as mean ± SE; Standard ‘t’ test; n=3; *p<0.05, **p<0.01). (F) Schematic of transwell migration assay. (G) DAPI staining on the lower side of the membrane from a transwell migration assay shows migrated adult primary SCs (scale bar, 100µm). (H) Quantification of transwell migration assay shows increased migration of primary SCs in MANF (50 ng/ml) supplemented group (data presented as mean ± SE; standard ‘t’ test; n=3; **p<0.01).

Article Snippet: ELISA was performed using the commercially available MANF ELISA Kit (Cat No. EKL58888- 96T, Biomatik) according to the manufacturer’s instructions.

Techniques: Immunostaining, Expressing, MTT Assay, Wound Healing Assay, Control, Transwell Migration Assay, Staining, Membrane, Migration

Journal: bioRxiv

Article Title: Schwann cells modified to secrete MANF is a potential cellular therapy for peripheral nerve regeneration

doi: 10.1101/2025.03.14.642904

Figure Lengend Snippet: (A) MTT assay using S16 SC line shows increased proliferation of cells in response to exogenous MANF (data presented as mean ± SE; One-Way ANOVA (Tukey’s multiple comparisons test); n=8; **p<0.01, ****p<0.0001). (B) DAPI staining on the lower side of the membrane from a transwell migration assay shows migrated S16 SCs at 48h in the control and MANF group (scale bar, 200µm). (C) Quantification of transwell migration assay shows increased migration of S16 SCs in MANF (100 ng/ml) supplemented group at 48h (data presented as mean ± SE; standard ‘t’ test; n=3; *p<0.05).

Article Snippet: ELISA was performed using the commercially available MANF ELISA Kit (Cat No. EKL58888- 96T, Biomatik) according to the manufacturer’s instructions.

Techniques: MTT Assay, Staining, Membrane, Transwell Migration Assay, Control, Migration

Journal: bioRxiv

Article Title: Schwann cells modified to secrete MANF is a potential cellular therapy for peripheral nerve regeneration

doi: 10.1101/2025.03.14.642904

Figure Lengend Snippet: (A) Schematic of in vivo nerve crush injury and local supplementation of MANF in adult mice. (B) Schematic of axon quantification approach. The number of axons crossing the perpendicular lines drawn at the shown distances are manually counted to tabulate the axon count at each distance. (C) βIII tubulin staining of sections of injured sciatic nerves from adult mice shows degenerated (blue arrows) and intact (green arrows) axons in the control and MANF group, respectively. White arrows show the crush site (scale bar, 200µm). (D) Quantification of intact axons in the distal nerve segment shows an increased number of axons in the MANF group compared to the control at varying distances from the crush site (data presented as mean ± SE; Two-Way ANOVA (Sidak’s multiple comparisons test); n=3; **p<0.01).

Article Snippet: ELISA was performed using the commercially available MANF ELISA Kit (Cat No. EKL58888- 96T, Biomatik) according to the manufacturer’s instructions.

Techniques: In Vivo, Staining, Control

Journal: bioRxiv

Article Title: Schwann cells modified to secrete MANF is a potential cellular therapy for peripheral nerve regeneration

doi: 10.1101/2025.03.14.642904

Figure Lengend Snippet: Maps of (A) pLVX TetOne Puro (empty vector) and (B) pLVX TetOne Puro MANF constructs.

Article Snippet: ELISA was performed using the commercially available MANF ELISA Kit (Cat No. EKL58888- 96T, Biomatik) according to the manufacturer’s instructions.

Techniques: Plasmid Preparation, Construct

Journal: bioRxiv

Article Title: Schwann cells modified to secrete MANF is a potential cellular therapy for peripheral nerve regeneration

doi: 10.1101/2025.03.14.642904

Figure Lengend Snippet: (A) SC-MANF after seven days of puromycin selection in a culture flask. (B ) SC- MANF grows healthy on an artificial nerve conduit (15-day). (C) ELISA assay shows increased secretion of MANF in the SC-MANF group compared to L-EV transduced primary SCs after Dox supplementation (data presented as mean ± SE; standard ‘t’ test; n=4). (D) Schematic of nerve crush injury experiment using DRG-nerve explant. (E) Immunostaining of βIII tubulin in sections of crush injured DRG-nerve explant (day 6) showing intact axons (yellow arrows) in the L-EV and L-MANF transduced groups. The white arrow shows DRG (scale bar, 200µm). (F) Quantification of axons in DRG-nerve explants on day 6 shows an increased number of intact axons in the L- MANF group compared to the L-EV group (data presented as mean ± SE; Two-Way ANOVA (Sidak’s multiple comparisons test); n=3; *p<0.05, **p<0.01, ***p<0.001). (G) Immunostaining shows the expression of MANF in L-EV and L-MANF transduced DRG-nerve explants cultured in the presence of Dox (1µg/ml). White arrows show the expression of MANF (green dots) in the nerve segment, and the red arrow shows MANF expression in DRG (scale bar, 500µm). Enlarged areas are provided in the insets. (H) A representative image shows the expression of MANF in SCs (SC-MANF generation) in the nerve segment of a DRG-nerve explant transduced with L-MANF and cultured in the presence of Dox.

Article Snippet: ELISA was performed using the commercially available MANF ELISA Kit (Cat No. EKL58888- 96T, Biomatik) according to the manufacturer’s instructions.

Techniques: Selection, Enzyme-linked Immunosorbent Assay, Immunostaining, Expressing, Cell Culture, Transduction

Journal: bioRxiv

Article Title: Schwann cells modified to secrete MANF is a potential cellular therapy for peripheral nerve regeneration

doi: 10.1101/2025.03.14.642904

Figure Lengend Snippet: (A) Schematic and timeline of the nerve crush injury experiment using DRG-nerve explants. (E) Immunostaining of GAP-43 in sections of DRG-nerve explants (day 15) shows longer regenerating axons (yellow arrows) past crush site (white arrow) in the L-MANF group compared the L-EV group (scale bar, 500µm). (B) Quantification of regenerating proximal and distal axons in DRG-nerve explants on day 15 shows a significant increase in regenerating distal axons in the L-MANF group compared to L-EV group (data presented as mean ± SE; Two-Way ANOVA (Sidak’s multiple comparisons test); n=3; *p<0.05, **p<0.01). (C) GFAP staining in sections of DRG-nerve explants (day 15) shows healthy distribution of SCs in the distal nerve of L-MANF group (scale bar, 500µm). (D) Immunostaining shows the expression of MANF in L-EV and L-MANF transduced DRG-nerve explants cultured in the presence of Dox (1µg/ml) for 15 days. Yellow arrows show the expression of MANF (green dots) in the nerve segment (scale bar, 100µm).

Article Snippet: ELISA was performed using the commercially available MANF ELISA Kit (Cat No. EKL58888- 96T, Biomatik) according to the manufacturer’s instructions.

Techniques: Immunostaining, Staining, Expressing, Cell Culture